Essay typer – FCA Cleveland http://fcacleveland.org/ Thu, 21 Oct 2021 04:32:28 +0000 en-US hourly 1 https://wordpress.org/?v=5.8 https://fcacleveland.org/wp-content/uploads/2021/10/icon-4-120x120.png Essay typer – FCA Cleveland http://fcacleveland.org/ 32 32 Isolating Switch Market Growth Report Explores Industry Trends, Future Growth, By Types, Share, Analysis 2028 | Schneider Electric, ABB, Siemens, GE, Mitsubishi Electric Corporation, Eaton, Rockwell Automation, Inc., Legrand, etc. – Puck77 https://fcacleveland.org/isolating-switch-market-growth-report-explores-industry-trends-future-growth-by-types-share-analysis-2028-schneider-electric-abb-siemens-ge-mitsubishi-electric-corporation-eaton-rockwell/ https://fcacleveland.org/isolating-switch-market-growth-report-explores-industry-trends-future-growth-by-types-share-analysis-2028-schneider-electric-abb-siemens-ge-mitsubishi-electric-corporation-eaton-rockwell/#respond Thu, 21 Oct 2021 04:21:17 +0000 https://fcacleveland.org/isolating-switch-market-growth-report-explores-industry-trends-future-growth-by-types-share-analysis-2028-schneider-electric-abb-siemens-ge-mitsubishi-electric-corporation-eaton-rockwell/ “ Global Isolation Switch Market Report The global isolation switches market is expected to grow from a market size of USD XX billion in 2020 to USD XX billion by 2028, with a compound annual growth rate (CAGR) of XX% during the forecast period . This report gives a better understanding of the Global Isolation […]]]>

Global Isolation Switch Market Report

The global isolation switches market is expected to grow from a market size of USD XX billion in 2020 to USD XX billion by 2028, with a compound annual growth rate (CAGR) of XX% during the forecast period . This report gives a better understanding of the Global Isolation Switch Market on the basis of applications, end-users, and geographic territories along with the competitive environment of these parts is explained in more detail.

Market research provides data on the growth factors and the difficulties that occasionally arise. Further, more information can be observed in this report such as current manufacturing methods, supply chain and its other segments, sales and revenue in terms of market share and on the basis of scenario and competition values.

As for the global distribution of Global Isolation Switch Market, it varies region to region, However, our analyst assesses the scope based on market share and describes better understanding value. Further, this report includes the data of the powerful companies which are currently dominating the market along with the major manufacturers.

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The best players, including:

By application:

Commercial

Industrial

In addition, the distribution channels in various regions, the distribution of resources, the advertising and pricing strategies implemented by the market as well as the potential competitors have been clearly explained.

Global Isolation Switch Market Segmentation

By Product Types of Industrial Isolation Switches Market:

By types:

Less than 100A

100 to 500A

501 to 1000A

Above 1000A

By Industrial Isolation Switch Market Applications:

By market players: Verdors:

Schneider Electric

ABB

Siemens

GE

Mitsubishi electric corporation

Eaton

Rockwell Automation, Inc.

Great

Hager

Hubbell

Zhejiang CHINT Electrics Co., Ltd.

Delixi

Shenglong electric group

Chinefato

Shanghai Liangxin Electrical Co., Ltd.

SHANGHAI HUAKUN ELECTRICAL CO., LTD.

Shanghai Xinchi Electric Co., Ltd.

Chang Song Electric Co., Ltd.

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Regional analysis

  • North America
  • Latin America
  • Europe
  • Asia Pacific
  • Middle East and Africa
  • Geographic area has been covered in this report

The report mentions not only detailed analysis of the market but also the various formats and techniques such as SWOT, PESTLE, and Porter’s five forces analysis. Moreover, the report also provides detailed information on the impact of COVID-19 and a global perspective on how to restore the situation that the market will witness.

The report covers a completely revised survey which specifies the precise restoration scenario for the market with maximum precision.

Contents:

  • Introduction, market definitions
  • Market trends and difficulties
  • Opportunities and Risks
  • Market segment categories
  • Best players and manufacturers
  • Insulation Switch Market Size, Share, Sales, Revenue
  • Past forecasts
  • Impact of Covid-19

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Note: The figures mentioned in the report may vary due to uncertainties related to the pandemic.

IMPACT Covid-19

Report covers impact of COVID-19 coronavirus: Since the COVID-19 virus outbreak in December 2019, the disease has spread to almost every country in the world, as declared by the World Health Organization public health emergency. The global impacts of the 2019 coronavirus disease (Covid-19) are already starting to be felt and will have a significant impact on the Isolating Switch market in 2020. The COVID-19 shutdown has had effects on many aspects, such as flight cancellations; travel bans and quarantines; closed restaurants; all indoor / outdoor events are restricted; more than forty countries have declared a state of emergency; massive downturn in the supply chain; volatility of stock markets; declining business confidence, growing public panic and uncertainty about the future.

About Us

About Contrive Datum Insights:

Contrive Datum Insights is the most comprehensive market research database. Serious researchers around the world seeking up-to-date information on the latest market trends with in-depth analysis turn to Contrive Datum Insights. Our huge database contains authentic reports published by leading authors and publications. Contrive Datum Insights’ highly motivated and expert in-house team conduct a rigorous review of publisher and author credentials before accepting their submissions. Such verification is imperative for internal quality control.

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Key words

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Terns Pharmaceuticals to Present Positive Clinical Data on NASH Programs at AASLD The Liver … | Business https://fcacleveland.org/terns-pharmaceuticals-to-present-positive-clinical-data-on-nash-programs-at-aasld-the-liver-business/ https://fcacleveland.org/terns-pharmaceuticals-to-present-positive-clinical-data-on-nash-programs-at-aasld-the-liver-business/#respond Mon, 18 Oct 2021 20:05:00 +0000 https://fcacleveland.org/terns-pharmaceuticals-to-present-positive-clinical-data-on-nash-programs-at-aasld-the-liver-business/ FOSTER CITY, Calif., October 18, 2021 (GLOBE NEWSWIRE) – Terns Pharmaceuticals, Inc. (“Terns” or the “Company”) (Nasdaq: TERN), a clinical-stage biopharmaceutical company developing a portfolio of small molecule candidates for monotherapy and in combination for the treatment of non-alcoholic steatohepatitis (NASH) and other chronic liver diseases, today announced that three abstracts detailing clinical data from […]]]>

FOSTER CITY, Calif., October 18, 2021 (GLOBE NEWSWIRE) – Terns Pharmaceuticals, Inc. (“Terns” or the “Company”) (Nasdaq: TERN), a clinical-stage biopharmaceutical company developing a portfolio of small molecule candidates for monotherapy and in combination for the treatment of non-alcoholic steatohepatitis (NASH) and other chronic liver diseases, today announced that three abstracts detailing clinical data from several non-alcoholic steatohepatitis (NASH) programs within the portfolio of the Company were accepted for presentation at The Liver Meeting® Digital Experience 2021, the annual meeting of the American Association for the Study of Liver Diseases (AASLD), to be held virtually November 12-15, 2021. Oral summaries and posters were published in the Hepatology Supplement, the AASLD’s peer-reviewed journal.

The details of the presentations at the Liver Meeting are as follows:

Oral presentation

Title: FXR Liver-Delivered Agonist TERN-101 Demonstrates Favorable Safety and Efficacy Profile in NASH Phase 2a LIFT Study Publication Number: 143 Session Title: Parallel 21: NAFLD and NASH: Clinical Trials of Novel Therapeutics Presenting Author: Rohit Loomba Date and Time: Sunday, November 14, 6:30 p.m. ET

Poster presentations

Title: Single doses of the THR-β agonist TERN-501 are well tolerated and result in dose-dependent changes in LDL cholesterol and sex hormone binding globulin in a novel human clinical trial Presentation number: 1889 Session title: NAFLD and NASH: Experimental: Clinical Presenting author: D. Barry Crittenden

Title: Liver-distributed FXR Agonist TERN-101 Leads to Corrected T1 (cT1) Response and a Population Shift to Lower cT1 Risk Categories in NASH Phase 2a LIFT Study Publication Number: 1875 Session Title: NAFLD and NASH: Experimental: Clinical Presenting Author: Eric Lawitz

About TERN-101 TERN-101 is a non-bile acid, liver-distributed FXR agonist that has demonstrated a differentiated tolerance profile and improved target engagement, possibly due to its sustained activation of FXR in the liver. liver, but only its transient activation of FXR in the intestine. FXR is a nuclear receptor primarily expressed in the liver, intestine, and kidneys. FXR regulates the hepatic expression of various genes involved in lipid metabolism, inflammation and fibrosis. Clinical studies with other FXR agonists have shown significant histological improvements in NASH, but have also resulted in pruritus, unwanted lipid changes and discontinuation of treatment. Terns reported the first positive results from the Phase 2a LIFT study of TERN-101 in June 2021.

About TERN-501 TERN-501 is a thyroid hormone receptor (THR-β) beta agonist with high metabolic stability, improved hepatic distribution and greater selectivity for THR-β compared to other THR agonists -β under development. THR-β agonism increases fatty acid metabolism via mitochondrial oxidation and affects cholesterol synthesis and metabolism. As a result, stimulation of THR-β has the ability to reduce hepatic steatosis and improve serum lipid parameters, especially LDL cholesterol and triglycerides. In vivo NASH studies in a rodent model demonstrated that low doses of TERN-501 achieved complete resolution of steatosis and reductions in serum lipids, liver inflammation and fibrosis. TERN-501 has a high hepatic distribution and is 23 times more selective for activation of THR-β than for activation of THR-α in a cell-free assay, thus minimizing the risk of cardiotoxicity and other side effects. target associated with non-selective THR stimulation. Finally, TERN-501 has been designed to be metabolically stable and is therefore expected to exhibit low pharmacokinetic variability and low clinical dose, making it an attractive candidate for use in fixed dose combinations for the treatment of NASH.

About Terns Pharmaceuticals Terns Pharmaceuticals, Inc. is a clinical-stage biopharmaceutical company developing a portfolio of small molecule candidates in monotherapy and in combination for the treatment of non-alcoholic steatohepatitis, or NASH, and other chronic liver diseases . Terns’ portfolio includes three clinical phase development programs, including an FXR agonist, a VAP-1 inhibitor and a THR-β agonist, as well as a preclinical GLP-1 receptor agonist program. Terns is focused on the development of clinically validated, complementary mechanisms of action-based combination therapies to treat the multiple liver disease processes of NASH in order to generate significant clinical benefits for patients. For more information, please visit: www.ternspharma.com.

Caution Regarding Forward-Looking Statements This press release contains forward-looking statements about Terns Pharmaceuticals, Inc. (the “Company”, “we”, “us” or “our”) within the meaning of federal securities laws, including including those related to the Company’s expectations regarding the timing and potential results of clinical trials and other Company development activities, as well as the potential usefulness and progress of the Company’s product candidates in NASH. All statements other than statements of historical fact contained in this press release, including statements regarding the strategy of the Company, future financial condition, future operations, projected costs, prospects, plans, objectives of management and expected market growth are forward-looking statements. In some cases, you may identify forward-looking statements by words such as “aim”, “anticipate”, “assume”, “believe”, “contemplate”, “continue”, “could”, “conceive”, “,” “Estimate”, “expect”, “objective”, “intend”, “power”, “objective”, “plan”, “,”, “,”, “of of of” “target” , “Will”, “would” and other similar expressions which are predictions or indicate future events and future trends, or the negative of these terms or other comparable terminology. The Company has based these forward-looking statements in large part on its current expectations, estimates, forecasts and projections regarding future events and financial trends that it believes could affect its financial condition, results of operations, business strategy and financial needs. In light of the material uncertainties in these forward-looking statements, you should not rely on forward-looking statements as predictions of future events. These statements are subject to risks and uncertainties which could cause actual results and the implementation of the Company’s plans to vary materially, including risks associated with the launch, costs, timing, progress and results of operations. current and future research and development activities of the Company. and preclinical studies and clinical trials. In particular, the impact of the COVID-19 pandemic on the Company’s ability to advance its research, development, manufacturing and regulatory efforts, including the Company’s clinical trials for its product candidates, will depend on future developments that are highly uncertain and cannot be predicted with confidence at this time, such as the ultimate duration of the pandemic, travel restrictions, quarantines, social distancing requirements and state business closures -United and in other countries, and the effectiveness of measures taken globally to contain and treat the disease. These risks are not exhaustive. For a detailed discussion of risk factors that could affect the actual results of the Company, please refer to the risk factors identified in the Company’s SEC reports, including, but not limited to, its annual report on Form 10 -K for the fiscal year ended December 31, 2020. and its quarterly reports on form 10-Q for the periods ended March 31, 2021 and June 30, 2021. Unless required by law, the Company does not undertake to publicly update forward-looking statements for any reason.

Contacts for terns

Investors Justin Ng investors@ternspharma.com

Media Jenna Urban Berry & Company Public Relations media@ternspharma.com

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Apoptosis Test Kits Market Size, Outlook, and Key Companies – Abcom plc, Merck KGaA (Germany), Thermo Fisher Scientific (US), Bio-Rad Laboratories (US), GE Healthcare (US), Danaher Corporation (US) ) https://fcacleveland.org/apoptosis-test-kits-market-size-outlook-and-key-companies-abcom-plc-merck-kgaa-germany-thermo-fisher-scientific-us-bio-rad-laboratories-us-ge-healthcare-us-danaher-corporation-us/ https://fcacleveland.org/apoptosis-test-kits-market-size-outlook-and-key-companies-abcom-plc-merck-kgaa-germany-thermo-fisher-scientific-us-bio-rad-laboratories-us-ge-healthcare-us-danaher-corporation-us/#respond Sun, 17 Oct 2021 22:34:31 +0000 https://fcacleveland.org/apoptosis-test-kits-market-size-outlook-and-key-companies-abcom-plc-merck-kgaa-germany-thermo-fisher-scientific-us-bio-rad-laboratories-us-ge-healthcare-us-danaher-corporation-us/ New Jersey, United States, – The Apoptosis Test Kits Market The report comprises broadly analyzed data and information on the Apoptosis Test Kit market with detailed analysis of key models, emerging trends, infrastructure properties, industrial landscape, and key market segments. The report provides a comprehensive analysis of the market growth and factors influencing the market […]]]>

New Jersey, United States, – The Apoptosis Test Kits Market The report comprises broadly analyzed data and information on the Apoptosis Test Kit market with detailed analysis of key models, emerging trends, infrastructure properties, industrial landscape, and key market segments. The report provides a comprehensive analysis of the market growth and factors influencing the market growth including the latest technology and product developments. The Apoptosis Test Kit market report also provides an overview of the industry position in the international market and its contribution to generating global revenue. The report provides significant statistical sales data, revenue estimates based on types and applications, regions, and major market players.

The report covers the analysis of the impact of the COVID-19 pandemic on the global market. The pandemic has dynamically altered the economic landscape and drastically changed the trends and demands for products and services in the market. The report also covers the current and future impact of the pandemic and provides an overview of the post-COVID-19 scenario.

The market size for Apoptosis Test Kits was valued at USD 4.26 billion in 2020 and is expected to reach USD 9.26 billion by 2028, with a CAGR of 10.2% from 2021 to 2028.

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The report covers an in-depth analysis of the major market players in the market, along with their business overview, expansion plans, and strategies. The major players studied in the report include:

Abcom plc, Merck KGaA (Germany), Thermo Fisher Scientific (United States), Bio-Rad Laboratories (United States), GE Healthcare (United States), Danaher Corporation (United States), Becton, Dickinson and Company ( United States), Sartorius AG (Germany), Geno Technology (United States), Gene Copoeia, Inc. (United States), Promega Corporation (United States).

Additionally, the report provides insightful information on key manufacturers and players and the business sphere. It covers data on the latest trade movements, product launches, technological advancements, mergers and acquisitions, partnerships and joint ventures. In-depth assessment of production and manufacturing capacity, industry chain analysis, market share, size, revenue, sales, growth rate and market share are also offered. by the report. The market assessment and CAGR are also included in the report to provide a comprehensive portrait of the Apoptosis Test Kit market.

Apoptosis Test Kits Market Segmentation

Apoptosis Test Kits Market, By Product

• Kits
• Reagents
• Instruments
• Others

Apoptosis Test Kits Market, By Application

• Drug discovery and development
• Stem cell research
• Others

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Scope of the Apoptosis Test Kit Market Report

ATTRIBUTE DETAILS
ESTIMATED YEAR 2021
YEAR OF REFERENCE 2020
PLANNED YEAR 2028
HISTORICAL YEAR 2019
UNITY Value (million USD / billion)
COVERED SEGMENTS Types, applications, end users, etc.
COVER OF THE REPORT Revenue forecast, company ranking, competitive landscape, growth factors and trends
BY REGION North America, Europe, Asia-Pacific, Latin America, Middle East and Africa
CUSTOMIZATION SCOPE Free customization of the report (equivalent to 4 working days for analysts) with purchase. Add or change the scope of country, region and segment.

Geographic segment covered in the report:

The Apoptosis Test Kit report provides information about the market area which is further subdivided into sub-regions and countries / regions. In addition to the market share in each country and sub-region, this chapter of this report also contains information on profit opportunities. This chapter of the report mentions the market share and growth rate of each region, country and sub-region during the estimated period.

• North America (United States and Canada)
• Europe (UK, Germany, France and rest of Europe)
• Asia-Pacific (China, Japan, India and the rest of the Asia-Pacific region)
• Latin America (Brazil, Mexico and the rest of Latin America)
• Middle East and Africa (GCC and rest of Middle East and Africa)

Key questions answered in the report:

• What is the growth potential of the Apoptosis Test Kit market?
• Which product segment will take the lion’s share?
• Which regional market will emerge as a pioneer in the years to come?
• Which application segment will experience strong growth?
• What growth opportunities might arise in the Apoptosis Test Kits industry in the years to come?
• What are the most significant challenges facing the Apoptosis Test Kits market in the future?
• Who are the leading companies in the Apoptosis Assay Kits market?
• What are the main trends that positively impact the growth of the market?
• What growth strategies are the players planning to stay in the Apoptosis Test Kit market?

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FDA updates for week of October 11, 2021 https://fcacleveland.org/fda-updates-for-week-of-october-11-2021/ https://fcacleveland.org/fda-updates-for-week-of-october-11-2021/#respond Sat, 16 Oct 2021 12:08:51 +0000 https://fcacleveland.org/fda-updates-for-week-of-october-11-2021/ In COVID-19 News: Advisory Board recommends both J&J and Moderna recall, Regeneron seeks full approval for its treatment, and Merck requests EUA for treatment. In cancer, the FDA approves Verzenio for early breast cancer, Keytruda for advanced cervical cancer, and Tecentriq for early non-small cell lung cancer. Advisory committee recommends Moderna COVID-19 recall. The FDA’s […]]]>

In COVID-19 News: Advisory Board recommends both J&J and Moderna recall, Regeneron seeks full approval for its treatment, and Merck requests EUA for treatment. In cancer, the FDA approves Verzenio for early breast cancer, Keytruda for advanced cervical cancer, and Tecentriq for early non-small cell lung cancer.

Advisory committee recommends Moderna COVID-19 recall.

The FDA’s Advisory Committee on Vaccines and Related Biologics Recommended a Booster of Moderna’s COVID-19 Vaccine for High-Risk People Over 65 and 18-64 Exposed in the Workplace or high risk. All 19 committee members voted in favor of the booster dose, which will be given at least six months after the second dose in the first series. The booster will be at a dose level that is half of what the two doses are.

Separately, on August 13, the FDA approved an update to the emergency use authorization for the Moderna COVID-19 vaccine to include a third dose at the dose level of 100 µg for immunocompromised people aged 18 or over. most in the United States who have undergone solid organ transplants, or who are diagnosed with conditions that are considered to have an equivalent level of immunosuppression.

The FDA Advisory Committee recommends approval of the J&J COVID-19 recall.

The FDA’s Advisory Committee on Vaccines and Related Biologics has recommended authorizing a second dose of Johnson & Johnson vaccine as early as two months after the initial dose.

J&J had submitted data to support its emergency use authorization request in early October 2021. The submission includes recent results from the Phase 3 ENSEMBLE 2 study which found a booster of the J&J COVID- vaccine. 19 given 56 days after primary dose provided 94% protection against moderate to severe / critical COVID-19 and 100% protection against severe / critical COVID-19, at least 14 days after booster vaccination.

Regeneron is seeking full approval for COVID-19 treatment.

The FDA has accepted Regeneron’s request for full approval of REGEN-COV (casirivimab and imdevimab) to treat COVID-19 in outpatients and as prophylaxis in certain individuals. The FDA has set a target action date of April 13, 2022. The agency plans to hold an advisory committee meeting to discuss this request before that date.

The biologics license application is supported by two positive phase 3 trials involving more than 6,000 patients in outpatients and on prophylaxis. A second BLA submission focused on treating patients hospitalized with COVID-19 is expected to be submitted later this year.

REGEN-COV is currently available under emergency use authorization to treat people with mild to moderate COVID-19 who are at high risk of serious consequences from COVID-19 infection and who are either already infected (not hospitalized) or at a certain post. -exposure prophylaxis parameters

Merck seeks EUA treatment for COVID-19.

Merck has submitted an Emergency Use Clearance (EUA) application to the FDA for molnupiravir, an investigational oral antiviral drug, for the treatment of mild to moderate COVID-19 in adults at risk of progressing to COVID -19 severe and / or hospitalization.

Submission is based on positive results from a planned interim analysis of the MOVe-OUT Phase 3 clinical trial. In the interim analysis, molnupiravir reduced the risk of hospitalization or death by approximately 50%. The incidence of drug-related adverse events was 12% in the molnupiravir group versus 11% in the placebo cohort, and fewer subjects in the molnupiravir group discontinued treatment due to an adverse event compared to the placebo group.

The FDA approves Keytruda for advanced cervical cancer.

The FDA has approved Keytruda (pembrolizumab), Merck’s anti-PD-1 treatment, in combination with chemotherapy, with or without Roche’s Avastin (bevacizumab), for the treatment of patients with cervical cancer. persistent, recurrent or metastatic uterus.

The approval is based on the phase 3 KEYNOTE-826 trial, which found that Keytruda plus chemotherapy (paclitaxel plus cisplatin or paclitaxel plus carboplatin), with or without Avastin demonstrated overall survival and progression-free survival.

In addition, more patients responded in the Keytruda arm than to chemotherapy with or without Avastin, with an objective response rate of 68%. Among the patients who responded, the median duration of response was 18.0 months.

The FDA clears Verzenio de Lilly for early breast cancer.

The FDA has approved Eli Lilly and Company’s Verzenio (abemaciclib), the first and only CDK4 / 6 inhibitor approved for patients with specific types of breast cancer.

Verzenio, in combination with hormone therapy (tamoxifen or an aromatase inhibitor), is indicated for the adjuvant treatment of patients with positive hormone receptors (HR +), human epidermal growth factor receptors 2-negative (HER2-), lymph nodes, early breast cancer and high risk of recurrence a Ki-67 score of ≥20%, Lilly said in a press release.

In addition, the FDA has approved Agilent Technologies’ Ki-67 IHC MIB-1 pharmDx (Dako Omnis) as an aid in the identification of early breast cancer patients at high risk of disease recurrence. It is the first IHC test measuring Ki-67 expression to receive FDA approval for treatment with Verzenio and was developed in collaboration with Lilly.

The FDA approves Tecentriq for the early treatment of non-small cell lung cancer.

The FDA has approved Genentech’s Tecentriq (atezolizumab) as a post-surgery and chemotherapy treatment for adults with stage II-IIIA non-small cell lung cancer (NSCLC).

The approval is based on the results of an interim analysis from the phase 3 IMpower010 study which showed that treatment with Tecentriq after platinum-based surgery and chemotherapy reduced the risk of disease recurrence or death. 34% in people with stage II-IIIA NSCLC whose tumors express PD-L1≥1%, compared to best supportive care.

Tecentriq’s safety data was consistent with its known safety profile and no new safety signals were identified. Fatal and serious side effects occurred in 1.8% and 18%, respectively, of patients receiving Tecentriq. The most common serious side effects were pneumonia, pneumonia and fever.

Ocular Therapeutix ophthalmic inserts are gaining in indication for itchy eyes.

Ocular Therapeutix today announced that the FDA has expanded the approval of its dexamethasone ophthalmic inserts to include an indication for the treatment of itchy eyes associated with allergic conjunctivitis.

The inserts, sold under the Dextenza brand, were initially approved in November 2018 as a treatment for eye pain after ophthalmic surgery with post-surgical eye inflammation treatment added as an indication in June 2019.

FDA approves first treatment for rare immune disorders.

The FDA has approved Enzyvant Therapeutics’ Rethymic (allogenically-agdc-treated thymus tissue), a unique therapy for immune reconstitution in pediatric patients with congenital athymia. Rethymic is human thymic tissue designed to regenerate thymic function.

Pediatric congenital athymia is ultra-rare with an estimated incidence of approximately 17 to 24 live births each year in the United States. Children who have this condition are born without a thymus. The thymus is part of the lymphatic system and plays a central role in the immune system. Children born with this disease are immunocompromised and are very susceptible to life-threatening infections.

Otsuka and Lundbeck submit sNDA for Rexulti in adolescent with schizophrenia.

The FDA has accepted an additional new drug application (sNDA) for the treatment of schizophrenia in adolescents with Rexulti (brexpiprazole). The FDA has granted priority review to Otsuka and Lundbeck.

Up to a third of patients with schizophrenia develop the disease during adolescence. Currently, Rexulti is approved in the United States for the treatment of schizophrenia in adults and the adjunct of major depressive disorder in adults.

The submission was completed a year ahead of schedule. This was made possible by performing an extrapolation analysis using data from previous studies in adult patients, pharmacokinetic results from adult and pediatric trials, and six-month data from the long-term open-label trial. ongoing in adolescent patients with schizophrenia.


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New high-throughput test could help anti-COVID-19 drug development https://fcacleveland.org/new-high-throughput-test-could-help-anti-covid-19-drug-development/ https://fcacleveland.org/new-high-throughput-test-could-help-anti-covid-19-drug-development/#respond Wed, 13 Oct 2021 22:57:00 +0000 https://fcacleveland.org/new-high-throughput-test-could-help-anti-covid-19-drug-development/ The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes four structural proteins, including the core (N), tip (S), membrane (M), and envelope proteins. The S protein is essential for the pathogenicity of SARS-CoV-2 because the receptor binding domain (RBD) of the S1 subunit of this protein binds to the angiotensin-2 converting enzyme receptor ( […]]]>

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes four structural proteins, including the core (N), tip (S), membrane (M), and envelope proteins. The S protein is essential for the pathogenicity of SARS-CoV-2 because the receptor binding domain (RBD) of the S1 subunit of this protein binds to the angiotensin-2 converting enzyme receptor ( ACE2) on the host cell to allow viral entry. Comparatively, the N-terminal domain of the S2 subunit is required for membrane fusion.

For long-term drug development purposes, testing targeting only these few sites is likely to be insufficient. Many complicated viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus, can adapt quickly to new drugs; therefore, a combination of drugs is needed to combat them effectively. In a recent study published on the Preprint Server bioRxiv *, the researchers are discussing their development of a new high-throughput assay that targets a well-conserved macrodomain within a non-structural protein, allowing rapid screening for potential inhibitors.

To study: High throughput activity assay for screening for macrodomain inhibitors SARS-CoV-2 Mac1. Image Credit: souvikonline200521 / Shutterstock.com

Background

Mac1 is a highly conserved macrodomain ribosylhydrolase of adenosine diphosphate (ADP) within nonstructural protein 3 (nsp3). A macrodomain is a protein fold observed in humans, as well as in many pathogens. Almost all macrodomains bind to ADP-ribose.

SARS-CoV-2, along with two other human coronaviruses like SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), contain two or three macrodomains in which the first (Mac1) can bind to ADP-ribosylhydrolase. Similar domains are also seen in alphaviruses, which is another group of viruses that scientists say has the potential to cause a pandemic.

When these macrodomains are mutated, viral replication is inhibited and virulence decreased. The ability to fight multiple diseases, as well as inhibit pathogenesis, makes Mac1 an excellent drug target.

About the study

Initially, the researchers attempted to find out whether it was possible to inhibit the Mac1 viral domain without also inhibiting the very similar human MacroD2 domain. These two domains are classified as MacroD type domains.

Chikungunya virus has a similar macrodomain that can hydrolyze ADP-ribose from the PARP10 catalytic domain and the G3BP1 protein of cells. The tests confirmed that the human and SARS-CoV-2 domains could replicate this ability, solidifying them as active ADP-ribosylhydrolases.

The researchers then developed a luminescence-based test to measure enzyme activity. When ADP-ribose is released from the protein substrate by the macrodomain, phosphodiesterase NudF cleaves ADP-ribose, which leads to the production of phosphoribose and AMP.

As luminescence will only be visible when ADP-ribose is free, the luminescence signal can be controlled by the level of enzymatic activity of the macrodomain. Using this method, the scientists identified different binding and kinetic properties for both the human MacroD2 domain and the SARS-CoV-2 Mac1 domain, allowing the study of selective inhibitors of the Mac1 domain.

Study results

The researchers compared the structures of the two domains to find that about 60% of the residues at the binding sites are retained. Upon closer examination, distinct surfaces in the active site that binds ADP-ribose showed key differences, such as Mac1 is larger and has more charged surfaces than MacroD2.

Following this, the researchers identified 3,233 potential candidates for selective Mac1 inhibitors, which were then used for a pilot screening. A total of 37 of these compounds were found to exhibit reproducible inhibition of Mac1 at a concentration of 100 micromolar (µM).

The authors have created an assay that can successfully identify ADP-ribosyhydrolase inhibitors and easily distinguish between those that can selectively inhibit the target and those that are more general. While many of the compounds identified showed cytotoxicity at the concentrations used, the researchers confirmed the proof of concept for this test.

While the current pandemic is slowly being brought under control through the use of mass vaccination programs, as well as continued social distancing and lockdown restrictions, coronaviruses are sure to remain a threat for years to come. The test discussed here could therefore prove to be a powerful tool to help develop drugs, against coronaviruses, as well as alphaviruses. In addition, the researchers hope that this test could also be used to develop broad-spectrum antivirals, which is a currently neglected area of ​​research that has recently received significant investment.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information


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Rapid Advancements of Innovative Antiviral https://fcacleveland.org/rapid-advancements-of-innovative-antiviral/ https://fcacleveland.org/rapid-advancements-of-innovative-antiviral/#respond Tue, 12 Oct 2021 08:06:30 +0000 https://fcacleveland.org/?p=285 PALM BEACH,  Fla., Oct. 11, 2021 /PRNewswire/ — With the arrival of the COVID-19 virus, the antivirals market had all eyes watching the ongoing frenetic rush to find a cure or at least a therapy. That race has not yet abated… not at all! Substantial increase in number of patients suffering from viral infections and […]]]>

PALM BEACH,  Fla., Oct. 11, 2021 /PRNewswire/ — With the arrival of the COVID-19 virus, the antivirals market had all eyes watching the ongoing frenetic rush to find a cure or at least a therapy. That race has not yet abated… not at all! Substantial increase in number of patients suffering from viral infections and increasing R&D activities by major players for development of novel products are major factors expected to drive the growth of global antiviral drugs market. In US the proportion of outpatient visits for influenza-like illness increased. In fact, a report from Acumen Research and Consulting projected that the global antiviral drugs market is anticipated to grow at a CAGR of around 3.7% during the forecast period 2020 to 2027 and to reach around US$ 74.7 billion by 2027.  The report added: “Increase in pandemic effect across the globe and rise in mortality rate are pressurizing the government to take necessary steps for drug development. Major players are focused on conducting rapid clinical trials for development of new drug, along with early product approval from the government as a preventive measure for treatment of covid-19 patients are factors cumulatively expected to augment the market growth.”  Active Biotechs in the markets today include:  NanoViricides, Inc. (NYSE: NNVC), Pfizer Inc. (NYSE: PFE), Merck (NYSE: MRK), Regeneron Pharmaceuticals, Inc. (NASDAQ: REGN), Eli Lilly and Company (NYSE: LLY).

Acumen Research article continued with:  “High investment by major players for new drug development and business development activities in order to enhance the business presence and increase the customer base is expected to boost the growth of antiviral drugs market… increasing investment by major players for cost effective drug development and supporting government regulations related to product approval and business establishment are factors expected to create new opportunities for players operating in the antiviral drugs market. In addition, increasing partnership and agreements between regional and international players is expected to support the revenue transaction of the target market. The market in North America is expected to account for major revenue share in the global drugs market due to rise in number of patients suffering from chronic diseases. In addition, availability of advanced infrastructure in the country, increasing R&D activities, and focus on introduction of innovative drugs are factors expected to impact the growth of antiviral drugs market.”

NanoViricides, Inc. (NYSE American: NNVC) Breaking News: NanoViricides Announces COVID-19 Clinical Drug Candidate NV-CoV-2 was Effective Against SARS-CoV-2, Further Demonstrating Its Broad-Spectrum Pan-Coronavirus Activity NanoViricides, a leader in the development of highly effective antiviral therapies based on a novel nanomedicines technology, announced today that its Pan-Coronavirus COVID-19 Drug Candidate.

NV-CoV-2 was found to be effective against SARS-CoV-2 in a standard cell culture pseudovirion assay, demonstrating that the drug indeed has broad-spectrum pan-coronavirus activity. This pan-coronavirus activity implies that the drug NV-CoV-2 should remain active in spite of evolution of variants of

SARS-CoV-2 in the field, a highly sought-after characteristic to combat the current global pandemic. 

In this assay, both the drug candidate NV-CoV-2 and a positive control antibody specific to the Spike antigen S1 of the SARS-CoV-2 virus suppressed the infection by the SARS-CoV-2-pseudovirions in cell culture studies to virtually the same baseline levels.

We have now demonstrated that NV-CoV-2 is highly effective in cell cultures against SARS-CoV-2, human coronavirus NL-63, and human coronavirus 229E, all very different human coronaviruses. These results imply that the drug will remain active in spite of novel variants of SARS-CoV-2 evolution in the field, and indeed demonstrate the pan-coronavirus activity of our clinical drug candidate NV-CoV-2.

Additionally, the pseudovirion study also showed that NV-CoV-2 neutralizes the virus particles themselves, outside of the cells, validating our design mechanism.

“We are now preparing submission documents to enable initiation of human clinical trials,” commented Dr. Anil Diwan, Chairman and President of the Company, adding, “We believe that NV-CoV-2 may help end the pandemic if it is shown to be effective in human clinical trials.”

A strong SARS-CoV-2 infection inhibition activity of NV-CoV-2 was observed in this pseudovirion study. Pseudovirion assay is a standard method for evaluating virus entry-inhibitors in BSL2 laboratories and is primarily used for viruses that would otherwise require high security BSL3 or BSL4 laboratories.  In this study, SARS-CoV-2-pseudovirion virus particles were made that carry a green fluorescent protein (GFP) producer mRNA inside, and use the SARS-CoV-2 S1 protein on their surface to bind to ACE2 receptor protein on cells. They were incubated with NV-CoV-2 (test article), or a known neutralizing antibody (positive control), or just the vehicle buffer (negative control). Then these solutions were separately used to infect ACE2 positive cells and the cultures were incubated. Only the infected cells produced GFP and were visualized by green fluorescence in microscopy. In this well-known assay, NV-CoV-2 was as effective as the neutralizing antibody in reducing the virus infection. This study demonstrates that NV-CoV-2 attacks the SARS-CoV-2 pseudovirion particles and renders them incapable of binding to the ACE2 positive cells.

A “pseudovirion” is a virus particle made of a BSL-2 virus shell, but with its original cell-binding protein replaced by the cell binding protein of a BSL3 or BSL4 virus, in this case, the S1 antigen of SARS-CoV-2.  Additionally, the pseudovirion particle contains an mRNA that is packaged like the original virus, except that the mRNA is edited and redesigned so that it cannot produce infectious virus particles. In our study, this mRNA allowed expression and production of the green fluorescent protein (GFP) enabling visual detection of the infected cells (green) in microscopy.  Read the full press release by going to:  http://www.nanoviricides.com/companynews.html 

In other biotech news in the markets this week: 

Merck (NYSE: MRK) recently announced that molnupiravir (MK-4482, EIDD-2801), an investigational oral antiviral medicine, significantly reduced the risk of hospitalization or death at a planned interim analysis of the Phase 3 MOVe-OUT trial in at risk, non-hospitalized adult patients with mild-to-moderate COVID-19. At the interim analysis, molnupiravir reduced the risk of hospitalization or death by approximately 50%; 7.3% of patients who received molnupiravir were either hospitalized or died through Day 29 following randomization (28/385), compared with 14.1% of placebo-treated patients (53/377); p=0.0012. Through Day 29, no deaths were reported in patients who received molnupiravir, as compared to 8 deaths in patients who received placebo. At the recommendation of an independent Data Monitoring Committee and in consultation with the U.S. Food and Drug Administration (FDA), recruitment into the study is being stopped early due to these positive results. Merck plans to submit an application for Emergency Use Authorization (EUA) to the U.S. FDA as soon as possible based on these findings and plans to submit marketing applications to other regulatory bodies worldwide.

Pfizer Inc. (NYSE: PFE) and BioNTech SE (Nasdaq: BNTX) recently announced they have submitted data to the U.S. Food and Drug Administration (FDA) from the Phase 2/3 trial of their COVID-19 vaccine in children 5 to <12 years of age.

The Companies announced positive topline results from the pivotal trial on September 20, 2021. In the trial, which included 2,268 participants 5 to <12 years of age, the vaccine demonstrated a favorable safety profile and elicited robust neutralizing antibody responses using a two-dose regimen of 10 μg doses. These results – the first from a pivotal trial of any COVID-19 vaccine in this age group – were comparable to those recorded in a previous Pfizer-BioNTech study in people 16 to 25 years of age, who were immunized with 30 μg doses. The 10 μg dose was carefully selected as the preferred dose for safety, tolerability and immunogenicity in children 5 to <12 years of age.

Regeneron Pharmaceuticals, Inc. (NASDAQ: REGN) recently announced that the New England Journal of Medicine (NEJM) published positive detailed results from a Phase 3 trial that assessed the ability of REGEN-COV™ (casirivimab and imdevimab) to treat COVID-19 in infected high-risk non-hospitalized patients (outpatients). The trial met its primary and all secondary endpoints and showed treatment with REGEN-COV significantly reduced the risk of hospitalization or death, with a safety profile consistent with previously reported data.

“Results from this NEJM publication show that REGEN-COV reduced the risk of hospitalization or death by 70% in high-risk non-hospitalized patients infected with COVID-19,” said George D. Yancopoulos, M.D., Ph.D., President and Chief Scientific Officer at Regeneron. “This peer-reviewed publication further supports the growing evidence on REGEN-COV’s critical role to improve outcomes for patients, and reduce the significant burden on their communities and healthcare systems during this COVID-19 pandemic.”

Eli Lilly and Company (NYSE: LLY) recently announced that the U.S. Food and Drug Administration (FDA) has granted approval of a new indication for ERBITUX® (cetuximab injection) in combination with BRAFTOVI®(encorafenib), marketed by Pfizer, Inc., for the treatment of adult patients with metastatic colorectal cancer (CRC) with a BRAF V600E mutation, as detected by an FDA-approved test, after prior therapy.1ERBITUX is the first and only anti-EGFR antibody approved, in combination with encorafenib, for this indication and is based on results from Pfizer’s BEACON CRC trial, the only Phase 3 trial to specifically study patients with previously treated metastatic CRC with a BRAF V600E mutation. With this approval, ERBITUX has now received seven FDA approvals to treat certain types of CRC and squamous cell carcinoma of the head and neck.

“The BEACON study showed that the combination of ERBITUX and encorafenib significantly improved overall survival in patients with metastatic colorectal cancer with a BRAF V600E mutation – a subtype that typically has worse outcomes compared to those without the mutation,” said David Hyman, M.D., chief medical officer, oncology at Lilly. “We are grateful to Pfizer for their collaboration as we’ve worked to bring this treatment regimen to patients.”

DISCLAIMER:  FN Media Group LLC (FNM), which owns and operates Financialnewsmedia.com and MarketNewsUpdates.com, is a third- party publisher and news dissemination service provider, which disseminates electronic information through multiple online media channels.  FNM is NOT affiliated in any manner with any company mentioned herein.  FNM and its affiliated companies are a news dissemination solutions provider and are NOT a registered broker/dealer/analyst/adviser, holds no investment licenses and may NOT sell, offer to sell or offer to buy any security.  FNM’s market updates, news alerts and corporate profiles are NOT a solicitation or recommendation to buy, sell or hold securities.  The material in this release is intended to be strictly informational and is NEVER to be construed or interpreted as research material.  All readers are strongly urged to perform research and due diligence on their own and consult a licensed financial professional before considering any level of investing in stocks.  All material included herein is republished content and details which were previously disseminated by the companies mentioned in this release.  FNM is not liable for any investment decisions by its readers or subscribers.  Investors are cautioned that they may lose all or a portion of their investment when investing in stocks.  For current services performed FNM was compensated twenty five hundred dollars for news coverage of current press release issued by NanoViricides, Inc. by a non-affiliated third party.   FNM HOLDS NO SHARES OF ANY COMPANY NAMED IN THIS RELEASE.

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Adult Asthmatics With Urine Levels of Leukotriene E4 https://fcacleveland.org/adult-asthmatics-with-urine-levels-of-leukotriene-e4/ https://fcacleveland.org/adult-asthmatics-with-urine-levels-of-leukotriene-e4/#respond Tue, 12 Oct 2021 08:06:23 +0000 https://fcacleveland.org/?p=291 Introduction Asthma is a heterogeneous disease with many clinical phenotypes characterized by variable airflow obstruction, chronic airway inflammation and bronchial hyperresponsiveness. Asthma is usually controlled by the regular use of an inhaled corticosteroid (ICS) with or without a long-acting β2-agonist (LABA) or a leukotriene receptor antagonist (LTRA). Despite the standard asthma treatment, about 10% to […]]]>

Introduction

Asthma is a heterogeneous disease with many clinical phenotypes characterized by variable airflow obstruction, chronic airway inflammation and bronchial hyperresponsiveness. Asthma is usually controlled by the regular use of an inhaled corticosteroid (ICS) with or without a long-acting β2-agonist (LABA) or a leukotriene receptor antagonist (LTRA). Despite the standard asthma treatment, about 10% to 20% of patients still remain uncontrolled, often with frequent exacerbations.1,2

The pathophysiology of asthma is complex, and multicellular inflammatory processes including eosinophils, neutrophils, T helper cells, monocytes, mast cells, basophils and epithelial cells with various chemical mediators and cytokines are involved.3 Cysteinyl leukotrienes (CysLTs), namely LTC4, LTD4 and LTE4, have been suggested to be important inflammatory mediators enhancing airway inflammation and uncontrolled asthma, especially for patients with aspirin-exacerbated respiratory disease (AERD). They are produced in a cascade manner via the arachidonic acid (AA) pathway by mast cells and eosinophils in the airways.4

LTE4, a stable and reliable metabolite, has been detected in serum and urine, and used as an indicator for the activation of AA or CysLT pathways.5 Higher urine levels of LTE4 (uLTE4) have been reported in patients with moderate to severe asthma as well as with AERD, in whom activation of the CysLT pathway is related to eosinophil recruitment into the airways,6 and shows a negative correlation with FEV1.7 Leukotriene receptor antagonists (LTRAs), including montelukast, zafirlukast and pranlukast, have been developed to reduce CysLTs-mediated airway inflammation in asthmatics.8 CysLTs are known to cause bronchoconstriction, mucosal edema and mucus secretion.9 Moreover, they are involved in airway remodeling via activating epithelial cells. Previous studies have revealed the role of CysLTs in airway remodeling via TGF-β1 production10 and enhancing TGF-β1 response.11 The clinical efficacy of LTRAs in adult asthmatics has been reported in some studies, in which addition of LTRAs improves asthma control status in adult asthmatics whose symptoms remain uncontrolled despite the use of ICSs.12 However, some asthmatics do not respond adequately to LTRAs, even together with ICS and LABA, and the detection of high LTE4 levels in urine or serum has been suggested to enrich the responders. To date, there have been few studies to examine whether airway inflammatory responses remain uncontrolled with use of LTRAs in adult patients with AERD or aspirin-tolerant asthma (ATA).

The primary objective of the present study was to examine the levels of AA metabolites according to LTRA medication as well as anti-inflammatory medication including ICS, and to find whether the LTRA treatment could suppress the levels of LTE4. Furthermore, we evaluated whether the levels of AA metabolites are associated with asthma control status and the degree of eosinophilic inflammation in a real-world clinical setting involving patients with AERD and those with ATA.

Methods

Study Design and Study Subjects

This study was a prospective cross-sectional and noninterventional real-world study. We enrolled 47 patients with AERD, 90 patients with ATA and 20 normal healthy controls (HC) at Ajou University Hospital in Suwon, South Korea. Asthma was diagnosed according to the GINA by the allergy specialists. Exclusion criteria for enrollment were as follows: 1) asthmatics who had ever been treated with type 2 biologics, including omalizumab, mepolizumab, reslizumab and dupilumab, within 130 days of enrollment, 2) current smokers or ex-smokers who quit smoking within 30 days of enrollment; and 3) asthmatics whose controller medications were changed within 7 days of enrollment.

Study subjects were recruited according to current maintenance medications, especially LTRA as well as ICS-LABA. In addition, they were grouped by NSAID/aspirin hypersensitivity because the levels of AA metabolites are known to be different in patients with AERD compared to those with ATA.5 The ATA group was divided into 2 subgroups: ATA with ICS-LABA and ATA with ICS-LABA plus LTRA. In the AERD group, all the AERD patients enrolled had been taking ICS-LABA plus LTRA (Figure 1). The doses of ICS were grouped into low, medium and high doses as defined in the Global Initiative for Asthma guideline (GINA) guideline.13

Figure 1 Study subject group enrolled in the present study.

Abbreviations: AERD, aspirin-exacerbated respiratory disease; ATA, aspirin tolerant asthma; HC, healthy control; ICSLABA, inhaled corticosteroid/long-acting beta-agonist; LTRA, leukotriene receptor antagonist.

Note: Asthma control status was defined by the GINA guideline.

AERD was defined by a typical clinical history (recurrent exacerbation of upper or lower respiratory reactions after ingestion of aspirin/NSAIDs) and/or a positive response to the lysine-aspirin bronchial provocation test (Lys-ASA BPT).14 The Lys-ASA BPT was performed with increasing doses of Lys-ASA solution up to 300 mg/mL using the method previously reported.15 The result of the Lys-ASA BPT was considered “positive” if FEV1% was decreased by more than 20% after the challenge. Subjects who showed negative results to the Lys-ASA BPT or denied any changes in upper or lower respiratory tract symptoms after ingestion of aspirin/NSAIDs were defined as ATA.

To evaluate the levels of AA metabolites according to asthma control status, study subjects were grouped by asthma control status (well controlled, partly controlled or uncontrolled status) based on the GINA guideline. Well-controlled asthma was defined if patients experienced daytime symptoms and reliever use less than twice a week without night awakening or activity limitation in the past 4 weeks. Uncontrolled asthma was defined if asthmatics showed ACT ≤ 208,16 or ACQ-6 ≥ 1.5.17 Severe asthma was defined according to the American Thoracic Society Workshop.18 All subjects gave written informed consent at the time of enrolment, and the study was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-BMR-SUR-15-498). This study was conducted in accordance with the Declaration of Helsinki.

Collection of Clinical Data and Samples

Peripheral venous blood, sputum and urine samples were collected from the subjects between 8:00 AM and 11:00 AM when the patients were maintained on controller medications including ICS-LABA and LTRAs. Blood eosinophil counts, sputum eosinophil counts and total immunoglobulin E (IgE) levels were analyzed. The serum and urine levels of metabolites, including LTE4, 15-hydroxyeicosatetraenoic acid (15-HETE), 11-dehydro thromboxane B2 (TXB2) and prostaglandin F (PGF), were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). LTE4-d5, 15(S)-HETE-d8, TXB2-d4 and 8-iso PGF-d4 were used as internal standards. Chromatographic separation was performed using a Waters Acquity UPLC system (Waters) with a Hypersil GOLD column (2.1 x 100 mm, 1.9 μm: ThermoFisher Scientific, San Jose, CA, USA). Data were obtained using an API5500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) equipped with an ESI source. For the quantitative determination of creatinine in urine samples, 10 μL of the urine sample were applied to the Creatinine Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA).19 Pulmonary function test, fractional exhaled nitric oxide (FeNO) measurement, questionnaires survey using the asthma control test (ACT), and asthma quality of life (AQLQ) and asthma control questionnaire (ACQ-6: ACQ mean of 6 individual item scores) were performed on the same day of enrollment.

Measurement of Serum and Urine Levels of Metabolites

Serum and urine samples stored at −70°C were thawed and analyzed by LC-MS/MS. Chromatographic separations of metabolites in urine and serum were performed with a Hypersil GOLD (2.1 X 100 mm, 1.9 μm: ThermoScientific, San Jose, CA, USA). The eluent was introduced into an AB Sciex, API5500 Triple Quadrupole mass spectrometer. The following deuterated internal standards were used: LTE4-d5 for LTE4, 15(S)-HETE-d8 for 15-HETE, 11-dehydro TXB2-d4 for 11-dehydro TXB2 and 8-iso PGF2α-d4 for PGF (Cayman Chemical Company, Ann Arbor, MI, USA). In urine samples, creatinine was also quantified to normalize to the actual concentrations of each biomarker and 1,3-dimethyl-2-imidazolidinone (Sigma-Aldrich, St. Louis, MO, USA) and then used as the internal standard for creatinine.

Statistical Analysis

Continuous variables were compared using Student’s t-test, and Pearson’s chi-squared or Fisher’s exact test were used for categorical variables. Analysis of variance was performed for comparisons among the 3 groups. General linear regression analysis was performed to adjust for the confounding factors age and sex, and to compare metabolite levels between the groups. Pearson’s correlation analysis identified associations among continuous variables. All computations were performed using SPSS software, version 22.0 (IBM Corp., Armonk, NY, USA). GraphPad Prism 5.0 software (GraphPad Inc., San Diego, CA, USA) was used for the production of graphs.

Results

Clinical Characteristics and Asthma Control Status of the Study Subjects

Table 1 summarizes the demographic information of all the study subjects on the day of enrollment. ICSs that were prescribed to the study subjects were beclomethasone, budesonide and fluticasone. The proportion of low-, medium or high-dose ICS users was not significantly different between patients with AERD and ATA (low-dose: 25.5% vs 15.6%, medium-dose: 57.4% vs 59.4%, high-dose: 17.0% vs 25.0%; P>0.05 for all). LTRAs that were used in the study subjects were montelukast (10mg/day) and pranlukast (450mg/day). The AERD group showed higher blood eosinophil counts but lower levels of total IgE compared to the ATA group (P=0.011 and P=0.029, respectively), while no significant differences were observed in lung function parameters. There were no significant differences in asthma control status between the AERD and ATA groups. The ACT, ACQ or AQLQ scores showed no significant differences between the AERD and ATA groups (ACT:21.15 ± 3.11 vs 20.40 ± 4.06, ACQ: 4.60 ± 4.95 vs 6.32 ± 5.74, AQLQ: 172.72 ± 37.04 vs 165.82 ± 38.84; P>0.05 for all). Inflammatory parameters or lung functions showed no significant differences between ATA patients with ICS/LABA and with ICS/LABA plus LTRA. However, a higher proportion of well-controlled asthmatics, as defined by the GINA guideline, was observed in ATA patients with ICS/LABA than with ICS/LABA plus LTRA (P=0.031). The ACT and AQLQ scores were significantly higher in ATA patients with ICS-LABA than with ICS-LABA plus LTRA (ACT: 21.77 ± 3.18 vs 19.84 ± 4.26, P=0.041,AQLQ: 186.96 ± 23.61 vs 157.23 ± 40.63; P<0.001 for both). The ACQ score showed no significant difference (4.680 ± 6.04 vs 6.97 ± 5.53, P = 0.092) between the 2 groups. Regarding the comorbidities, chronic rhinosinusitis was more common in AERD than in ATA patients (40 [85.1%] in AERD and 35 [38.9%] in ATA patients, P<0.001). There were no significant differences in prevalence of gastroesophageal reflux disease and obesity between patients with AERD and those with ATA (4 [8.5%] vs 7 [7.8%], P=0.881; 12 [25.5] vs 32 [35.6], P=0.254).

Table 1 Clinical Characteristics of the Total Study Subjects

Serum and Urine Levels of LTE4, 15-HETE, TXB2 and PGF2α in the Study Subjects

In HCs, the serum levels of LTE4, 15-HETE, TXB2, PGF2α and LTE4/PGF2α ratio were found to be 0.03 ± 0.06 ng/mL, 0.97 ± 0.97 ng/mL, 0.26 ± 0.70 ng/mL, 0.07 ± 0.15 ng/mL and 0.09 ± 0.19 (mean ± SD), respectively; the urine levels of LTE4, 15-HETE, TXB2, PGF2α and LTE4/PGF2α ratio were 469.11 ± 1390.33 pg/mg creatinine, 412.06 ± 1423.53 pg/mg creatinine, 381.92 ± 703.50 pg/mg creatinine, 781.11 ± 454.50 pg/mg creatinine and 0.22 ± 0.48 (Figure 2).

Figure 2 The serum and urine levels of lipid mediators in the 3 study groups. (A) LTE4. (B) 15-HETE. (C) TXB2. (D) PGF2α. (E) LTE4/PGF2α.

Abbreviations: AERD, aspirin-exacerbated respiratory disease; ATA, aspirin tolerant asthma; LT, leukotriene; HETE, hydroxyeicosatetraenoic acid; TXB2, 11-dehydro thromboxane B2; PG, prostaglandin.

Note: General linear regression analysis was performed to compare metabolite levels between the groups.

The serum levels of LTE4, LTE4/PGF2α ratio and the urine levels of LTE4, PGF2α and LTE4/PGF2α ratio showed significantly different among groups of AERD, ATA, and HC (ANOVA, P=0.014, P=0.016, P=0.007, P=0.010, and P<0.001, respectively) (Figure 2). The urine levels of LTE4, and the serum and urine levels of LTE4/PGF2α ratio were significantly higher in the AERD group than in the ATA group (urine: LTE4, 539.25 ± 789.53 pg/mg creatinine vs 161.57 ± 328.94 pg/mg creatinine, serum: LTE4/PGF2α, 0.62 ± 0.81 vs 0.39 ± 0.46, urine: LTE4/PGF2α, 0.39 ± 0.46 vs 0.12 ± 0.21; P<0.001, P=0.009 and P<0.001, respectively), while marginal differences were noted in the serum levels of TXB2; no significant differences were noted in the levels of 15-HETE (Figure 2). Subsequently, we further analyzed the association of the levels of LTE4 and LTE4/PGF ratio with clinical parameters. Receiver operating characteristic analysis (Figure S1) revealed that the urine levels of LTE4 and LTE4/PGF ratio could discriminate AERD patients from ATA patients (P<0.001, AUC=0.746 and P<0.001, AUC=0.747, respectively). Significant positive correlations were observed between the serum and urine levels of LTE4, but no correlation was found between the serum and urine levels of other metabolites (Figure S2).

Serum and Urine Levels of LTE4 in Asthmatics According to LTRA Medication

The serum and urine levels of LTE4 and LTE4/PGF ratio were found to be 0.15 ± 0.23 ng/mL, 0.43 ± 0.59, 324.44 ± 600.14 pg/mg creatinine and 0.23 ± 0.36 in asthmatics who had been taking ICS/LABA plus LTRA. The serum levels of LTE4/PGF2α ratio and the urine levels of LTE4, and LTE4/PGF2α ratio showed significantly different among groups (ANOVA, P=0.031, P=0.001, and P<0.001, respectively) as shown in Figure 3. The urine levels of LTE4, and the serum and urine levels of LTE4/PGF ratio were significantly higher in the AERD patients than in the ATA patients who were on LTRA medication (P=0.001, P=0.023 and P<0.001, respectively) (Figure 3A and B). Neither serum nor urine levels of LTE4 or LTE4/PGF ratio showed any significant differences in the ATA group between ATA patients on ICS-LABA and on ICS-LABA plus LTRA (P>0.05 for all). Considering the higher proportion of well-controlled asthmatics in the ATA group with ICS-LABA use, multivariate linear regression analysis was performed to adjust for asthma control status. After adjusting for asthma control status (regardless of GINA guideline asthma control status, ACT, ACQ and AQLQ score), neither serum or urine levels of LTE4 or LTE4/PGF2α ratio showed any significant differences according to LTRA medication in ATA patients (P>0.05 for all). There were no differences in the serum or urine levels of 15-HETE, TXB2 or PGF between the AERD and ATA groups according to asthma control status or clinical parameters.

Figure 3 The serum (A) and urine (B) levels of LTE4 in asthmatics according to LTRA medication.

Abbreviations: AERD, aspirin-exacerbated respiratory disease; ATA, aspirin tolerant asthma; LT, leukotriene; PG, prostaglandin; ICSLABA, inhaled corticosteroid/long-acting beta agonist; LTRA, leukotriene receptor antagonist.

Note: General linear regression analysis was performed to compare metabolite levels between the groups.

Serum and Urine LTE4 Levels in Asthmatics According to Asthma Control Status and Severity

Although the serum and urine levels of LTE4 and LTE4/PGF2α ratio were not significantly different by ANOVA, uncontrolled AERD patients showed significantly higher serum and urine levels of LTE4 compared to controlled or partly controlled AERD patients (Figure 4A). The serum levels of 15-HETE and TXB2 were significantly higher in uncontrolled AERD patients than in controlled or partly controlled patients (P=0.002 and P=0.011, respectively), while no differences were noted in the serum levels of PGF (data not shown). When asthma control status was classified according to ACQ scores, the AERD patients with ACQ ≥ 1.5 showed significantly higher serum levels of LTE4 and LTE4/PGF ratio compared to controlled AERD patients (P=0.025 and P=0.011, respectively) (Figure S3).

Figure 4 The levels of LTE4 in patients according to asthma control status in AERD (A) and ATA (B) patients.

Abbreviations: AERD, aspirin-exacerbated respiratory disease; ATA, aspirin tolerant asthma; LT, leukotriene; PG, prostaglandin.

Notes: Asthma control status was defined by GINA guideline. General linear regression analysis was performed to compare metabolite levels between the groups.

No differences in the serum or urine levels of LTE4 or LTE4/PGF ratio were observed in ATA patients according to asthma control status as defined by the GINA guideline, ACT ≤ 20 or ACQ-6 ≥ 1.5 (Figure 4B). However, ACQ scores significantly associated with the urine levels of LTE4 after adjusting for age and sex using multivariate linear regression analysis in ATA patients (P=0.032, B=11.931, Exp[B]=151,979.07).

There were no significant differences in the serum or urine levels of LTE4, 15-HETE, TXB2 PGF or LTE4/PGF2α ratio between non-severe asthma and severe asthma (data not shown).

Correlation Between LTE4 Levels and Type 2 Inflammatory Markers

The serum and urine levels of LTE4 and LTE4/PGF2α ratio were found to be significantly higher in patients with blood eosinophil counts ≥300/µL (serum, P=0.001 and P=0.020 respectively; urine, P<0.001 for each) (Figure 5). There were weak but significant positive associations of the serum and urine levels of LTE4 with blood eosinophil counts and FeNO levels (serum: r=0.426, P<0.001, and r=0.243, P=0.005, respectively; urine: r=0.386, P<0.001, and r=0.356, P<0.001, respectively) (Figure 6A), These correlations were seen in both AERD and ATA patients (AERD: serum, r=0.446, P=0.002, and P=0.168; urine, r=0.389, P=0.007 and r=0.524, P<0.001; ATA: serum, r=0.367, P<0.001, and r=0.263, P=0.014; urine, r=0.274, P=0.009 and P=0.566, respectively).

Figure 5 The levels of LTE4 in patients with eosinophilic asthma and non-eosinophilic asthma.

Abbreviations: LT, leukotriene; PG, prostaglandin.

Notes: Eosinophilic asthma was defined when the blood eosinophil count ≥300 /µL. General linear regression analysis was performed to compare metabolite levels between the groups.

Figure 6 Correlation between the levels of LTE4 and clinical parameters ((A) eosinophil markers, (B) pulmonary function).

Abbreviations: LT, leukotriene; FeNO, fractional exhaled nitric oxide; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity.

Note: P values were obtained by Pearson’s correlation test.

Patients with chronic rhinosinusitis (CRS) had significantly higher serum and urine levels of LTE4 and LTE4/PGF ratio than those without (serum: P=0.001 and P=0.018, respectively; urine: P=0.001 and P<0.001, respectively) (data not shown). No significant associations were found between the serum and urine levels of LTE4 and lung function parameters, including FEV1, FVC and MMEF (Figure 6B), in all asthmatics.

However, when study subjects were classified into the high (≥the median level of LTE4) vs low (<the median level of LTE4) LTE4 groups, significantly lower predicted values of FEV1, FVC and MMEF were noted in subjects with high LTE4 within the AERD group (P=0.006, P=0.015 and P=0.016, respectively) (Tables S1 and S2). Also, blood eosinophil counts were significantly higher in the high LTE4 group than in the low LTE4 group (P<0.05). In addition, to investigate factors affecting the serum and urine levels of LTE4, multivariate analyses of aspirin sensitivity, presence of CRS, asthma control status by the GINA guideline, blood eosinophil counts, FeNO levels age and sex were performed. Blood eosinophil count was the only factor significantly affecting the serum levels of LTE4 (P<0.001, B=0.000336, Exp[B]=1.000336) (Table S3). Aspirin sensitivity, blood eosinophil counts and FeNO levels significantly affected the urine levels of LTE4 (P=0.009, B=257.246, Exp[B]=5.26E+111; P<0.001, B=0.674, Exp[B]=1.961, and P=0.001, B=4.444, Exp[B]=85.094, respectively) (Table S4).

Discussion

This is the first study to demonstrate that LTE4 levels remain high and show a strong association with blood eosinophil counts, FeNO levels and asthma control status in asthmatic patients despite maintenance medications with ICS-LABA plus LTRA. First, we showed that, in a subpopulation of AERD patients with high LTE4 levels, airway inflammation could not be fully suppressed by maintenance medications according to current treatment guidelines, ie ICS-LABA plus LTRA, suggesting that CysLTs-mediated inflammation does not solely drive disease severity in these patients. Secondly, high LTE4 levels in ATA patients did not show association with lung function, and the proportion of well-controlled patients was lower in patients treated with ICS-LABA plus LTRA when compared to those treated with ICS-LABA alone. Thirdly, in both AERD and ATA patients, high LTE4 levels were linked with persistent eosinophilia. Based on these findings, we propose that earlier eosinophilia-targeting treatment could be a better option than LTRA when a combination of ICS-LABA does not lead to disease control.

ICS-LABA is known to be the mainstay of treatment options for asthma.13 LTRA provides an additive benefit for asthmatics whose asthma is not adequately controlled with ICS alone or ICS-LABA, which is derived from the limited capability of corticosteroids to inhibit the leukotriene synthesis pathway.8,20–23 Clinical studies on the efficacy of LTRA treatment have been discrepant, and targeting of the LTRA treatment to asthmatic patients with high LTE4 levels has been suggested to improve the efficacy. The present study performed in a real-world clinical setting clearly demonstrated that the serum and urine levels of LTE4 were high in a considerable number of adult asthmatics, and the higher in the AERD group than in the ATA group despite ICS-LABA plus LTRA medications, suggesting that LTRA medication does not modulate the levels of LTE4.5 LTRA, a selective antagonist of the CysLT1 receptor, is known to directly block the action of CysLTs, but not their synthesis. However, it has been speculated that the synthesis of CysLTs could be reduced in the chronic use of LTRAs.24 LTD4 up-regulates IL-13 and its receptor, and IL-13 production up-regulates the synthesis of CysLTs and then the expression of the CysLT1 receptor.25 Furthermore, the anti-inflammatory activity of LTRAs could decrease the influx of inflammatory cells in the lungs. Accordingly, the cross-talk between CysLTs and type 2 cytokines could result in the self-perpetuating circuit of airway inflammation.20 Blocking of CysLT1 receptor could interrupt this and secondarily down-regulate the levels of LTE4. However, this kind of modulation of the levels of CysLTs by LTRA was not supported by our findings.

Human eosinophils are the main source of CysLTs, and CysLTs exert autocrine signals regulating IL-4 secretion and activating eosinophils.26 Recent studies suggest that CysLTs also induce innate immune responses via stimulating release of thymic stromal lymphopoietin (TSLP) and IL-33 from type 2 alveolar cells and epithelial cells.14,27,28 Therefore, TSLP/IL-33 and CysLTs synergistically activate group 2 innate lymphoid cells, provoking IL-5 release and persistent eosinophilic inflammation.29 The present study showed that there were significant associations between the serum/urine levels of LTE4 and blood eosinophil counts/FeNO levels, with a negative correlation between uLTE4 and FEV1/FVC, indicating a close link between LTE4 and type 2/eosinophilic airway inflammation.

The strength of the present study is that this study was performed in a real-world clinical setting, which also has its limitations in design. Although the critical factors that may affect the levels of LTE4 were controlled (eg asthmatics who used biologics or showed recent asthma exacerbation were excluded, LTRA users were separately analyzed), other factors that could affect the levels of LTE4 including medications for comorbidities, ICS adherence or diet pattern were not completely controlled. Because of the cross-sectional study design, further studies are needed to confirm our results. Altogether, our findings support earlier reports that a subgroup of patients with asthma, both in the ATA and AERD groups, is not adequately controlled with a combination of ICS-LABA plus LTRA.30 In addition, targeting LTRA treatment to patients with high LTE4 levels does not necessarily improve the efficacy, since persistent eosinophilia, lung inflammation and progressive lung function decline were seen in patients with high LTE4 despite LTRA treatment along with ICS-LABA. Another plausible explanation for the poor efficacy of LTRA treatment may be that an unidentified LTE4 receptor could mediate airway inflammation in asthma and resist currently available LTRAs.31,32 However, no such recent evidence exists to support this interpretation.

In conclusion, our results emphasize the need for other therapeutic options in asthmatic patients with high LTE4 levels, such as targeting eosinophils, TSLP or IL-33, and suggest the role of CysLTs as a major driver of eosinophilic airway inflammation and remodeling.33

Abbreviations

AA, arachidonic acid; ACQ, asthma control questionnaire; ACT, asthma control test; AERD, aspirin-exacerbated respiratory disease; AQLQ, asthma quality of life; ATA, aspirin-tolerant asthma; CysLTs, Cysteinyl leukotrienes; FeNO, Fractional exhaled NO; GINA, Global Initiative for Asthma guideline; IgE, immunoglobulin E; ICS, inhaled corticosteroid; LABA, long-acting β2-agonist; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LTRA, leukotriene receptor antagonist; Lys-ASA BPT, lysine-aspirin bronchial provocation test; PGF2α, prostaglandin F2α; TSLP, thymic stromal lymphopoietin; TXB2, 11-dehydro thromboxane B2; 15-HETE, 15-hydroxyeicosatetraenoic acid.

Acknowledgments

We thank Fanyi Jiang, Katerina Pardali, Outi Vaarala and Joo-Youn Cho for their cooperation and contribution to this research.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study was supported by AstraZeneca, a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HR16C0001) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No: 2020R1I1A3051800).

Disclosure

The authors declare that they have no competing interests.

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Knockdown of LINC01224 Suppresses Colon Cancer Progression by Sponging https://fcacleveland.org/knockdown-of-linc01224-suppresses-colon-cancer-progression-by-sponging/ https://fcacleveland.org/knockdown-of-linc01224-suppresses-colon-cancer-progression-by-sponging/#respond Tue, 12 Oct 2021 08:06:14 +0000 https://fcacleveland.org/?p=297 Introduction Colon cancer (CC) is the most commonly occurring malignant tumor in the world and leads to a major global health problem.1 In China, its incidence is rapidly rising.2 Until now, several therapies: operation, radiotherapy, and chemotherapy were major solid tumor treatments of CC. Moreover, multi-modality therapy has been previously used in the treatment of […]]]>

Introduction

Colon cancer (CC) is the most commonly occurring malignant tumor in the world and leads to a major global health problem.1 In China, its incidence is rapidly rising.2 Until now, several therapies: operation, radiotherapy, and chemotherapy were major solid tumor treatments of CC. Moreover, multi-modality therapy has been previously used in the treatment of CC patients, which made the incidence and mortality rate decreased in recent years.3 However, most of the diagnosed CC cases are always at an advanced stage, leading to a poor prognosis.4 Several common chemotherapeutic agents used for CC treatment have been confirmed with limited effectiveness, toxicity, and drug resistance.5 Therefore, more effective therapies are urgently needed. Accumulating evidence has verified that several biomarkers as molecular targets participate in cancer progression. In this study, we aim to determine potential diagnostic biomarkers and molecular therapeutic targets.

Long noncoding RNAs (lncRNAs) are a class of transcribed RNA molecules longer than 200 nucleotides.6 In recent years, the roles of many lncRNAs have been extensively studied in cancer progression and carcinogenesis.7 More and more attention has been focused on their effects on tumors. Multiple lncRNAs have been reported to be abnormally expressed in cancer tumors, and have been regarded as oncogenes or tumor suppressors.8,9 In the process of exploring the molecular therapeutic targets of CC, several lncRNAs such as lncRNA LINC00460,10 lncRNA NEAT1,11 and lncRNA STEAP3-AS112 have been verified as oncogenes. Notably, mostly lncRNAs act as a sponge for microRNAs (miRNAs) to regulate the expression of the target gene in CC treatment. Previous studies have confirmed that lncRNAs can serve as competing endogenous RNAs (ceRNAs) or sponges for miRNAs, further acting on the target mRNAs.13 Hence, exploring the lncRNA-miRNA axis is essential for the development of molecular therapeutic targets.

The lncRNA LINC01224 reportedly promotes Epithelial Ovarian Cancer progression by sponging miR-485-5p and consequently increasing PAK4 expression.14 Whereas the role of LINC01224 in CC has not been investigated till now. In this study, for the first time, we systematically explored the role of LINC01224 in CC progression and further investigated the underlying mechanism.

Materials and Methods

Samples

The tumoral tissues and adjacent normal tissues from 52 patients with CC were obtained from the institutional review board of Taizhou Hospital of Zhejiang Province. The informed consent of the patients with CC was provided according to the declaration of Helsinki. And this study was approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province, the approval number is [KY-E-2019-10-20]. The clinical parameters of the patients were shown in Table 1.

Table 1 The Clinical Parameters of the Studied CC Patients

Cells and Plasmids

The CC cell lines (HCT116, HT-29, SW620, and SW480) and normal human colonic mucosa cells (NCM460) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The si-RNAs (si-LINC01224#1, si-LINC01224#2, si-NC) were designed and synthesized by TransGen Biotech Co., Ltd. (Beijing, China). The miR-485-5p mimics/inhibitor was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell transfection was achieved by using the Lipofectamine 2000 according to the manufacturer’s information (Invitrogen, Carlsbad, CA, USA).

Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted by Trizol reagent according to the manufacturer’s information (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was achieved by using PrimeScript™RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The qRT-PCR analysis was performed by using the SYBR Green Premix ExTaq PCR kit (Beijing Cowin Biotech., China) on the ABI7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers used in this study were designed by Primer 5.0.

Cell Counting Kit- 8 (CCK- 8) Assay

CCK-8 assays were performed to explore the effect of LINC01224 knockdown on HT-29 and SW480 cell proliferation abilities. In brief, cells in different groups were plated on a 96-well plate. CCK-8 reagent (Keygen, Jiangsu, China) was added at 0 h, 24 h, 48 h, and 72 h after transfection. Afterward, the absorbance at 450 nm was detected by a microplate reader.

Colony Formation Assays

Colony formation assay was performed to investigate the effect of LINC01224 knockdown on CC cell clonogenic activities. After transfection for 48 h, the cells (5×103 cells per well) were seeded on a 6-well plate. Following culture for two weeks, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and stained with 0.1% crystal violet dye (Sigma-Aldrich, St. Louis, MO, USA). Colony number was counted using an inverted microscope.

Transwell Assay

The migration or invasion abilities of CC cells were determined using transwell chambers (Corning Company, NY, USA). Briefly, cells were resuspended and planted into upper chambers (about 1×104 cells) with or without the pre-coated Matrigel (BD Bioscience, Waltham, MA, USA). Meanwhile, an 800 μL medium with 30% FBS was planted into the lower chamber. The chamber was incubated for 24 h at 37 °C. Subsequently, the transmigrated cells were fixed with stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The migrated or invaded cells were photographed and counted by microscopy (Nikon, Tokyo, Japan).

Animal Experiments and Xenograft Collection

The nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). A suspension of HT29 cells (about 5×106 cells) was injected subcutaneously into the nude mice. The tumor volume was greater than 100 mm3, and the mice were selected for further experiments. Following the selection, intratumoral multipoint injection with si-NC or si-LINC01224#1 (50 nM) was performed. Each group contained six mice. After treatment, the mice were separately sacrificed at 7 days, 14 days, 21 days, and 28 days, then the tumors were excised for evaluation. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and the welfare of the laboratory animals strictly follows “The Guidance of The Good Treatment of Laboratory Animals” issued by the National Defense Science and Technology, as well as Five Freedom-Principles. The animal experiment authorized by the Animal Ethics Committee of Taizhou Hospital of Zhejiang Province.

Western Blot

Cells were lysed with RIPA buffer and quantified with a BCA Protein Assay Kit (Beyotime, Beijing, China). Total protein (40 μg) were loaded to SDS-PAGE gel and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk and incubated with MCL1 antibody (1: 1000; Beyotime, Beijing, China)(catalog:AF1390). After incubation, the secondary antibody HRP-labeled Goat Anti-Rabbit IgG (H+L)) (1: 1000; Beyotime, Beijing, China) (catalog:A0208) was used to incubate the membrane. The membrane was consequently visualized with an ECL reagent (Beyotime, Beijing, China). GAPDH was used as an internal control.

Dual-Luciferase Reporter Assay

Through starbase 2.0, we found the miR-485-5p binding site in LINC01224. The LINC01224 wide type (LINC01224 WT) and mutant type (LINC01224 Mut) reporter vectors were established by Beijing TransGen Biotech Co., (Beijing, China). The reporter plasmids were co-transfected with either miR-485-5p mimics or a negative control into H293T cells. After transfection for 48 h, the cells were lysed and the relative luciferase activities were detected using a dual-luciferase reporter assay system (Promega, Madison, WI, USA).

RNA Pull-Down Assay

For RNA pull-down assay, the streptavidin-coated magnetic beads (Life Technologies, CA, USA) were covered by biotinylated LINC01224 (Bio-LINC01224) and Bio-NC according to its instruction and transfected into l × 106 HT29 cells at a final concentration of 50 nM for 48 h before harvest. Subsequently, 0.7 mL lysis buffer (5 mM MgClz, 100 mM KCl, 20 mM Tris (pH 7.5), 0.3% NP-40) and complete protease inhibitor cocktail (Roche Applied Science, IN) were added into the cell pellets, then the cell lysates were incubated together with the RNA-tagged beads for the co-immunoprecipitation (Invitrogen, Carlsbad, CA, USA). The RNA-RNA complexes were subsequently collected by centrifugation at 10,000r for 10 min and then the miR-485-5p enrichment level was detected with qRT-PCR analysis.

Statistical Analysis

The statistical analysis was performed using SPSS 20.0 (SPSS, Chicago, IL, USA). The difference between two groups was calculated by Student’s t-test. The significance among multiple groups was calculated by ANOVA with Bonferroni correction. The survival curves were assessed by Kaplan–Meier analysis. p < 0.05 was considered statistically significant.

Results

Expression Level of LINC01224 in CC Tumor Tissues and Several Cell Lines

The qRT-PCR assay revealed that the expression level of LINC01224 was higher in several CC cell lines (HCT116, HT-29, SW620, and SW480) and CC tumor tissues, as compared with levels observed in NCM460 cells and normal tissues (Figure 1A and B). The patients were divided into low expression level group (n=26) and high expression level group (n=26). The KM survival curve indicated that patients with higher LINC01224 levels have a lower survival percent (Figure 1C). The clinicopathological data was assessed by chi-square test and summarized in Table 1. The analysis demonstrated that LINC01224 level is associated with stage, lymph node metastasis, and tumor size, not with age and sex.

Figure 1 Expression level of LINC01224 in CC tumor tissues and several cell lines. (A) Expression levels of LINC01224 in several colon cancer cell lines (HCT116, HT-29, SW620 and SW480) and control NCM460 cells were detected by qRT-PCR. (B) Expression levels of LINC01224 in colon cancer tumor tissues and adjacent normal tissues were detected by qRT-PCR. (C) Kaplan–Meier survival curves for patients with colon cancer were plotted according to high or low LINC01224 expression level. **p < 0.01. ***p < 0.001.

Knockdown of LINC01224 Suppressed Proliferation, Migration, and Invasion of CC Cells

As shown in Figure 2A, two si-RNAs against LINC01224 effectively repressed the LINC01224 level (Figure 2A). CCK-8 assay illustrated that LINC01224 knockdown significantly suppressed the OD 450 values at 72 h in HT29 and SW480 cells (Figure 2B). Colony formation assay showed that silencing LINC01224 significantly decreased the colony numbers as compared with si-NC groups (Figure 2C). Transwell assays demonstrated that LINC01224 knockdown significantly suppressed migration and invasion abilities of HT29 and SW480 cells (Figure 2D and E).

Figure 2 Knockdown of LINC01224 suppressed proliferation, migration and invasion of CC cells. (A) qRT-PCR revealed that si-RNA LINC01224#1 or si-RNA LINC01224#2 effectively suppressed the level of LINC01224 in HT29 and SW480 cells. (B) CCK-8 assay illustrated that LINC01224 knockdown significantly suppressed the OD 450 values at 72 h in HT29 and SW480 cells (C) Colony formation assay showed that LINC01224 knockdown significantly decreased the colony numbers as compared with si-NC group. (D) Transwell assays demonstrated that LINC01224 knockdown significantly suppressed migration ability of HT29 and SW480 cells. (E) Transwell assays demonstrated that LINC01224 knockdown significantly suppressed invasion ability of HT29 and SW480 cells. (F and G) LINC01224 knockdown markedly suppressed tumor volume and weight. **p < 0.01. ***p < 0.001.

Xenografts volume and weight were measured for evaluation. Measurements of the tumor xenografts revealed that LINC01224 knockdown markedly reduced tumor volume and weight (Figure 2F and G).

LINC01224 Acts as a Sponge for miR-485-5p

Through the Starbase online database, we found a binding site of miR-485-5p in LINC01224 sequence (Figure 3A). For evaluation of the interaction between miR-485-5p and LINC01224, a series of functional trials were performed. Dual-luciferase reporter assay showed that miR-485-5p overexpression markedly suppressed the luciferase activity of H293T cells in LINC01224 WT group, but not work in LINC01224 Mut group (Figure 3B). RNA pull-down assay found a significant enrichment of miR-485-5p with bio-LINC01224, compared with input control (Figure 3C). qRT-PCR revealed that miR-485-5p levels were markedly up-regulated in si-LINC01224 pool group cells, compared with levels observed in si-NC group (Figure 3D). The expression level of miR-485-5p was lower in CC tumor tissues than that in normal tissues (Figure 3E).

Figure 3 LINC01224 acts as a sponge for miR-485-5p. (A) Starbase online database predicted a binding site of miR-485-5p in LINC01224 sequence. (B) Dual-luciferase reporter assay was used to evaluate the interaction between miR-485-5p and LINC01224. The results showed that miR-485-5p overexpression markedly suppressed the luciferase activity of H293T cells in LINC01224 widetype (WT) group, but not work in LINC01224 mutant (Mut) group. (C) The binding of LINC01224 to miR-485-5p detected by RNA pull-down. RNA pull-down assay found a significant enrichment of miR-485-5p with bio-LINC01224, compared with input control. (D) qRT-PCR revealed that miR-485-5p levels were markedly up-regulated in si-LINC01224 pool group cells, compared with levels observed in si-NC pool group. (E) qRT-PCR detected that the expression level of miR-485-5p in colon cancer tumor tissues and adjacent normal tissues. The expression level of miR-485-5p was lower in CC tumor tissues than that in normal tissues. **p < 0.01.

Interaction Between MCL1 and miR-458-5p

Starbase online database predicted that a binding site of miR-458-5p in MCL1 sequence (Figure 4A). Dual-luciferase reporter assay results showed that MCL1 WT reporter was strongly inhibited by miR-485-5p, whereas MCL1 Mut reporter was not affected by miR-485-5p (Figure 4B). In miR-485-5p mimics groups, the RNA and the protein levels of MCL1 were markedly inhibited in HT29 and SW480 cells (Figure 4C and D). The level of MCL1 was obviously higher in CC tumor tissues than that in normal tissues (Figure 4E).

Figure 4 Interaction between MCL1 and miR-458-5p. (A) Starbase online database predicted that a binding site of miR-458-5p in MCL1 sequence. (B) Dual-luciferase reporter assay was used to evaluate the interaction between miR-485-5p and MCL1. The results showed that MCL1 WT reporter was strongly inhibited by miR-485-5p, whereas MCL1 Mut reporter was not affected by miR-485-5p. (C and D) The mRNA and the protein levels of MCL1 in HT29 and SW480 cells were markedly inhibited in miR-485-5p mimics groups. (E) qRT-PCR results revealed that the level of MCL1 was obviously higher in CC tumor tissues than that in normal tissues. **p < 0.01.

LINC01224 Inhibited MCL1 Expression by Sponging miR-455-5p

In HT29 and SW480 cell lines, si-LINC01224 pool markedly suppressed the mRNA and protein level of MCL1, whereas miR-485-5p inhibitor (miR-485-5p inh) or MCL1 overexpression (MCL1 oe) restored these effects of si-LINC01224 pool (Figure 5A and B). Furthermore, the functional experiments displayed that miR-485-5p inh or MCL1 oe markedly restored the repressive effect of si-LINC01224 pool on the cell proliferation (Figure 5C and D), cell migration (Figure 5E) and invasion (Figure 5F). These data suggest that LINC01224 may be a potential competing endogenous RNA to miR-485-5p. And silence of LINC01224 can aggravate the miR-485-5p-induced MCL1 downregulation.

Figure 5 LINC01224 inhibited MCL1 expression by sponging miR-455-5p. (A and B) In HT29 and SW480 cell lines, si-LINC01224 pool markedly suppressed the mRNA and protein level of MCL1, whereas miR-485-5p inhibitor (miR-485-5p inh) or MCL1 overexpression (MCL1 oe) restored the effects of si-LINC01224 pool. (CF) CCK-8 assay, colony formation assay, transwell assays with Matrigel displayed that miR-485-5p inh or MCL1 oe markedly restored the repressive effect of si-LINC01224 pool on the cell proliferation (C), cell lone formation (D), cell migration (E) and invasion (F). **p < 0.01 versus si-NC group. #p < 0.01, ##p < 0.001 versus si-LINC01224 pool group.

Discussion

This study was primarily conducted to identify possible molecular therapeutic target for CC treatment. LncRNAs have been shown to be involved in the regulation of various cellular processes. Studies have shown that lncRNA can effectively regulate the epithelial-mesenchymal transition (EMT) by sponging miR-433-3p.10 It has also been shown to regulate the proliferation, migration and arrest of colon cancer cells at the G0–G1 phase,12 and also regulate the invasion and migration of CC cells.11

In this study, we first investigated the role of LINC01224 in CC progression. We found a high level of LINC01224 in CC tumors and cells, which consequently resulted in a poor prognosis. We confirmed that LINC01224 level was related with tumor size, histological grade and lymph node metastasis. These results indicated that LINC01224 can be regarded as a biomarker for CC. Furthermore, we found that an in vitro knockdown of LINC01224 markedly suppressed the proliferation, migration and invasion of HT29 and SW480 cells. Our in vivo study also showed that the expression level of LINC01224 positively correlated with the tumor volume and weight, indicating that the LINC01224 is an oncogenic lncRNA involved in CC progression and repressing its expression could significantly inhibit the progression of colon cancer.

Mostly, lncRNAs serve as ceRNAs to sponge miRNAs, consequently regulating the target mRNA. In this study, we confirmed that LINC01224 acted as a sponge for miR-485-5p and further regulated the expression level of its target mRNA myeloid cell leukemia sequence 1 (MCL1). Accumulating studies have revealed the critical roles of many miRNAs in cancer development, as they serve as oncogenes or cancer suppressor genes. Recently, miR-485-5p has been studied in multiple tumors, including hepatocellular carcinoma,15,16 ovarian cancer,14 papillary thyroid cancer,17 breast cancer,18 colorectal cancer19,20 and so on. From previous studies, we found that the levels of miR-485-5p were down-regulated in multiple cancer tumors and cells, and overexpression of miR-485-5p could suppress tumor cell metastasis, migration, invasion and proliferation, indicating that miR-485-5p could function as a tumor suppressor in multiple cancers including colorectal cancer. In this study, the results confirmed that miR-485-5p level was markedly lower in CC tumor tissues than adjacent normal tissues, which is consistent with previous researches. MCL1, the miR-485-5p target mRNA observed in this study, role in tumorigenesis was also studied in previous research.21 MCL1 was one important anti-apoptotic member of the BCL-2 family.22 Its involvement in multiple myeloma,23,24 osteosarcoma,25 and colon cancer21,26,27 has been reported. The overexpression of miR-125a-5p, the tumor suppressor, significantly suppressed the expression level of MCL1, and restoration of MCL1 in CC cells reversed the cell proliferation inhibition and apoptosis stimulation caused by miR-125-5p.21 Lu et al reported that MCL1 si-RNA could be delivered by DOTAP and MPEG-PCL hybrid micelles for the treatment of CC in vivo and in vitro, which was proved to induce the apoptosis of C26 CC cells and suppress the growth of C26 cells with high safety.26 Hence, we concluded that MCL1 is a target gene involved in CC progression.

In summary, in this study, we identified the LINC01224/miR-485-5p/MCL1 axis involvement in CC progression and demonstrated that LINC01224 knockdown could increase the level of miR-485-5p (the tumor suppressor), and consequently inhibit the level of MCL1 (an oncogene for CC progression). Overall, these results highlighted LINC01224/miR-485-5p/MCL1 axis as a possible molecular therapeutic target involved in the progression of CC.

Disclosure

The authors report no conflicts of interest in this work.

References

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https://fcacleveland.org/knockdown-of-linc01224-suppresses-colon-cancer-progression-by-sponging/feed/ 0
Effect of triptolide on thyroid cancer https://fcacleveland.org/effect-of-triptolide-on-thyroid-cancer/ https://fcacleveland.org/effect-of-triptolide-on-thyroid-cancer/#respond Tue, 12 Oct 2021 08:06:07 +0000 https://fcacleveland.org/?p=324 Introduction Thyroid cancer is a type of cancer that develops in the thyroid gland.1 According to the newest thyroid cancer epidemiology, the incidence of thyroid cancer was 586,202 cases and number of new deaths was 43,646 in 2020, worldwide.2 Women aged between 35 and 65 are commonly affected by thyroid cancer. The causes of thyroid […]]]>

Introduction

Thyroid cancer is a type of cancer that develops in the thyroid gland.1 According to the newest thyroid cancer epidemiology, the incidence of thyroid cancer was 586,202 cases and number of new deaths was 43,646 in 2020, worldwide.2 Women aged between 35 and 65 are commonly affected by thyroid cancer. The causes of thyroid cancer are unclear, although environmental and genetic factors contribute to thyroid cancer. The subtypes of thyroid cancer according to histopathological characteristics include papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, etc.3 Papillary thyroid cancer is the most common type of thyroid cancer. In addition, papillary thyroid cancer has the best overall prognosis among the subtypes of thyroid cancer.4 The symptoms of thyroid cancer are atypical in the early stage, and the first symptom might be a nodule in the thyroid region.5 The other symptoms of thyroid cancer include hoarseness, difficulty swallowing, neck pain, and swollen lymph nodes. The treatment methods include radiotherapy, surgery, thermotherapy, thyroid hormone therapy, targeted drug therapy, etc. However, treatments for thyroid cancer have some adverse effects. Therefore, it is important to investigate new therapeutic methods for thyroid cancer.

Triptolide is extracted from thunder god vine, Tripterygium wilfordii. It has been demonstrated to be useful in treating various types of cancer.6 A previous study found that triptolide was useful against lung, liver, and breast cancer.7,8 Triptolide was found to suppress lung cancer invasion, migration, and metastasis.9 Triptolide was also found to inhibit breast cancer cell proliferation and invasion. However, the mechanism of triptolide in the treatment of thyroid cancer has not been clarified.

In this study, we used network pharmacology and molecular docking analyses to investigate the antithyroid cancer function of triptolide. First, we evaluated triptolide targets and thyroid cancer targets. Second, we generated the protein-protein interaction (PPI) networks of the triptolide targets and thyroid cancer targets. Cytoscape software was used to merge the two PPI networks. Then, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the core PPI network were obtained. Via molecular docking analysis, we evaluated the binding of triptolide with the four core targets. Finally, we demonstrated the function of triptolide in treating thyroid cancer in vitro. Apoptosis assays, real-time polymerase chain reaction (RT-PCR) and Western blotting were used to evaluate the anticancer functions of triptolide. We aimed to reveal the mechanism of triptolide in treating thyroid cancer in this study. Our research could provide theoretical and experimental findings to support future studies.

Methods

Evaluation of Triptolide Targets and Thyroid Cancer Targets

In this study, we used traditional Chinese medicine systems pharmacology (TCMSP) database to evaluate the targets of triptolide. The TCMSP database is a useful pharmacological database containing chemical, drug and target networks. In addition, we used the Genetic Association Database (GAD), Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and Pharmacogenomics Knowledge Base (PharmGKB) databases to investigate the targets of thyroid cancer. The GAD database provides information about the relationships between diseases and genes. The OMIM database also contains data on diseases and genes. The TTD database provides information about proteins, nucleic acids and related targets. The PharmGKB database includes information on the effects of genetic variation on drug responses.

PPI Network Construction

In this study, a PPI network was constructed with the BisoGenet plugin. The databases providing data for the PPI network included Database for Interacting Proteins (DIP), Biological General Repository for Interaction Datasets (BioGRID), IntAct, Biomolecular Interaction Network Database (BIND), Human Protein Reference Database (HPRD), and Molecular INTeraction Database (MINT). Then, Cytoscape software was used to visualize the PPI network. Cytoscape was also used to merge the triptolide PPI network and the thyroid cancer PPI network. Furthermore, topological features were evaluated by parameters including the eigenvector centrality (EC), closeness centrality (CC), local average connectivity centrality (LAC), betweenness centrality (BC), degree centrality (DC), and neighbourhood centrality (NC). The CytoNCA plugin was used in the topological feature analysis.

GO and KEGG Analyses

In this study, GO and KEGG analyses were conducted with the DAVID database. GO analysis included the biological process (BP), molecular function (MF), and cellular component (CC) categories. KEGG analysis revealed correlated genes, drugs, molecular pathways, etc.

Molecular Docking Analysis

We used CB-Dock and AutoDock to investigate the physical interactions between triptolide and the target proteins. CB-Dock can analyze the center, size, and binding sites. CB-Dock uses a cavity detection approach. In CB-Dock, ligands and receptors are visualized as space-filling representations and cartoons. The colors of the receptors and ligands are determined by the chains and elements. Discovery Studio contains tools for structure design, ligand design, and simulation. The ligand design tools include library optimization and molecular library construction. The simulations include quantum and molecular mechanics simulations. AutoDock is a suite of automated docking tools to predict the hydrogen bonds of small molecules. These two are powerful models to explore the relationships between drugs and proteins.

Cells and Reagents

The thyroid cancer cell line TPC-1 was selected for this study. TPC-1 cells were purchased from Bena Culture Collection (Henan, China). RPMI 1640 medium (Corning, 10-040-cv) was supplemented with 10% fetal bovine serum (FBS) (Gibco, 10099141), Penicillin-Streptomycin solution (Corning, 30-001-CI). TPC-1 cells were maintained under humidified conditions with 5% CO2 at 37°C. Triptolide was purchased from Merck (T3652). Cell apoptosis kits containing propidium iodide (PI) and Annexin V- Fluorescein isothiocyanate (FITC) were purchased from Abcam (ab14085). The anti- cyclin-dependent kinase inhibitor 1A (CDKN1A) primary antibody was purchased from Sigma-Aldrich (SAB5700742). Anti-phospho-c-JUN (CST, 3270s), anti-phospho-p53 (CST, 2521s), anti-phospho-NF-κB p65 (CST, 3031s), and anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (CST, 5174s) antibodies were purchased from Cell Signaling Technology. Goat anti-rabbit secondary antibody was purchased from Abcam (ab6721).

Treatment Protocol of Cells

In the subculture of cells, we viewed cultures using an inverted microscope to assess the degree of confluency. After washing the culture flask with PBS, 0.25% Trypsin/ Ethylenediaminetetraacetic acid (EDTA) (Gibco, 2186970) was pipetted. After pouring the excess trypsin, the culture flask was returned to the incubator and leave for 5 minutes. Then, we resuspend the cells in a small volume of fresh serum-containing medium to inactivate the trypsin. Cells were counted and required number of cells were transferred in new labelled culture flask.

In the apoptosis assay, cells were cultured in 6-well plates with 1.2×105 cells in every well. After 24 h culture, PBS was used to wash the plate and different concentrations of triptolide were added into labelled wells. After 24 h treatment, 0.25% Trypsin without EDTA (Gibco, 2195440) was used to digest the cells. Then, cells were collected to conduct the apoptosis assay. In Western blot and RT-PCR analysis, cells were cultured in 6-well plates with 4×105 cells in every well. After 24 h culture, PBS was used to wash the plate and different concentrations of triptolide were added into labelled wells. After 24 h treatments, cells were collected to conduct the experiments.

Cell Proliferation Assay

In this study, we used an MTT assay (Biorigin, BN20390) to evaluate the proliferation of thyroid cancer cells treated with triptolide. TPC-1 cells were treated with various concentrations of triptolide in 96-well plates. The plates were incubated with MTT solution after 48 h of incubation alone. Finally, the absorbance of each plate well was evaluated with a microplate reader.

Western Blot Analysis

After treatment with triptolide for 24 h, cells were lysed with RIPA buffer (Beyotime, P0013B) containing phenylmethylsulfonyl fluoride (PMSF) for 20 min. The proteins were separated at 120 V for 80 min. After transfer at 120 V for 150 min, the membrane was incubated with 5% milk for 2 h. Then, the membrane was incubated with primary antibodies overnight at 4°C. After washing with phosphate buffered saline with Tween 20 (PBST) 3 times, the membrane was incubated with the secondary antibody. Finally, immunoreactions on the membrane were detected.

RT-PCR Analysis

SYBR Green Master Mix was used in this experiment. The primers were as follows: CDKN1A (forward: 5’-gggctgggagtagttgtctt-3’, reverse: 5’- attgtgggaggagctgtgaa-3’), c-JUN (forward: 5’- tttcaggaggctggaggaag-3’, reverse: 5’- ctgccaccaattcctgcttt-3’), RELA (forward: 5’- agaagcaggctggaggtaag-3’, reverse: 5’- gccatggttgagcaaggaaa-3’), tumor protein p53 (TP53) (forward: 5’- tggccatctacaagcagtca-3’, reverse: 5’- ggtacagtcagagccaacct-3’), and GAPDH (forward: 5’- ccaaggagtaagacccctgg-3’, reverse: 5’- tggttgagcacagtctt-3’). TRIzol (Invitrogen, 15596018) was used to acquire total RNA. Then, ReverTra Ace qPCR RT Master Mix (Toyobo, FSQ-201) was conducted to reverse transcribe 1 μg RNA to cDNA. SYBR Green RT-PCR Master Mix (Toyobo, QPK-201) was used to perform RT-PCR. 3-step cycle was used in cycling condition of Real-Time PCR System with 40 cycles. 2−ΔΔCt method was conducted to evaluate relative expression. β-actin expression was used to normalize gene expression.

Apoptosis Assay

TPC-1 thyroid cancer cells were tested with a cell apoptosis kit. After centrifugation, cells were resuspended in 500 µL of 1X Annexin V binding buffer. Then, we added 5 µL of Annexin V-FITC and 5 µL of propidium iodide. Furthermore, we incubated the cells at room temperature for 5 min in the dark. Finally, we analyzed the cells by flow cytometry.

Statistical Analysis

GraphPad Prism was used to analyze the results. Student’s two-tailed t-test was used to evaluate statistical significance. P < 0.05 was considered statistically significant.

Results

Evaluation of Triptolide Targets and Thyroid Cancer Targets

There were 34 targets of triptolide in the TCMSP dataset. The triptolide targets included B-cell lymphoma 2 (Bcl2), CDKN1A, C-X-C Motif Chemokine Ligand 8 (CXCL8), c-JUN, Mitogen-Activated Protein Kinase 8 (MAPK8), etc. (Figure 1A). There were 210 targets of thyroid cancer in the GAD, OMIM, TTD, and PharmGKB databases. The thyroid cancer targets included A-Kinase Anchoring Protein 9 (AKAP9), AKT1, Cholecystokinin B Receptor (CCKBR), CD80, Cyclin Dependent Kinase Inhibitor 1B (CDKN1B), 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), Janus Kinase 1 (JAK1), etc. (Figure 1B).

Figure 1 Evaluation of triptolide targets and thyroid cancer targets. (A) The potential targets of triptolide. (B) The potential targets of thyroid cancer.

Construction of PPI Networks

In this study, we constructed PPI networks of triptolide and thyroid cancer targets. First, we constructed the triptolide target PPI network. The triptolide target PPI network had 2551 nodes and 52,255 edges (Figure 2A). The thyroid cancer target PPI network had 6694 nodes and 164,435 edges (Figure 2B). Then, the triptolide PPI network and thyroid cancer PPI network were merged. The merged PPI network contained 2058 nodes and 47,470 edges (Figure 2C). The filtering parameters included DC>60, BC>0.000837712025133824, CC>0.531353135313531, EC>0.0340744964778423, LAC>18.0862068965517, and NC>19.7636224602856. Finally, the core PPI network included 164 nodes and 4513 edges (Figure 2D).

Figure 2 Construction of PPI networks. (A) Triptolide target PPI network. (B) Thyroid cancer target PPI network. (C) Merged PPI network. (D) Core PPI network.

Functional Enrichment Analysis of Triptolide in Treating Thyroid Cancer

We conducted GO and KEGG enrichment analyses to discover the mechanism of triptolide in treating thyroid cancer. GO analysis showed the biological processes related to the treatment of thyroid cancer by triptolide; these biological process terms included apoptotic process, cell cycle arrest, NF-kappa B transcription factor activity, stress-activated mitogen-activated protein kinases (MAPK) cascade, G2/M transition of mitotic cell cycle, signal transduction by p53 class mediator, I-kappaB kinase/NF-kappa B signaling, G1/S transition of mitotic cell cycle, etc. (Figure 3A). KEGG analysis showed that the pathways related to the treatment of thyroid cancer by triptolide included the cell cycle, pathways in cancer, MAPK signaling pathway, ubiquitin-mediated proteolysis, thyroid hormone signaling pathway, apoptosis, PI3K-Akt signaling pathway, NF-kappa B signaling pathway, and p53 signaling pathway (Figure 3B).

Figure 3 Functional enrichment analysis. (A) GO biological process (BP) analysis. Enrichment scores are shown on the X axis, while GO terms enriched with target genes are shown on the Y axis. (B) The related pathways of triptolide in treating thyroid cancer. The enrichment scores are listed on the X axis, while target gene-enriched items are shown on the Y axis.

Molecular Docking Analysis

We used CB-Dock to evaluate the interactions between triptolide and the core targets. We combined the triptolide targets and the core PPI network to obtain the core targets (Figure 4). The four core targets were CDKN1A, c-JUN, RELA, and TP53. Then, we analyzed the Vina scores for the binding of triptolide with CDKN1A, c-JUN, RELA, and TP53 (Figure 5). The results showed that the lowest Vina score for the binding of triptolide to CDKN1A was −6.9 kcal/mol. The lowest Vina score for the binding of triptolide to c-JUN was −5.7 kcal/mol. The lowest Vina score for the binding of triptolide to RELA was −6.9 kcal/mol. The lowest Vina score for the binding of triptolide to TP42 was −8.9 kcal/mol. These results indicated that triptolide can bind stably to the core targets.

Figure 4 Venn diagram of triptolide targets and the core PPI network.

Figure 5 Molecular docking analysis. (A) The binding affinity of triptolide for CDKN1A (lowest Vina score = −6.9, cavity size = 18). (B) The binding affinity of triptolide for c-JUN (lowest Vina score = −5.7, cavity size = 24). (C) The binding affinity of triptolide for RELA (lowest Vina score = −7.5, cavity size = 304). (D) The binding affinity of triptolide for TP53 (lowest Vina score = −8.9, cavity size = 1320).

Triptolide Suppressed the Growth of Thyroid Cancer Cells

The thyroid cancer cell line TPC-1 was used to investigate the antithyroid cancer function of triptolide. We used various concentrations of triptolide (50 nM to 200 nM) to treat TPC-1 cells. The results of the MTT assay showed that triptolide inhibited TPC-1 cell proliferation in a dose-dependent manner (Figure 6). Triptolide inhibited thyroid cancer cell growth.

Figure 6 MTT assay of triptolide-treated TPC-1 cells. The curve shows that triptolide inhibited TPC-1 cell growth in a dose-dependent manner. *P<0.05, ***P<0.001.

Triptolide Regulated the mRNA Expression of the Core Targets

RT-PCR analysis was used to measure the effects of triptolide on the mRNA expression of the core targets. Triptolide increased the mRNA expression of CDKN1A and TP53 in a dose-dependent manner (50 nM, 100 nM, 200 nM). Triptolide suppressed the expression of c-JUN and RELA in a dose-dependent manner (50 nM, 100 nM, 200 nM) (Figure 7).

Figure 7 RT-PCR analysis of the regulatory effects of triptolide on the mRNA expression of the core targets. (A) CDKN1A, (B) c-JUN, (C) RELA, (D) TP53. Triptolide regulated the mRNA expression of the core targets in a dose-dependent manner. *P<0.05, **P<0.01.

Triptolide Regulated the Protein Levels of the Core Targets in TPC-1 Cells

We used Western blotting to evaluate the function of triptolide in regulating the core targets. Triptolide increased the protein levels of CDKN1A and phospho-p53 in a dose-dependent manner (50 nM, 100 nM, 200 nM). In addition, triptolide decreased the levels of phospho-c-JUN and phospho-NF-κB p65 in a dose-dependent manner (50 nM, 100 nM, 200 nM) (Figure 8).

Figure 8 Western blot analysis of the regulatory effects of triptolide on the protein levels of the core targets. CDKN1A, phospho-p53, phospho-c-JUN, phospho-NF-κB p65. Triptolide regulated the protein levels of the core targets in a dose-dependent manner (50 nM, 100 nM, 200 nM).

Triptolide Induced the Apoptosis of Thyroid Cancer Cells

TPC-1 cells were incubated with different concentrations of triptolide (50 nM, 100 nM, 200 nM) for 24 h. We used a cell apoptosis detection kit to evaluate the apoptosis of TPC-1 cells. After incubation, TPC-1 cells were subjected to Annexin V-FITC and PI staining with the kit reagents. We found that triptolide induced thyroid cancer cell apoptosis in a dose-dependent manner (50 nM, 100 nM, 200 nM) (Figure 9).

Figure 9 Triptolide induced the apoptosis of thyroid cancer cells. A cell apoptosis kit was used to measure the apoptosis of TPC-1 cells by flow cytometry. (A) TPC-1 cells were incubated with 0 nM triptolide as control. (B) TPC-1 cells were incubated with 50 nM triptolide. (C) TPC-1 cells were incubated with 100 nM triptolide. (D) TPC-1 cells were incubated with 200 nM triptolide. Triptolide induced the apoptosis of TPC-1 cells (quadrant Q4; control: 3.4%, 50 nM: 7%, 100 nM: 10.9%, 200 nM: 23.5%) in a dose-dependent manner.

Discussion

In this study, we evaluated the function of triptolide in treating thyroid cancer with in silico and in vitro experiment. In network pharmacology, we found that inflammatory pathways, cell cycle and apoptosis were involved in triptolide treating thyroid cancer. Molecular docking analysis showed that triptolide directly interacted with core targets. In vitro experiment showed triptolide inhibited the proliferation and induced the apoptosis of thyroid cancer cells. Triptolide could regulate the mRNA expression and protein levels of the core targets in thyroid cancer cells.

In the treatment of thyroid cancer, surgery is initial step in treatment of thyroid cancer in the majority of cases. In more demolitive surgery, interventions are affected by more severe complications. Previous studies have tried to solve this situation. Calò et al used intraoperative neuromonitoring (IONM) in predicting postoperative nerve function during thyroid surgery.10 It was highly predictive of postoperative nerve function. Docimo et al found that oral calcium and vitamin D supplements could participate in the prevention of postoperative hypocalcemia.11 Conzo et al believed the neck dissection in papillary thyroid cancers management remains controversial which needs to be more investigated.12

Triptolide is a kind of diterpenoid epoxide extracted from thunder god vine, Tripterygium wilfordii.13 It has been useful in the treatment of various kinds of inflammatory diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), lupus nephritis, etc.14 The functions of triptolide in treating inflammatory diseases are multifactorial. Triptolide can inhibit the production of inflammatory cytokines such as interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α). In addition, it can reduce the production of reactive oxygen species to alleviate inflammation.15 Triptolide also suppresses the activities of inflammatory cells, including neutrophils, macrophages, dendritic cells, and T cells. In addition, triptolide inhibits signaling related to the NF-κB, MAPK, and PI3K-Akt signaling pathways. Furthermore, triptolide exhibits anticancer activities. Previous studies have found that triptolide can treat thyroid cancer. Zhu et al demonstrated that triptolide inhibits the invasion and angiogenesis of human anaplastic thyroid carcinoma cells.16 Zhu et al also revealed that triptolide exerts antiangiogenic activity by regulating vascular endothelial and tumor cells.17 Although previous studies have revealed that triptolide is useful for treating thyroid cancer, the mechanism underlying this effect has not been thoroughly investigated.

In this study, KEGG analysis showed that the pathways of triptolide in treating thyroid cancer included the cell cycle pathway. GO analysis showed that the biological processes included the terms of cell cycle arrest, G2/M transition of mitotic cell cycle, and G1/S transition of mitotic cell cycle. Therefore, we considered that triptolide could regulate the cell cycle to treat thyroid cancer. The cell cycle, the process by which cells divide and proliferate, is the key mediator of cancer. In normal cells, the cell cycle is controlled by a complex series of signaling pathways through which a cell grows, undergoes DNA replication and divides.18 This process also includes mechanisms to ensure that errors are corrected and that cells commit suicide (undergo apoptosis) if they are not corrected. In cancer, this regulatory process malfunctions as a result of genetic mutations and leads to uncontrolled cell proliferation.19 Cell cycle progression is accelerated by cyclin-dependent kinases stimulated by cyclins, including cyclin D1, and inactivated by cyclin-dependent kinase inhibitors (CDKIs).20 Thus, triptolide could influence the G2/M and G1/S transitions in the mitotic cell cycle to regulate the cell cycle in thyroid cancer cells. Furthermore, we found that triptolide could interact with CDKN1A to influence the cell cycle. CDKN1A is implicated in the regulation of cell growth and the cellular response to DNA damage.21 It inhibits cell cycle progression in G1 phase by binding to G1 cyclin-CDK complexes and may also induce G2 arrest.22 In response to DNA damage, p53 induces CDKN1A expression, which is responsible for cell cycle arrest at the G1 checkpoint.23 Our RT-PCR and Western blot results showed that triptolide increased the expression of CDKN1A. Therefore, triptolide could inhibit thyroid cancer proliferation by regulating the cell cycle through binding to CDKN1A.

In addition to the involvement of the cell cycle, KEGG analysis showed that inflammatory pathways were also involved in the antithyroid cancer function of triptolide. The inflammatory pathways included NF-kappa B transcription factor activity, the stress-activated MAPK cascade, and I-kappaB kinase/NF-kappa B signaling. GO analysis also showed that the biological processes were related to NF-kappa B transcription factor activity, the stress-activated MAPK cascade, and I-kappaB kinase/NF-kappa B signaling. Thus, the NF-κB and MAPK signaling pathways were considered to participate in the antithyroid cancer function of triptolide. Inflammation is a characteristic of cancer that plays an important role in its initiation and development. NF-κB signaling pathway activation can induce the production of inflammatory cytokines, such as TNF-α, IL-1, and IL-6.24 These inflammatory cytokines can enhance the proliferation and survival of cancer cells. In addition, NF-κB pathway activation can increase cancer cell invasion, angiogenesis, metastasis, and therapeutic resistance.25 In addition to the NF-κB pathway, the MAPK pathway was also involved in the function of triptolide. The MAPK signaling pathway plays an essential role in regulating gene expression, cell proliferation and the survival of cancer cells.26 This pathway regulates oncogenesis, cancer cell growth, and chemotherapeutic resistance. c-JUN may function as a sensor for ERK signaling dynamics. In our study, we discovered that triptolide can directly regulate the expression of c-JUN and NF-κB p65. c-JUN is the regulator of the MAPK pathway, while NF-κB p65 is the regulator of the NF-κB pathway. Therefore, we inferred that triptolide could modulate the NF-κB and MAPK signaling pathways by interacting directly with c-JUN and NF-κB p65.

Finally, we found that triptolide induced the apoptosis of thyroid cancer cells. Apoptosis is characterized by cell membrane blebbing, chromatin condensation, and cell shrinkage. Reduced apoptosis plays an important role in cancer cell development and carcinogenesis.27 The mechanisms underlying the reduction in apoptosis include reduced caspase function and an impaired balance of antiapoptotic and proapoptotic proteins. Apoptotic proteins include the Bcl-2 family of proteins, p53 (or TP53), and inhibitor of apoptosis proteins (IAPs). The Bcl-2 family of proteins is a group of proapoptotic and antiapoptotic proteins, such as Bcl-2, B-cell lymphoma-extra large (Bcl-xL), Mcl-1, and Bcl-w.28 Inhibitors of apoptosis proteins, including BIRC1, BIRC2, BIRC3, BIRC4, and BIRC5, can modulate signal transduction, cytokine production, and apoptosis. The P53 protein is a tumor suppressor protein that can induce the apoptosis of cancer cells.29 P53 is able to induce apoptosis via transcription-dependent and transcription-independent mechanisms. Disruption of P53 activates tumor development and progression. P53 can regulate the transcription of Bcl-2 family members. In addition, P53 transactivates some components of the apoptotic effector machinery.30 It also induces genes in short-circuit antiapoptotic pathways. In our study, we found that triptolide induced the apoptosis of thyroid cancer cells. Molecular docking analysis showed that triptolide can directly interact with P53. RT-PCR and WB analyses demonstrated that triptolide increased the expression of P53. Thus, we inferred that triptolide could increase the expression of P53 to induce the apoptosis of thyroid cancer cells by influencing the transcription of Bcl-2 family genes, transactivating some components of the apoptotic effector machinery, and inducing apoptotic pathways. Apart from apoptosis, P53 plays important role in several fundamental cellular signaling pathways including cell cycle arrest. P53 indirectly regulates many important genes expression which are essential in cell division cycle. As the CDK inhibitor, CDKN1A was the transcriptional target of P53. P53 activation could transcriptionally up-regulates CDKN1A expression. Then, CDKN1A acts as cell cycle checkpoint control to inhibit cell cycle progression in G1 phase by binding to G1 cyclin-CDK complexes and may also induce G2 arrest. So, P53 might also participate in cell cycle arrest except for apoptosis.

Conclusion

In conclusion, we discovered the function of triptolide in treating thyroid cancer in this study. Using the network pharmacology method, we found that inflammatory pathways, including the NF-κB and MAPK signaling pathways, were involved in the antithyroid cancer mechanism of triptolide. In addition, triptolide influenced the cell cycle and apoptosis of thyroid cancer cells. Molecular docking analysis showed that triptolide directly interacted with four core targets: CDKN1A, c-JUN, RELA, and TP53. CDKN1A is related to the cell cycle, while TP53 is related to apoptosis. c-JUN and RELA are related to the NF-κB and MAPK signaling pathways. Then, we explored the function of triptolide in vitro. Triptolide inhibited the proliferation and induced the apoptosis of thyroid cancer cells. Triptolide increased the mRNA expression levels of CDKN1A and TP53 but reduced those of c-JUN and RELA in a dose-dependent manner. Triptolide increased the protein levels of CDKN1A and phospho-p53 but reduced those of phospho-c-JUN and phospho-NF-κB p65 in a dose-dependent manner. Therefore, we considered that triptolide could treat thyroid cancer by inhibiting cell proliferation, inducing apoptosis and inhibiting inflammatory pathways such as the NF-κB and MAPK signaling pathways. CDKN1A, c-JUN, RELA, and TP53 were involved in the mechanism of triptolide in the treatment of thyroid cancer.

Abbreviations

AKAP9, A-Kinase Anchoring Protein 9; BC, betweenness centrality; Bcl-2, B-cell lymphoma 2; Bcl-Xl, B-cell lymphoma-extra large; BIND, Biomolecular Interaction Network Database; BioGRID, Biological General Repository for Interaction Datasets; BP, biological process; CC, closeness centrality; CC, cellular component; CCKBR, Cholecystokinin B Receptor; CKDN1A, cyclin-dependent kinase inhibitor 1A; CDKN1B, Cyclin Dependent Kinase Inhibitor 1B; CXCL8, C-X-C Motif Chemokine Ligand 8; DC, degree centrality; DIP, Database for Interacting Proteins; EC, eigenvector centrality; EDTA, Ethylenediaminetetraacetic acid; FBS, fetal bovine serum; FITC, Fluorescein isothiocyanate; GAD, Genetic Association Database; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GO, Gene ontology; HMGCR, 3-Hydroxy-3-Methylglutaryl-CoA Reductase; HPRD, Human Protein Reference Database; IBD, inflammatory bowel disease; IL-1β, interleukin 1 beta; IL-6, interleukin 6; IONM, intraoperative neuromonitoring; JAK1, Janus Kinase 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; LAC, local average connectivity centrality; MAPK, Mitogen-Activated Protein Kinase; MF, molecular function; MINT, Molecular INTeraction Database; NC, neighbourhood centrality; OMIM, Online Mendelian Inheritance in Man; PBST, phosphate buffered saline with Tween 20; PharmGKB, Pharmacogenomics Knowledge Base; PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride; PPI, protein-protein interaction; RA, rheumatoid arthritis; RT-PCR, real-time polymerase chain reaction; TCMSP, traditional Chinese medicine systems pharmacology; TNF-α, tumor necrosis factor alpha; TP53, tumor protein p53; TTD, Therapeutic Target Database.

Acknowledgments

This work was supported by the National Natural Science Foundation of China [grant numbers 81500689].

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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