Skip to main content
  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • "Best of" Collection
      • Editors' Picks
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • My Cart

Search

  • Advanced search
Cancer Prevention Research
Cancer Prevention Research
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • "Best of" Collection
      • Editors' Picks
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Research Article

The Novel Akt Inhibitor API-1 Induces c-FLIP Degradation and Synergizes with TRAIL to Augment Apoptosis Independent of Akt Inhibition

Bo Li, Hui Ren, Ping Yue, Mingwei Chen, Fadlo R. Khuri and Shi-Yong Sun
Bo Li
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hui Ren
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ping Yue
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mingwei Chen
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Fadlo R. Khuri
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shi-Yong Sun
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1158/1940-6207.CAPR-11-0548 Published April 2012
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Additional Files
  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1.

    API-1 inhibits the (A) growth of and (B) induces apoptosis including caspase (Casp) activation (C) in human cancer cells. A, the indicated cancer cell lines were seeded in 96-well cell culture plates and treated the next day with API-1 at 0.625, 1.25, 2.5, 5, and 10 μmol/L. After 3 days, cell numbers were estimated by the SRB assay. Cell survival was expressed as the percentage of control (DMSO treated) cells. Data are the means of 4 replicate determinations. Bars, ±SDs. B and C, the indicated cell lines were treated with the given concentrations of API-1 for 24 hours and then harvested for measurement of apoptosis using Annexin V staining (A) and for detection of caspase activation with Western blotting (C). Data are the means of duplicate experiments. Bars, ±SDs. CF, cleaved fragment.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2.

    API-1 downregulates c-FLIP levels without increasing DR4 and DR5 expression. A, the given cell lines were treated with 5 μmol/L API-1 for 24 hours. B, H157 cells were treated with different concentrations of API-1 as indicated for 12 hours. C, H157 cells were treated with 5 μmol/L API-1 for the indicated times. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for the given proteins. LE, longer exposure.

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3.

    API-1 enhances TRAIL-induced apoptosis as evaluated by cell survival (A), caspase activation (B), and Annexin V staining (C). A, the indicated cell lines were seeded in 96-well cell culture plates and treated the next day with the given concentrations of API-1 alone, TRAIL alone, or their respective combinations. After 24 hours, cell numbers were estimated by the SRB assay. Data are the means of 4 replicate determinations. Bars, ±SDs. B and C, the indicated cell lines were treated with 20 ng/mL TRAIL alone, 5 μmol/L API-1 alone, or their combination (A + T). After 24 hours, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis (B) or for measurement of apoptosis using Annexin V staining (C). CF, cleaved fragment. Columns, means of duplicate determinations; bars, ±SDs.

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4.

    Enforced expression of ectopic c-FLIP attenuates the ability of API-1 to decrease cell survival (A) and induce apoptosis (B) including activation of caspases (C). A, the indicated cell lines were seeded in 96-well cell culture plates and treated the next day with DMSO, different concentrations of API-1 alone, 20 ng/mL TRAIL alone, or API-1 plus TRAIL. After 24 hours, cell numbers were estimated by the SRB assay. Cell survival was expressed as the percentage of control (DMSO treated) cells. Data are the means of 4 replicate determinations. Bars, ±SDs. B and C, the indicated cell lines were treated with DMSO, 5 μmol/L API-1 alone, 20 ng/mL TRAIL alone, or API-1 plus TRAIL. After 24 hours, the cells were harvested for detection of apoptosis with Annexin V staining (B) and for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect the given proteins (C). Columns, means of duplicate experiments; bars, ±SDs. CF, cleaved fragment.

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5.

    API-1 promotes ubiquitin/proteasome-mediated c-FLIP degradation (A and D) and decreases c-FLIP stability (B and C). A, H157 cells were pretreated with 20 μmol/L MG132 for 30 minutes and then cotreated with 5 μmol/L API-1 for another 4 hours. The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. B and C, H157 cells were treated with DMSO or 5 μmol/L API-1 for 5 hours. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 μg/mL cycloheximide (CHX). At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis (B). Protein levels were quantified with the NIH ImageJ software and were normalized to actin. The results were plotted as the relative c-FLIP levels compared with those at time 0 of cycloheximide treatment (C). D, H157-FLIPL-21 cells, which stably express ectopic flag-FLIPL, were transfected with HA-ubiquitin plasmid with FuGENE 6 transfection reagent for 24 hours. The cells were then pretreated with 20 μmol/L MG132 for 30 minutes and then cotreated with 5 μmol/L API-1 for 4 hours. Whole-cell protein lysates were then prepared for immunoprecipitation (IP) using anti-Flag antibody followed by Western blot (WB) analysis using anti-HA antibody for detection of ubiquitinated FLIPL (Ub-FLIPL) and anti-Flag antibody for detection of ectopic FLIPL. LE, longer exposure.

  • Figure 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 6.

    MK2206 and API-2 do not reduce c-FLIP levels (A and B) and only minimally enhance TRAIL-mediated cell killing (C and D). A and B, the indicated cell lines were treated with different concentrations of MK2206 or API-2 as indicated for 24 hours and then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for the given proteins. C and D, the indicated cell lines were seeded in 96-well cell culture plates and treated the next day with the given concentrations of MK2206 or API-2 alone, TRAIL alone, or TRAIL plus MK2206 or API-2. After 24 hours, cell numbers were estimated by the SRB assay. Data are the means of 4 replicate determinations. Bars, ±SDs.

Additional Files

  • Figures
  • Supplementary Data

    Files in this Data Supplement:

    • Supplementary Figure 1 - PDF file - 76K
    • Supplementary Figure 2 - PDF file - 186K
PreviousNext
Back to top
Cancer Prevention Research: 5 (4)
April 2012
Volume 5, Issue 4
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Cancer Prevention Research article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
The Novel Akt Inhibitor API-1 Induces c-FLIP Degradation and Synergizes with TRAIL to Augment Apoptosis Independent of Akt Inhibition
(Your Name) has forwarded a page to you from Cancer Prevention Research
(Your Name) thought you would be interested in this article in Cancer Prevention Research.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
The Novel Akt Inhibitor API-1 Induces c-FLIP Degradation and Synergizes with TRAIL to Augment Apoptosis Independent of Akt Inhibition
Bo Li, Hui Ren, Ping Yue, Mingwei Chen, Fadlo R. Khuri and Shi-Yong Sun
Cancer Prev Res April 1 2012 (5) (4) 612-620; DOI: 10.1158/1940-6207.CAPR-11-0548

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
The Novel Akt Inhibitor API-1 Induces c-FLIP Degradation and Synergizes with TRAIL to Augment Apoptosis Independent of Akt Inhibition
Bo Li, Hui Ren, Ping Yue, Mingwei Chen, Fadlo R. Khuri and Shi-Yong Sun
Cancer Prev Res April 1 2012 (5) (4) 612-620; DOI: 10.1158/1940-6207.CAPR-11-0548
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Disclosure of Potential Conflicts of Interest
    • Authors' Contributions
    • Grant Support
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF
Advertisement

Related Articles

Cited By...

More in this TOC Section

  • IgG Glycosylation and Esophageal Precancerosis
  • Liver cancer treatment and HCV eradication
  • Role of FoxQ1 in bCSC Inhibition by Withaferin A
Show more Research Articles
  • Home
  • Alerts
  • Feedback
  • Privacy Policy
Facebook   Twitter   LinkedIn   YouTube   RSS

Articles

  • Online First
  • Current Issue
  • Past Issues

Info for

  • Authors
  • Subscribers
  • Advertisers
  • Librarians

About Cancer Prevention Research

  • About the Journal
  • Editorial Board
  • Permissions
  • Submit a Manuscript
AACR logo

Copyright © 2021 by the American Association for Cancer Research.

Cancer Prevention Research
eISSN: 1940-6215
ISSN: 1940-6207

Advertisement