Effect of (-)-gossypol (AT-101) on transcriptional regulation of Noxa and Puma and on Mcl-1-mediated cancer cell resistance to apoptosis

Abstract

e14611 Background: AT-101 is being evaluated in Phase II clinical trials for the treatment of human cancers and demonstrates anticancer activity as a single agent and in combination with docetaxel. Despite extensive investigations, the mechanism of action for its antitumor activity is not completely understood. In this study, we have performed detailed investigations using a panel of human cancer cell lines on the mechanism of action for AT-101. Methods: A series of human breast and prostate cancer cell lines were utilized to evaluate the antitumor activity of AT-101. Cell growth inhibition, cell viability and apoptosis were determined by WST assay, trypan blue assay and flow cytometry, respectively. Gene specific siRNAs were used to knockdown target gene expression. Quantitative RT- PCR was performed to detect the mRNA levels of Puma and Noxa. Co-immunoprecipitation was carried out to detect the association between Mcl-1 and Noxa or Puma. Results: AT-101 induced apoptosis in both Bax/Bak-dependent and -independent manners in a variety of human cancer cell lines concomitant with increased expression of Noxa and Puma in a p53-independent manner. The up-regulation of Noxa was not observed in normal primary breast epithelial cells or normal-like human breast epithelial MCF-12F cells. Transcription inhibitor actinomycin D blocked AT-101-induced up-regulation of Puma and Noxa. Knockdown of Noxa or Puma attenuated AT-101-induced apoptosis. Furthermore, AT-101 effectively overcame Mcl-1-mediated cancer cell resistance to apoptosis. Co-immunoprecipitation studies demonstrated that Mcl-1 specifically associated with Noxa but not Puma. Conclusions: Our findings suggest that transcriptional up-regulation of pro-apoptotic Noxa and Puma contributes to the antitumor activity of AT-101, which plays a dominant role in antagonizing Mcl-1 and overcoming Mcl-1-mediated resistance to apoptosis of cancer cells. AT-101 functions as an antagonist of Bcl-2, Bcl-xL and Mcl-1 through direct binding to these proteins, as well as through upregulation of Noxa and Puma. [Table: see text]