Activation of the Fas-FasL Signaling Pathway by MDA-7/IL-24 Kills Human Ovarian Cancer Cells

Began Gopalan,Anya Litvak,Sikha Sharma,Rajagopal Ramesh,Sunil Chada,Abner M Mhashilkar

doi:10.1158/0008-5472.can-04-3758

Began Gopalan, Anya Litvak + Show 4 more

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https://doi.org/10.1158/0008-5472.can-04-3758

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Abstract

The tumor-suppressive activity of melanoma differentiation-associated gene-7 (mda-7), also known as interleukin 24 (IL-24), has been shown in a spectrum of human cancer cells in vitro and in vivo. However, mechanisms responsible for antitumor activity of mda-7 in human ovarian cancer cells have not been identified. We investigated the therapeutic activity and underlying mechanisms of adenovirus-mediated mda-7 gene (Ad-mda7) transfer in human ovarian cancer cells. Ad-mda7 treatment resulted in overexpression of MDA-7/IL-24 protein in both ovarian cancer and normal ovarian epithelial cells. However, Ad-mda7 significantly (P = 0.001) inhibited cell proliferation and induced apoptosis only in tumor cells and not in normal cells. Studies addressing the mechanism of action of Ad-mda7-induced tumor cell apoptosis revealed early activation of the transcription factors c-Jun and activating transcription factor 2, which in turn stimulated the transcription of an immediate downstream target, the death-inducer Fas ligand (FasL), and its cognate receptor Fas. Associated with the activation of Fas-FasL was the activation of nuclear factor kappaB and induction of Fas-associated factor 1, Fas-associated death domain, and caspase-8. Promoter-based reporter gene analyses showed that Ad-mda7 specifically activated the Fas promoter. Inhibition of Fas using small interfering RNA resulted in a significant decrease in Ad-mda7-mediated tumor cell death. Additionally, blocking of FasL with NOK-1 antibody abrogated Ad-mda7-mediated apoptosis. Collectively, these results show that Ad-mda7-mediated killing of human ovarian cancer cells involves activation of the Fas-FasL signaling pathway, a heretofore unrecognized mediator of MDA-7 apoptosis induction.

Highlights

Ovarian cancer is the second most common gynecologic malignancy in the United States and is the fifth leading cause of death among women [1]
Exogenous MDA-7 protein expression that increased over time was observed in both tumor and normal cells treated with adenovirus-mediated mda-7 gene (Ad-mda7) but not in cells treated with PBS or Ad-luc (Fig. 1A)
Significant (P = 0.001) inhibition of cell proliferation was observed in tumor cells but not in normal cells treated with Ad-mda7 (Fig. 1B)

Summary

Introduction

Ovarian cancer is the second most common gynecologic malignancy in the United States and is the fifth leading cause of death among women [1]. Cells were harvested at 48 hours after Ad-mda7 treatment, washed with PBS twice, and analyzed for apoptotic cells by flow cytometry as described above and for caspase-8 activity by Western blotting. These results show that Ad-mda7 effectively and selectively inhibits ovarian cancer cell proliferation by inducing G2-M cell cycle arrest and apoptosis, while sparing normal cells.

Results

Conclusion