Enhanced apoptosis in melanoma cells following treatment with bortezomib (Velcade ®, PS-341) and Interferon Alpha

Abstract

The 26S proteasome is a protein complex that plays a critical role in the degradation of cellular proteins including transcription factors, cyclins, and other proteins required for cell cycle progression in normal and malignant cells. Bortezomib (PS-341, Velcade) is a specific and selective inhibitor of the 26S proteasome that has cytotoxic activity against tumor cells. Treatment with this compound has been shown to induce apoptosis in malignant cells. Interferon-alpha (IFN-alpha) is an immunostimulatory cytokine that is known to inhibit proliferation and to promote apoptosis in tumor cells. Apoptosis of human melanoma cells was evaluated following treatment with PBS, bortezomib (10nM), IFN-alpha (104 U/mL), or both agents combined by Annexin V/ propidium iodide (PI) staining and demonstrated that cell death was synergistic in nature. The pro-apoptotic activity of this treatment combination was evident in multiple malignant melanoma cell lines (1259 MEL, 18105 MEL, A375) indicating that this effect was not cell line specific. Cell death induced by bortezomib and IFN-alpha was associated with cleavage of caspase proteins (caspase-3, -7) and poly ADP-ribose polymerase (PARP). Interestingly, levels of the cyclin dependent kinase inhibitor, p21, were increased slightly following treatment with bortezomib plus IFN-alpha. Levels of the pro-survival mitochondrial membrane protein Bcl-2 were decreased in a synergistic manner following treatment of human melanoma cells with bortezomib and IFN-alpha combined. Real-Time PCR analysis demonstrated that pre-treatment of melanoma cells with bortezomib enhanced IFN-alpha induced gene expression. Together these data suggest that bortezomib and IFN-alpha exert complimentary pro-apoptotic effects on the tumor cell and that pre-treatment with bortezomib can sensitize melanoma cells to the direct effects of IFN-alpha. The safety of this treatment combination is being evaluated in a Phase I clinical trial in patients with metastatic melanoma at The Ohio State University (OSU 04105; PI: Carson).