Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells by stabilization of death receptors

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin (ADAM) activity. We have previously shown that adenovirally expressed tissue inhibitor of metalloproteinases-3 (TIMP-3) induces apoptosis in melanoma cells and inhibits growth of human melanoma xenografts. Here, we have studied the role of death receptors in apoptosis of melanoma cells induced by TIMP-3. Our results show, that the exposure of three metastatic melanoma cell lines (A2058, SK-Mel-5, and WM-266-4) to recombinant TIMP-3, N-terminal MMP inhibitory domain of TIMP-3, as well as to adenovirally expressed TIMP-3 results in stabilization of tumor necrosis factor receptor-1 (TNF-RI), FAS, and TNF-related apoptosis inducing ligand receptor-1 (TRAIL-RI) on melanoma cell surface and sensitizes these cells to apoptosis induced by TNF-alpha, anti-Fas-antibody and TRAIL. Stabilization of death receptors by TIMP-3 results in activation of caspase-8 and caspase-3, and subsequent apoptosis is blocked by specific caspase-8 inhibitor (Z-IETD-FMK) and by pan-caspase inhibitor (Z-DEVD-FMK). Adenovirus-mediated expression of TIMP-3 in human melanoma xenografts in vivo resulted in increased immunostaining for TNF-RI, FAS, and cleaved caspase-3, and in apoptosis of melanoma cells. Taken together, these results show that TIMP-3 promotes apoptosis in melanoma cells through stabilization of three distinct death receptors and activation of their apoptotic signaling cascade through caspase-8.