In vivo tumor regression achieved by a potent bivalent Smac mimetic (SM-164) in combination with TRAIL
2007
C228 Background: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in some but not in all cancer cells. X-linked Inhibitor of Apoptosis Protein (XIAP) and other IAPs are believed to cause TRAIL-resistance of cancer cells by blocking caspases activity. Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI) binds to XIAP and other IAP proteins and overcomes the anti-apoptotic activity of IAP proteins. Recently, a potent bivalent Smac mimetic, SM-164 was developed in our laboratory, which binds to XIAP and other IAP proteins with extremely high affinity. The aim of this study was to determine whether SM-164 can achieve synergistic activity with TRAIL in human breast and prostate cancer models in vitro and in vivo .
Procedures: The levels of XIAP and other IAPs were detected using Western blotting method in a panel of human breast and prostate cancer cell lines. Cellular targets of SM-164 were probed by biotin-streptavidin pull-down assays using a biotinylated SM-164 (BL-SM-164) and competition assays using unlabeled Smac mimetics. A WST-based assay and trypan blue exclusion analysis were used to determine the cellular activities of TRAIL, Smac mimetics and their combination on cell growth inhibition and cell viability in a panel of cancer cell lines. Cleavage of caspase-3, -8, -9, and PARP was analyzed by Western blotting and the role of these caspases was further analyzed using pharmacological inhibitors of these caspases and small intensifying RNA technique. The in vivo antitumor activity of SM-164 was determined using human cancer xenografts in nude mice. The pharmacodynamic study of the tumor tissue was performed using TUNEL method.
Results: IAPs were expressed with high levels in all the tested cancer cell lines. BL-SM-164 dose-dependently pulled-down XIAP and cIAP1 and cIAP2 in the 2LMP cell line, while SM-164 efficiently competed off these IAPs from the binding with BL-SM-164. Furthermore, 2LMP cell line was sensitive to TRAIL, while MDA-MB-453, BT549, PC-3, DU-145 and CL-1 were resistant to TRAIL in both cell growth and cell viability assays. SM-164 dramatically sensitized all these cancer cell lines to TRAIL in apoptosis induction and activation of caspases -3, -9 and -8 and cleavage of PARP. SM-164 in combination with subtoxic concentration of TRAIL completely induced tumor regression on human 2LMP breast cancer in xenografts nude mice. The TUNEL study showed that SM-164 induced apoptosis in tumor cells but not in normal tissues.
Conclusion: Our study demonstrates that SM-164 is highly effective in overcoming TRAIL-resistance in human breast and prostate cancer cell lines. SM-164 represents a promising lead compound and warrants further investigation for its therapeutic potential for the treatment of human cancer.
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