B. D. Babcock1, M. F. Shaikh1, K. Davitt1, Y. Piazza2, T. Meckmongkol1, V. Purohit1, R. Patel1, M. Estioko1, E. Gleeson1, M. R. Pincus3, W. B. Bowne1 1Drexel University College Of Medicine,Surgery,Philadelphia, Pa, USA 2Drexel University College Of Medicine,Pathology,Philadelphia, Pa, USA 3New York Harbor Healthcare System VAMC,Pathology,New York, NY, USA
Introduction: PNC-27 is a p53-derived peptide construct from the MDM-2 binding domain that possesses anti-cancer activity while sparing normal untransformed cells. To further elucidate anti-cancer activity and mechanism we tested PNC-27 in a murine colon cancer (MCC) peritoneal carcinomatosis (PC) model.
Methods: 1 x 104 CT-26 MCC cells and untransformed colonic fibroblasts (CF) were treated with PNC-27 and control peptide. Cellular proliferation (MTT), necrosis (LDH), and apoptosis (caspase-3) were measured. Quantification of MDM-2 in CT-26 and CF was performed using western blot analysis. Confocal microscopy localized PNC-27/MDM-2 interaction. Nu/Nu mice with established PC determined by in-vivo bioluminescent imaging (IVIS-BLI) were treated by intra-peritoneal peptide injection for 14 days. BLI, tumor volume, and histology were studied.
Results: PNC-27 showed no elevation of pro-apoptotic proteins but induced rapid (4h) dose-dependent CT-26 specific cellular necrosis with 3-fold increase in LDH release (p<0.001) and 92.3% decrease in proliferation (p<0.001). This observed anti-cancer activity may, in part, be due to increased expression of MDM-2 detected in CT-26 plasma membranes compared to CF. Moreover, confocal microscopy demonstrated specific co-localization of PNC-27 and MDM-2 along CT-26 plasma membrane. Importantly, tumor volume decreased by nearly 40% in PNC-27 treated mice (versus controls; p = 0.1) which was in accordance to observed reduction in BLI and histologically confirmed tumor specific coagulation necrosis. No toxicity was observed in PNC-27 treated mice.
Conclusion: Our results suggest a mechanism of PNC-27 binding to MDM-2 on the CT-26 plasma membrane, leading to rapid targeted necrosis with a potential role in the treatment of colon cancer PC.