M. F. Shaikh1, B. Babcock1, E. Gleeson1, K. Davitt1, P. Love1, A. Desai1, D. Zimmerman1, M. R. Pincus2, W. B. Bowne1 1Drexel University College Of Medicine,Philadelphia, Pa, USA 2New York Harbor Healthcare System VAMC,New York, NY, USA
Introduction: PNC-27 is an anti-cancer peptide derived from the MDM-2 binding domain of p53, residues 12-26, attached to a membrane residency peptide. Previously, this peptide has been shown to be effective against murine colon cancer. We now test PNC-27 against a human colon cancer cell line, HCT-116, to further characterize anti-cancer activity and mechanism.
Methods: 1 x 104 HCT-116 cells and untransformed colonic fibroblasts (CF) were treated with PNC-27 and control peptide. Anti-cancer activity was assessed using the MTT cell proliferation assay. Mechanism of cancer cell death (necrosis vs. apoptosis) was assessed using the LDH and caspase-3 assays, respectively. Western blot analysis determined HDM2 expression in both HCT-116 and CF. PNC-27/HDM-2 interaction was studied using immunofluorescent staining and confocal microscopy.
Results: PNC-27 demonstrated specific anti-cancer effects in HCT-116 cells with a greater than 80% reduction in cell proliferation after treatment (p<.007). Mechanism studies revealed no elevation of pro-apoptotic proteins but did reveal rapid cell death by 5-fold increase in LDH release (p<.0001), signifying necrosis. Western blot analysis showed increased expression in HCT-116 cells as compared to untransformed CF cells. Moreover, confocal microscopy demonstrated specific co-localization of PNC-27 and MDM-2 along the cancer plasma membrane.
Conclusion: Our results suggest that PNC-27 targets HDM-2 on the HCT-116 cancer cell membrane, leading to specific anti-cancer necrosis. This peptide shows promise in the treatment of colon cancer.