Targeting Membrane HDM-2 by PNC-27 Induces Necrosis in Leukemia Cells But Not in Normal Hematopoietic Cells

Abstract

Background/Aim: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. Materials and Methods: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. Results: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. Conclusion: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.

Molecular Targeting of H/MDM-2 Oncoprotein in Human Colon Cancer Cells and Stem-like Colonic Epithelial-derived Progenitor Cells

“…As shown in these previous studies, binding of PNC-27 to HDM-2 in cancer cell membranes induces the formation of transmembrane pores resulting in cell death (6,8,12). This mechanism of PNC-27induced cancer cell death for the cells tested in this study was very likely due to pore formation given that the rise in cytosolic LDH was rapid and, at higher concentrations of peptide, reached maximal levels within four hours as observed in prior studies (9)(10)(11). Our findings that PNC-27 induced rapid cancer cell death without expression of early (annexin V staining of exposed phospholipid) or late (caspase 3) markers for apoptosis in any of the cell lines tested in this study (Figures 5) strongly support tumor cell necrosis and not apoptosis as the mechanism of cell death.…”

“…On the other hand, our positive control, staurosporine, a known activator of apoptosis, resulted in significant annexin Vpositive cell and caspase-3 activity ( Figure 5). Therefore, PNC-27 induces its cytotoxic effects via necrosis independent of apoptosis, as we found for all of the other cell lines that we have studied previously (5)(6)(7)(8)(9)(10)(11)21). Figure 6A and B.…”

“…Further evidence that apoptosis was not the cause of PNC-27-induced cancer cell death is that staurosporine, that is known to be a strong inducer of apoptosis, required from 10-24 h to induce total cancer cell death in each of the cell lines that we tested while PNC-27 induced total cancer cell death in 4 h for each cell line tested and induced maximal release of LDH over this time course. In prior studies, we have found that PNC-27 actually induces immediate release of LDH into the medium suggesting early cancer cell membrane damage (5)(6)(7)(8)(9)(10)(11)21). Electron microscopic analysis of these cancer cells treated with PNC-27 after several minutes of incubation shows transmembrane pore formation in these cells (6,8).…”

“…Confocal microscopy. Colocalization experiments using confocal microscopy were performed on all cell lines in a manner identical to the procedure used in a prior publication (10). Briefly, cells grown in confocal dishes overnight to 50-60% density were treated for 1h and maintained in 5% CO 2 humidified air at 37˚C.…”

“…Formation of this peptide-protein complex induces transmembrane pore formation resulting in rapid tumor cell necrosis (6)(7)(8). Subsequent studies have further confirmed that PNC-27 selectively induces tumor cell necrosis by co-localizing with H/MDM-2 resulting in rapid tumor cell necrosis (9)(10)(11).…”