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Thadi A , Gleeson EM , Khalili M , Shaikh MF , Goldstein E , Morano WF , Daniels LM , Grandhi N , Glatthorn H , Richard SD , Campbell PM , Sarafraz-Yazdi E , Pincus MR , Bowne WB
Anti-Cancer Tumor Cell Necrosis of Epithelial Ovarian Cancer Cell Lines Depends on High Expression of HDM-2 Protein in Their Membranes
Ann Clin Lab Sci. 2020 Sep;50(5) :611-624
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OBJECTIVE: Patients with epithelial ovarian cancers experience the highest fatality rates among all gynecological malignancies which require development of novel treatment strategies. Tumor cell necrosis was previously reported in a number of cancer cell lines following treatment with a p53-derived anti-cancer peptide called PNC-27. This peptide induces necrosis by transmembrane pore formation with HDM-2 protein that is expressed in the cancer cell membrane. We aimed to extend these studies further by investigating expression of membrane HDM-2 protein in ovarian cancer as it relates to susceptibility to PNC-27. PROCEDURES: Herein, we measured HDM-2 membrane expression in two ovarian cancer cell lines (SKOV-3 and OVCAR-3) and a non-transformed control cell line (HUVEC) by flow cytometric and western blot analysis. Immunofluorescence was used to visualize colocalization of PNC-27 with membrane HDM-2. Treatment effects with PNC-27 and control peptide were assessed using a MTT cell proliferation assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers; annexin V and caspase-3. RESULTS: HDM-2 protein was highly expressed and frequently detected in the membranes of SKOV-3 and OVCAR-3 cells; a prominent 47.6 kDa HDM-2 plasma membrane isoform was present in both cell lines whereas 25, 29, and 30 kDa isoforms were preferentially expressed in OVCAR-3. Notably, PNC-27 colocalized with HDM-2 in the membranes of both cancer cell lines that resulted in rapid cellular necrosis. In contrast, no PNC-27 colocalization and cytotoxicity was observed with non-transformed HUVEC demonstrating minimal expression of membrane HDM-2. CONCLUSIONS: Our results suggest that HDM-2 is highly expressed in the membranes of these ovarian cancer cell lines and colocalizes with PNC-27. We therefore conclude that the association of PNC-27 with preferentially expressed membrane HDM-2 isoforms results in the proposed model for the formation of transmembrane pores and epithelial ovarian cancer tumor cell necrosis, as previously described in a number of solid tissue and hematologic malignancies.
1550-8080 Thadi, Anusha Gleeson, Elizabeth M Khalili, Marian Shaikh, Mohammad F Goldstein, Eve Morano, William F Daniels, Lynsey M Grandhi, Nikhil Glatthorn, Haley Richard, Scott D Campbell, Paul M Sarafraz-Yazdi, Ehsan Pincus, Matthew R Bowne, Wilbur B Journal Article United States Ann Clin Lab Sci. 2020 Sep;50(5):611-624.