Abstract NTOC-095: THE SYNERGISTIC EFFECTS OF CARBOPLATIN AND PF–477736, A SMALL MOLECULE CHECKPOINT KINASE 1 INHIBITOR ON A PLATINUM RESISTANT OVARIAN CANCER CELL LINE
Authors: Jessica Lee, Stephanie V. Blank, Robert J. Schneider
Journal: Clinical Cancer Research (2017)

Abstract PURPOSE: To investigate the effects of PF-477736, a small molecular checkpoint kinase 1 (Chk1) inhibitor with and without carboplatin on cell apoptosis and proliferation in a p53-mutated platinum resistant ovarian cancer cell line. METHODS: OVCAR3 cells were treated with PF-477736 alone and in combination with carboplatin. Western blot analyses were performed to assess the protein expression of Chk1, phosphorylated Chk1, H2AX, and phosphorylated H2AX. Cell proliferation was evaluated with colony formation assays 10 days following treatment and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assays over the course of 5 days following treatment. Cell apoptosis was measured with flow cytometry after staining with FITC Annexin V and propidium iodide. Paired t-tests were performed for comparative analyses. RESULTS: PF-477736 decreased the expression of phosphorylated Chk1 on serine 249 within 24 hours following treatment as seen on Western blot assay. Total Chk1 expression was unchanged after treatment with PF-477736. The combination treatment of PF-477736 and carboplatin effectively decreased OVCAR3 cell proliferation. Colony formation assays revealed fewer colonies after dual treatment with PF-477736 and carboplatin compared to PF-477736 alone (average colony count 1.5 vs 13.6, p = 0.03) and compared to carboplatin alone (average colony count 1.5 vs 9.7, p &amp;lt; 0.001). MTT assays demonstrated a synergistic effect with PF-477736 and carboplatin: dual treated cells had lower mean dye absorbance values than PF-477736 alone (mean absorbance fold change 1.7 vs 3.2, p = 0.009) and carboplatin alone (mean absorbance fold change 1.7 vs 5.6, p &amp;lt; 0.001). OVCAR3 cells after treatment with PF-477736 and carboplatin had higher rates of apoptosis. On flow cytometry, a higher proportion of cells stained with the apoptotic markers Annexin V and propidium iodide after treatment with PF-477736 and carboplatin compared to PF-477736 alone (percentage of apoptotic cells 34.9% vs 13.0%, p = 0.004) and carboplatin alone (percentage of apoptotic cells 34.9% vs 16.0%, p = 0.008). In addition, western blot analysis of the cell lysates following dual treatment of PF-477736 and carboplatin showed increased expression of phosphorylated H2AX, a marker of double-stranded DNA damage while there was minimal expression with PF-477736 alone and carboplatin alone. CONCLUSIONS: Carboplatin and PF-477736, a small molecule Chk1 inhibitor, synergistically decreased cell proliferation and increased cell apoptosis in OVCAR3 cells, an in vitro model of platinum resistant ovarian cancer. PF-477736 may be a potential new therapeutic strategy for the resensitization of platinum resistant ovarian cancer. Citation Format: Jessica Lee MD, Stephanie V. Blank MD, Robert J. Schneider MD. THE SYNERGISTIC EFFECTS OF CARBOPLATIN AND PF–477736, A SMALL MOLECULE CHECKPOINT KINASE 1 INHIBITOR ON A PLATINUM RESISTANT OVARIAN CANCER CELL LINE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-095.