Search

SIGNALING THROUGH THE HEDGEHOG PATHWAY IN OVARIAN CANCER Sneha S, Nagare R. P, Krishna Priya S and Ganesan TS. Departments of Medical Oncology & Clinical Research, Cancer Institute (WIA), Chennai tsganesan@gmail.com Background: The signaling pathways that cancer stem cells (CSCs) employ may be similar to those in embryonic stem cells. These are 'wnt', 'notch' and 'hedgehog'. Objective: Evaluate signaling by CSCs, using specific small molecule inhibitors in serous ovarian cancer cells. Methods: Serous ovarian cancer cell lines (N=7) were cultured as spheroids under stem cell conditions in the presence or absence of small molecule inhibitors and counted after two weeks. The effect on cell proliferation was evaluated by the MTT assay. In addition, cells from malignant ascites from patients with serous adenocarcinoma of the ovary (PMC) were cultured as monolayer in the presence or absence of inhibitors. Assays were performed to analyse effect on CSCs. These were ALDH1A1 assay (N=20), Hoechst exclusion assay (N=3), expression of surface markers (N=20), wound healing assay (N=6), and colony assay (N=7). The effect on the cell cycle of CSCs was analysed using Hoechst and Pyronin Y. Results: Spheroid formation in OVCAR3 and UC1101 cells was inhibited completely by the smoothened inhibitor (SMO) GDC0449 (3 μM) despite lack of cytotoxic effect when evaluated by the MTT assay. Flow cytometry analysis of PMC cultured in the presence of GDC0449 showed significant reduction in the expression of ALDH1A1 (2.5±0.9), CD44 (18.6±5.5), CD9 (1.6±0.8), CD133 (0.5±0.3), CD117 (18.8±13.4), CD24 (0.1±0). Similar results were obtained with inhibitors, LDE225 (SMO, 2.5nM) and GANT61 (Gli, 5 μM). Significant reduction in migration of PMC was observed in the scratch assay and clonogenic assay in the presence of inhibitors (P< 0.001). Side population assay revealed a reduction in CSCs following treatment with GANT61 (P< 0.05). In PMC, GANT61 also decreased the quiescent (Hoechst33342low/PyroninYlow) cell population (P< 0.05). Discussion and Conclusion: These results demonstrate that the hedgehog pathway is important in maintaining the integrity of CSCs in ovarian cancer. [ABSTRACT FROM AUTHOR]