Josephine S Kim1, Jane Turbov2, Rebecca Rosales2, Larry G Thaete1, Gustavo C Rodriguez3. 1. Division of Gynecologic Oncology, NorthShore University HealthSystem, 2650 Ridge Avenue, Suite 1507, Walgreen Building, Evanston, IL 60201, USA; Section of Gynecologic Oncology, The University of Chicago Medicine, 5841 S. Maryland Avenue, MC 2050, Chicago, IL 60637, USA. 2. Division of Gynecologic Oncology, NorthShore University HealthSystem, 2650 Ridge Avenue, Suite 1507, Walgreen Building, Evanston, IL 60201, USA. 3. Division of Gynecologic Oncology, NorthShore University HealthSystem, 2650 Ridge Avenue, Suite 1507, Walgreen Building, Evanston, IL 60201, USA; Section of Gynecologic Oncology, The University of Chicago Medicine, 5841 S. Maryland Avenue, MC 2050, Chicago, IL 60637, USA. Electronic address: grodriguez@northshore.org.
Abstract
OBJECTIVE: Recent data show that simvastatin (SIM) and metformin (MET) have anti-proliferative effects in endometrial cancer cells. The combination (MET+SIM) inhibits tumor growth and metastasis in prostate cancer cells which possess similar molecular alterations to many early endometrial cancers. We tested the hypothesis that the anti-proliferative effects of MET+SIM in endometrial cancer cells would be greater than the effects of each agent alone. METHODS: RL95-2, HEC1B, and Ishikawa endometrial cancer cell lines were treated with MET and/or SIM. Growth inhibition was measured by MTS cell proliferation assays. Apoptosis was evaluated by caspase-3, Annexin V, and TUNEL assays and by apoptosis markers (BAX, Bcl-2, Bim) using western blot. Bim was silenced using Bim siRNA to confirm this apoptotic pathway. Treatment effects on the mTOR pathway were investigated by western blot using antibodies to phosphorylated (phospho)-AMPK and phospho-S6. RESULTS: MET+SIM synergistically inhibited growth in all three cell lines. The combination induced apoptosis as measured by TUNEL, Annexin V, and caspase-3 assays. Bim siRNA transfection abrogated this effect-silencing Bim in MET+SIM-treated RL95-2 cells rescued cell viability in MTS assays and reduced caspase-3 activity compared with control siRNA-transfected cells. Combination treatment upregulated phosphorylated AMPK and downregulated downstream phosphorylated S6, suggesting mTOR inhibition as a mechanism for these anti-proliferative effects. CONCLUSIONS: MET+SIM treatment synergistically inhibits endometrial cancer cell viability. This may be mediated by apoptosis and mTOR pathway inhibition. Our results provide preclinical evidence that the combination of these well-tolerated drugs may warrant further clinical investigation for endometrial cancer treatment.
OBJECTIVE: Recent data show that simvastatin (SIM) and metformin (MET) have anti-proliferative effects in endometrial cancer cells. The combination (MET+SIM) inhibits tumor growth and metastasis in prostate cancer cells which possess similar molecular alterations to many early endometrial cancers. We tested the hypothesis that the anti-proliferative effects of MET+SIM in endometrial cancer cells would be greater than the effects of each agent alone. METHODS: RL95-2, HEC1B, and Ishikawa endometrial cancer cell lines were treated with MET and/or SIM. Growth inhibition was measured by MTS cell proliferation assays. Apoptosis was evaluated by caspase-3, Annexin V, and TUNEL assays and by apoptosis markers (BAX, Bcl-2, Bim) using western blot. Bim was silenced using Bim siRNA to confirm this apoptotic pathway. Treatment effects on the mTOR pathway were investigated by western blot using antibodies to phosphorylated (phospho)-AMPK and phospho-S6. RESULTS:MET+SIM synergistically inhibited growth in all three cell lines. The combination induced apoptosis as measured by TUNEL, Annexin V, and caspase-3 assays. Bim siRNA transfection abrogated this effect-silencing Bim in MET+SIM-treated RL95-2 cells rescued cell viability in MTS assays and reduced caspase-3 activity compared with control siRNA-transfected cells. Combination treatment upregulated phosphorylated AMPK and downregulated downstream phosphorylated S6, suggesting mTOR inhibition as a mechanism for these anti-proliferative effects. CONCLUSIONS:MET+SIM treatment synergistically inhibits endometrial cancer cell viability. This may be mediated by apoptosis and mTOR pathway inhibition. Our results provide preclinical evidence that the combination of these well-tolerated drugs may warrant further clinical investigation for endometrial cancer treatment.