Abstract 2122: The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian cancer cells.

Introduction: Cell stiffness (Young9s E modulus), as measured via Atomic Force Microscopy (AFM) is a newly recognized characteristic of cancer cells. We previously demonstrated that cisplatin-resistant ovarian cancer cells have a stiffer cell membrane as compared to their syngeneic cisplatin-sensitive counterpart, and that cisplatin increases cell stiffness in cisplatin-sensitive cells. Here we examine the role of Rho GTPase in cell stiffness and cisplatin resistance. Experimental Procedures: Prior to AFM analysis, cells were stained with Annexin V-FLUOS and only non-apoptotic cells were chosen for measurements. Cell stiffness was measured via AFM on live cells in physiological buffer. The actin cytoskeleton was visualized using phalliodin-ATTO 647N. Cells were imaged with a Leica TCS SP5 STED confocal system or a Nikon Eclispe 90i. Cisplatin sensitivity was determined via the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] cell viability assay. A nonlinear regression curve fit (one phase exponential decay) was used to analyze cisplatin dose response experiments and determine the IC50 (GraphPad Prism). Rho inhibition and activation were accomplished using Rho Inhibitor I and Rho Activator II, respectively (Cytoskeleton, Inc.). Results: The cisplatin IC50 of cisplatin-sensitive (A2780, OVCAR5, SKOV3) and cisplatin-resistant (CP70, OVCAR5-CisR, SKOV3-CisR, RMG-1) cells correlated with Young9s E modulus, with increasing IC50 corresponding to a stiffer cell phenotype. Cells that are resistant to cisplatin displayed an actin cytoskeleton that consisted of long stress fibers that spanned the width of the cell. Rho inhibition in CP70 and OVCAR5-CisR cells abolished this network of actin stress fibers, decreased cell stiffness, and increased cisplatin sensitivity. In contrast, Rho activation in A2780 and OVCAR5 cells induced the reorganization of the actin cytoskeleton into long stress fibers, increased cell stiffness, and decreased cisplatin sensitivity. We demonstrated previously that cisplatin increases cell stiffness in cisplatin-sensitive OVCAR5 cells, and here we show that cisplatin induces actin stress fiber formation in these cells. Conclusions: Rho-mediated actin stress fiber formation increases cell stiffness and mediates cisplatin resistance in ovarian cancer cells. Citation Format: Chintda Santiskulvong, Shivani Sharma, Laurent Bentolila, Jian Y. Rao, James Gimzewski, Oliver Dorigo. The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2122. doi:10.1158/1538-7445.AM2013-2122