Abstract 2559: Development of carboplatin, docetaxel and combined carboplatin/docetaxel resistant ovarian cancer cell lines
Authors: Stephen Armstrong, Baoqing Guo, Amadeo Parissenti, Carita Lannér
Journal: Cancer Research (2010)

Abstract Carboplatin and docetaxel are two standard chemotherapeutics used to treat ovarian cancer. Patients often exhibit resistance to single agent therapy, and dual agent therapy was developed to overcome this resistance. However, resistance still commonly occurs in dual agent therapy. It is known that specific changes in gene expression occur in single agent resistance, but it is not known whether resistance to combined chemotherapy involves novel gene expression changes or if resistance is only the sum of changes seen in single agent resistant cells. Using the chemotherapy-naïve A2780 ovarian cancer cell line, we have generated three resistant cell lines including carboplatin and docetaxel single agent resistant lines (A2780CBN, and A2780DXL respectively), and a carboplatin/docetaxel dual resistant line (A2780CBNDXL) by exposing A2780 cells to increasing concentrations of drug until a dose is reached where resistance could no longer be developed. The A2780 parent line was co-cultured alongside selection to control for gene expression changes due to continuous culture and not drug resistance. The concentration of drug at which 50% of cells die (IC50) was measured for reach resistant cell line using a clonogenic assay. Cells were cultured in varying drug concentrations and plated in methylcellulose. Colonies were counted and normalized to the no drug control. The IC50 was calculated as a 50% survival fraction plotted against the log of drug (M). The IC50 of A2780CBN is 8×10−5M, 13-fold more resistant than the parent line. The IC50 of A2780DXL is 3×10−7M, 5000-fold more resistant than the parent line. The IC50 of A2780CBNDXL is 1×10−5M for carboplatin and 1×10−8M for docetaxel, 1000-fold more resistant than the parent line. This dual resistance may be caused by a synergistic effect of carboplatin with docetaxel leading to increased sensitivity of the co-cultured control to combination treatment. Total RNA was isolated from A2780DXL and its co-cultured counterpart and hybridized to Agilent 4×44k total human genome arrays to study gene expression. Agilent Feature Extraction software was used to scan the arrays and Partek Genomics suite was used to find statistical significance. A total of 1827 gene expression changes occurred, with 1061 genes up regulated and 766 genes down regulated in the resistant line. Signal intensity differences ≥ 2-fold and P ≤ 0.01 were considered significantly changed. Up regulated genes include ABCB4 (141) and ABCB1 (34) that encode two drug transporters implicated in taxane resistance. Down regulated genes include TUBB2B (−16), a beta-tubulin encoding gene, and growth factor receptors PDGFRB (−10), and PDGFRA (−8). Microarray analysis will also be performed on the A2780CBN and A2780CBNDXL lines. Gene expression changes associated with dual drug resistance may be used to develop clinically relevant biomarkers for carboplatin/docetaxel resistance in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2559.