Proteomic analysis of 17-DMAG effects on colon cancer HT-29 cells

Abstract

17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) is an inhibitor of heat shock protein 90 (HSP 90) that stabilizes a variety of proteins required for survival of cancer cells. 17-DMAG was developed as chemotherapeutic agents, having an increasing importance in cancer treatment. However, their effect on colon cancer and on the regulated proteins remains largely unknown. In this study, 17-DMAG was used and its effects on colon cancer cell line HT-29 were examined. 17-DMAG can induce apoptosis and inhibit the proliferation of HT-29 cells in a dose-and time-dependent manner. Through two-dimensional electrophoresis (2DE), we detected 19 differentially expressed protein spots in the HT-29 cells after 17-DMAG (1 µM) treating for 24 hours (h) compared with the un-treated. Of which, 2 up- and 14 down-regulated non-redundant proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS). Furthermore real-time RT-PCR showed that 4 differentially expressed proteins were down-regulated in mRNA level with consistent regulation in protein level. Two of them (cortactin (SRC8) and Heat shock 70 kDa protein 1A/1B (HSP71) were verified by western blotting to have consistent change with 2DE based proteomic study. In conclusion, we demonstrated that 17-DMAG inhibits proliferation and induces apoptosis. Moreover, 17-DMAG altered the expression profile of 16 proteins including cortactin and HSP71. This data suggest that SRC8 or HSP71 may be potential HSP90 client proteins and new targets for anti-cancer drug development.

Keywords: 17-DMAG, proteomics, colon cancer, HT-29, cortactin, HSP71