VEGF expression is inhibited by cisplatin at the transcriptional level through HIF-1 alpha expression.
Zhong-X; Skinner-H; Ding-M; Lopez-LA; Reed-E; Jiang-BH
Proceedings of the American Association for Cancer Research (AACR) 94th Annual Meeting, July 11-14, 2003, Washington, DC. 2nd edition. Philadelphia, PA: American Association for Cancer Research, 2003 Jul; 44:404
Cisplatin (CDDP) is an effective anti-neoplastic agent against a variety of human solid tumors, particularly those of the ovary. However, the molecular mechanism of its action is poorly understood. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis, and is upregulated in many human cancers. The expression of VEGF is regulated by hypoxia-inducible factor-1 (HIF-1). HIF-1 is a hetrerodimeric basic helix-loop-helix transcription factor, composed of HIF-1a and HIF-1B subunits. HIF-1 activity is regulated by the level of HIF-1a expression in the cells. In. this study, we demonstrated that CDDP inhibited VEGF expression in human ovarian cancer cells. To determine whether CDDP inhibited VEGF expression at the transcriptional level, we analyzed the effects of CDDP on a reporter containing human VEGF 5'-flanking sequences inserted into pGL2-basic luciferase vector, and found that CDDP inhibited the reporter activity in a dose dependent manner in ovarian cancer cells, indicating that CDDP inhibited transcriptional activation of VEGF. To determine whether CDDP inhibited transcriptional activation of VEGF through the activation of HIF-1, the HIF-1 binding site at the VEGF 5'-flanking sequences was mutated. We found that: 1) the luciferase activity mediated by VEGF reporter containing the mutation of HIF-1 binding site was much lower than the reporter containing the wild-type HIF-1 binding site in the ovarian cancer cells. This result confirms that HIF-1 is a major transcriptional regulator for VEGF expression. 2) CDDP greatly inhibited the VEGF reporter activity containing the wild-type HIF-1 binding site, but increased the reporter activity containing the mutation at the HIF-1 binding site. This result indicates that CDDP inhibited VEGF transcriptional activation specifically by decreasing HIF-1 activity. To further confirm the role of HIF-1 in CDDP -mediated VEGF expression, A2780-CP70 cells were co-transfected with VEGF reporter and a dominant negative construct of HIF-1. Co-transfection of a dominant negative construct of HIF-1 inhibited VEGF reporter activity in a dose dependent manner in ovarian cancer cells. CDDP inhibited HIF-1 activity through the expression of HIF-a, while level of HIF-1B did not change by the treatment. HIF-1a expression in OVCAR.-3 cells was inhibited by CDDP at much lower dose than in A2780-CP70 cells which are more resistant to CDDP, indicating that HIF-1a expression is also correlated with the sensitization of cells to CDDP. Taken together, these results suggest a novel mechanism of CDDP 's anti-tumor activity in ovarian cancer cells through the transcriptional regulation of VEGF expression and HIF-1 activation in the cells.
Tumors; Tumorigenesis; Reproductive-system-disorders; Cancer; Cell-biology; Cell-damage; Cell-function; Cellular-function
Abstract; Conference/Symposia Proceedings
Proceedings of the American Association for Cancer Research (AACR) 94th Annual Meeting, July 11-14, 2003, Washington, DC. 2nd edition