The Mechanism of Bladder Tumor Cell Line T24 Against Genistein Induced Growth Inhibition

• Main Authors: Yi-chun Tsai, 蔡宜鈞
• Other Authors: Biehuoy Hsieh, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/55999010203763586664

碩士 === 中山醫學院 === 生物化學研究所 === 89 === Abstrate Soybean isoflavone genistein is a plant estrogen. Many papers had reported that genistein could suppress the cancer development and inhibit the growth of many cancer cell lines. In our previous studies, we had observed a bladder cancer cell line T24 with a single H-ras mutation (V12G) that resisted to the effect of genistein. In this research project, we are tring to understand the molecular mechanisms involving in the genistein resistant of the T24 cells. Recent studies have demonstrated that Egr-1 mediates cell proliferation and differentiation. Egr-1 transcription transiently induced in T24 cells-treated with genistein, thus, we used the Egr-1 antisense(0, 0.1, 5, 10μM) to block the Egr-1 translation, followed by genistein treatment in T24 cells. The T24 cells proliferation were not suppressed clearly, the results shown Egr-1 has no influence in the resistant to genistein in T24 cells. Since, the mutated H-ras was constitutively active in T24 cells, in order to understand the Ras regulated signaling pathway, we used the MEK inhibitors PD98059(0, 1, 20μM), U0126(0, 5, 10μM) and c-fos antisense(0, 0.1, 5, 10μM) assay to study the molecular mechanisms of Ras modulated in T24 cells, two other signal transduction pathways; PI3K inhibitor LY294002(0-20μM) and PKC inhibitor H7(50nM-5μM) were also used as a control in this experiment. The cell growth of T24 cells was significantly suppressed when T24 cells was treated with MEK inhibitors(down to 40%) or c-fos antisense(down to 10%) followed by 50μM genistein stimulation. There was no obvious cell growth inhibition when T24 cells treated with PI3K or PKC inhibitors. The results demonstrated that Ras plays an important role in the resistant to the effect of genistein in T24 cells. Finally, we had used the microarray hybridization to study the gene expression patterns in T24 cells treated with H-ras antisense ODNs and genistein. The experiment include the following three groups: (1)Ras antisense ODNs only: T24 cells treated with only H-ras antisense ODNs; (2)Ras antisense ODNs + genistein: T24 cells treated with H-ras antisense ODNs eight hours followed by 50μM genistein; (3)Control ODNs + genistein: T24 cells treated with control ODNs eight hours followed by 50μM genistein. From the microarray hybridization analysis, we have identified a number of genes, whose expression were substantially changed during the treatment. Among them , the expression of Egr-1, c-fos, topoisomerase II α, VDUP1, interferon-inducible protein 9-27, and PML have also been confirmed by RT-PCR. The results suggested that c-fos and topoisomerase II α may play a role in resistant to the genistein treatment in T24 cells. The physiological role of the other genes in mediated the genistein resistance in T24 cells were need further characterized.