| Abstract: | Genistein是一個存在於豆類的植物性雌激素，目前已有許多的報導指出其具有抑制癌症的效果，且許多癌化的細胞株都對其有一定的敏感性。然而在先前的實驗發現T24這株H-ras突變的膀胱癌細胞株，它對genistein則是有resistant的效應，且在抑制H-ras的表現後，T24細胞生長有明顯的受genistein抑制。於是我們想探討，T24對抗genistein是否是因為其本身H-ras持續活化所導致的結果。因為在先前的實驗中發現，T24細胞在處理genistein時期Egr-1及c-fos會有短暫而大量的表現，之後在回歸到基本的表現值，因此我們先以Egr-1的antisense ONDs 抑制T24細胞Egr-1的表現後，再加genistein處理，發現Egr-1對T24細胞抵抗genistein的效應沒有顯著的影響。再分別以MEK抑制劑PD98059(0、10、20μM)、U0126(0、5、10μM )抑制MEK的活性及以Ras下游c-fos 的antisense ODNs抑制c-fos的表現後，發現MEK的活化與c-fos的表現對T24細胞抵抗genistein的效應有顯著的影響。接著分別以PI3K及PKC的抑制劑LY294002(0-20μM)、H7(0-10μM)，發現兩者對T24細胞抵抗genistein的效應沒有顯著的影響，亦即T24細胞抵抗genistein的效應應該是Ras過度活化所導致的結果。最後以Ras的antisense ONDs抑制T24細胞Ras的表現後，再加50μM genistein分別處理不同的時間，共分成三組即：(1)Ras antisense ODNs only；(2)Ras antisense ODNs + genistein;；(3)Control ODNs + genistein，收集其mRNA做microarray hybridizaion分析，再挑選其中6個基因(Egr-1、c-fos、topoisomerase II α、interfron-inducible protein 9-27、VDUP1、及PML)以RT-PCR做確認，其結果與microarray hybridization相符。其中c-fos的表現，從前面的結果顯示確實與T24細胞抵抗genistein效應有關。而Topoisomerase II α的表現量與細胞的分裂生長有關，因此我們推測它應該也與T24細胞抵抗genistein效應有關。Egr-1從前面的結果得知它與T24細胞抵抗genistein效應無關；其他3個基因目前則無報導指出與細胞分裂有關。從以上實驗我們可以推測：T24細胞對抗genistein確實是因H-ras處在一個持續表現的狀態，造成其下游c-fos持續表現，進一步促使與細胞增生有關的基因持續活化，使得T24細胞對genistein有一個抵抗的效應產生。
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. |
