细胞外信号调节激酶在不同转移性人前列腺癌细胞中被激活的调节机制

目的　探讨不同转移潜能的人前列腺癌细胞中与增殖分化密切相关的细胞外信号调节激酶（ERK）信号传导途径被激活的机制。方法　用细胞计数及MTT法检测外源性P2嘌呤受体激动剂ATP对人前列腺癌细胞PC-3M亚系1E8（高转移）和2B4（低转移）体外生长的影响。用特异性识别双磷酸化ERK1/2（p44/p42）的抗体及蛋白质印迹方法，检测细胞经ATP作用后ERK1/2的活化情况并研究其调节机制。结果　ATP可明显抑制1E8和2B4细胞的体外生长，第6和第8天的抑制率分别为1E854％和59％；2B467％和39％。ATP可激活1E8和2B4细胞内的ERK1/2激酶。ATP诱导的ERK1/2活化可被P2嘌呤受体拮抗剂苏拉明抑制，抑制率1E882%±9％；2B481%±6％。ERK1/2上游激酶MEK抑制剂PD98059可有效抑制ATP对ERK1/2的激活，抑制率1E894%±4％；2B491%±4％。ATP对ERK的激活受到G蛋白活性调节剂PTX的抑制，抑制率1E850%±3％，2B451%±4％。ATP对1E8细胞ERK1/2的激活水平高于2B4细胞。两种细胞对蛋白激酶C活性调节剂TPA作用的反应性不同。结论　不同转移性的人前列腺癌细胞亚系对外源性ATP激活ERK1/2信号传导通路的反应性间存在差异，提示肿瘤转移受到不同信号传导机制调节。

Mechanism of the activation of extracellular signal-regulated kinase (ERK) in prostate cancer cell lines with different metastatic potential

OBJECTIVE: To investigate the mechanism of the activation of signal transduction of ERK induced by purinergic receptor agonist ATP in prostate cancer cells with different metastatic potential. METHODS: Cell counts and MTT method were used to detect the influence of ATP on the growth of 1E8 (metastatic) and 2B4 (non-metastatic) cells derived from human prostate cancer cell line PC3M. The activity of ERK1/2 was analyzed with phosphospecific antibodies directed against the dually phosphorylated, active forms of ERK1/2 (p44/p42) by Western Blot. RESULTS: ATP can significantly inhibit the growth of 1E8 and 2B4 cells in vitro (inhibition rate in the 6th and the 8th day were 54% and 59% for 1E8 and 67% and 39% for 2B4 respectively). ATP activated both ERK1 and ERK2 in 1E8 and 2B4 cells with a time and dose dependent pattern. The activation of ERK1/2 by ATP was blocked by the P2 purinoceptor antagonist, suramin with an inhibitory rate of 82% +/- 9% for 1E8 and an inhibitory rate of 81% +/- 6% for 2B4. The activation of ERK1/2 by ATP can be inhibited by the inhibitor of the upstream kinase MEK- PD098059 with an inhibitory rate of 94% +/- 4% for 1E8 and 91% +/- 4% for 2B4,which suggests a link between the G protein coupled P2 purinoceptor and activation of Ras MEK MAPK pathway. ATP-stimulated ERK activation was sensitive to treatment with G protein modulator pertussis toxin (PTX) with an inhibitory rate of 50% +/- 3% for 1E8 and 51% +/- 4% for 2B4.The activating potential of ATP to ERK1/2 in metastatic 1E8 cells is greater than that to nonmetastatic 2B4 cells, and the response of 1E8 cells to TPA was quite different from the response of 2B4 cells, thus implying a potential signaling mechanism in regulating metastasis phenotypes. CONCLUSION: The metastatic 1E8 and non-metastatic 2B4 cells show differential response to ATP-induced ERK activation. This may provide an instructive clue to cancer metastasis research.