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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 :1E8:5 4%和 5 9%;2B4:6 7%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 :1E8:82 %± 9%;2B4:81%± 6 %。ERK1/ 2上游激酶MEK抑制剂PD980 5 9可有效抑制ATP对ERK1/ 2的激活 ,抑制率 :1E8:94%± 4%;2B4:91%±4%。ATP对ERK的激活受到G蛋白活性调节剂PTX的抑制 ,抑制率 :1E8:5 0 %± 3%,2B4:5 1%± 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。
Objective To investigate the mechanism of activation of extracellular signal-regulated kinase (ERK) signaling pathway closely related to proliferation and differentiation in human prostate cancer cells with different metastatic potentials. Methods The effects of exogenous P2 purinergic receptor agonist ATP on the growth of human prostate cancer cell line PCM subline 1E8 (high metastasis) and 2B4 (low metastasis) in vitro were detected by cytometry and MTT assay. The specific ERK1 / 2 (p44 / p42) antibody and Western blotting were used to detect the activation of ERK1 / 2 after ATP treatment and to investigate its regulation mechanism. Results ATP inhibited the growth of 1E8 and 2B4 cells in vitro. The inhibitory rates of 1E8: 5 4% and 5 9% and 2B4: 6 7% and 39% on the 6th and 8th days, respectively. ATP activates ERK1 / 2 kinase in 1E8 and 2B4 cells. ATP-induced activation of ERK1 / 2 was inhibited by the P2 purinergic receptor antagonist suramin, with inhibition rates of 1E8: 82% ± 9% and 2B4: 81% ± 6%. ERK1 / 2 upstream kinase MEK inhibitor PD98059 can effectively inhibit the activation of ERK1 / 2 by ATP. The inhibition rate is 1E8: 94% ± 4%; 2B4: 91% ± 4%. The activation of ERK by ATP was inhibited by PTX, a modulator of G protein activity. The inhibition rates were 1E8: 50% ± 3%, 2B4: 5 1% ± 4%. The activation level of ERK1 / 2 by ATP on 1E8 cells was higher than that of 2B4 cells. The reactivity of the two cells to TPA, a modulator of protein kinase C activity, is different. Conclusions There are differences in the reactivity of exogenous ATP-activated ERK1 / 2 signaling in different metastatic human prostate cancer cell sublines, suggesting that tumor metastasis is regulated by different signal transduction mechanisms.