MEK-MAPK信号通路在P2Y嘌呤受体活化对人前列腺癌细胞效应中的作用

目的 探讨细胞外信号调节激酶(ERK1／2)及p38通路在P2Y嘌呤受体活化对前列腺癌细胞产生的生物学效应中所起的作用。方法 脂质体法将负显性MAPK激酶1(KA—MEK1)转染高转移性前列腺癌1E8细胞;Western印迹法检测ERK1／2活化水平;应用细胞计数、软琼脂集落形成实验、体外侵袭实验检测ERK1／2及p38通路在P2Y嘌呤受体活化对前列腺癌细胞生长、集落形成、体外侵袭效应中的作用;用流式细胞术检测ATP对凋亡的影响。结果 转染KA—MEK1抑制了细胞的ERK1／2活性。常规培养6d时,KA—MEK1细胞的活细胞数比对照组细胞减少了约71％;以100μmol／L ATP连续刺激6d,KA—MEK1细胞的生长被进一步抑制17.2％;用10μmol／L p38抑制剂SB203580预处理细胞,可以封闭ATP所诱导的额外生长抑制效应。说明持续P2Y受体活化可抑制前列腺癌细胞生长,ERK1／2和p38通路均在其中起作用。细胞凋亡并非ATP生长抑制作用的主要机制。在软琼脂集落形成实验中,KA—MEK1细胞比对照组细胞形成的集落小,而且数目减少了约75％;在抑制了ERK1／2活性后,以ATP预处理并不显著改变细胞的集落形成能力;进一步在抑制了：ERK1／2活性的基础上,以SB203580处理细胞,对细胞集落形成能力也无明显影响。说明在影响癌细胞集落形成方面,主要是ERK1／2通路起作用。在体外侵袭实验中,KA—MEK1细胞的穿膜细胞数比对照组细胞减少了41％;以ATP刺激12h可以促进前列腺癌细胞体外侵袭,其中KA—MEK1细胞的穿膜细胞增加率低于对照组细胞;进一步以SB203580处理细胞,可以抑制ATP诱导的促侵袭效应。说明ERK1／2和p38通路在ATP促侵袭效应中均发挥主要作用。结论P2Y嘌呤受体激活程度的差异可导致不同效应,并涉及不同信号传导通路。持续P2Y嘌呤受体活化抑制前列腺癌细胞生长,其中有ERK1／2及p38通路的参与;短暂P2Y嘌呤受体活化抑制前列腺癌细胞集落形成但促进前列腺癌细胞体外侵袭,在前者ERK1／2起重要的作用,后者有ERK1／2和p38通路的参与。

Effects of P2Y receptor activation on prostatic cancer cells requiring ERK1/2 or p38 cascade

OBJECTIVE: To determine the role of extracellular signal-regulated kinase (ERK1/2) and p38 cascades in P2Y receptor-evoked effects on prostatic cancer cells. METHODS: Highly metastatic prostatic cancer cells 1E8 were transfected with dominant-negative MAPK kinase 1 (KA-MEK1). The activation of ERK1/2 was determined by Western blot technique. The role of ERK1/2 and p38 cascades in P2Y receptor-evoked effects on in vitro growth, colony formation and in vitro invasion was detected by cell count, soft agar colony formation assay and in vitro invasion assay. The effect of ATP on apoptosis was detected by flow cytometry. RESULTS: ERK1/2 activity in 1E8-KA-MEK1 transfectants was significantly suppressed by dominant-negative MEK1 transfection. After culture of 6 days, 1E8-KA-MEK1 transfectants exhibited a growth inhibition of 71% as compared with 1E8-pcDNA3 control. Moreover, after continuous treatment with 100 micro mol/L ATP for 6 days, the growth of 1E8-KA-MEK1 transfectants was further inhibited by an additional 17.2%. Pretreatment with 10 micro mol/L p38 inhibitor SB203580 antagonized the effect of ATP-induced additional growth inhibition, suggesting that ERK1/2 and p38 pathways play an important role in ATP-induced growth inhibition. In soft agar assay, 1E8-KA-MEK1 transfectants formed smaller colonies and exhibited a 75% decrease in colony formation (as compared with control). Further treatment with ATP or SB203580 plus ATP did not show significant effect on colony formation of 1E8-KA-MEK1 cells, implying a potential role of ERK1/2, instead of p38, in P2Y receptor-mediated inhibitory effect on colony formation. In in vitro invasion assay, 1E8-KA-MEK1 cells showed a 41% decrease in passing through matrigel-coated membranes, as compared with control. Treatment with ATP could restore their invasive ability, and this effect by ATP could be blocked by pretreatment with SB203580, indicating the involvement of both ERK1/2 and p38 pathways in invasive ability of prostatic cancer cells. CONCLUSIONS: The effects of ATP on in vitro growth, invasion and colony formation of prostatic cancer cells depend on the status of P2Y receptor activation by different treatment protocols. Continuous activation of P2Y receptor results in growth inhibition and transient activation of P2Y receptor stimulates in vitro invasion of prostatic cancer cells. Both ERK1/2 and p38 pathways are responsible for these effects; but only the ERK1/2 pathway is involved in regulation of colony formation of prostatic cancer cells.