摘要
目的研究 P2Y 嘌呤受体激动剂对前列腺癌细胞 PI-3K 信号通路的激活及对肿瘤细胞生长和侵袭的影响。方法以高转移的人前列腺癌细胞系1E8为研究对象,以 Western blot 法,用特异性识别磷酸化 Akt 的抗体检测 PI-3K信号通路的活化情况;以细胞计数、流式细胞术、Matrigel 侵袭实验等方法检测 P2Y 受体激活的 PI-3K/Akt 信号通路在前列腺癌细胞生长和侵袭中的作用。结果P2Y 嘌呤受体激动剂以时间和剂量依赖的方式激活了1E8细胞中的 PI-3K/Akt 信号通路。其持续刺激导致的生长抑制作用主要通过将细胞阻滞在 S 期实现(刺激组 S 期细胞分数比对照组提高22.3%)。应用特异性抑制剂 LY294002阻断 PI-3K 通路明显影响1E8细胞的生长,但不能逆转 P2Y受体介导的生长抑制作用。P2Y 受体瞬时激活对前列腺癌细胞体外侵袭的促进作用主要通过增强细胞的运动能力(刺激组划痕修复率比对照组提高21.2%),而非提高基质金属蛋白酶(MMP)-2和MMP-9的酶解活性实现,且此促侵袭作用可被 LY294002明显抑制。结论 P2Y 嘌呤受体可以激活人前列腺癌细胞的 PI-3K 信号通路,并通过这条通路促进肿瘤细胞的运动能力进而促进侵袭;该通路是前列腺癌细胞生长的重要调节通路,但不参与 P2Y 嘌呤受体对前列腺癌细胞生长抑制的调节。
Objective To investigate P2Y purinergic receptor activated PI-3K/Akt signaling pathway in the regulation of growth and invasion of prostate cancer in vitro. Methods Western blot was used to detect phosphorylation of Akt （a downstream target molecule of PI-3K） by P2Y receptor agonist in 1E8 cells （a highly metastatic subclone derived from PC-3 prostatic cancer cell line）. Cell counts, flow cytometry, Matrigel invasion assay, wound healing assay and gelatin zymography were used to detect changes of biological behaviors of 1E8 cells after P2Y receptor activation. Results AMP-PNP, one non-hydrolysis ATP analogue and P2Y receptor agonist, induced significant phosphorylation of Akt in a time- and dose- dependent manner in IE8 cells. LY294002, a specific inhibitor of PI-3K, effectively blocked Akt phosphorylation induced by AMP-PNP. Continuous exposure to AMP-PNP induced significant growth inhibition of 1E8 cells （inhibition rate at 50. 2% at the 8 th day）, and this inhibition was mainly due to an arrest at S phase of the cell cycle （the S phase fraction of AMP-PNP treated cells was 22.3% higher than that of the control）. Application of LY294002 did not reverse the growth inhibition effect of AMP-PNP. Matrigel invasion assay showed that AMP-PNP stimulation increased invasive ability of 1E8 cells, and this effect was effectively blocked by LY294002. No significant changes in the activation of MMP-2 and MMP-9 were detected by gelatin zymography, although wound healing assay showed 21.2% increase in cell migration after AMP-PNP treatment. Conclusions PI-3K/Akt signaling pathway participates in P2Y receptor-stimulated prostate cancer invasion by enhancing cell motility, rather than up-regulating MMP-2 and MMP-9 activities. PI-3K signaling pathway plays an important role in prostate cancer proliferation, but is not involved in P2Y receptor mediated growth inhibition.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2007年第10期681-686,共6页
Chinese Journal of Pathology
基金
国家自然科学基金(30471936)
