目的：探讨桔皮素（ TGN）对非小细胞肺癌（ NSCLC）细胞生长和侵袭的影响及其分子机制。方法：体外培养非小细胞肺癌A549细胞，分别用不同浓度的TGN处理，MTT比色法检测细胞活性，Annexin V-FITC／PI染色及流式细胞术检测细胞凋亡率， Transwell检测细胞侵袭， RT-PCR分析MMP-2和MMP-9的mRNA表达水平， Western blotting检测Ki67、Cyt C、caspase-3、cleaved caspase-3、MMP-2、MMP-9、Akt、p-Akt以及p-PI3K表达水平。结果：桔皮素剂量依赖性地抑制A549细胞增殖（ P＜0.05），同时伴随有增殖标记分子Ki67表达水平的下调。分析发现，桔皮素诱导细胞中Cyt C、caspase-3和cleaved caspase-3的表达上调（ P＜0.01），加速A549细胞的凋亡。此外，桔皮素作用后，A549细胞中侵袭相关蛋白MMP-2和MMP-9的表达量下降，且侵袭数目随桔皮素浓度增加而减少。进一步研究表明，桔皮素作用后A549细胞中p-Akt和p-PI3K表达水平降低（P＜0.05），阻断PI3K／Akt信号通路后，不同浓度TGN对细胞活性影响没有变化。结论：桔皮素能抑制A549细胞生长及侵袭，促进细胞凋亡，可能通过抑制PI3 K／Akt信号通路的激活起作用。因此，本研究将为非小细胞肺癌的防治提供新的研究方向。%[ ABSTRACT] AIM: To study the influences of tangeretin ( TGN) on the growth and invasion of non-small-cell lung cancer ( NSCLC) cells, and to explore the molecular mechanisms.METHODS: The A549 cells were treated with different concentrations of TGN in vitro.The relative cell activity was determined by MTT assay.The apoptotic rate was an-alyzed by flow cytometry with Annexin V-FITC/PI staining.The number of the invasive cells was measured by Transwell as-say.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR, and the protein levels of Ki67, Cyt C, caspase-3, cleaved caspase-3, MMP-2, MMP-9, Akt, p-Akt and p-PI3K were determined by Western blotting analysis. RESULTS:TGN inhibited the proliferation of A549 cells in a dose-dependent manner ( P<0.05) along with the low ex-pression level of proliferation biomarker Ki67.TGN up-regulated the protein levels of Cyt C, caspase-3 and cleaved caspase-3 (P<0.01) and promoted the apoptosis of A549 cells in a dose-dependent manner.Moreover, TGN down-regu-lated the invasion-related molecules MMP-2 and MMP-9 at the mRNA and protein levels, and the number of invasive cells reduced with the increase in the concentration of TGN.The protein levels of p-Akt and p-PI3K in the A549 cells was re-duced (P<0.05), and no difference of the cell viability in the cells treated with different concentrations of TGN was ob-served after blocking PI3K/Akt signaling pathway using LY294002.CONCLUSION: TGN inhibits the growth and inva-sion of A549 cells and promotes the cell apoptosis by potentially inhibiting PI3K/Akt signaling pathway activation.There-fore, this study will provide a new target for the prevention and control of NSCLC.
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