抑制PI3K/Akt信号转导通路逆转喉癌耐药的作用及其机制

effect and mechanism of suppressing PI3K/Akt signal pathway to reverse drug resistance of laryngocarcinoma

目的探讨磷脂酰肌醇-3激酶(PI3K)/Akt信号转导通路抑制剂LY294002对脂多糖(LPS)刺激喉癌细胞引发耐药反应的影响。方法实验分为空白组、LPS组和LPS+LY294002抑制剂组,收集对数生长期人喉癌Hep-2细胞,接种于6孔培养板中,待密度为5×105个/孔时,LPS+LY294002抑制剂组加入10 mmol/L LY294002抑制剂刺激1 h,随后LPS组和LPS+LY294002抑制剂组均加入1 mg/m L LPS刺激5 h。通过实时定量PCR检测各组中肿瘤细胞多药耐药性基因mRNA的表达情况,倒置荧光显微镜观察顺铂处理后各组细胞的形态,MTT法检测各组细胞生长抑制率,流式细胞仪技术检测各组细胞凋亡率。结果 LPS组BCRP及MRP3/5 mRNA表达量均明显高于空白组和LY294002抑制剂组(P均<0.05)。顺铂刺激24 h后,空白组中细胞数量明显减少,有较多漂浮的小圆形细胞和坏死的细胞碎片;LPS组细胞形态无明显改变,漂浮的小圆细胞和碎片较少;LPS+LY294002抑制剂组中漂浮的小圆细胞和碎片多于LPS组。LPS组细胞生长抑制率和细胞凋亡率均明显低于空白组(P均<0.05),LPS+LY294002组细胞生长抑制率和细胞凋亡率均明显高于LPS组(P均<0.05)。结论 PI3K/Akt信号转导通路抑制剂LY294002可逆转Hep-2细胞对顺铂的耐药性,可能与降低肿瘤细胞多药耐药基因mRNA的表达有关。

Objective It is to investigate the influence of PI3K/Akt inhibitor of LY294002 on Hep-2 mediated drug resist- ance responses with the interference of lipopolysaccharide. Methods Cultured human laryngeal cancer Hep-2 cells were divided into control group, LPS group and LY294002 inhibitor group. The expression of muhidrug resistance gene mRNA in each groups were detected by real-time quantitative PCR, the morphology of cells in each groups was observed after cisplatin treatment by inverted fluorescence microscope, the cell growth inhibition rate in each groups were detected by MTT assay, and the apoptosis of cells in each groups were detected by flow cytometry. Results Compared with the control group and LY294002 in- hibitor group, the expression of BCRP and MRP3/5 mRNA in the LPS group was significantly increased （ all P 〈 0.05）. After treated with cisplatin for 24 hours, the number of cells in the control group was decreased significantly, with more floating small round cells and necrotic cell debris; the morphology of ceils in the LPS group did not change significantly, and the floating small round cells and fragments were less; in the LY294002 inhibitor group, floating small round ceils and fragments were more numerous than the LPS group. The growth inhibition rate and apoptosis rate of LPS group were significantly lower than that of the control group （ all P 〈 0.05 ） , and these of the LY294002 group were significantly higher than LPS group （ P 〈 0.05）. Conclusion PI3K/Akt signaling pathway inhibitor LY294002 can reverse the resistance of Hep-2 cells to eisplatin, it may be related to decreasing the expression of muhidrug resistance gene mRNA in tumor cells.