GANT61阻断Hedgehog信号通道调控肺腺癌A549/DDP细胞耐药的机制研究

目的:探讨GANT61阻断Hedgehog信号通道调控肺腺癌A549/DDP细胞增殖、细胞周期及耐药的机制研究。方法:应用CCK8法检测顺铂(DDP)对肺腺癌细胞株A549和A549/DDP的半数抑制浓度(IC50),从而计算A549/DDP细胞的耐药指数;同时采用Western blot检测A549、A549/DDP细胞中Gli1、Ptch1、Shh、XRCC1蛋白的表达水平。用不同浓度的GANT61干预A549/DDP细胞后,CCK8法检测增殖活力。流式细胞术检测GANT61对A549/DDP细胞凋亡率和细胞周期的影响。Western blot检测GANT61对A549/DDP细胞周期和凋亡相关蛋白表达的影响。采用20μmol/L GANT61联合不同浓度的顺铂干预A549/DDP细胞,CCK8法检测各组细胞增殖抑制率变化及Western blot检测各组细胞中XRCC1蛋白的表达水平。结果:A549/DDP细胞的耐药指数为5. 34。A549/DDP细胞与A549细胞相比,Gli1、Ptch1、Shh、XRCC1在蛋白表达水平明显升高,差异有统计学意义(P <0. 01或P <0. 05)。使用GANT61处理A549/DDP细胞,当其浓度分别≥20μmol/L、≥10μmol/L时,分别培养24 h和72 h,与对照组相比肿瘤细胞增殖活性下降(P<0. 01),且呈现剂量依赖性。GANT61可通过下调c-myc、cyclin D1表达(P<0. 01),阻滞细胞周期于G1期(P<0. 01)。GANT61能够明显诱导A549/DDP细胞发生凋亡(P<0. 05),其分子机制可能与促进caspase-3的剪切活化相关(P<0. 05)。GANT61联合顺铂用药后使A549/DDP细胞抑制率显著增加,呈协同效应,且伴随XRCC1蛋白表达下降(P<0. 01)。结论:Gli1在人肺腺癌耐药细胞A549/DDP中的表达明显高于顺铂敏感株A549,提示Gli1可能与肺腺癌顺铂耐药相关,且XRCC1蛋白在A549/DDP细胞高表达。故采用Gli1抑制剂GANT61能够明显抑制A549/DDP细胞增殖,阻滞细胞周期,并诱导细胞凋亡。体外试验中,GANT61与顺铂联合用药可以协同抑制增殖作用,其机制可能与下调XRCC1蛋白表达有关。

Mechanism of GANT61 blocking Hedgehog signal pathway regulating drug resistance in lung adenocarcinoma A549/DDP cells

Objective:Study on the mechanism of GANT61 blocking the Hedgehog signaling pathway to regulate the proliferation,cell cycle and drug resistance of A549/DDP cells in lung adenocarcinoma.Methods:The CCK8 method was used to detect the half maximal inhibitory concentration(IC 50)of cisplatin on A549 and A549/DDP cells,and the resistance index of A549/DDP cells was calculated.Western blot was used to detect the expression of Gli1,Ptch1,Shh and XRCC1 proteins in A549 and A549/DDP cells.Treated with different concentrations of GANT61,the viability of A549/DDP cells was detected by CCK8.The effect of GANT61 on the apoptosis rate and cell cycle of A549/DDP cells was measured by flow cytometry.The expression levels of apoptosis-related proteins and cell cycle-related proteins in A549/DDP cells treated with GANT61 were measured by Western blot.The A549/DDP cells were intervened by 20μmol/L GANT61 combining with different concentrations of cisplatin,the proliferation inhibition rate of each group was detected,and the expression level of XRCC1 proteins in each group was detected by Western blot.Results:The drug resistance index of A549/DDP cells was 5.34.Compared with A549 cells,the protein expression levels of Gli1,Ptch1,Shh and XRCC1 were significantly increased in A549/DDP cells,and the difference was statistically significant(P<0.01 or P<0.05).The A549/DDP cells were treated with GANT61 for 24 h or 72 h respectively,which its concentration was≥20μmol/L,or≥10μmol/L,the proliferation of A549/DDP cells was significantly inhibited,compared with the control group(P<0.01),and there was the dose dependence.Treated with GANT61,the cell cycle was arrested at S phase by down-regulating the expression of c-myc and cyclinD1(P<0.01).GANT61 significantly induced the cell apoptosis(P<0.05)by activating caspase-3 protein(P<0.05).Treated with the combined using of GANT61 and cisplatin,the cell proliferation of A549/DDP was significantly inhibited,which showed a synergistic effect,accompanying with the low expression of XRCC1 protein(P<0.01).Conclusion:The expression of Gli1 in human lung adenocarcinoma cell line A549/DDP was significantly higher than that of cisplatin-sensitive A549 cell line,suggesting that Gli1 may be associating with cisplatin-resistant lung cancer and XRCC1 protein is highly expressed in A549/DDP cell line.The GANT61,Gli1 inhibitor,can significantly inhibit the proliferation of A549/DDP cells,blocking cell cycle and induce apoptosis.There is a synergistic anti-proliferative effect in vitro under combined use of GANT61 and cisplatin,and its mechanism may be relating to down-regulating the expression of XRCC1 protein.