GANT61阻断Hedgehog信号通道调控肺腺癌A549/DDP细胞耐药的机制研究
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摘要
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。
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( IC50) 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 20 μmol/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 ≥20 μmol/L,or ≥ 10 μmol/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 cyclin D1( 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.
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( IC50) 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 20 μmol/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 ≥20 μmol/L,or ≥ 10 μmol/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 cyclin D1( 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.
引文
[1]Siegel RL,Miller KD,Jemal A.Cancer statistics,2017[J].CA,2017,67(1):7-30.
[2]Fennell DA,Summers Y,Cadranel J,et al.Cisplatin in the modern era:The backbone of first-line chemotherapy for non-small cell lung cancer[J].Cancer Treat Rev,2016,44:42-50.
[3]Chang A.Chemotherapy,chemoresistance and the changing treatment landscape for NSCLC[J].Lung Cancer,2011,71(1):3-10.
[4]Xu M,Gong A,Yang H,et al.Sonic hedgehog-glioma associated oncogene homolog 1 signaling enhances drug resistance in CD44(+)/Musashi-1(+)gastric cancer stem cells[J].Cancer Lett,2015,369(1):124-133.
[5]Huang L,Walter V,Hayes DN,et al.Hedgehog-GLI signaling inhibition suppresses tumor growth in squamous lung cancer[J].Clin Cancer Res,2014,20(6):1566-1575.
[6]Gonnissen A,Isebaert S,Haustermans K.Targeting the Hedgehog signaling pathway in cancer:beyond Smoothened[J].Oncotarget,2015,6(16):13899-13913.
[7]Fu J,Rodova M,Roy SK,et al.GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft[J].Cancer Lett,2013,330(1):22-32.
[8]Yan R,Peng X,Yuan X,et al.Suppression of growth and migration by blocking the Hedgehog signaling pathway in gastric cancer cells[J].Cell Oncol(Dordr),2013,36(5):421-435.
[9]Yan M,Wang L,Zuo H,et al.HH/GLI signalling as a new therapeutic target for patients with oral squamous cell carcinoma[J].Oral Oncol,2011,47(6):504-509.
[10]Long B,Wang LX,Zheng FM,et al.Targeting GLI1 suppresses cell growth and enhances chemosensitivity in CD34+enriched acute myeloid leukemia progenitor cells[J].Cell Physiol Biochem,2016,38(4):1288-1302.
[11]Kiesslich T,Mayr C,Wachter J,et al.Activated hedgehog pathway is a potential target for pharmacological intervention in biliary tract cancer[J].Mol Cell Biochem,2014,396(1-2):257-268.
[12]Zahreddine HA,Culjkovic-Kraljacic B,Assouline S,et al.The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation[J].Nature,2014,511(7507):90-93.
[13]Giroux LE,Vieira T,Antoine M,et al.Sonic hedgehog pathway activation is associated with resistance to platinum-based chemotherapy in advanced non-small-cell lung carcinoma[J].Clin Lung Cancer,2016,17(4):301-308.
[14]达丽隽,李华,张献全.沉默Gli1基因对人非小细胞肺癌A549/DDP顺铂耐药性的影响[J].中国免疫学杂志,2014,30(8):1064-1068.Da LJ,Li H,Zhang XQ.Impacts of Gli1 silencing on cisplatin resistance of lung cancer cell A549/DDP[J].Chin J Immunol,2014,30(8):1064-1068.
[15]Infante P,Alfonsi R,Botta B,et al.Targeting GLI factors to inhibit the Hedgehog pathway[J].Trends Pharmacol Sci,2015,36(8):547-558.
[16]Vlckova K,Ondrusova L,Vachtenheim J,et al.Survivin,a novel target of the Hedgehog/GLI signaling pathway in human tumor cells[J].Cell Death Dis,2016,7:e2048.
[17]Lauth M,Bergstrom A,Shimokawa T,et al.Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists[J].Proc Natl Acad Sci U S A,2007,104(20):8455-8460.
[18]Xie C,Pan Y,Hao F,et al.C-Myc participates in beta-cateninmediated drug resistance in A549/DDP lung adenocarcinoma cells[J].APMIS,2014,122(12):1251-1258.
[19]Fu YZ,Yan YY,He M,et al.Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway[J].Oncol Rep,2016,35(2):912-922.
[20]Wickstrom M,Dyberg C,Shimokawa T,et al.Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo[J].Int J Cancer,2013,132(7):1516-1524.
[21]Graab U,Hahn H,Fulda S.Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma[J].Oncotarget,2015,6(11):8722-8735.
[22]Abdel-Fatah T,Sultana R,Abbotts R,et al.Clinicopathological and functional significance of XRCC1 expression in ovarian cancer[J].Int J Cancer,2013,132(12):2778-2786.
[23]Zhang R,Niu Y,Zhou Y.Increase the cisplatin cytotoxicity and cisplatin-induced DNA damage in HepG2 cells by XRCC1 abrogation related mechanisms[J].Toxicol Lett,2010,192(2):108-114.
[24]Kudo K,Gavin E,Das S,et al.Inhibition of Gli1 results in altered c-Jun activation,inhibition of cisplatin-induced upregulation of ERCC1,XPD and XRCC1,and inhibition of platinum-DNAadduct repair[J].Oncogene,2012,31(44):4718-4724.
[2]Fennell DA,Summers Y,Cadranel J,et al.Cisplatin in the modern era:The backbone of first-line chemotherapy for non-small cell lung cancer[J].Cancer Treat Rev,2016,44:42-50.
[3]Chang A.Chemotherapy,chemoresistance and the changing treatment landscape for NSCLC[J].Lung Cancer,2011,71(1):3-10.
[4]Xu M,Gong A,Yang H,et al.Sonic hedgehog-glioma associated oncogene homolog 1 signaling enhances drug resistance in CD44(+)/Musashi-1(+)gastric cancer stem cells[J].Cancer Lett,2015,369(1):124-133.
[5]Huang L,Walter V,Hayes DN,et al.Hedgehog-GLI signaling inhibition suppresses tumor growth in squamous lung cancer[J].Clin Cancer Res,2014,20(6):1566-1575.
[6]Gonnissen A,Isebaert S,Haustermans K.Targeting the Hedgehog signaling pathway in cancer:beyond Smoothened[J].Oncotarget,2015,6(16):13899-13913.
[7]Fu J,Rodova M,Roy SK,et al.GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft[J].Cancer Lett,2013,330(1):22-32.
[8]Yan R,Peng X,Yuan X,et al.Suppression of growth and migration by blocking the Hedgehog signaling pathway in gastric cancer cells[J].Cell Oncol(Dordr),2013,36(5):421-435.
[9]Yan M,Wang L,Zuo H,et al.HH/GLI signalling as a new therapeutic target for patients with oral squamous cell carcinoma[J].Oral Oncol,2011,47(6):504-509.
[10]Long B,Wang LX,Zheng FM,et al.Targeting GLI1 suppresses cell growth and enhances chemosensitivity in CD34+enriched acute myeloid leukemia progenitor cells[J].Cell Physiol Biochem,2016,38(4):1288-1302.
[11]Kiesslich T,Mayr C,Wachter J,et al.Activated hedgehog pathway is a potential target for pharmacological intervention in biliary tract cancer[J].Mol Cell Biochem,2014,396(1-2):257-268.
[12]Zahreddine HA,Culjkovic-Kraljacic B,Assouline S,et al.The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation[J].Nature,2014,511(7507):90-93.
[13]Giroux LE,Vieira T,Antoine M,et al.Sonic hedgehog pathway activation is associated with resistance to platinum-based chemotherapy in advanced non-small-cell lung carcinoma[J].Clin Lung Cancer,2016,17(4):301-308.
[14]达丽隽,李华,张献全.沉默Gli1基因对人非小细胞肺癌A549/DDP顺铂耐药性的影响[J].中国免疫学杂志,2014,30(8):1064-1068.Da LJ,Li H,Zhang XQ.Impacts of Gli1 silencing on cisplatin resistance of lung cancer cell A549/DDP[J].Chin J Immunol,2014,30(8):1064-1068.
[15]Infante P,Alfonsi R,Botta B,et al.Targeting GLI factors to inhibit the Hedgehog pathway[J].Trends Pharmacol Sci,2015,36(8):547-558.
[16]Vlckova K,Ondrusova L,Vachtenheim J,et al.Survivin,a novel target of the Hedgehog/GLI signaling pathway in human tumor cells[J].Cell Death Dis,2016,7:e2048.
[17]Lauth M,Bergstrom A,Shimokawa T,et al.Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists[J].Proc Natl Acad Sci U S A,2007,104(20):8455-8460.
[18]Xie C,Pan Y,Hao F,et al.C-Myc participates in beta-cateninmediated drug resistance in A549/DDP lung adenocarcinoma cells[J].APMIS,2014,122(12):1251-1258.
[19]Fu YZ,Yan YY,He M,et al.Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway[J].Oncol Rep,2016,35(2):912-922.
[20]Wickstrom M,Dyberg C,Shimokawa T,et al.Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo[J].Int J Cancer,2013,132(7):1516-1524.
[21]Graab U,Hahn H,Fulda S.Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma[J].Oncotarget,2015,6(11):8722-8735.
[22]Abdel-Fatah T,Sultana R,Abbotts R,et al.Clinicopathological and functional significance of XRCC1 expression in ovarian cancer[J].Int J Cancer,2013,132(12):2778-2786.
[23]Zhang R,Niu Y,Zhou Y.Increase the cisplatin cytotoxicity and cisplatin-induced DNA damage in HepG2 cells by XRCC1 abrogation related mechanisms[J].Toxicol Lett,2010,192(2):108-114.
[24]Kudo K,Gavin E,Das S,et al.Inhibition of Gli1 results in altered c-Jun activation,inhibition of cisplatin-induced upregulation of ERCC1,XPD and XRCC1,and inhibition of platinum-DNAadduct repair[J].Oncogene,2012,31(44):4718-4724.