异甘草素对人肾透明细胞癌786-O细胞抗癌作用及分子机制
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摘要
目的:研究异甘草素(isoliquiritigenin,ISL)对人肾透明细胞癌786-O细胞的抗癌作用,并探讨其可能存在的分子机制。方法:通过噻唑蓝(thiazolyl blue tetrazolium bromide,MTT)比色法检测ISL(0,10,25,50,75,100μmol·L~(-1))对786-O细胞增殖的作用;细胞划痕和Transwell实验检测ISL对786-O细胞迁移、侵袭能力的影响;吖啶橙染色,Ad-GFP-LC3转染实验观察细胞自噬状态;蛋白免疫印迹法(Western blot)检测自噬相关蛋白的表达,并分析磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素蛋白(mammalian target of rapamycin,m TOR)信号通路变化,探讨其可能的作用机制。结果:MTT比色法结果显示,与空白组比较,ISL能够明显抑制786-O细胞的增殖,且呈时间-剂量依赖性(P<0.05);划痕和Transwell实验结果表明,与空白组比较,ISL能够抑制785-O细胞的迁移和侵袭(P<0.05);吖啶橙染色和Ad-GFP-LC3转染实验表明ISL能够诱导786-O细胞发生自噬。此外,与空白组比较,ISL能够诱导细胞内自噬标志物-Ⅱ(LC3-Ⅱ),自噬相关蛋白Beclin1,Atg5蛋白表达(P<0.05),并显著下调p62蛋白表达(P<0.05),当加入自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)可逆转上述现象(P<0.05);同时ISL能够抑制Akt,m TOR的磷酸化水平(P<0.05),加入抑制剂LY294002和雷帕霉素(rapamycin,Rap),LC3-Ⅱ,Beclin1,Atg5蛋白表达明显上调(P<0.05),p62蛋白表达明显下调(P<0.05)。结论:ISL能够抑制肾透明细胞癌786-O细胞的增殖、迁移和侵袭,并通过抑制PI3K/Akt/m TOR信号通路诱导细胞发生自噬。
Objective:To investigate the anticancer effect of isoliquiritigenin(ISL)on human clear cell renal cell carcinoma 786-O cells,and explore its possible molecular mechanism.Method:Thiazolyl blue tetrazolium bromide(MTT)assay was used to detect effect of ISL(0,10,25,50,75,100μmol·L~(-1))on proliferation of 786-O cells.The effect of ISL on migration and invasion of 786-O cells was detected by cell scratch test and Transwell assay.The autophagy was observed under the fluorescence microscope through acridine orange staining and Ad-GFP-LC3 transfection experiment.Western blot was used to detect the expression of autophagy related protein and analyze the changes of phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(m TOR)signaling pathway to explore the possible mechanism.Result:MTT results showed that ISL could significantly inhibit the proliferation of 786-O cells in a time-dose dependent manner(P<0.05).The results of scratch and Transwell experiments indicated that ISL could inhibit the migration and invasion of 786-O cells(P<0.05).Acridine orange staining and ad-GFP-LC3 transfection showed that ISL can induce autophagy in 786-O cells.Besides,ISL can induce LC3-Ⅱ,Beclin1,Atg5 protein expressions(P<0.05),and significantly reduce p62 protein expression(P<0.05);autophagy inhibitor 3-methyl adenine(3-MA)can be added to reverse the above phenomena(P<0.05).Meanwhile,ISL can inhibit phosphorylation of Akt,m TOR levels(P<0.05),inhibitor LY294002 and rapamycin(Rap)can be added to significantly reregulate LC3-Ⅱ,Beclin1,Atg5 protein expressions(P<0.05)and significantly down-regulate p62 protein expression(P<0.05).Conclusion:ISL can inhibit the proliferation,migration and invasion of clear cell renal carcinoma 786-O cells,and induce autophagy by inhibiting the PI3K/Akt/m TOR signaling pathway.
Objective:To investigate the anticancer effect of isoliquiritigenin(ISL)on human clear cell renal cell carcinoma 786-O cells,and explore its possible molecular mechanism.Method:Thiazolyl blue tetrazolium bromide(MTT)assay was used to detect effect of ISL(0,10,25,50,75,100μmol·L~(-1))on proliferation of 786-O cells.The effect of ISL on migration and invasion of 786-O cells was detected by cell scratch test and Transwell assay.The autophagy was observed under the fluorescence microscope through acridine orange staining and Ad-GFP-LC3 transfection experiment.Western blot was used to detect the expression of autophagy related protein and analyze the changes of phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(m TOR)signaling pathway to explore the possible mechanism.Result:MTT results showed that ISL could significantly inhibit the proliferation of 786-O cells in a time-dose dependent manner(P<0.05).The results of scratch and Transwell experiments indicated that ISL could inhibit the migration and invasion of 786-O cells(P<0.05).Acridine orange staining and ad-GFP-LC3 transfection showed that ISL can induce autophagy in 786-O cells.Besides,ISL can induce LC3-Ⅱ,Beclin1,Atg5 protein expressions(P<0.05),and significantly reduce p62 protein expression(P<0.05);autophagy inhibitor 3-methyl adenine(3-MA)can be added to reverse the above phenomena(P<0.05).Meanwhile,ISL can inhibit phosphorylation of Akt,m TOR levels(P<0.05),inhibitor LY294002 and rapamycin(Rap)can be added to significantly reregulate LC3-Ⅱ,Beclin1,Atg5 protein expressions(P<0.05)and significantly down-regulate p62 protein expression(P<0.05).Conclusion:ISL can inhibit the proliferation,migration and invasion of clear cell renal carcinoma 786-O cells,and induce autophagy by inhibiting the PI3K/Akt/m TOR signaling pathway.
引文
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[9]Baba M,Asano R,Takigami I,et al. Studies on cancer chemoprevention by traditional folk medicinesⅩⅩⅤ.Inhibitory effect of isoliquiritigenin on azoxymethaneinduced murine colon aberrant crypt focus formation and carcinogenesis[J]. Biol Pharm Bull,2002,25(2):247-250.
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[13]Hsia S M,YU C C,Shih Y H,et al. Isoliquiritigenin as a cause of DNA damage and inhibitor of ataxiatelangiectasia mutated expression leading to G2/M phase arrest and apoptosis in oral squamous cell carcinoma[J]. Head Neck,2016,38(Suppl 1):360-371.
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[15]MA J,FU N Y,PANG D B,et al. Apoptosis induced by isoliquiritigenin in human gastric cancer MGC-803cells[J]. Planta Med,2001,67(8):754-757.
[16]LIN Y, SUN H, DANG Y, et al. Isoliquiritigenin inhibits the proliferation and induces the differentiation of human glioma stem cells[J]. Oncol Rep,2018,39(2):687-694.
[17]ZHANG B,LAI Y,LI Y,et al. Antineoplastic activity of isoliquiritigenin, a chalcone compound, in and androgen-independent human prostate cancer cells linked to G2/M cell cycle arrest and cell apoptosis[J].Eur J Pharmacol,2018,821(1):57-67.
[18]WU M, WU Y, DENG B, et al. Isoquiritigenin decreases the incidence of colitis-associated colorectal cancer by modulating the intestinal microbiota[J].Oncotarget,2016,7(51):85318-85331.
[19]Hirchaud F, Hermetet F, Ablise M, et al.Isoliquiritigenin induces caspase-dependent apoptosis via downregulation of HPV16 E6 expression in cervical cancer Ca Ski cells[J]. Planta Med,2013,79(17):1628-1635.
[20]CHEN H, ZHANG B, YUAN X, et al.Isoliquiritigenin-induced effects on Nrf2 mediated antioxidant defence in the HL-60 cell monocytic differentiation[J]. Cell Biol Int,2013,37(11):1215-1224.
[21]Kobayashi S. Choose delicately and reuse adequately:the newly revealed process of autophagy[J]. Biol Pharm Bull,2015,38(8):1098-1103.
[22]Levine B,Mizushima N,Virgin H W. Autophagy in immunity and inflammation[J]. Nature,2011,469(7330):325-335.
[23]CAO Y,Klionsky D J. Physiological functions of Atg6/Beclin 1:a unique autophagy-related protein[J]. Cell Res,2007,17(10):839-849.
[24]Pankiv S,Clausen T H, Lamark T, et al. p62/SQSTMQ binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33):24131-24145.
[25]PENG J,ZHANG R,CUI Y,et al. Atg5 regulates late endosome and lysosome biogenesis[J]. China Life Sci,2014,57(1):59-68.
[26]Rameh L E,Cantley L C. The role of phosphoinositide3-kinase lipid produccts in cell function[J]. J Biol Chem,1999,274(13):8347-8350.
[27]Stal O, Perez-Tenorio G, Akerberg L, et al. Akt kinases in breast cancer and the results of adjuvant therapy[J]. Breast Cancer Res, 2003, 5(2):R37-R44.
[28]Kennedy S G,Kandel E S,Cross T K,et al. Akt/protein kinase B inhibits cell death by preventing the release of cytochrome C from mitochondria[J]. Mol Cell Biol,1999,19(8):5800-5810.
[29]Ersahin T,Tuncbag N,Cetin-Atalay R. The PI3K/Akt/m TOR interactive pathway[J]. Mol Biosyst,2015,11(7):1946-1954.
[2]Asl M N,Hosseinzadeh H. Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds[J]. Phytother Res,2008,22(6):709-724.
[3]Takahashi T, Takasuka N, Iigo M, et al.Isoliquiritigenin, a flavonoid from licorice, reduces prostaglandin E2 and nitricoxide,causes apoptosis,and suppresses aberrant crypt foci development[J]. Cancer Sci,2004,95(5):448-453.
[4]JIN X Y, Sohn D H, Lee S H. Isoliquiritigenin suppresses tumor necrosis factor-α-induced inflammation via peroxisome proliferator-activated receptor-γin intestinal epithelial cells[J]. Arch Pharm Res,2016,39(10):1465-1471.
[5]Traboulsi H,Cloutier A, Boyapelly K, et al. The flavonoid isoliquiritigenin reduces lung inflammation and mouse morbidity during influenza virus infection[J].Antimicrob Agents Chemother, 2015, 59(10):6317-6327.
[6]WU T Y, Khor T O, Saw C L, et al. Antiinflammatory/anti-oxidative stress activities and differential regulation of Nrf2-mediated genes by nonpolar fractions of tea Chrysanthemum zawadskii and licorice Glycyrrhiza uralensis[J]. Aaps J,2010,13(1):1-13.
[7]Cronin H,Draelos Z D. Top 10 botanical ingredients in2010 anti-aging creams[J]. J Cosmet Dermatol,2010,9(3):218-225.
[8]Yerra V G,Kalvala A K,Kumar A. Isoliquiritigenin reduces oxidative damage and alleviates mitochondrial impairment by SIRT1 activation in experimental diabetic neuropathy[J]. J Nutr Biochem,2017,47(1):41-52.
[9]Baba M,Asano R,Takigami I,et al. Studies on cancer chemoprevention by traditional folk medicinesⅩⅩⅤ.Inhibitory effect of isoliquiritigenin on azoxymethaneinduced murine colon aberrant crypt focus formation and carcinogenesis[J]. Biol Pharm Bull,2002,25(2):247-250.
[10]Yamazaki S,Morita T,Endo H,et al. Isoliquiritigenin suppresses pulmonary metastasis of mouse renal cell carcinoma[J]. Cancer Lett,2002,183(1):23-30.
[11]PENG F,TANG H,LIU P,et al. Isoliquiritigenin modulates miR-374a/PTEN/Akt axis to suppress breast cancer tumorigenesis and metastasis[J]. Sci Rep,2017,7(1):9022-9036.
[12]SI L,YANG X,YAN X,et al. Isoliquiritigenin induces apoptosis of human bladder cancer T24 cells via a cyclin-dependent kinase-independent mechanism[J].Oncol Lett,2017,14(1):241-249.
[13]Hsia S M,YU C C,Shih Y H,et al. Isoliquiritigenin as a cause of DNA damage and inhibitor of ataxiatelangiectasia mutated expression leading to G2/M phase arrest and apoptosis in oral squamous cell carcinoma[J]. Head Neck,2016,38(Suppl 1):360-371.
[14]Jung S K,Lee M H,Lim D Y,et al. Isoliquiritigenin induces apoptosis and inhibits xenograft tumor growth of human lung cancer cells by targeting both wild type and L858R/T790M mutant EGFR[J]. J Biol Chem,2014,289(52):35839-35848.
[15]MA J,FU N Y,PANG D B,et al. Apoptosis induced by isoliquiritigenin in human gastric cancer MGC-803cells[J]. Planta Med,2001,67(8):754-757.
[16]LIN Y, SUN H, DANG Y, et al. Isoliquiritigenin inhibits the proliferation and induces the differentiation of human glioma stem cells[J]. Oncol Rep,2018,39(2):687-694.
[17]ZHANG B,LAI Y,LI Y,et al. Antineoplastic activity of isoliquiritigenin, a chalcone compound, in and androgen-independent human prostate cancer cells linked to G2/M cell cycle arrest and cell apoptosis[J].Eur J Pharmacol,2018,821(1):57-67.
[18]WU M, WU Y, DENG B, et al. Isoquiritigenin decreases the incidence of colitis-associated colorectal cancer by modulating the intestinal microbiota[J].Oncotarget,2016,7(51):85318-85331.
[19]Hirchaud F, Hermetet F, Ablise M, et al.Isoliquiritigenin induces caspase-dependent apoptosis via downregulation of HPV16 E6 expression in cervical cancer Ca Ski cells[J]. Planta Med,2013,79(17):1628-1635.
[20]CHEN H, ZHANG B, YUAN X, et al.Isoliquiritigenin-induced effects on Nrf2 mediated antioxidant defence in the HL-60 cell monocytic differentiation[J]. Cell Biol Int,2013,37(11):1215-1224.
[21]Kobayashi S. Choose delicately and reuse adequately:the newly revealed process of autophagy[J]. Biol Pharm Bull,2015,38(8):1098-1103.
[22]Levine B,Mizushima N,Virgin H W. Autophagy in immunity and inflammation[J]. Nature,2011,469(7330):325-335.
[23]CAO Y,Klionsky D J. Physiological functions of Atg6/Beclin 1:a unique autophagy-related protein[J]. Cell Res,2007,17(10):839-849.
[24]Pankiv S,Clausen T H, Lamark T, et al. p62/SQSTMQ binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33):24131-24145.
[25]PENG J,ZHANG R,CUI Y,et al. Atg5 regulates late endosome and lysosome biogenesis[J]. China Life Sci,2014,57(1):59-68.
[26]Rameh L E,Cantley L C. The role of phosphoinositide3-kinase lipid produccts in cell function[J]. J Biol Chem,1999,274(13):8347-8350.
[27]Stal O, Perez-Tenorio G, Akerberg L, et al. Akt kinases in breast cancer and the results of adjuvant therapy[J]. Breast Cancer Res, 2003, 5(2):R37-R44.
[28]Kennedy S G,Kandel E S,Cross T K,et al. Akt/protein kinase B inhibits cell death by preventing the release of cytochrome C from mitochondria[J]. Mol Cell Biol,1999,19(8):5800-5810.
[29]Ersahin T,Tuncbag N,Cetin-Atalay R. The PI3K/Akt/m TOR interactive pathway[J]. Mol Biosyst,2015,11(7):1946-1954.