Wnt/β-catenin/TCF-4信号通路调节livin表达在肾癌细胞增殖凋亡中的研究
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摘要
目的:研究人肾癌细胞系786-0中Wnt/β-catenin/TCF-4通路与livin的关系,探索Wnt/β-catenin/TCF-4通路在肾癌细胞中的凋亡调节机制。方法:不同浓度吲哚美辛处理肾癌786-0细胞,CCK-8法检测各组细胞活性;流式细胞术检测各组细胞凋亡改变;荧光定量PCR检测β-catenin、TCF-4、caspase-3及livin转录水平变化;Western blot检测β-catenin、caspase-3及livin蛋白表达水平变化。结果:随吲哚美辛浓度增加,细胞活力降低,呈剂量-效应关系(P=0.000)。流式结果显示伴随吲哚美辛浓度升高,细胞凋亡逐渐增加,25μmol/L组、50μmol/L组和100μmol/L组凋亡率分别为(32.07±1.01)%、(40.03±1.95)%和(72.33±0.02)%,与对照组比较差异存在统计学意义(P=0.000,P=0.002,P=0.000)。Real-time PCR结果显示,β-catenin、TCF-4和livin相对表达水平均呈下降趋势(P=0.002,P=0.000,P=0.000),而caspase-3相对表达呈明显上升趋势(P=0.001)。Western blot分析显示在25μmol/L组、50μmol/L组和100μmol/L组中β-catenin相对表达量分别为0.568±0.020(P=0.001)、0.396±0.030(P=0.000)、0.142±0.038(P=0.000);livin相对表达量分别为0.139±0.016(P=0.005)、0.050±0.006(P=0.000)、0.011±0.001(P=0.000);而caspase-3相对表达量分别为0.278±0.035(P=0.046)、0.396±0.071(P=0.000)、0.518±0.015(P=0.000)。结论:吲哚美辛介导的肾癌细胞中β-catenin/TCF-4降解可能导致livin的转录抑制和caspase-3表达水平的上调,进而诱导肾癌细胞凋亡。
Objective:To investigate the relationship between the Wnt/β-catenin/TCF-4 pathway and livin expression in human renal cancer cell line 786-0,and to explore the apoptosis regulation mechanism of this pathway. Methods:786-0 cells were treated with different concentrations of indomethacin. Cell viability was then determined by CCK-8 assay. Flow cytometry was used to determine apoptosis change. The transcriptional changes of β-catenin,TCF-4,caspase-3,and livin were measured by quantitative real-time PCR,while the expression levels of β-catenin,caspase-3,and livin were measured by Western blot. Results:With the increase in the concentration of indomethacin,the cell viability decreased in a dose-response manner(P=0.000). Flow cytometry showed that the apoptosis rate increased with the increase in the concentration of indomethacin. The apoptosis rates of the 25 μmol/L group,50 μmol/L group,and 100 μmol/L group were(32.07±1.01)%,(40.03±1.95)%,and(72.33±0.02)%,respectively,all showing significant differences compared with those of the control group(P=0.000,0.002,and 0.000,respectively). Real-time PCR showed that the relative expression levels of β-catenin,TCF-4,and livin decreased significantly(P =0.002,0.000,and 0.000,respectively),while the level of caspase-3 increased significantly(P=0.001). Western blot revealed that,in the 25 μmol/L group,50 μmol/L group,and100 μmol/L group,the relative expression levels of β-catenin were 0.568±0.020(P=0.001),0.396±0.030(P=0.000),and 0.142±0.038(P=0.000),respectively;the levels of livin were 0.139±0.016(P=0.005),0.050±0.006(P=0.000),and 0.011±0.001(P=0.000),respectively;and the levels of caspase-3 were 0.278 ±0.035(P=0.046),0.396 ±0.071(P=0.000),and 0.518 ±0.015(P=0.000),respectively. Conclusion:Indomethacin-mediated degradation of β-catenin/TCF-4 in renal cancer cells may lead to the transcriptional repression of livin and the up-regulation of caspase-3 expression,thereby inducing apoptosis of renal cancer cells.
Objective:To investigate the relationship between the Wnt/β-catenin/TCF-4 pathway and livin expression in human renal cancer cell line 786-0,and to explore the apoptosis regulation mechanism of this pathway. Methods:786-0 cells were treated with different concentrations of indomethacin. Cell viability was then determined by CCK-8 assay. Flow cytometry was used to determine apoptosis change. The transcriptional changes of β-catenin,TCF-4,caspase-3,and livin were measured by quantitative real-time PCR,while the expression levels of β-catenin,caspase-3,and livin were measured by Western blot. Results:With the increase in the concentration of indomethacin,the cell viability decreased in a dose-response manner(P=0.000). Flow cytometry showed that the apoptosis rate increased with the increase in the concentration of indomethacin. The apoptosis rates of the 25 μmol/L group,50 μmol/L group,and 100 μmol/L group were(32.07±1.01)%,(40.03±1.95)%,and(72.33±0.02)%,respectively,all showing significant differences compared with those of the control group(P=0.000,0.002,and 0.000,respectively). Real-time PCR showed that the relative expression levels of β-catenin,TCF-4,and livin decreased significantly(P =0.002,0.000,and 0.000,respectively),while the level of caspase-3 increased significantly(P=0.001). Western blot revealed that,in the 25 μmol/L group,50 μmol/L group,and100 μmol/L group,the relative expression levels of β-catenin were 0.568±0.020(P=0.001),0.396±0.030(P=0.000),and 0.142±0.038(P=0.000),respectively;the levels of livin were 0.139±0.016(P=0.005),0.050±0.006(P=0.000),and 0.011±0.001(P=0.000),respectively;and the levels of caspase-3 were 0.278 ±0.035(P=0.046),0.396 ±0.071(P=0.000),and 0.518 ±0.015(P=0.000),respectively. Conclusion:Indomethacin-mediated degradation of β-catenin/TCF-4 in renal cancer cells may lead to the transcriptional repression of livin and the up-regulation of caspase-3 expression,thereby inducing apoptosis of renal cancer cells.
引文
[1] Jaquith JB. Targeting the inhibitor of apoptosis protein BIR3binding domains[J]. Pharm Pat Anal,2014,3(3):297-312.
[2] Liu C,Wu X,Luo C,et al. Antisense oligonucleotide targeting Livin induces apoptosis of human bladder cancer cell via a mechanism involving caspase 3[J]. J Exp Clin Cancer Res,2010,29:63.
[3] Yuan D,Liu L,Gu D. Transcriptional regulation of livin by betacatenin/TCF signaling in human lung cancer cell lines[J]. Mol Cell Biochem,2007,306(1-2):171-178.
[4] Cojocaru E,Lozneanu L,Giusca SE,et al. Renal carcinogenesis--insights into signaling pathways[J]. Rom J Morphol Embryol,2015,56(1):15-19.
[5] Tai D,Wells K,Arcaroli J,et al. Targeting the WNT signaling pathway in cancer therapeutics[J]. Oncologist,2015,20(10):1189-1198.
[6] Sareddy GR,Geeviman K,Panigrahi M,et al. Increased betacatenin/Tcf signaling in pilocytic astrocytomas:a comparative study to distinguish pilocytic astrocytomas from low-grade diffuse astrocytomas[J]. Neurochem Res,2012,37(1):96-104.
[7] Rosenbluh J,Wang X,Hahn WC. Genomic insights into WNT/betacatenin signaling[J]. Trends Pharmacol Sci,2014,35(2):103-109.
[8] Ying Y,Tao Q. Epigenetic disruption of the WNT/beta-catenin signaling pathway in human cancers[J]. Epigenetics,2009,4(5):307-312.
[9] Kassab SE,Khedr MA,Ali HI,et al. Discovery of new indomethacinbased analogs with potentially selective cyclooxygenase-2 inhibition and observed diminishing to PGE2 activities[J]. Eur J Med Chem,2017,141:306-321.
[10] Sever B,Altintop MD,Kus G,et al. Indomethacin based new triazolothiadiazine derivatives:synthesis,evaluation of their anticancer effects on T98 human glioma cell line related to COX-2 inhibition and docking studies[J]. Eur J Med Chem,2016,113:179-186.
[11] Kim HS,Sharma A,Ren WX,et al. COX-2 Inhibition mediated anti-angiogenic activatable prodrug potentiates cancer therapy in preclinical models[J]. Biomaterials,2018,185:63-72.
[12] Liu Z,Hu J,Huang Z,et al. Effect of indomethacin on BCR/ABLWnt/beta-catenin pathway in K562 cells[J]. Nan Fang Yi Ke Da Xue Xue Bao,2015,35(7):998-1002.
[13] Yin L,Liu S,Li C,et al. CYLD downregulates Livin and synergistically improves gemcitabine chemosensitivity and decreases migratory/invasive potential in bladder cancer:the effect is autophagy-associated[J]. Tumour Biol,2016,37(9):12731-12742.
[14] Li CJ,Cong Y,Liu XZ,et al. Research progress on the livin gene and osteosarcomas[J]. Asian Pac J Cancer Prev,2014,15(20):8577-8579.
[15] Cheng T,Zhang JG,Cheng YH,et al. Relationship between PTEN and Livin expression and malignancy of renal cell carcinomas[J]. Asian Pac J Cancer Prev,2012,13(6):2681-2685.
[16] Han Y,Zhang L,Wang W,et al. Livin promotes the progression and metastasis of breast cancer through the regulation of epithelialmesenchymal transition via the p38/GSK3beta pathway[J]. Oncol Rep,2017,38(6):3574-3582.
[17] Liu GH,Wang WL,Wang C,et al. Livin modulates the apoptotic effects of vesicular stomatotitis virus in lung adenocarcinoma[J]. Int J Oncol,2015,47(5):1775-1782.
[18] Su QB,Wang LY,Wei GN,et al. Livin serves as a prognostic marker for mid-distal rectal cancer and a target of mid-distal rectal cancer treatment[J]. Oncol Lett,2017,14(6):7759-7766.
[19] Yoon TM,Kim SA,Lee DH,et al. Livin enhances chemoresistance in head and neck squamous cell carcinoma[J]. Oncol Rep,2017,37(6):3667-3673.
[20] Wang Z,Liu S,Ding K,et al. Silencing Livin induces apoptotic and autophagic cell death,increasing chemotherapeutic sensitivity to cisplatin of renal carcinoma cells[J]. Tumour Biol,2016,37(11):15133-15143.
[21] Crnkovic-Mertens I,Wagener N,Semzow J,et al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J]. Cell Mol Life Sci,2007,64(9):1137-1144.
[22] Leal LF,Bueno AC,Gomes DC,et al. Inhibition of the Tcf/betacatenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis[J]. Oncotarget,2015,6(40):43016-43032.
[23] Stewart DJ,Chang DW,Ye Y,et al. Wnt signaling pathway pharmacogenetics in non-small cell lung cancer[J]. Pharmacogenomics J,2014,14(6):509-522.
[24] Joosten SC,Smits KM,Aarts MJ,et al. Epigenetics in renal cell cancer:mechanisms and clinical applications[J]. Nat Rev Urol,2018,15(7):430-451.
[25] Gao ZH,Lu C,Wang MX,et al. Differential beta-catenin expression levels are associated with morphological features and prognosis of colorectal cancer[J]. Oncol Lett,2014,8(5):2069-2076.
[26] VON Schulz-Hausmann SA,Schmeel LC,Schmeel FC,et al. Targeting the Wnt/beta-catenin pathway in renal cell carcinoma[J]. Anticancer Res,2014,34(8):4101-4108.
[27] Kawakami K,Hirata H,Yamamura S,et al. Functional significance of Wnt inhibitory factor-1 gene in kidney cancer[J]. Cancer Res,2009,69(22):8603-8610.
[28] Kawamoto K,Hirata H,Kikuno N,et al. DNA methylation and histone modifications cause silencing of Wnt antagonist gene in human renal cell carcinoma cell lines[J]. Int J Cancer,2008,123(3):535-542.
[29] Wallmen B,Schrempp M,Hecht A. Intrinsic properties of Tcf1and Tcf4 splice variants determine cell-type-specific Wnt/beta-catenin target gene expression[J]. Nucleic Acids Res,2012,40(19):9455-9469.
[30] Wang L,Zhang Q,Liu B,et al. Challenge and promise:roles for Livin in progression and therapy of cancer[J]. Mol Cancer Ther,2008,7(12):3661-3669.
[31] Dihlmann S,Siermann A,Von Knebel Doeberitz M. The nonsteroidal anti-inflammatory drugs aspirin and indomethacin attenuate beta-catenin/TCF-4 signaling[J]. Oncogene,2001,20(5):645-653.
[32] Nagaraj AB,Wang QQ,Joseph P,et al. Using a novel computational drug-repositioning approach(DrugPredict)to rapidly identify potent drug candidates for cancer treatment[J]. Oncogene,2018,37(3):403-414.
[33] Zheng Q,Zhang Y,Ren Y,et al. Antiproliferative and apoptotic effects of indomethacin on human retinoblastoma cell line Y79 and the involvement of beta-catenin,nuclear factor-kappaB and Akt signaling pathways[J]. Ophthalmic Res,2014,51(2):109-115.
[2] Liu C,Wu X,Luo C,et al. Antisense oligonucleotide targeting Livin induces apoptosis of human bladder cancer cell via a mechanism involving caspase 3[J]. J Exp Clin Cancer Res,2010,29:63.
[3] Yuan D,Liu L,Gu D. Transcriptional regulation of livin by betacatenin/TCF signaling in human lung cancer cell lines[J]. Mol Cell Biochem,2007,306(1-2):171-178.
[4] Cojocaru E,Lozneanu L,Giusca SE,et al. Renal carcinogenesis--insights into signaling pathways[J]. Rom J Morphol Embryol,2015,56(1):15-19.
[5] Tai D,Wells K,Arcaroli J,et al. Targeting the WNT signaling pathway in cancer therapeutics[J]. Oncologist,2015,20(10):1189-1198.
[6] Sareddy GR,Geeviman K,Panigrahi M,et al. Increased betacatenin/Tcf signaling in pilocytic astrocytomas:a comparative study to distinguish pilocytic astrocytomas from low-grade diffuse astrocytomas[J]. Neurochem Res,2012,37(1):96-104.
[7] Rosenbluh J,Wang X,Hahn WC. Genomic insights into WNT/betacatenin signaling[J]. Trends Pharmacol Sci,2014,35(2):103-109.
[8] Ying Y,Tao Q. Epigenetic disruption of the WNT/beta-catenin signaling pathway in human cancers[J]. Epigenetics,2009,4(5):307-312.
[9] Kassab SE,Khedr MA,Ali HI,et al. Discovery of new indomethacinbased analogs with potentially selective cyclooxygenase-2 inhibition and observed diminishing to PGE2 activities[J]. Eur J Med Chem,2017,141:306-321.
[10] Sever B,Altintop MD,Kus G,et al. Indomethacin based new triazolothiadiazine derivatives:synthesis,evaluation of their anticancer effects on T98 human glioma cell line related to COX-2 inhibition and docking studies[J]. Eur J Med Chem,2016,113:179-186.
[11] Kim HS,Sharma A,Ren WX,et al. COX-2 Inhibition mediated anti-angiogenic activatable prodrug potentiates cancer therapy in preclinical models[J]. Biomaterials,2018,185:63-72.
[12] Liu Z,Hu J,Huang Z,et al. Effect of indomethacin on BCR/ABLWnt/beta-catenin pathway in K562 cells[J]. Nan Fang Yi Ke Da Xue Xue Bao,2015,35(7):998-1002.
[13] Yin L,Liu S,Li C,et al. CYLD downregulates Livin and synergistically improves gemcitabine chemosensitivity and decreases migratory/invasive potential in bladder cancer:the effect is autophagy-associated[J]. Tumour Biol,2016,37(9):12731-12742.
[14] Li CJ,Cong Y,Liu XZ,et al. Research progress on the livin gene and osteosarcomas[J]. Asian Pac J Cancer Prev,2014,15(20):8577-8579.
[15] Cheng T,Zhang JG,Cheng YH,et al. Relationship between PTEN and Livin expression and malignancy of renal cell carcinomas[J]. Asian Pac J Cancer Prev,2012,13(6):2681-2685.
[16] Han Y,Zhang L,Wang W,et al. Livin promotes the progression and metastasis of breast cancer through the regulation of epithelialmesenchymal transition via the p38/GSK3beta pathway[J]. Oncol Rep,2017,38(6):3574-3582.
[17] Liu GH,Wang WL,Wang C,et al. Livin modulates the apoptotic effects of vesicular stomatotitis virus in lung adenocarcinoma[J]. Int J Oncol,2015,47(5):1775-1782.
[18] Su QB,Wang LY,Wei GN,et al. Livin serves as a prognostic marker for mid-distal rectal cancer and a target of mid-distal rectal cancer treatment[J]. Oncol Lett,2017,14(6):7759-7766.
[19] Yoon TM,Kim SA,Lee DH,et al. Livin enhances chemoresistance in head and neck squamous cell carcinoma[J]. Oncol Rep,2017,37(6):3667-3673.
[20] Wang Z,Liu S,Ding K,et al. Silencing Livin induces apoptotic and autophagic cell death,increasing chemotherapeutic sensitivity to cisplatin of renal carcinoma cells[J]. Tumour Biol,2016,37(11):15133-15143.
[21] Crnkovic-Mertens I,Wagener N,Semzow J,et al. Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis[J]. Cell Mol Life Sci,2007,64(9):1137-1144.
[22] Leal LF,Bueno AC,Gomes DC,et al. Inhibition of the Tcf/betacatenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis[J]. Oncotarget,2015,6(40):43016-43032.
[23] Stewart DJ,Chang DW,Ye Y,et al. Wnt signaling pathway pharmacogenetics in non-small cell lung cancer[J]. Pharmacogenomics J,2014,14(6):509-522.
[24] Joosten SC,Smits KM,Aarts MJ,et al. Epigenetics in renal cell cancer:mechanisms and clinical applications[J]. Nat Rev Urol,2018,15(7):430-451.
[25] Gao ZH,Lu C,Wang MX,et al. Differential beta-catenin expression levels are associated with morphological features and prognosis of colorectal cancer[J]. Oncol Lett,2014,8(5):2069-2076.
[26] VON Schulz-Hausmann SA,Schmeel LC,Schmeel FC,et al. Targeting the Wnt/beta-catenin pathway in renal cell carcinoma[J]. Anticancer Res,2014,34(8):4101-4108.
[27] Kawakami K,Hirata H,Yamamura S,et al. Functional significance of Wnt inhibitory factor-1 gene in kidney cancer[J]. Cancer Res,2009,69(22):8603-8610.
[28] Kawamoto K,Hirata H,Kikuno N,et al. DNA methylation and histone modifications cause silencing of Wnt antagonist gene in human renal cell carcinoma cell lines[J]. Int J Cancer,2008,123(3):535-542.
[29] Wallmen B,Schrempp M,Hecht A. Intrinsic properties of Tcf1and Tcf4 splice variants determine cell-type-specific Wnt/beta-catenin target gene expression[J]. Nucleic Acids Res,2012,40(19):9455-9469.
[30] Wang L,Zhang Q,Liu B,et al. Challenge and promise:roles for Livin in progression and therapy of cancer[J]. Mol Cancer Ther,2008,7(12):3661-3669.
[31] Dihlmann S,Siermann A,Von Knebel Doeberitz M. The nonsteroidal anti-inflammatory drugs aspirin and indomethacin attenuate beta-catenin/TCF-4 signaling[J]. Oncogene,2001,20(5):645-653.
[32] Nagaraj AB,Wang QQ,Joseph P,et al. Using a novel computational drug-repositioning approach(DrugPredict)to rapidly identify potent drug candidates for cancer treatment[J]. Oncogene,2018,37(3):403-414.
[33] Zheng Q,Zhang Y,Ren Y,et al. Antiproliferative and apoptotic effects of indomethacin on human retinoblastoma cell line Y79 and the involvement of beta-catenin,nuclear factor-kappaB and Akt signaling pathways[J]. Ophthalmic Res,2014,51(2):109-115.