冬凌草甲素对结肠癌LOVO细胞增殖和EMT影响的实验研究
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摘要
目的探讨冬凌草甲素(oridonin,ORI)对结肠癌细胞株LOVO细胞增殖和上皮间质转化(epithelialmesenchymal transition,EMT)的影响及可能的机制。方法不同药物浓度(0、2、4、8、12和16μg/m L)冬凌草甲素处理结肠癌LOVO细胞,不同时间点(24、48和72 h)采用CCK-8法观察细胞增殖情况,RT-PCR检测E-cadherin和Vimentin mRNA表达水平,Western blot检测磷酸化糖原合成酶激酶-3β(phospharylated glycogen synthase kinase-3β,p-GSK-3β)、β-catenin、E-cadherin和Vimentin蛋白表达水平。结果冬凌草甲素对LOVO细胞增殖抑制率呈时间和剂量依赖性(均P <0. 05)。4、8和12μg/m L冬凌草甲素作用48 h,增加LOVO细胞E-cadherin基因表达量,减少Vimentin的基因表达量(均P <0. 05),也增加LOVO细胞p-GSK-3β(Tyr216)蛋白表达量,减少p-GSK-3β(Ser9)蛋白表达量,同时下调β-catenin蛋白表达量(均P <0. 05)。GSK-3β特定的抑制剂CHIR 2μmol/L作用1 h后,再加入12μg/m L冬凌草甲素共同作用24 h,能逆转冬凌草甲素对β-catenin、E-cadherin和Vimentin蛋白表达的影响(均P <0. 05)。结论冬凌草甲素可能通过Wnt/β-catenin信号通路抑制结肠癌LOVO细胞的生长增殖和EMT过程。
Objective To investigate the effect of oridonin on the proliferation and epithelial-mesenchymal transition( EMT) of colon cancer LOVO cell line and possible mechanisms. Methods CCK-8 method was used to observe the effect of( 0,2,4,8,12,16 μg/m L) oridonin on the proliferation of colon cancer LOVO cells at different time points( 24,48 and72 h); and the mRNA expressions of E-cadherin and Vimentin were detected by RT-PCR; and the protein expressions of phospharylated glycogen synthase kinase-3β( p-GSK-3β),β-catenin,E-cadherin and Vimentin were analyzed by Western blot. Results CCK-8 showed the proliferation inhibition rate of LOVO cells was increased gradually in oridonin-treated groups in a concentration and time dependent manner( all P < 0. 05). RT-PCR showed that the gene expression of the Ecadherin was increased and the gene expression of Vimentin was reduced in LOVO cells treated with 4,8 and 12 μg/m L oridonin for 48 h( all P < 0. 05). The results of Western blot showed that the expression level of the p-GSK-3β( Tyr216) protein was up-regulated and the expression level of the p-GSK-3β( Ser9) and β-catenin protein were down-regulated in LOVO cells treated with 4,8 and 12 μg/m L oridonin for 48 h( all P < 0. 05). One-hour pretreatment of 2 μmol/L GSK-3β specific inhibitor CHIR and 24 h treatment of 2 μmol/L CHIR and 12 μg/m L oridonin could reverse the effect of oridonin on the expression of β-catenin,E-cadherin and Vimentin( all P < 0. 05). Conclusion Oridonin can inhibit the proliferation and EMT of colon cancer LOVO cells by Wnt/β-catenin signaling pathway.
Objective To investigate the effect of oridonin on the proliferation and epithelial-mesenchymal transition( EMT) of colon cancer LOVO cell line and possible mechanisms. Methods CCK-8 method was used to observe the effect of( 0,2,4,8,12,16 μg/m L) oridonin on the proliferation of colon cancer LOVO cells at different time points( 24,48 and72 h); and the mRNA expressions of E-cadherin and Vimentin were detected by RT-PCR; and the protein expressions of phospharylated glycogen synthase kinase-3β( p-GSK-3β),β-catenin,E-cadherin and Vimentin were analyzed by Western blot. Results CCK-8 showed the proliferation inhibition rate of LOVO cells was increased gradually in oridonin-treated groups in a concentration and time dependent manner( all P < 0. 05). RT-PCR showed that the gene expression of the Ecadherin was increased and the gene expression of Vimentin was reduced in LOVO cells treated with 4,8 and 12 μg/m L oridonin for 48 h( all P < 0. 05). The results of Western blot showed that the expression level of the p-GSK-3β( Tyr216) protein was up-regulated and the expression level of the p-GSK-3β( Ser9) and β-catenin protein were down-regulated in LOVO cells treated with 4,8 and 12 μg/m L oridonin for 48 h( all P < 0. 05). One-hour pretreatment of 2 μmol/L GSK-3β specific inhibitor CHIR and 24 h treatment of 2 μmol/L CHIR and 12 μg/m L oridonin could reverse the effect of oridonin on the expression of β-catenin,E-cadherin and Vimentin( all P < 0. 05). Conclusion Oridonin can inhibit the proliferation and EMT of colon cancer LOVO cells by Wnt/β-catenin signaling pathway.
引文
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[29]Gao ZH,Lu C,Wang MX,et al.Differentialβ-catenin expression levels are associated with morphological features and prognosis of colorectal cancer[J].Oncol Lett,2014,8(5):2069-2076.
[30]Yang D,Zhang X,Zhang W,et al.Vicenin-2 inhibits Wnt/β-catenin signaling and induces apoptosis in HT-29human colon cancer cell line[J].Drug Des Devel Ther,2018,12(18):1303-1310.
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[2]Gustavsson B,Carlsson G,Machover D,et al.A review of the evolution of systemic chemotherapy in the management of colorect[J].Clin Colorectal Cancer,2015,14(1):1-10.
[3]Weaver BA.How Taxol/paclitaxel kills cancer cells[J].Mol Biol Cell,2014,25(17):2677-2681.
[4]Owona BA,Schluesener HJ.Molecular insight in the multifunctional effects of oridonin[J].Drugs in R&D,2015,15(3):233-244.
[5]Liu QQ,Chen K,Ye Q,et al.Oridonin inhibits pancreatic cancer cell migration and epithelial-mesenchymal transition by suppressing Wnt/β-catenin signaling pathway[J].Cancer Cell Int,2016,16(11):57.
[6]Guo Z,Zhou Y,Yang J,et al.Dendrobium candidum extract inhibits proliferation and induces apoptosis of liver cancer cells by inactivating Wnt/β-catenin signaling pathway[J].Biomed Pharmacother,2019,110(2):371-379.
[7]Srivastava NS,Srivastava RAK.Curcumin and quercetin synergistically inhibit cancer cell proliferation in multiple cancer cells and modulate Wnt/β-catenin signaling and apoptotic pathways in A375 cells[J].Phytomedicine,2019,52(1):117-128.
[8]Xian X,Tang L,Wu C,et al.miR-23b-3p and miR-130a-5p affect cell growth,migration and invasion by targeting CB1R via the Wnt/β-catenin signaling pathway in gastric carcinoma[J].Onco Targets Ther,2018,11(8):7503-7512.
[9]Zhang Z,Chang Y,Zhang J,et al.HMGB3 promotes growth and migration in colorectal cancer by regulating WNT/β-catenin pathway[J].PLo S One,2017,12(7):e0179741.
[10]Hu HZ,Yang YB,Xu XD,et al.Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma He La cell line[J].Acta Pharmacol Sin,2007,28(11):1819-1826.
[11]Li X,Li X,Wang J,et al.Oridonin up-regulates expression of P21 and induces autophagy and apoptosis in human prostate cancer cells[J].Int J Biol Sci,2012,8(6):901-912.
[12]Liang J,Wang W,Wei L,et al.Oridonin inhibits growth and induces apoptosis of human neurocytoma cells via the Wnt/β-catenin pathway[J].Oncol Lett,2018,16(3):3333-3340.
[13]Liu Y,Liu YZ,Zhang RX,et al.Oridonin inhibits the proliferation of human osteosarcoma cells by suppressing Wnt/β-catenin signaling[J].Int J Oncol,2014,45(2):795-803.
[14]中国医师协会外科医师分会多学科综合治疗专业委员会,中国抗癌协会大肠癌专业委员会,结直肠癌肺转移多学科综合治疗专家共识[J].实用肿瘤杂志,2018,33(6):487-501.
[15]Wang J,Du Y,Liu X,et al.MicroRNAs as regulator of signaling networks in metastatic colon cancer[J].Biomed Res Int,2015,2015(823620):1-12.
[16]Liu DM,Thakor AS,Baerlocher M,et al.A review of conventional and drug-eluting chemoembolization in the treatment of colorectal liver metastases:principles and proof[J].Future Oncol,2015,11(9):1421-1428.
[17]Liu RX,Ma Y,Hu XL,et al.Anticancer effects of oridonin on colon cancer are mediated via BMP7/p38 MAPK/p53 signaling[J].Int J Oncol,2018,53(5):2091-2101.
[18]Ren CM,Li Y,Chen QZ,et al.Oridonin inhibits the proliferation of human colon cancer cells by upregulating BMP7 to activate p38 MAPK[J].Oncol Rep,2016,35(5):2691-2698.
[19]Gao FH,Liu F,Wei W,et al.Oridonin induces apoptosis and senescence by increasing hydrogen peroxide and glutathione depletion in colorectal cancer cells[J].Int JMol Med,2012,29(4):649-655.
[20]Zhang D,Zhou Q,Huang D,et al.ROS/JNK/c-Jun axis is involved in oridonin-induced caspase-dependent apoptosis in human colorectal cancer cells[J].Biochem Biophys Res Commun,2019,513(3):594-601.
[21]Gao FH,Hu XH,Li W,et al.Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16,p21,p27and c-myc[J].BMC Cancer,2010,10(6):610-621.
[22]Mitra A,Mishra L,Li S.EMT,CTCs and CSCs in tumor relapse and drug-resistance[J].Oncotarget,2015,6(13):10697-10711.
[23]陈韡,于悦,孙艳霞,等.Id1和Id3协同诱导结肠癌SW620细胞EMT并影响其侵袭与迁移[J].中国肿瘤生物治疗杂志,2018,25(10):987-993.
[24]Yang YJ,Li ZB,Zhang GR,et al.Snail-induced epithelial-mesenchymal transition in gastric carcinoma cells and generation of cancer stem cell characteristics[J].Genet Mol Res,2016,15(3):1-13.
[25]Kim JH,Hwang YJ,Han SH,et al.Dexamethasone inhibits hypoxia-induced epithelial-mesenchymal transition in colon cancer[J].World J Gastroenterol,2015,21(34):9887-9899.
[26]Lee SC,Kim OH,Lee SK,et al.IWR-1 inhibits epithelial-mesenchymal transition of colorectal cancer cells through suppressing Wnt/β-catenin signaling as well as survivin expression[J].Oncotarget,2015,6(29):27146-27159.
[27]Clevers H,Nusse R.Wnt/β-catenin signaling and disease[J].Cell,2012,149(6):1192-205.
[28]Zhen T,Dai S,Li H,et al.MACC1 promotes carcinogenesis of colorectal cancer viaβ-catenin signaling pathway[J].Oncotarget,2014,5(11):3756-3769.
[29]Gao ZH,Lu C,Wang MX,et al.Differentialβ-catenin expression levels are associated with morphological features and prognosis of colorectal cancer[J].Oncol Lett,2014,8(5):2069-2076.
[30]Yang D,Zhang X,Zhang W,et al.Vicenin-2 inhibits Wnt/β-catenin signaling and induces apoptosis in HT-29human colon cancer cell line[J].Drug Des Devel Ther,2018,12(18):1303-1310.
[31]Bikkavilli RK,Feigin ME,Malbon CC.p38 mitogen-activated protein kinase regulates canonical Wnt/β-catenin signaling by inactivation of GSK3β[J].J Cell Sci,2008,121(21):3598-3607.
[32]Gao CX,Chen GM,Kuan SF.et al.FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β[J].Elife.2015,4(e):10072.
[33]Tejeda-Mu1oz N,Robles-Flores M.Glycogen synthase kinase 3 in Wnt signaling pathway and cancer[J].IUB-MB Life,2015,67(12):914-922.