GW4064激活法尼酯X受体抑制结肠癌细胞生长浸润及基质细胞衍生因子1的表达
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摘要
目的探讨法尼酯X受体(FXR)特异性激动剂GW4064抑制结肠癌细胞生长浸润的机制。方法体外培养人结肠癌细胞系HT-29,用浓度为0、0.1、1、3、5、7、10μmol/L的GW4064分别处理HT-29细胞72 h,采用MTT法检测细胞活力;用浓度为0、1、5μmol/L的GW4064分别处理HT-29细胞24 h,用相差显微镜观察细胞形态;用浓度为0、1μmol/L的GW4064分别处理HT-29细胞24 h,采用细胞划痕实验检测细胞浸润的变化;用浓度为0、1、5μmol/L的GW4064分别处理HT-29细胞24 h,采用PCR检测FXR mRNA和基质细胞衍生因子1(SDF-1)m RNA表达的变化;用浓度为0、1、5、7μmol/L的GW4064分别处理24 h,采用ELISA法检测细胞培养上清液中SDF-1表达的变化。于裸鼠皮下接种HT-29细胞建立裸鼠成瘤模型,灌胃给予GW4064或溶剂DMSO,16 d后检测肿瘤生长情况及瘤体中FXR与SDF-1 mRNA的表达。结果 GW4064处理HT-29细胞后,细胞生长受到抑制,且呈剂量依赖性,1、3、5、7、10μmol/L GW4064组HT-29细胞的生长活力与对照组(0μmol/L)相比差异均有统计学意义(P均<0.05)。用相差显微镜观察可见GW4064处理HT-29细胞后,细胞收缩变圆,从瘦长的细胞向表皮样细胞改变。细胞划痕实验结果显示GW4064处理HT-29细胞后,细胞迁移距离变短,与对照组(0μmol/L)相比差异有统计学意义(P<0.05)。PCR检测结果显示GW4064处理后FXR mRNA表达呈剂量依赖性增加,而SDF-1m RNA表达则相反。ELISA检测结果显示细胞培养上清液中SDF-1的表达量随着GW4064浓度的增加而逐渐下降,1、5、7μmol/L GW4064组与对照组(0μmol/L)相比差异均有统计学意义(P均<0.05)。给予GW4064后,荷瘤小鼠肿瘤体积变小,与对照组(给予DMSO)相比差异有统计学意义(P<0.05),并且瘤体内FXR mRNA表达增加、SDF-1 mRNA表达减少。结论 FXR激活后抑制了结肠癌细胞生长浸润,同时抑制了结肠癌细胞SDF-1的表达分泌。
Objective To explore the mechanism by which speci?c agonist of farnesoid X receptor(FXR) GW4064 inhibits the growth and invasion of colon cancer cells. Methods Human colon cancer cell lines HT-29 were in vitro cultured.After treatment with GW4064 of 0, 0.1, 1, 3, 5, 7 and 10 μmol/L for 72 h, cell viability was measured by MTT assay. After treatment with GW4064 of 0, 1 and 5 μmol/L for 24 h, the cell morphology was observed under phase contrast microscope,and the mRNA expression levels of FXR and stromal cell-derived factor 1(SDF-1) were detected by PCR. After treatment with GW4064 of 0 and 1 μmol/L for 24 h, the cell invasive ability was detected by cell scratch test. After treatment with GW4064 of 0, 1, 5 and 7 μmol/L for 24 h, the SDF-1 expression in the culture medium was detected by ELISA. Nude mouse tumorigenesis model was established by subcutaneous inoculation of HT-29 cells. After intragastric administration with GW4064 or DMSO for 16 d, the tumor growth and the mRNA expression of FXR and SDF-1 in the tumors were determined.Results GW4064 inhibited the growth of HT-29 cells in a dose-dependent manner, and there was signi?cant difference in the cell viability of HT-29 cells between the GW4064 groups(1, 3, 5, 7 and 10 μmol/L) and the control group(0 μmol/L, all P<0.05). After treatment with GW4064, phase contrast microscopy showed contracted and rounded colon cancer cells and slender cells transforming into epidermoid cells. The cell scratch test showed that the invasion ability of the colon cancer cells was signi?cantly reduced after treatment with GW4064 compared with the control group(0 μmol/L, P<0.05). PCR results showed that the mRNA expression level of FXR was increased in a dose-dependent manner after GW4064 treatment, while the expression of SDF-1 mRNA changed in the opposite way. ELISA results showed that the SDF-1 expression in the cell culture supernant was decreased with the increase of GW4064 concentrations, and there were signi?cant differences between the GW4064(1, 5 and 7 μmol/L) groups and the control group(0 μmol/L, P<0.05). GW4064 signi?cantly reduced tumor size compared with the control group(DMSO, P<0.05). In addition, the mRNA expression of FXR in the tumors was increased,and the mRNA expression of SDF-1 was decreased. Conclusion The activation of FXR can inhibit the invasive growth of colon cancer cells and the expression of SDF-1.
Objective To explore the mechanism by which speci?c agonist of farnesoid X receptor(FXR) GW4064 inhibits the growth and invasion of colon cancer cells. Methods Human colon cancer cell lines HT-29 were in vitro cultured.After treatment with GW4064 of 0, 0.1, 1, 3, 5, 7 and 10 μmol/L for 72 h, cell viability was measured by MTT assay. After treatment with GW4064 of 0, 1 and 5 μmol/L for 24 h, the cell morphology was observed under phase contrast microscope,and the mRNA expression levels of FXR and stromal cell-derived factor 1(SDF-1) were detected by PCR. After treatment with GW4064 of 0 and 1 μmol/L for 24 h, the cell invasive ability was detected by cell scratch test. After treatment with GW4064 of 0, 1, 5 and 7 μmol/L for 24 h, the SDF-1 expression in the culture medium was detected by ELISA. Nude mouse tumorigenesis model was established by subcutaneous inoculation of HT-29 cells. After intragastric administration with GW4064 or DMSO for 16 d, the tumor growth and the mRNA expression of FXR and SDF-1 in the tumors were determined.Results GW4064 inhibited the growth of HT-29 cells in a dose-dependent manner, and there was signi?cant difference in the cell viability of HT-29 cells between the GW4064 groups(1, 3, 5, 7 and 10 μmol/L) and the control group(0 μmol/L, all P<0.05). After treatment with GW4064, phase contrast microscopy showed contracted and rounded colon cancer cells and slender cells transforming into epidermoid cells. The cell scratch test showed that the invasion ability of the colon cancer cells was signi?cantly reduced after treatment with GW4064 compared with the control group(0 μmol/L, P<0.05). PCR results showed that the mRNA expression level of FXR was increased in a dose-dependent manner after GW4064 treatment, while the expression of SDF-1 mRNA changed in the opposite way. ELISA results showed that the SDF-1 expression in the cell culture supernant was decreased with the increase of GW4064 concentrations, and there were signi?cant differences between the GW4064(1, 5 and 7 μmol/L) groups and the control group(0 μmol/L, P<0.05). GW4064 signi?cantly reduced tumor size compared with the control group(DMSO, P<0.05). In addition, the mRNA expression of FXR in the tumors was increased,and the mRNA expression of SDF-1 was decreased. Conclusion The activation of FXR can inhibit the invasive growth of colon cancer cells and the expression of SDF-1.
引文
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[14]MARGOLIN D A,MYERS T,ZHANG X,BERTONID M,REUTER B A,OBOKHARE I,et al.The critical roles of tumor-initiating cells and the lymph node stromal microenvironment in human colorectal cancer extranodal metastasis using a unique humanized orthotopic mouse model[J].FASEB J,2015,29:3571-3581.
[15]WANG B,WANG W,NIU W,LIU E,LIU X,WANG J,et al.SDF-1/CXCR4 axis promotes directional migration of colorectal cancer cells through upregulation of integrinαvβ6[J].Carcinogenesis,2014,35:282-291.
[16]MA J,SU H,YU B,GUO T,GONG Z,QI J,et al.CXCL12gene silencing down-regulates metastatic potential via blockage of MAPK/PI3K/AP-1 signaling pathway in colon cancer[J].Clin Transl Oncol,2018,20:1035-1045.
[17]MA J C,SUN X W,SU H,CHEN Q,GUO T K,LI Y,et al.Fibroblast-derived CXCL12/SDF-1αpromotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer[J].World J Gastroenterol,2017,23:5167-5178.
[18]STANISAVLJEVI?L,A?MUS J,STORLI K E,LEH S M,DAHL O,MYKLEBUST M P.CXCR4,CXCL12 and the relative CXCL12-CXCR4 expression as prognostic factors in colon cancer[J].Tumour Biol,2016,37:7441-7452.
[19]PENG Z,RAUFMAN J P,XIE G.Src-mediated crosstalk between farnesoid X and epidermal growth factor receptors inhibits human intestinal cell proliferation and tumorigenesis[J/OL].PLoS One,2012,7:e48461.doi:10.1371/journal.pone.0048461.
[20]MASSAFRA V,VAN MIL S W C.Farnesoid X receptor:a“homeostat”for hepatic nutrient metabolism[J].Biochim Biophys Acta,2018,1864:45-59.
[2]OCVIRK S,O’KEEFE S J.Influence of bile acids on colorectal cancer risk:potential mechanisms mediated by diet-gut microbiota interactions[J].Curr Nutr Rep,2017,6:315-322.
[3]DI CIAULA A,WANG D Q,MOLINA-MOLINAE,LUNARDIBACCETT O R,CALAMITA G,PALMIERI V O,et al.Bile acids and cancer:direct and environmental-dependent effects[J].Ann Hepatol,2017,16(Suppl 1:s3-105.):s87-s105.
[4]KOUTSOUNAS I,GIAGINIS C,THEACHARIS S.Farnesoid X receptor(FXR)from normal to malignant state[J].Histol Histopathol,2012,27:835-853.
[5]LALMAHOMED Z S,BR?KER M E,VAN HUIZEN NA,COEBERGH VAN DEN BRAAK R R,DEKKER L J,RIZOPOULOS D,et al.Hydroxylated collagen peptide in urine as biomarker for detecting colorectal liver metastases[J].Am J Cancer Res,2016,6:321-330.
[6]GADALETA R M,CARIELL O M,SABBàC,MOSCHETTA A.Tissue-specific actions of FXR in metabolism and cancer[J].Biochim Biophys Acta,2015,1851:30-39.
[7]MODICA S,MURZILLI S,SALVATORE L,SCHMIDTD R,MOSCHETTA A.Nuclear bile acid receptor FXRprotects against intestinal tumorigenesis[J].Cancer Res,2008,68:9589-9594.
[8]MARAN R R,THOMAS A,ROTH M,SHENG Z,ESTERLY N,PINSON D,et al.Farnesoid X receptor deficiency in mice leads to increased intestinal epithelial cell proliferation and tumor development[J].J Pharmacol Exp Ther,2009,328:469-477.
[9]DE GOTTARDI A,TOURI F,MAURER C A,PEREZA,MAURHOFER O,VENTRE G,et al.The bile acid nuclear receptor FXR and the bile acid binding protein IBABP are differently expressed in colon cancer[J].Dig Dis Sci,2004,49:982-989.
[10]LAX S,SCHAUER G,PREIN K,KAPITAN M,SILBERTD,BERGHOLD A,et al.Expression of the nuclear bile acid receptor/farnesoid X receptor is reduced in human colon carcinoma compared to nonneoplastic mucosa independent from site and may be associated with adverse prognosis[J].Int J Cancer,2012,130:2232-2239.
[11]张超峰,王坚.法尼酯X受体在胆管癌中的表达特点[J].外科理论与实践,2009,14:171-173.
[12]SMITH D L,KESHAVAN P,AVISSAR U,AHMED K,ZUCJER S D.Sodium taurocholate inhibits intestinal adenoma formation in APCMin/+mice,potentially through activation of the farnesoid X receptor[J].Carcinogenesis,2010,31:1100-1109.
[13]SHIN H N,MOON H H,KU J L.Stromal cell-derived factor-1αand macrophage migration-inhibitory factor induce metastatic behavior in CXCR4-expressing colon cancer cells[J].Int J Mol Med,2012,30:1537-1543.
[14]MARGOLIN D A,MYERS T,ZHANG X,BERTONID M,REUTER B A,OBOKHARE I,et al.The critical roles of tumor-initiating cells and the lymph node stromal microenvironment in human colorectal cancer extranodal metastasis using a unique humanized orthotopic mouse model[J].FASEB J,2015,29:3571-3581.
[15]WANG B,WANG W,NIU W,LIU E,LIU X,WANG J,et al.SDF-1/CXCR4 axis promotes directional migration of colorectal cancer cells through upregulation of integrinαvβ6[J].Carcinogenesis,2014,35:282-291.
[16]MA J,SU H,YU B,GUO T,GONG Z,QI J,et al.CXCL12gene silencing down-regulates metastatic potential via blockage of MAPK/PI3K/AP-1 signaling pathway in colon cancer[J].Clin Transl Oncol,2018,20:1035-1045.
[17]MA J C,SUN X W,SU H,CHEN Q,GUO T K,LI Y,et al.Fibroblast-derived CXCL12/SDF-1αpromotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer[J].World J Gastroenterol,2017,23:5167-5178.
[18]STANISAVLJEVI?L,A?MUS J,STORLI K E,LEH S M,DAHL O,MYKLEBUST M P.CXCR4,CXCL12 and the relative CXCL12-CXCR4 expression as prognostic factors in colon cancer[J].Tumour Biol,2016,37:7441-7452.
[19]PENG Z,RAUFMAN J P,XIE G.Src-mediated crosstalk between farnesoid X and epidermal growth factor receptors inhibits human intestinal cell proliferation and tumorigenesis[J/OL].PLoS One,2012,7:e48461.doi:10.1371/journal.pone.0048461.
[20]MASSAFRA V,VAN MIL S W C.Farnesoid X receptor:a“homeostat”for hepatic nutrient metabolism[J].Biochim Biophys Acta,2018,1864:45-59.