SET蛋白诱导人乳腺癌紫杉醇耐药的机制研究
|
摘要
目的探讨patient SE translation(SET)蛋白诱导人乳腺癌细胞(MCF-7/S)紫杉醇耐药的机制。方法分别采用小干扰RNA(small interfering RNA,siRNA)技术降低乳腺癌紫杉醇耐药细胞株(MCF-7/PTX)中SET的表达,质粒转染技术升高人乳腺癌野生细胞株(MCF-7/S)中SET的表达,噻唑蓝、流式细胞术检测两株细胞对PTX的敏感性变化以及凋亡情况,Westernblot和Real-time PCR检测经典的肿瘤化疗耐药相关ATP结合盒(adenosine-triphosphate binding cassette,ABC)转运体的蛋白表达和mRNA水平。结果 MCF-7/PTX细胞基因敲除SET后,对紫杉醇的敏感性显著增加,紫杉醇诱导的细胞凋亡增加,ATP结合盒转运体的蛋白表达和mRNA水平显著降低;另一方面,MCF-7/S细胞高表达SET后,对紫杉醇的敏感性降低,紫杉醇诱导的细胞凋亡减少。并且,升高SET显著促进ATP结合盒转运体的表达,诱导耐药。结论发现SET通过调节ATP结合盒转运体的表达诱导乳腺癌紫杉醇耐药的形成,为探讨乳腺癌的紫杉醇耐药机制提供新的依据,SET有望成为临床上逆转乳腺癌化疗耐药的分子靶标。
OBJECTIVE To investigate the resistance mechanisms related to SET in paclitaxel-induced human breast cancer cells.METHODS The different expressions of SET and ABC transporters between MCF-7 / S and paclitaxel resistant MCF-7 / PTX cells were identified using Western blot. We adopted siRNA method to knockdown SET in MCF-7 / PTX cells and plasmid transfection analysis to upregulated SET in MCF-7 / S cells. The cell viability to paclitaxel was assessed by MTT assay. The cell apoptosis was analyzed by flow cytometry. The levels of ABC transporters were analyzed using Western blot and Real-time PCR,respectively. RESULTS We found that higher levels of SET and ABC transporters in MCF-7 / PTX cells. Knockdown of SET not only significantly sensitized MCF-7 / PTX cells to paclitaxel,but also induced cell apoptosis. The levels of the ABC transporters were also reduced. Upregulated SET in MCF-7 / S cells expressed resistant to paclitaxel and decreased cell apoptosis. High expression of the SET significantly promotes the mRNA and protein level of ABC transporters. CONCLUSION The above results demonstrate that SET is associated with paclitaxel resistance in MCF-7 / PTX cells. The SET is expected to be one of novel biological targets of overcoming paclitaxel resistant in breast cancer treatment.
OBJECTIVE To investigate the resistance mechanisms related to SET in paclitaxel-induced human breast cancer cells.METHODS The different expressions of SET and ABC transporters between MCF-7 / S and paclitaxel resistant MCF-7 / PTX cells were identified using Western blot. We adopted siRNA method to knockdown SET in MCF-7 / PTX cells and plasmid transfection analysis to upregulated SET in MCF-7 / S cells. The cell viability to paclitaxel was assessed by MTT assay. The cell apoptosis was analyzed by flow cytometry. The levels of ABC transporters were analyzed using Western blot and Real-time PCR,respectively. RESULTS We found that higher levels of SET and ABC transporters in MCF-7 / PTX cells. Knockdown of SET not only significantly sensitized MCF-7 / PTX cells to paclitaxel,but also induced cell apoptosis. The levels of the ABC transporters were also reduced. Upregulated SET in MCF-7 / S cells expressed resistant to paclitaxel and decreased cell apoptosis. High expression of the SET significantly promotes the mRNA and protein level of ABC transporters. CONCLUSION The above results demonstrate that SET is associated with paclitaxel resistance in MCF-7 / PTX cells. The SET is expected to be one of novel biological targets of overcoming paclitaxel resistant in breast cancer treatment.
引文
[1]QU C,ZHANG W,ZHENG G,et al.Metformin reverses multidrug resistance and epithelial-mesenchymal transition(EMT)via activating AMP-activated protein kinase(AMPK)in human breast cancer cells[J].Mol Cell Biochem,2014,386(1-2):63-71.
[2]CARRARA L,GUZZO F,ROQUE D M,et al.Differential in vitro sensitivity to patupilone versus paclitaxel in uterine and ovarian carcinosarcoma cell lines is linked to tubulin-beta-III expression[J].Gynecol Oncol,2012,125(1):231-236.
[3]YIN S,ZENG C,HARI M,et al.Random mutagenesis of betatubulin defines a set of dispersed mutations that confer paclitaxel resistance[J].Pharm Res,2012,29(11):2994-3006.
[4]ZHANG J,ZHAO J,ZHANG W,et al.Establishment of paclitaxel-resistant cell line and the underlying mechanism on drug resistance[J].Int J Gynecol Cancer,2012,22(9):1450-1456.
[5]BHATTACHARYA R,CABRAL F.Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance[J].J Biol Chem,2009,284(19):13023-13032.
[6]YU J W,LU Z H,GAO J,et al.Research progress in chemoresistance mechanism of paclitaxel and the future direction[J].Chin Pharm J(中国药学杂志),2013,48(14):1147-1150.
[7]FUKUDA Y,SCHUETZ J D.ABC Transporters and their role in nucleoside and nucleotide drug resistance[J].Biochem Pharmacol,2012,83(8):1073-1083.
[8]KOVALEV A A,TSVETAEVA D A,GRUDINSKAJA T V.Role of ABC-cassette transporters(MDR1,MRP1,BCRP)in the development of primary and acquired multiple drug resistance in patients with early and metastatic breast cancer[J].Exp Oncol,2013,35(4):287-290.
[9]CRISTOBAL I,BLANCO F J,GARCIA-ORTI L,et al.SETBP1Overexpression is a novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia[J].Blood,2010,115(3):615-625.
[10]AVET C,GARREL G,DENOYELLE C,et al.SET Protein interacts with intracellular domains of the gonadotropin-releasing hormone receptor and differentially regulates receptor signaling to c AMP and calcium in gonadotrope cells[J].J Biol Chem,2013,288(4):2641-2654.
[11]CHEN S,DONG Q,HU S,et al.Proteomic analysis of the proteins that are associated with the resistance to paclitaxel in human breast cancer cells[J].Mol Biosyst,2014,10(2):294-303.
[12]CHEN S Y,HU S S,DONG Q,et al.Establishment of paclitaxel-resistant breast cancer cell line and nude mice models,and underlying multidrug resistance mechanisms in vitro and in vivo[J].Asian Pac J Cancer Prev,2013,14(10):6135-6140.
[13]LU X,WU X L,LI Y,et al.Inhibitory effect of wogonin on gastric cancer SGC7901 cell line in vitro[J].Chin Pharm J(中国药学杂志),2011,46(7):512-517.
[14]LIU H,GU Y,YIN J,et al.SET-mediated NDRG1 inhibition is involved in acquisition of epithelial-to-mesenchymal transition phenotype and cisplatin resistance in human lung cancer cell[J].Cell Signal,2014,26(12):2710-2720.
[15]NAKANISHI T,ROSS D D.Breast cancer resistance protein(BCRP/ABCG2):Its role in multidrug resistance and regulation of its gene expression[J].Chin J Cancer,2012,31(2):73-99.
[16]CHAE S,KIM Y B,LEE J S,et al.Resistance to paclitaxel in hepatoma cells is related to static JNK activation and prohibition into entry of mitosis[J].Am J Physiol Gastrointest Liver Physiol,2012,302(9):1016-1024.
[17]LEE J P,HAHN H S,HWANG S J,et al.Selective cyclooxygenase inhibitors increase paclitaxel sensitivity in taxane-resistant ovarian cancer by suppressing P-glycoprotein expression[J].J Gynecol Oncol,2013,24(3):273-279.
[18]BANERJEE A.Increased levels of tyrosinated alpha-,beta(III)-,and beta(IV)-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells[J].Biochem Biophys Res Commun,2002,293(1):598-601.
[19]XU B Q,DAI X N,CUI Y G,et al.Expression of SET protein in the ovaries of patients with polycystic ovary syndrome[J].Int J Endocrinol,2013:367956.
[20]LEOPOLDINO A M,SQUARIZE C H,GARCIA C B,et al.SET Protein accumulates in HNSCC and contributes to cell survival:Antioxidant defense,Akt phosphorylation and AVOs acidification[J].Oral Oncol,2012,48(11):1106-1113.
[21]CERVONI N,DETICH N,SEO S B,et al.The oncoprotein Set/TAF-1beta,an inhibitor of histone acetyltransferase,inhibits active demethylation of DNA,integrating DNA methylation and transcriptional silencing[J].J Biol Chem,2002,277(28):25026-25031.
[22]ROOSWINKEL R W,VAN DE KOOIJ B,DE VRIES E,et al.Anti-apoptotic potency of Bcl-2 proteins primarily relies on their stability,not binding selectivity[J].Blood,2014,123(18):2806-2815.
[23]MAO Z,ZHOU J,LUAN J,et al.Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Aktsignaling pathway in ER-negative human gastric cancer cells[J].Biomed Pharmacother,2014,68(2):179-183.
[24]KAZI A A,GILANI R A,SCHECH A J,et al.Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer[J].Breast Cancer Res,2014,16(1):1-18.
[25]CHENG L,LUO S,JIN C,et al.FUT Family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway[J].Cell Death Dis,2013,4:e923.
[26]SWITZER C H,CHENG R Y,VITEK T M,et al.Targeting SET/I(2)PP2A oncoprotein functions as a multi-pathway strategy for cancer therapy[J].Oncogene,2011,30(22):2504-2513.
[2]CARRARA L,GUZZO F,ROQUE D M,et al.Differential in vitro sensitivity to patupilone versus paclitaxel in uterine and ovarian carcinosarcoma cell lines is linked to tubulin-beta-III expression[J].Gynecol Oncol,2012,125(1):231-236.
[3]YIN S,ZENG C,HARI M,et al.Random mutagenesis of betatubulin defines a set of dispersed mutations that confer paclitaxel resistance[J].Pharm Res,2012,29(11):2994-3006.
[4]ZHANG J,ZHAO J,ZHANG W,et al.Establishment of paclitaxel-resistant cell line and the underlying mechanism on drug resistance[J].Int J Gynecol Cancer,2012,22(9):1450-1456.
[5]BHATTACHARYA R,CABRAL F.Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance[J].J Biol Chem,2009,284(19):13023-13032.
[6]YU J W,LU Z H,GAO J,et al.Research progress in chemoresistance mechanism of paclitaxel and the future direction[J].Chin Pharm J(中国药学杂志),2013,48(14):1147-1150.
[7]FUKUDA Y,SCHUETZ J D.ABC Transporters and their role in nucleoside and nucleotide drug resistance[J].Biochem Pharmacol,2012,83(8):1073-1083.
[8]KOVALEV A A,TSVETAEVA D A,GRUDINSKAJA T V.Role of ABC-cassette transporters(MDR1,MRP1,BCRP)in the development of primary and acquired multiple drug resistance in patients with early and metastatic breast cancer[J].Exp Oncol,2013,35(4):287-290.
[9]CRISTOBAL I,BLANCO F J,GARCIA-ORTI L,et al.SETBP1Overexpression is a novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia[J].Blood,2010,115(3):615-625.
[10]AVET C,GARREL G,DENOYELLE C,et al.SET Protein interacts with intracellular domains of the gonadotropin-releasing hormone receptor and differentially regulates receptor signaling to c AMP and calcium in gonadotrope cells[J].J Biol Chem,2013,288(4):2641-2654.
[11]CHEN S,DONG Q,HU S,et al.Proteomic analysis of the proteins that are associated with the resistance to paclitaxel in human breast cancer cells[J].Mol Biosyst,2014,10(2):294-303.
[12]CHEN S Y,HU S S,DONG Q,et al.Establishment of paclitaxel-resistant breast cancer cell line and nude mice models,and underlying multidrug resistance mechanisms in vitro and in vivo[J].Asian Pac J Cancer Prev,2013,14(10):6135-6140.
[13]LU X,WU X L,LI Y,et al.Inhibitory effect of wogonin on gastric cancer SGC7901 cell line in vitro[J].Chin Pharm J(中国药学杂志),2011,46(7):512-517.
[14]LIU H,GU Y,YIN J,et al.SET-mediated NDRG1 inhibition is involved in acquisition of epithelial-to-mesenchymal transition phenotype and cisplatin resistance in human lung cancer cell[J].Cell Signal,2014,26(12):2710-2720.
[15]NAKANISHI T,ROSS D D.Breast cancer resistance protein(BCRP/ABCG2):Its role in multidrug resistance and regulation of its gene expression[J].Chin J Cancer,2012,31(2):73-99.
[16]CHAE S,KIM Y B,LEE J S,et al.Resistance to paclitaxel in hepatoma cells is related to static JNK activation and prohibition into entry of mitosis[J].Am J Physiol Gastrointest Liver Physiol,2012,302(9):1016-1024.
[17]LEE J P,HAHN H S,HWANG S J,et al.Selective cyclooxygenase inhibitors increase paclitaxel sensitivity in taxane-resistant ovarian cancer by suppressing P-glycoprotein expression[J].J Gynecol Oncol,2013,24(3):273-279.
[18]BANERJEE A.Increased levels of tyrosinated alpha-,beta(III)-,and beta(IV)-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells[J].Biochem Biophys Res Commun,2002,293(1):598-601.
[19]XU B Q,DAI X N,CUI Y G,et al.Expression of SET protein in the ovaries of patients with polycystic ovary syndrome[J].Int J Endocrinol,2013:367956.
[20]LEOPOLDINO A M,SQUARIZE C H,GARCIA C B,et al.SET Protein accumulates in HNSCC and contributes to cell survival:Antioxidant defense,Akt phosphorylation and AVOs acidification[J].Oral Oncol,2012,48(11):1106-1113.
[21]CERVONI N,DETICH N,SEO S B,et al.The oncoprotein Set/TAF-1beta,an inhibitor of histone acetyltransferase,inhibits active demethylation of DNA,integrating DNA methylation and transcriptional silencing[J].J Biol Chem,2002,277(28):25026-25031.
[22]ROOSWINKEL R W,VAN DE KOOIJ B,DE VRIES E,et al.Anti-apoptotic potency of Bcl-2 proteins primarily relies on their stability,not binding selectivity[J].Blood,2014,123(18):2806-2815.
[23]MAO Z,ZHOU J,LUAN J,et al.Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Aktsignaling pathway in ER-negative human gastric cancer cells[J].Biomed Pharmacother,2014,68(2):179-183.
[24]KAZI A A,GILANI R A,SCHECH A J,et al.Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer[J].Breast Cancer Res,2014,16(1):1-18.
[25]CHENG L,LUO S,JIN C,et al.FUT Family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway[J].Cell Death Dis,2013,4:e923.
[26]SWITZER C H,CHENG R Y,VITEK T M,et al.Targeting SET/I(2)PP2A oncoprotein functions as a multi-pathway strategy for cancer therapy[J].Oncogene,2011,30(22):2504-2513.