结直肠癌奥沙利铂耐药相关蛋白的筛选与鉴定
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摘要
目的:采用蛋白质组学技术筛选结直肠癌(colorectal cancer,CRC)中对化疗药物奥沙利铂(oxaliplatin,L-OHP)耐药的相关蛋白,以期为CRC的个体化治疗提供生物标志物。方法:建立L-OHP耐药细胞株HT-29/L-OHP,提取细胞总蛋白,采用二维凝胶电泳(two-dimensional gel electrophoresis,2-DE)和基质辅助激光解吸电离-飞行时间质谱(matrix assisted laser desorption-ionization time-of-flight tandem masss pectrometry,MALDI-TOF-MS)筛选并鉴定与亲本HT-29细胞差异表达的蛋白,并应用蛋白质印迹法对鉴定获得的部分蛋白进行验证。结果:建立CRC亲本细胞株HT-29和L-OHP耐药细胞株HT-29/L-OHP蛋白的2-DE图谱,共获得表达差异2倍以上的蛋白质点38个,经MALDI-TOF-MS分析,有37个得到鉴定,与亲本细胞相比,表达上调的有17个,表达下调的20个;蛋白质印迹法检测结果显示,多聚胞嘧啶结合蛋白1[poly(C)-binding protein-1,PCBP1]和TUBB2A(tubulin beta-2A)蛋白在HT-29/L-OHP细胞中表达上调,膜联蛋白A3(annexin A3,ANXA3)和磷酸化应激诱导蛋白(stress-induced-phosphoprotein 1,STIP1)表达下调,与MALDI-TOF-MS的实验结果相一致。结论:筛选获得37个与CRC对L-OHP耐药的相关蛋白,为进一步研究CRC对L-OHP耐药机制提供了有力的实验依据。
Objective: To screen and identify oxaliplatin-resistance-associated proteins in CRC (colorectal cancer) cell lines using proteomics technologies in order to find new biomarkers for individual therapy of CRC. Methods: Oxaliplatin-resistant human CRC cell line HT-29/L-OHP (oxaliplatin) was established. The total proteins in HT-29 and HT-29/L-OHP cells were extracted. The differentially expressed proteins between HT-29 and HT-29/L-OHP cells were screened and identified using 2-DE (two-dimensional gel electrophoresis) and MALDI-TOF-MS (matrix assisted laser desorption-ionization time-of-flight tandem mass spectrometry). Some proteins obtained were validated by Western blotting. Results: The 2-DE maps of total proteins in HT-29 and HT-29/L-OHP cells were established. Of the 38 protein spots identified as differentially expressed proteins (over two-fold, P < 0.05) between HT-29 and HT-29/L-OHP cells, 37 protein spots were positively identified by MALDI-TOF-MS (17 proteins were up-regulated and 20 proteins were down-regulated as compared with the parental HT-29 cells). The result of Western blotting showed that the PCBP1 [poly (C)-binding protein-1] and TUBB2A (tubulin beta 2A ) proteins were up-regulated while ANXA3 (annexin A3) and STIP1 (stress-induced-phosphoprotein 1) proteins were down-regulated in HT-29/L-OHP cells. The result of Western blotting was consistent with that of proteomics. Conclusion: There were 37 oxaliplatin-resistance-associated proteins in CRC identified in this study which may provide useful evidence in further research on mechanism of oxaliplatin-resistance in CRC.
Objective: To screen and identify oxaliplatin-resistance-associated proteins in CRC (colorectal cancer) cell lines using proteomics technologies in order to find new biomarkers for individual therapy of CRC. Methods: Oxaliplatin-resistant human CRC cell line HT-29/L-OHP (oxaliplatin) was established. The total proteins in HT-29 and HT-29/L-OHP cells were extracted. The differentially expressed proteins between HT-29 and HT-29/L-OHP cells were screened and identified using 2-DE (two-dimensional gel electrophoresis) and MALDI-TOF-MS (matrix assisted laser desorption-ionization time-of-flight tandem mass spectrometry). Some proteins obtained were validated by Western blotting. Results: The 2-DE maps of total proteins in HT-29 and HT-29/L-OHP cells were established. Of the 38 protein spots identified as differentially expressed proteins (over two-fold, P < 0.05) between HT-29 and HT-29/L-OHP cells, 37 protein spots were positively identified by MALDI-TOF-MS (17 proteins were up-regulated and 20 proteins were down-regulated as compared with the parental HT-29 cells). The result of Western blotting showed that the PCBP1 [poly (C)-binding protein-1] and TUBB2A (tubulin beta 2A ) proteins were up-regulated while ANXA3 (annexin A3) and STIP1 (stress-induced-phosphoprotein 1) proteins were down-regulated in HT-29/L-OHP cells. The result of Western blotting was consistent with that of proteomics. Conclusion: There were 37 oxaliplatin-resistance-associated proteins in CRC identified in this study which may provide useful evidence in further research on mechanism of oxaliplatin-resistance in CRC.
引文
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[8]ZHANG Z,HUANG L,ZHAO W,et al.Annexin1induced by anti-inflammatory drugs binds to NF-kappaB and inhibits its activation:anticancer effects in vitro and in vivo[J].Cancer Res,2010,70(6):2379-2388.
[9]TONG S W,YANG Y X,HU H D,et al.Proteomic investigation of5-fluorouracil resistance in a human hepatocellular carcinoma cell line[J].J Cell Biochem,2012,113(5):1671-1680.
[10]GUO W,ZHANG Y,CHEN T,et al.Efficacy of RNAi targeting of pyruvate kinase M2combined with cisplatin in a lung cancer model[J].J Cancer Res Clin Oncol,2011,137(1):65-72.
[11]WANG T H,CHAO A,TSAI C L,et al.Stress-induced phosphoprotein1as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation[J].Mol Cell Proteomics,2010,9(9):1873-1884.
[12]陆海,孙珏,许建华,等.人结肠癌奥沙利铂耐药细胞HCT116/L-OHP的建立及其耐药机制初探[J].肿瘤,2011,31(8):675-681.
[2]WU J,WANG F,GONG Y,et al.Proteomic analysis of changes induced by nonylphenol in Sprague-Dawley rat Sertoli cells[J].Chem Res Toxicol,2009,22(4):668-675.
[3]WAGGONER S A,JOHANNES G J,LIEBHABER S A.Depletion of the poly(C)-binding proteins alphaCP1and alphaCP2from K562cells leads to p53-independent induction of cyclin-dependent kinase inhibitor(CDKN1A)and G1arrest[J].J Biol Chem,2009,284(14):9039-9049.
[4]LEANDRO-GARCíA L J,LESKEL S,JARA C,et al.Regulatory polymorphisms inβ-tubulinⅡa are associated with paclitaxel-induced peripheral neuropathy[J].Clin Cancer Res,2012,18(16):4441-4448.
[5]YAO Y,JIA X Y,TIAN H Y,et al.Comparative proteomic analysis of colon cancer cells in response to oxaliplatin treatment[J].Biochim Biophys Acta,2009,1794(10):1433-1440.
[6]YANG Y X,SUN X F,CHENG A L,et al.Increased expression of HSP27linked to vincristine resistance in human gastric cancer cell line[J].J Cancer Res Clin Oncol,2009,135(2):181-189.
[7]ZHU F,WANG Y,ZENG S,et al.Involvement of Annexin A1in multidrug resistance of K562/ADR cells identified by the proteomic study[J].OMICS,2009,13(6):467-476.
[8]ZHANG Z,HUANG L,ZHAO W,et al.Annexin1induced by anti-inflammatory drugs binds to NF-kappaB and inhibits its activation:anticancer effects in vitro and in vivo[J].Cancer Res,2010,70(6):2379-2388.
[9]TONG S W,YANG Y X,HU H D,et al.Proteomic investigation of5-fluorouracil resistance in a human hepatocellular carcinoma cell line[J].J Cell Biochem,2012,113(5):1671-1680.
[10]GUO W,ZHANG Y,CHEN T,et al.Efficacy of RNAi targeting of pyruvate kinase M2combined with cisplatin in a lung cancer model[J].J Cancer Res Clin Oncol,2011,137(1):65-72.
[11]WANG T H,CHAO A,TSAI C L,et al.Stress-induced phosphoprotein1as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation[J].Mol Cell Proteomics,2010,9(9):1873-1884.
[12]陆海,孙珏,许建华,等.人结肠癌奥沙利铂耐药细胞HCT116/L-OHP的建立及其耐药机制初探[J].肿瘤,2011,31(8):675-681.