Guan Haitao, Zhao Ping, Dai Zhijun, Liu Xiaoxu, Wang Xijing
Department of Oncology, The Second Affiliated Hospital of Xi'an Jiaotong University, 157 West NO. 5 Road, Xi'an, Shaanxi Province, 710004, China.
Department of Gastroenterology, The Second Affiliated Hospital of Xi'an Jiaotong University, 157 West NO.5 Road, Xi'an, Shaanxi Province, 710004, China.
Tumour Biol. 2016 Sep;37(9):12153-12160. doi: 10.1007/s13277-016-5092-0. Epub 2016 May 24.
Multidrug resistance is one of the major reasons colorectal cancer (CRC) chemotherapy-based treatments fail, and novel biologically based therapies are urgently needed. Src homology 3 (SH3)-domain GRB2-like protein 1 (SH3GL1) is a membrane-bound protein which was found to be involved in tumor formation, progression, and metastasis. In this study, immunohistochemistry staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis revealed a high expression of SH3GL1 in human CRC tumor specimens and several CRC cells resistant to chemotherapeutics. Cell Counting Kit-8 (CCK-8) assay showed that transfection of pCDNA3.1(+)-SH3GL1 increased while transfection of SH3GL1 siRNA decreased cell viability in response to 5-fluorouracil (5-FU) treatment (P < 0.05). Further studies indicated that transfection of SH3GL1 siRNA significantly downregulated multidrug resistance protein 1 (MDR1)/P-glycoprotein expression (P < 0.05), decreased MDR1 promoter activity and activator protein-1 (AP-1) binding activity (P < 0.05), and inhibited the activation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling (P < 0.05) in CRC cells resistant to chemotherapeutics. Transfection of pCDNA3.1(+)-SH3GL1 caused the opposite effect. Additionally, pre-treatment with either EGFR kinase inhibitor PD153035 or ERK1/2 kinase inhibitor PD98059 in HCT116/5-FU cells partly inhibits P-glycoprotein expression and AP-1 binding activity (P < 0.05). In conclusion, we confirmed that inhibition of SH3GL1 reverses multidrug resistance through declining P-glycoprotein expression via the EGFR/ERK/AP-1 pathway.
多药耐药是基于化疗的结直肠癌（CRC）治疗失败的主要原因之一，因此迫切需要新型生物疗法。Src同源3（SH3）结构域类GRB2蛋白1（SH3GL1）是一种膜结合蛋白，已发现其与肿瘤形成、进展和转移有关。在本研究中，免疫组织化学染色、定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹分析显示，SH3GL1在人CRC肿瘤标本和几种对化疗药物耐药的CRC细胞中高表达。细胞计数试剂盒-8（CCK-8）检测表明，转染pCDNA3.1(+)-SH3GL1可提高细胞活力，而转染SH3GL1 siRNA则可降低5-氟尿嘧啶（5-FU）处理后的细胞活力（P < 0.05）。进一步研究表明，转染SH3GL1 siRNA可显著下调多药耐药蛋白1（MDR1）/P-糖蛋白的表达（P < 0.05），降低MDR1启动子活性和激活蛋白-1（AP-1）结合活性（P < 0.05），并抑制化疗耐药CRC细胞中表皮生长因子受体（EGFR）和细胞外信号调节激酶1/2（ERK1/2）信号通路的激活（P < 0.05）。转染pCDNA3.1(+)-SH3GL1则产生相反的效果。此外，在HCT116/5-FU细胞中用EGFR激酶抑制剂PD153035或ERK1/2激酶抑制剂PD98059预处理可部分抑制P-糖蛋白表达和AP-1结合活性（P < 0.05）。总之，我们证实抑制SH3GL1可通过EGFR/ERK/AP-1途径降低P-糖蛋白表达来逆转多药耐药。
