He Qing-Chun, Zhang Gui-Ying, Cao Wei-Jun
Department of Digestive Diseases, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, PR China.
Ai Zheng. 2008 Apr;27(4):337-42.
BACKGROUND & OBJECTIVE: Vincristine (VCR)-resistant gastric cancer cell line SGC7901/VCR is a typical multidrug resistant (MDR) cell line with high expression of P-glycoprotein (P-gp). However, verapamil (VRP), the inhibitor of P-gp, can not totally reverse the drug resistance, indicating that additional mechanisms must contribute to the MDR phenotype. Our previous study showed that sorcin, a calcium-binding protein, is significantly up-regulated in SGC7901/VCR cells. This study was to explore the role of sorcin in the development of MDR in human gastric cancer cell line SGC7901.
The full length sorcin cDNA was isolated by reverse transcription-polymerase chain reaction (RT-PCR). The FLAG-sorsin-pcDNA3.1 plasmid was constructed and transfected into SGC7901 cells. The mRNA and protein levels of sorcin in stable clones were detected by RT-PCR and Western blot. The sensitivity of SGC7901 cells to chemotherapeutic drugs was detected using MTT assay. Then sorcin-transfected SGC7901 cells (SGC-F-Sor) were transfected with sorcin antisense oligonucleotides (ASO). The VCR-sensitivity of SGC7901 cells was determined by MTT assay.
The full-length sorcin cDNA (616 bp) was amplified by RT-PCR. The FLAG-sorsin-pcDNA3.1 plasmid was constructed successfully. The mRNA and protein levels of sorcin were up-regulated in SGC-F-Sor cells. Overexpression of sorcin produced 8.87 folds of VCR-resistance, 6.13 folds of adriamycin (ADM)-resistance, 6.67 folds of taxol-resistance, and 2.80 folds of 5-fluorouracil (5-FU)-resistance. However, when SGC-F-Sor cells were transfected with sorcin ASO, down-regulation of sorcin expression and increased sensitivity to VCR were observed.
Overexpression of sorcin could induce low level of MDR in SGC7901 cells, indicating that sorcin is associated with MDR of SGC7901 cells. Sorcin maybe be a target of MDR reversal in gastric cancer cells.
长春新碱(VCR)耐药胃癌细胞系SGC7901/VCR是一种典型的多药耐药(MDR)细胞系,P-糖蛋白(P-gp)高表达。然而,P-gp抑制剂维拉帕米(VRP)并不能完全逆转耐药性,这表明还有其他机制参与了MDR表型的形成。我们之前的研究表明,钙结合蛋白索菌素(sorcin)在SGC7901/VCR细胞中显著上调。本研究旨在探讨索菌素在人胃癌细胞系SGC7901多药耐药发生中的作用。
通过逆转录聚合酶链反应(RT-PCR)分离索菌素全长cDNA。构建FLAG-索菌素-pcDNA3.1质粒并转染至SGC7901细胞。采用RT-PCR和蛋白质印迹法检测稳定克隆中索菌素的mRNA和蛋白水平。采用MTT法检测SGC7901细胞对化疗药物的敏感性。然后用索菌素反义寡核苷酸(ASO)转染索菌素转染的SGC7901细胞(SGC-F-Sor)。采用MTT法测定SGC7901细胞对VCR的敏感性。
通过RT-PCR扩增出全长616 bp的索菌素cDNA。成功构建了FLAG-索菌素-pcDNA3.1质粒。SGC-F-Sor细胞中索菌素的mRNA和蛋白水平上调。索菌素过表达产生了8.87倍的VCR耐药、6.13倍的阿霉素(ADM)耐药、6.67倍的紫杉醇耐药和及2.80倍的5-氟尿嘧啶(5-FU)耐药。然而,当用索菌素ASO转染SGC-F-Sor细胞时,观察到索菌素表达下调且对VCR的敏感性增加。
索菌素过表达可诱导SGC7901细胞产生低水平的多药耐药,表明索菌素与SGC7901细胞的多药耐药有关。索菌素可能是胃癌细胞多药耐药逆转的靶点。