Wang Guowen, Rong Jiesheng, Zhou Zhihong, Duo Jian
Department of Orthopedic Oncology, Tianjin Cancer Institute, Hospital, Tianjin Medical University, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Huanhuxi Road, Hexi District, Tianjin, 300060 China.
Mol Biol (Mosk). 2010 Nov-Dec;44(6):1010-7.
As previously reported, a novel gene P28GANK conferred a multidrug resistant phenotype in gastric cancer cells. The aim of this study was to explore the role of P28GANK in the development of multidrug resistance (MDR) in osteosarcoma cells. P28GANK gene was found to be overexpressed at the mRNA level and the protein level in a cisplatin induced MDR osteosarcoma cell line Saos-2/CDDP compared to its parent cell line Saos-2. Here, we transfected the osteosarcoma cell line Saos-2 with eukaryotic expression vector of P28GANK. In vitro drug sensitivity assay suggested that Saos-2-P28GANK cells conferred resistance to both P-glycoprotein (P-gp)-related and P-gp-nonrelated drugs. Blocking P28GANK expression in MDR osteosarcoma cells Saos-2/CDDP by P28GANK-specific small interfering RNA (siRNA) increased the cell sensitivity to various chemotherapeutic drugs. Flow cytometry examination suggested that P28GANK gene expression could suppress Adriamycin-induced apoptosis accompanied by decreased accumulation and increased release of Adriamycin. Semiquantitative RT-PCR, Western blot and Luciferase reporter assay suggested that P28GANK gene could significantly up-regulate the expression of MDR-1 and Bcl-2, transcription of the MDR-1 gene and down-regulate the expression of Bax. In addition, inhibition of P28GANK expression by RNA interference or P-gp inhibition could partially reverse P28GANK-mediated MDR. Taken together, our findings suggest that down-regulation of P28GANK gene expression could sensitize osteosarcoma cells to chemotherapeutic drugs by down-regulation of the MDR-1 and Bcl-2 and up-regulation of Bax gene expression, without altering the glutathione S-transferase activity, or intracellular glutathione content in osteosarcoma cells. Further study on biological function of P28GANK may be helpful for understanding MDR mechanism of osteosarcoma and developing a strategy for osteosarcoma treatment.
如先前报道，一种新基因P28GANK在胃癌细胞中赋予了多药耐药表型。本研究的目的是探讨P28GANK在骨肉瘤细胞多药耐药（MDR）发生发展中的作用。与亲本细胞系Saos-2相比，在顺铂诱导的MDR骨肉瘤细胞系Saos-2/CDDP中发现P28GANK基因在mRNA水平和蛋白水平均过表达。在此，我们用P28GANK的真核表达载体转染骨肉瘤细胞系Saos-2。体外药敏试验表明，Saos-2-P28GANK细胞对P-糖蛋白（P-gp）相关和P-gp非相关药物均具有耐药性。用P28GANK特异性小干扰RNA（siRNA）阻断MDR骨肉瘤细胞Saos-2/CDDP中P28GANK的表达可增加细胞对各种化疗药物的敏感性。流式细胞术检测表明，P28GANK基因表达可抑制阿霉素诱导的细胞凋亡，同时伴随阿霉素积累减少和释放增加。半定量RT-PCR、蛋白质印迹法和荧光素酶报告基因检测表明，P28GANK基因可显著上调MDR-1和Bcl-2的表达、MDR-1基因的转录，并下调Bax的表达。此外，RNA干扰或P-gp抑制对P28GANK表达的抑制可部分逆转P28GANK介导的MDR。综上所述，我们的研究结果表明，下调P28GANK基因表达可通过下调MDR-1和Bcl-2以及上调Bax基因表达使骨肉瘤细胞对化疗药物敏感，而不改变骨肉瘤细胞中的谷胱甘肽S-转移酶活性或细胞内谷胱甘肽含量。进一步研究P28GANK的生物学功能可能有助于理解骨肉瘤的MDR机制并制定骨肉瘤治疗策略。
