Department of Biochemistry and Molecular Biology, Graduate Institute of Biomedical Sciences, Chang Gung University, 259 Wen-Hua, 1st Road, Gueishan, Taoyuan 333, Taiwan, ROC.
Biochem Pharmacol. 2010 Sep 15;80(6):827-37. doi: 10.1016/j.bcp.2010.05.026.
Cisplatin is a widely used anti-cancer drug which targets DNA in replicating cells. In the present study, we found that NAPA--a protein found in the endoplasmic reticulum (ER) and implicated in protein trafficking--protects cells against cisplatin. Accordingly, knockdown of NAPA using lentivirus-encoded shRNA (shNAPA) induced ER stress similar to cisplatin treatment in HEK293 cells. A low dose of cisplatin also elicited a mild ER stress response associated with the accumulation of the protective proteins BiP and NAPA. Remarkably, knockdown of NAPA induced apoptosis and enhanced cisplatin-induced cytotoxicity/apoptosis, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of NAPA increased resistance to cisplatin by reducing cisplatin-induced ER stress and apoptosis. The modulatory effects of shNAPA required the tumor suppressor p53 since the effects of NAPA knockdown were reduced by the p53 inhibitor PFT-alpha and in H1299 cells which are p53-null. A partial reversal of cisplatin resistance was also observed in cisplatin-resistant HeLa cells following knockdown of NAPA. Our results also indicated that calpain is required for ER-mediated apoptosis. Importantly, combined cisplatin/shNAPA treatment suppressed tumor growth in vivo in xenograph experiments performed in nude mice. Taken together, these observations suggest that NAPA represents a target of cisplatin, and that knockdown of NAPA may improve cisplatin-based cancer therapy.
顺铂是一种广泛应用于临床的抗癌药物，其主要作用靶点是处于有丝分裂期的肿瘤细胞 DNA。本研究发现内质网（ER）中的 NAPA 蛋白在蛋白质运输中起作用，它可以保护细胞免受顺铂的损伤。因此，使用慢病毒编码 shRNA（shNAPA）敲低 NAPA 会在 HEK293 细胞中诱导类似于顺铂处理的内质网应激。低剂量的顺铂也会引起轻度的内质网应激反应，伴随着保护性蛋白 BiP 和 NAPA 的积累。值得注意的是，敲低 NAPA 会诱导细胞凋亡，并增强顺铂诱导的细胞毒性/凋亡，从而使癌细胞对顺铂更加敏感。另一方面，NAPA 的过表达通过减少顺铂诱导的内质网应激和细胞凋亡来增加对顺铂的耐药性。shNAPA 的调节作用需要肿瘤抑制因子 p53，因为 p53 抑制剂 PFT-α和 p53 缺失的 H1299 细胞均可降低 NAPA 敲低的作用。在顺铂耐药的 HeLa 细胞中，敲低 NAPA 也部分逆转了顺铂耐药性。我们的结果还表明钙蛋白酶是内质网介导的细胞凋亡所必需的。重要的是，在裸鼠异种移植实验中，顺铂/ shNAPA 联合治疗可抑制肿瘤生长。综上所述，这些结果表明 NAPA 是顺铂的作用靶点，敲低 NAPA 可能会改善基于顺铂的癌症治疗。
