Department of Gynecology, Jiangsu Province Hospital, The first Affiliated Hospital with Nanjing Medical University, Nanjing, Jiangsu, China (mainland).
Department of Obstetrics and Gynecology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China (mainland).
Med Sci Monit. 2018 Apr 13;24:2197-2209. doi: 10.12659/msm.906413.
BACKGROUND GRP78, the 78-kDa glucose-regulated protein, occupies a significant position in endoplasmic reticulum stress. Emerging evidences have shown that GRP78 induces chemoresistance in several tumors; however, the role of GRP78 in cervical cancer (CVC) still needs to be elucidated clearly. MATERIAL AND METHODS In the present study, we determined the expression levels of GRP78 in CVC tissues collected from patients through immuocytochemistry, western blot and real-time PCR. To evaluate the exact role of GRP78 in CVC cells in the presence of cisplatin, we generated GRP78 knock-down cervical cancer cells through small interfering RNA. After successful transfection, the apoptosis rate was assessed with flow cytometry. The expression levels of caspase-3, CHOP and Bcl-2 in GRP78 knock-down cells were determined by western blot. RESULTS The GRP78 levels in CVC tissues were increased significantly. Three types of CVC cells HeLa, SiHa, and C33A were treated with different concentrations of cisplatin and cultured for 12 hours, 24 hours, and 48 hours respectively. And SiHa cells exhibited the highest resistance to cisplatin at all time. Specifically, after 25 μM cisplatin treatment, more than 80% of C33A cells underwent apoptosis, whereas the apoptotic rate of SiHa cells was only 30-40%. Data suggested that GRP78 silencing increased chemo-sensitivity and improved the effects of cisplatin-induced apoptosis in SiHa cells. Moreover, inhibition of GRP78 could upregulate caspase-3 and CHOP expression and downregulate Bcl-2 expression. CONCLUSIONS GRP78 may represent a key bio-marker of CVC and silencing GRP78 may strengthen the resistance against cisplatin. GRP78 may be a potential molecular target for CVC therapies in future.
背景
GRP78，即 78kDa 葡萄糖调节蛋白，在内质网应激中占有重要地位。越来越多的证据表明，GRP78 可诱导几种肿瘤产生化疗耐药性；然而，GRP78 在宫颈癌（CVC）中的作用仍需进一步阐明。
材料与方法
在本研究中，我们通过免疫细胞化学、western blot 和实时 PCR 测定了患者 CVC 组织中 GRP78 的表达水平。为了评估 GRP78 在顺铂存在下对 CVC 细胞的确切作用，我们通过小干扰 RNA 生成了 GRP78 敲低的宫颈癌细胞。转染成功后，用流式细胞术评估细胞凋亡率。western blot 检测 GRP78 敲低细胞中 caspase-3、CHOP 和 Bcl-2 的表达水平。
结果
CVC 组织中 GRP78 水平明显升高。用不同浓度的顺铂处理三种 CVC 细胞系 HeLa、SiHa 和 C33A，并分别培养 12 小时、24 小时和 48 小时。结果显示，所有时间点 SiHa 细胞对顺铂的耐药性最高。具体来说，在 25μM 顺铂处理后，超过 80%的 C33A 细胞发生凋亡，而 SiHa 细胞的凋亡率仅为 30-40%。数据表明，沉默 GRP78 可增强 C33A 细胞对顺铂的化疗敏感性，并改善顺铂诱导的凋亡作用。此外，抑制 GRP78 可上调 caspase-3 和 CHOP 的表达，下调 Bcl-2 的表达。
结论
GRP78 可能是 CVC 的关键生物标志物，沉默 GRP78 可能增强顺铂的耐药性。GRP78 可能成为未来 CVC 治疗的潜在分子靶点。
