Department of general surgery, the affiliated hospital of Jinan central hospital, Shandong university, No105, Jiefang Road, District Lixia, Jinan, 250013, RP China.
J Exp Clin Cancer Res. 2012 Sep 11;31(1):73. doi: 10.1186/1756-9966-31-73.
To study the hypothesis that gemcitabine treatment augments the chemoresistance to gemcitabine by clusterin (sCLU) upregulation. Clusterin inhibition could augment the chemosensitivity of human pancreatic cancer cells by inhibition of clusterin-dependent pERK1/2 activation.
Clusterin was silenced by serial concentration of OGX-011 transfection in pancreatic cancer MIAPaCa-2 and BxPC-3 cell lines, then treated with serial concentration of gemcitabine. After the cells were treated with OGX-011 for 8 h, the cells were then treated with 5 μM ERK inhibitor PD98059 for 18 h or transfected with a wt-pERK-expressing plasmid into these cells for 24 h, after which the cells were treated with 1.0 uM gemcitabine for 24-72 h. Cell proliferation was determined by MTT. Apoptosis was quantified by flow cytometry,.sCLU and pERK1/2 production was analyzed by western blot, and sCLU mRNA was analyzed by RT-PCR. Xenograft of established tumors was used to evaluate primary tumor growth and apoptosis after treatment with gemcitabine alone or in combination with OGX-011. Phosphorylated ERK1/2 and sCLU levels in tumor tissues were measured by TUNEL analysis.
As detected by MTT and FACS assay, a combination of gemcitabine + OGX-011 reflected the chemotherapeutic sensitivity and increased the gemcitabine -induced apoptosis in MIAPaCa-2 and BxPC-3 cells. Western blotting and RT-PCR analysis revealed that the expression of clusterin was higher in gemcitabine -resistant MIAPaCa-2 cells, however, decreased significantly after pretreatment with OGX-011. Furthermore, the OGX-011 or combination of gemcitabine + OGX-011 decreased the gemcitabine -induced activation of pERK1/2. wt-pERK-re-expression decreased OGX-011+ gemcitabine -induced apoptosis. Finally, OGX-011 in combination with gemcitabine substantially decreased the in vivo tumor growth and promoted apoptosis. Taken together, clusterin confers gmcitabine resistance in pancreatic cancer cells.
Knockdown of clusterin by OGX-011 transfection sensitizes pancreatic cancer cells to gemcitabine by inhibition of gemcitabine -induced clusterin-pERK1/2 activation.
研究假设，即吉西他滨治疗通过上调聚集蛋白（sCLU）增强吉西他滨的化学耐药性。通过抑制聚集蛋白依赖性 pERK1/2 激活，聚集蛋白抑制可增强人胰腺癌细胞的化疗敏感性。
通过系列浓度的 OGX-011 转染在胰腺癌细胞 MIAPaCa-2 和 BxPC-3 系中沉默聚集蛋白，然后用系列浓度的吉西他滨处理。用 OGX-011 处理细胞 8 小时后，用 5 μM ERK 抑制剂 PD98059 处理细胞 18 小时，或用 wt-pERK 表达质粒转染这些细胞 24 小时，然后用 1.0 μM 吉西他滨处理 24-72 小时。用 MTT 测定细胞增殖。通过流式细胞术定量细胞凋亡，用 Western blot 分析 sCLU 和 pERK1/2 的产生，用 RT-PCR 分析 sCLU mRNA。用建立的肿瘤的异种移植评估吉西他滨单独或联合 OGX-011 治疗后的原发性肿瘤生长和凋亡。通过 TUNEL 分析测量肿瘤组织中磷酸化 ERK1/2 和 sCLU 水平。
通过 MTT 和 FACS 分析检测，吉西他滨+ OGX-011 的组合反映了化疗敏感性，并增加了 MIAPaCa-2 和 BxPC-3 细胞中吉西他滨诱导的细胞凋亡。Western blot 和 RT-PCR 分析显示，吉西他滨耐药的 MIAPaCa-2 细胞中聚集蛋白的表达更高，但用 OGX-011 预处理后显著降低。此外，OGX-011 或吉西他滨+ OGX-011 的组合降低了吉西他滨诱导的 pERK1/2 激活。wt-pERK 再表达降低了 OGX-011+吉西他滨诱导的细胞凋亡。最后，OGX-011 联合吉西他滨显著降低了体内肿瘤生长并促进了凋亡。总之，聚集蛋白赋予胰腺癌细胞对吉西他滨的耐药性。
通过 OGX-011 转染敲低聚集蛋白可通过抑制吉西他滨诱导的聚集蛋白-pERK1/2 激活使胰腺癌细胞对吉西他滨敏感。
