Department of Gastroenterology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Digestive Disease, Shanghai, China.
J Dig Dis. 2011 Apr;12(2):110-6. doi: 10.1111/j.1751-2980.2011.00478.x.
To investigate whether RNA interference (RNAi) of the ubiquitin fusion-degradation 1-like protein (Ufd1) could sensitize hydroxycamptothecin (HCPT)-resistant colon cancer cell line SW1116/HCPT to the cytotoxic effect of HCPT.
SW1116/HCPT cells were transfected with plasmids containing Ufd1-specific small interfering RNA (siRNA) (Ufd1 knockdown cells) and non-specific siRNA (control cells). A drug sensitivity analysis, 3-(4,5)-dimethylthiahiazol (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed on Ufd1 knockdown cells and control cells. After treating the cells with HCPT, a caspase-3 and caspase-4 activity assay, flow cytometric analysis and Western blot for detecting phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated protein kinases B (p-Akt), P53, ubiquitin, GADD 153 and Grp78/Bip were performed.
According to the MTT assay, the survival rate of knockdown cells was significantly lower than that of the control cells (P < 0.01). Both caspase-3 and caspase-4 activity assay showed higher activation level in Ufd1 knockdown cells than that in the control cells (P < 0.01). A flow cytometric analysis revealed more severe S-phase arrest in the Ufd1 knockdown cells than that in the control cells (P < 0.05). The Western blot showed that increasing the concentration of HCPT resulted in a higher expression level of p-JNK, P53, ubiquitin, GADD 153 and Grp78/Bip in the Ufd1 knockdown cells than that in the control cells.
Ufd1 plays a key role in HCPT resistance of SW1116/HCPT and RNAi of Ufd1 can sensitize SW1116/HCPT to the cytotoxic effect of HCPT via strengthening the activation of caspase-3 pathway and disturbing endoplasmic reticulum functions.
研究泛素融合降解蛋白 1 样蛋白（Ufd1）的 RNA 干扰（RNAi）是否能使耐羟喜树碱（HCPT）的结肠癌细胞系 SW1116/HCPT 对 HCPT 的细胞毒性作用更敏感。
用含有 Ufd1 特异性小干扰 RNA（siRNA）（Ufd1 敲低细胞）和非特异性 siRNA（对照细胞）的质粒转染 SW1116/HCPT 细胞。对 Ufd1 敲低细胞和对照细胞进行药物敏感性分析、3-(4,5)-二甲基噻唑 (-z-y1)-3,5-二苯基四唑溴盐（MTT）检测。用 HCPT 处理细胞后，进行 caspase-3 和 caspase-4 活性检测、流式细胞术分析和检测磷酸化 c-Jun N 末端激酶（p-JNK）、磷酸化蛋白激酶 B（p-Akt）、P53、泛素、GADD 153 和 Grp78/Bip 的 Western blot。
根据 MTT 检测，敲低细胞的存活率明显低于对照细胞（P < 0.01）。Ufd1 敲低细胞中的 caspase-3 和 caspase-4 活性检测显示其激活水平均高于对照细胞（P < 0.01）。流式细胞术分析显示 Ufd1 敲低细胞的 S 期阻滞更为严重，与对照细胞相比差异有统计学意义（P < 0.05）。Western blot 显示，随着 HCPT 浓度的增加，Ufd1 敲低细胞中 p-JNK、P53、泛素、GADD 153 和 Grp78/Bip 的表达水平均高于对照细胞。
Ufd1 在 SW1116/HCPT 的 HCPT 耐药中起关键作用，通过增强 caspase-3 途径的激活和干扰内质网功能，Ufd1 的 RNAi 可使 SW1116/HCPT 对 HCPT 的细胞毒性作用更敏感。
