OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.