ABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Camptothecin/analogs & derivatives , Colonic Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Proteins/metabolism , RNA Interference/physiology , Adaptor Proteins, Vesicular Transport , Blotting, Western , Camptothecin/therapeutic use , Caspase 3/analysis , Caspases, Initiator/analysis , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Humans , Intracellular Signaling Peptides and Proteins , Proteins/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: Apoptosis is frequently found in oral lichen planus (OLP) lesions, but the pathways leading to apoptosis are unknown. STUDY DESIGN: This study focused on analysis of caspase expression which is essential for apoptosis. Expression of caspases 2, 3, 8, 9, and 12 was studied in 70 biopsy samples from atrophic OLP to identify which cascade pathway, extrinsic or intrinsic, is of importance in apoptosis in OLP. RESULTS: Caspase-2 expression was present in every sample, and >70% of the epithelial cells were positive in 33% of the lesions. More than 70% of the epithelial cells expressed caspase-12 in 84% of the specimens. Caspase-8 expression was shown totally in 87% of the specimens. No caspase-3 expression was found in 57% of the samples, and caspase-9 expression was absent in the entire OLP specimen. CONCLUSIONS: The high frequency of intrinsic apoptotic pathway markers caspases 2 and 12 indicates intracellular stress in atrophic OLP epithelial cells.
