title: RNA interference (RNAi) of Ufd1 protein can sensitize a hydroxycamptothecin-resistant colon cancer cell line SW1116/HCPT to hydroxycamptothecin.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Gastroenterology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Digestive Disease, Shanghai, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.forename: Zhi Hua
authors.initials: ZH
authors.lastname: Ran
authors.forename: Jin Lu
authors.initials: JL
authors.lastname: Tong
authors.forename: Fang
authors.initials: F
authors.lastname: Nie
authors.forename: Ming Ming
authors.initials: MM
authors.lastname: Zhu
authors.forename: Xi Tao
authors.initials: XT
authors.lastname: Xu
authors.forename: Shu Dong
authors.initials: SD
authors.lastname: Xiao
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D000972:Antineoplastic Agents, Phytogenic / Q000627:therapeutic use*; D015153:Blotting, Western; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000627:therapeutic use; D053148:Caspase 3 / Q000032:analysis; D053471:Caspases, Initiator / Q000032:analysis; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D000972:Antineoplastic Agents, Phytogenic; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C491497:UFD1 protein, human; C527042:hydroxycamptothecinum; C093516:CASP4 protein, human; D053148:Caspase 3; D053471:Caspases, Initiator; D002166:Camptothecin