title: Knockdown of clusterin sensitizes pancreatic cancer cells to gemcitabine chemotherapy by ERK1/2 inactivation.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Knockdown of clusterin by OGX-011 transfection sensitizes pancreatic cancer cells to gemcitabine by inhibition of gemcitabine -induced clusterin-pERK1/2 activation.
authors.affiliation: Department of general surgery, the affiliated hospital of Jinan central hospital, Shandong university, No105, Jiefang Road, District Lixia, Jinan, 250013, RP China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tang
authors.forename: Fenghua
authors.initials: F
authors.lastname: Liu
authors.forename: Chunning
authors.initials: C
authors.lastname: Zheng
authors.forename: Shaochuan
authors.initials: S
authors.lastname: Sun
authors.forename: Yingsheng
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D051152:Clusterin / Q000037:antagonists & inhibitors / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D000093542:Gemcitabine
chemical_list: D051152:Clusterin; D005419:Flavonoids; C503781:OGX-011; D013873:Thionucleotides; D003841:Deoxycytidine; D048052:Mitogen-Activated Protein Kinase 3; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000093542:Gemcitabine