title: HuR mediated post-transcriptional regulation as a new potential adjuvant therapeutic target in chemotherapy for pancreatic cancer.
abstract: OBJECTIVE
  To investigate the expression of HuR in pancreatic ductal adenocarcinoma (PDA) and to assess the effects of HuR silencing on the expression of cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) and the in vitro response to gemcitabine (GEM) treatment in pancreatic cell lines.
  
  
  METHODS
  We compared the expression of HuR, COX-2, and HO-1 in PDA and normal pancreatic tissue using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. In addition, the HuR, COX-2 and HO-1 were analyzed in four types of cancer cell lines (MiaPaca2, Su.86.86, Capan-1, and Capan-2) with and without GEM treatment. Immunocytofluorescence analysis was used to investigate HuR localization in cells. Cell viability and response to GEM after HuR silencing were determined with the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide test and the crystal violet clonogenic assay, respectively. To measure apoptosis, activation of caspases 3/7 was evaluated using immunofluorescence.
  
  
  RESULTS
  In PDA tissue obtained from patients not treated with GEM, HuR mRNA expression was 3.2 times lower (P < 0.05) and COX-2 and HO-1 mRNA expression was 2.3-fold and 7.2-fold higher (P < 0.05), respectively, than normal pancreatic tissue (from organ donor). qRT-PCR analysis showed that HuR, COX-2, and HO-1 mRNA were overexpressed in all cancer cell lines treated with the half maximal inhibitory concentration (IC50) dose of GEM compared with control cells (P < 0.05). Western blot analysis revealed that COX-2 and HO-1 levels were significantly decreased in cancer cells after HuR silencing. Furthermore, HuR silencing increased the response to GEM treatment and decreased cell viability by 11.6%-53.7% compared to control cell lines. Caspases 3 and 7 were activated after HuR silencing and GEM treatment in all pancreatic cancer cell lines. In comparison, treatment with GEM alone did not activate caspases 3 and 7 in the same cell lines.
  
  
  CONCLUSIONS
  HuR mediated post-transcriptional upregulation of COX-2 and HO-1 expression after GEM treatment in pancreatic cancer cells. HuR silencing significantly increased the effectiveness of GEM treatment in vitro.
authors.affiliation: Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas, Institute for Digestive System Research, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania.
authors.forename: Aldona
authors.initials: A
authors.lastname: Jakstaite
authors.affiliation: Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas, Institute for Digestive System Research, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania.
authors.forename: Aurelija
authors.initials: A
authors.lastname: Maziukiene
authors.affiliation: Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas, Institute for Digestive System Research, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania.
authors.forename: Giedre
authors.initials: G
authors.lastname: Silkuniene
authors.affiliation: Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas, Institute for Digestive System Research, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kmieliute
authors.affiliation: Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas, Institute for Digestive System Research, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania.
authors.forename: Antanas
authors.initials: A
authors.lastname: Gulbinas
authors.affiliation: Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas, Institute for Digestive System Research, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania.
authors.forename: Zilvinas
authors.initials: Z
authors.lastname: Dambrauskas
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003131:Combined Modality Therapy; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D000067780:ELAV-Like Protein 1 / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D014162:Transfection; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D003841:Deoxycytidine; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D000093542:Gemcitabine
keywords: Chemotherapy; Cyclooxygenase-2; Heme oxygenase-1; HuR; Pancreatic cancer; Post-transcriptional regulation