title: Synergistic cytotoxicity, inhibition of signal transduction pathways and pharmacogenetics of sorafenib and gemcitabine in human NSCLC cell lines.
abstract: BACKGROUND
  Lung cancer is one of the most lethal tumors and, although standard chemotherapy produces clinical response, there has been little improvement in prognosis. Therefore, research effort has focused on target-specific agents, such as sorafenib, which blocks both the RAF/MEK/ERK signalling pathways and receptors involved in neovascularization and tumor progression, including VEGFR-2 and c-Kit. We investigated whether sorafenib would be synergistic with gemcitabine against NSCLC cell lines.
  
  
  METHODS
  Human lung cancer cells A549, CALU-1, CALU-6, H23 and HCC 827 were treated with sorafenib and gemcitabine, alone or in combination, and the cytotoxicity was assessed with CellTiter 96 Non-radioactive cell proliferation kit. Cell cycle and apoptosis were investigated with flow cytometry and fluorescence microscopy, respectively. Moreover, the effects of drugs on Akt (S473), c-Kit (Y823) and ERK (pTpY185/187) phosphorylation were studied with ELISA. Finally, quantitative PCR analysis was performed to assess whether sorafenib and gemcitabine modulated the expression of genes related to drug activity.
  
  
  RESULTS
  Gemcitabine and sorafenib synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index. Sorafenib reduced c-Kit and ERK activation and gemcitabine inhibited Akt phosphorylation. Moreover quantitative PCR showed that sorafenib modulated the expression of targets related to gemcitabine activity, while gemcitabine induced the expression of RKIP.
  
  
  CONCLUSIONS
  These data demonstrate that sorafenib and gemcitabine synergistically interact against NSCLC cells, through suppression of Akt, c-Kit and ERK phosphorylation, induction of apoptosis and modulation of dCK, RRM1, RRM2 and RKIP gene expression. The association between traditional cytotoxic agents with new target-specific agents, such as sorafenib, is a challenge for both clinical and preclinical future investigations in lung cancer treatments.
authors.affiliation: Clinical Pharmacology Centre for Drug Experimentation, University Hospital of Pisa, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pasqualetti
authors.forename: Simona
authors.initials: S
authors.lastname: Ricciardi
authors.forename: Valentina
authors.initials: V
authors.lastname: Mey
authors.forename: Mario
authors.initials: M
authors.lastname: Del Tacca
authors.forename: Romano
authors.initials: R
authors.lastname: Danesi
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001557:Benzenesulfonates / Q000008:administration & dosage / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D004357:Drug Synergism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D009389:Neovascularization, Pathologic; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D049488:Phosphatidylethanolamine Binding Protein / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019009:Proto-Oncogene Proteins c-kit / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000008:administration & dosage / Q000494:pharmacology; D040262:Receptors, Vascular Endothelial Growth Factor / Q000037:antagonists & inhibitors; D000077157:Sorafenib; D048490:raf Kinases / Q000037:antagonists & inhibitors; D000093542:Gemcitabine
chemical_list: D001557:Benzenesulfonates; C511482:PEBP1 protein, human; D010671:Phenylurea Compounds; D049488:Phosphatidylethanolamine Binding Protein; D011725:Pyridines; D003841:Deoxycytidine; D009536:Niacinamide; D000077157:Sorafenib; D019009:Proto-Oncogene Proteins c-kit; D040262:Receptors, Vascular Endothelial Growth Factor; D048490:raf Kinases; D000093542:Gemcitabine