title: [Bufalin reverses hepatocyte growth factor-induced resistance to afatinib in H1975 lung cancer cells].
abstract: OBJECTIVE
  To investigate the effects of bufalin in reversing hepatocyte growth factor (HGF)-induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism.
  
  
  METHODS
  The afatinib-resistant H1975 lung cancer cells (H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad-HGF-GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway-related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot.
  
  
  RESULTS
  The results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67 ± 8.76)%, significantly lower than the growth rate of (63.45 ± 12.65)% in the H1975 cells treated with HGF alone (P < 0.05). The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber (48.98 ± 11.43), significantly lower than the 118.92 ± 37.29 of afatinib-treated or the 88.84 ± 19.53 of bufalin-treated cells (P < 0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p-EGFR, p-cMet, p-AKT, p-ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down-regulated markedly, and the expression of E-cadherin was up-regulated markedly.
  
  
  CONCLUSIONS
  Combination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial-mesenchymal transition.
authors.affiliation: Department of Oncology, the First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Kang
authors.forename: Ping
authors.initials: P
authors.lastname: Lu
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Cui
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Qingqin
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yabin
authors.initials: Y
authors.lastname: Gong
authors.affiliation: Email: xuzhenye1947@126.com.
authors.forename: Zhenye
authors.initials: Z
authors.lastname: Xu
mesh_terms: D000077716:Afatinib; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D002018:Bufanolides / Q000494:pharmacology*; D015820:Cadherins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004396:Coloring Agents; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D066246:ErbB Receptors; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System; D009363:Neoplasm Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D011799:Quinazolines / Q000494:pharmacology*; D015398:Signal Transduction; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D000970:Antineoplastic Agents; D002018:Bufanolides; D015820:Cadherins; D004396:Coloring Agents; C066855:HGF protein, human; D009363:Neoplasm Proteins; D011799:Quinazolines; D013778:Tetrazolium Salts; D013844:Thiazoles; D000077716:Afatinib; D017228:Hepatocyte Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; C022616:thiazolyl blue; C022777:bufalin