title: Ubiquitin ligase c-Cbl is involved in tamoxifen-induced apoptosis of MCF-7 cells by downregulating the survival signals.
abstract: BACKGROUND
  Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been widely used in the treatment of breast cancer through its anti-estrogen activity. Recent studies show that TAM is cytotoxic to both estrogen receptor (ER)-positive and ER-negative cells via the induction of apoptosis. However, the molecular mechanisms of this effect are not well understood. In the present study, we investigated the roles of c-Src, ERK, AKT and c-Cbl ubiquitin ligases during TAM-induced apoptosis of MCF-7 cells.
  
  
  METHODS
  MCF-7 cell proliferation and apoptosis were measured by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometry, respectively. c-Cbl expression, and the activity of c-Src, ERK, AKT were assayed by Western blotting. Overexpression of the wild and the dominant-negative type of c-Cbl (70Z/Cbl) were achieved by transient transfection of plasmids encoding c-Cbl and 70Z/Cbl, respectively, and were confirmed by Western blotting. Statistical analysis was performed using the t-test, and a p-value <0.05 was considered to be statistically significant.
  
  
  RESULTS
  A high concentration of TAM (25 μM) induced a time-dependent apoptosis of MCF-7 cells. ERK1/2 and AKT were activated during TAM-induced apoptosis. The ERK1/2 inhibitor PD98059, the PI3K/Akt inhibitor LY294002, and the c-Src inhibitor PP2 all enhanced TAM action. Moreover, the ubiquitin ligase c-Cbl was up-regulated during this process. Over-expression of c-Cbl significantly enhanced the apoptosis-inducing effects of TAM, while 70Z/Cbl suppressed the apoptosis-inducing effects of TAM. Further investigation revealed that, overexpression of c-Cbl significantly downregulated the c-Src protein levels and TAM-induced AKT activity. But 70Z/Cbl significantly upregulated TAM-induced ERK and AKT activity.
  
  
  CONCLUSIONS
  This study demonstrates that c-Src, ERK, and AKT played a protective role during TAM-induced apoptosis, and that c-Cbl sensitized MCF-7 cells to TAM by modulating the expression of c-Src, and TAM-induced ERK and AKT activity.
authors.affiliation: Department of Medical Oncology, The First Hospital, China Medical University, Heping District, Shenyang, Liaoning Province, China.
authors.forename: Shun-Chao
authors.initials: SC
authors.lastname: Yan
authors.forename: Yun-Peng
authors.initials: YP
authors.lastname: Liu
authors.forename: Ling-Yun
authors.initials: LY
authors.lastname: Zhang
authors.forename: Jing-Lei
authors.initials: JL
authors.lastname: Qu
authors.forename: Ling
authors.initials: L
authors.lastname: Xu
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhang
authors.forename: Ke-Zuo
authors.initials: KZ
authors.lastname: Hou
authors.forename: Yue-E
authors.initials: YE
authors.lastname: Teng
authors.forename: Xiu-Juan
authors.initials: XJ
authors.lastname: Qu
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D004965:Estrogen Antagonists / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D006801:Humans; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013629:Tamoxifen / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000502:physiology
chemical_list: D004965:Estrogen Antagonists; D013629:Tamoxifen; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; C067419:CBL protein, human