title: [p14ARF enhances chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway].
abstract: OBJECTIVE
  To explore the effects of tumor suppressor p14ARF on chemosensitivity of human osteosarcoma U2OS cells to cisplatin and elucidate its molecular mechanism.
  
  
  METHODS
  U2OS cells expressing no p14ARF and U2OS-ARF cells expressing p14ARF stably through stable transfection were treated with cisplatin. Cell viability and IC50 were assayed with methyl thiazolyl tetrazolium (MTT). Apoptosis was examined by fluorescence-activated cell sorting and Hoechst33258 staining. The expressions of p53, Bax, p21, Mdm2 and Fas were detected by Western blot. And colorimetry was used to determine the activities of caspase-3, caspase-8 and caspase-9.
  
  
  RESULTS
  The viability was 84.8% ± 4.4%, 86.9% ± 5.0% and 66.7% ± 4.6% respectively in U2OS, U2OS-vec and U2OS-ARF cells. The values of IC50 were (15.8 ± 0.9) µmol/L, (16.3 ± 0.6) and (8.9 ± 0.8) µmol/L respectively in U2OS, U2OS-vec and U2OS-ARF cells. The levels of viability and IC50 obviously decreased in U2OS-ARF cells in response to cisplatin (P < 0.05). There were higher apoptotic rate and more obvious apoptotic morphological changes in U2OS-ARF cells than U2OS and U2OS-vec cells. The basal levels of p53, Mdm2 and p21 in U2OS-ARF cells were slightly higher than those in U2OS-vec cells. Cisplatin up-regulated p53, Mdm2 and p21 in both cell lines. However, the up-regulation was more pronounced in U2OS-ARF cells. Cisplatin did not change the levels of Bax and Fas in U2OS-vec cells. Bax protein was up-regulated in U2OS-ARF cells while the level of Fas remained constant. p14ARF also enhanced the activities of caspase-9 and caspase-3 in response to cisplatin.
  
  
  CONCLUSIONS
  p14ARF enhances the chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway. And intrinsic mitochondrial apoptosis is involved.
authors.affiliation: Department of Orthopedic Surgery, Affiliated Jiangmen Hospital, Sun Yat-sen University, Jiangmen 529030, China. Email: doctoryuanxw@163.com.
authors.forename: Xiangwei
authors.initials: X
authors.lastname: Yuan
authors.forename: Xiufang
authors.initials: X
authors.lastname: Huang
authors.forename: Zhongxian
authors.initials: Z
authors.lastname: Chen
authors.forename: Shenggen
authors.initials: S
authors.lastname: Liang
authors.forename: Weiming
authors.initials: W
authors.lastname: Liao
mesh_terms: D017209:Apoptosis; D020169:Caspases; D045744:Cell Line, Tumor; D002470:Cell Survival; D002945:Cisplatin; D016147:Genes, Tumor Suppressor; D006801:Humans; D015513:Oncogene Proteins; D012516:Osteosarcoma; D051736:Proto-Oncogene Proteins c-mdm2; D016159:Tumor Suppressor Protein p53; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein
chemical_list: C401561:CDK2AP2 protein, human; D015513:Oncogene Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D020169:Caspases; D002945:Cisplatin