title: c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells.
abstract: OBJECTIVE
  To investigate the role of c-Jun N-terminal kinase (JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.
  
  
  METHODS
  Human gastric cancer SGC-7901 cells were cultured in vitro. Following thermotherapy at 43°C for 0, 0.5, 1, 2 or 3 h, the cells were cultured for a further 24 h with or without the JNK specific inhibitor, SP600125 for 2 h. Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)] and flow cytometry (Annexin vs propidium iodide). Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The production of p-JNK, Bcl-2, Bax and caspase-3 proteins was evaluated by Western blotting. The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction.
  
  
  RESULTS
  The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy, and was 32.7%, 30.6%, 43.8% and 52.9% at 0.5, 1, 2 and 3 h post-thermotherapy, respectively. Flow cytometry analysis revealed an increased population of SGC-790l cells in G0/G1 phase, but a reduced population in S phase following thermotherapy for 1 or 2 h, compared to untreated cells (P < 0.05). The increased number of SGC-790l cells in G0/G1 phase was consistent with induced apoptosis (flow cytometry) following thermotherapy for 0.5, 1, 2 or 3 h, compared to the untreated group (46.5% ± 0.23%, 39.9% ± 0.53%, 56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%, P < 0.01), respectively. This was supported by the TUNEL assay (48.2% ± 0.4%, 40.1% ± 0.2%, 61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%, P < 0.01) respectively. More importantly, the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment (P < 0.01), and peaked at 2 h. A similar pattern was detected for Bax and caspase-3 proteins. Bcl-2 increased at 0.5 h, peaked at 1 h, and then decreased. Furthermore, the JNK specific inhibitor, SP600125, suppressed p-JNK, Bax and caspase-3 at the protein level in SGC790l cells following thermotherapy, compared to mock-inhibitor treatment, which was in line with the decreased rate of apoptosis. The expression of Bcl-2 was consistent with thermotherapy alone.
  
  
  CONCLUSIONS
  Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels, and up-regulated the expression of Bax and caspase-3 proteins. Bcl-2 may play a protective role during thermotherapy. Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.
authors: Department of Pathology, Medical School, Nanchang University, Nanchang 330006, Jiangxi Province, China.
authors: Feng
authors: F
authors: Xiao
authors: Bin
authors: B
authors: Liu
authors: Qing-Xian
authors: QX
authors: Zhu
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006979:Hyperthermia, Induced / Q000379:methods*; D007150:Immunohistochemistry; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000201:enzymology / Q000473:pathology*; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C494826:BAX protein, human; D051028:bcl-2-Associated X Protein; D048031:JNK Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3
keywords: Apoptosis; Apoptosis-related protein; Gastric cancer; Thermotherapy; c-Jun N-terminal kinase