c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells

Abstract

Aim: 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.

Conclusion: 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.

Keywords: Apoptosis; Apoptosis-related protein; Gastric cancer; Thermotherapy; c-Jun N-terminal kinase.

Figure 1

Cell cycle change in SGC-7901 cells following thermotherapy for various time periods. A-C: Cell cycle distribution in SGC-7901 cells following thermotherapy for 0 h, 1 h, 2 h respectively. Thermotherapy increased the number of SGC-790l cells in G0/G1 phase, but reduced the number in S phase (P < 0.05) at 1 h and 2 h, compared with that at 0 h.

Figure 2

Apoptotic rate of SGC7901 cells with or without SP600125 treatment following thermotherapy for various time periods. A1, B1, C1, D1, E1: Apoptotic rate of SGC-7901 cells without SP600125 treatment, significantly increased after thermotherapy for 0.5, 1, 2 and 3 h, compared to that at 0 h (P < 0.01); A2, B2, C2, D2, E2: Apoptotic rate of SGC-7901 cells with SP600125 treatment, significantly inhibited cellular apoptosis induced by thermotherapy at 0.5, 1, 2 and 3 h (P < 0.01), compared to that at the same time points in the control groups. PI: Propidium iodine; FITC: Fluoresceine isothiocyanate.

Figure 3

Apoptosis of SGC-7901 cells with or without SP600125 treatment following thermotherapy for various time periods (×400). Apoptotic cells showing yellow fluorescence. A1, B1, C1, D1, E1: Apoptosis of SGC-7901 cells without SP600125 treatment, significantly increased after thermotherapy for 0.5, 1, 2 and 3 h, compared to that at 0 h (P < 0.01); A2, B2, C2, D2, E2: Apoptosis with SP600125 treatment, significantly inhibited cellular apoptosis induced by thermotherapy at 0.5, 1, 2 and 3 h (P < 0.01), compared to that at the same time points in the control groups.

Figure 4

Expression of p-c-Jun N-terminal kinase, Bax, caspase-3 and Bcl-2 proteins in SGC-7901 cells with or without SP600125 treatment following thermotherapy for various time periods. A1, B2, C1, D1: Expression of p-JNK, Bax, caspase-3 and Bcl-2 proteins in SGC-7901 cells without SP600125 treatment; A2, B2, C2, D2: Expression of p-JNK, Bax, caspase-3 and Bcl-2 proteins in SGC-7901 cells with SP600125 treatment; A3, B3, C3, D3: Comparison of p-JNK, Bax, caspase-3 and Bcl-2 proteins in SGC-7901 cells with or without SP600125 treatment; Expression of p-JNK, Bax, caspase-3 proteins in SGC-7901 cells with SP600125 treatment was significantly inhibited compared with that in SGC-7901 cells without SP600125 treatment (^bP < 0.01); Bcl-2 protein did not show an obvious change in SGC-7901 cells with or without SP600125 treatment (P > 0.05). JNK: c-Jun N-terminal kinase.

Figure 5

Expression of c-Jun N-terminal kinase at the mRNA level in SGC-7901 cells with or without SP600125 treatment following thermotherapy for various time periods. A: Expression of c-Jun N-terminal kinase (JNK) at the mRNA level in SGC-7901 cells without SP600125 treatment; B: Expression of JNK at the mRNA level in SGC-7901 cells with SP600125 treatment; C: Comparison of JNK at the mRNA level in SGC-7901 cells with or without SP600125 treatment; Expression of JNK at the mRNA level in SGC-7901 cells with SP600125 treatment was significantly inhibited compared with that in SGC7-901 cells without SP600125 treatment (^bP < 0.01).