Exogenous hydrogen sulfide promotes hepatocellular carcinoma cell growth by activating the STAT3-COX-2 signaling pathway

Abstract

The effects of hydrogen sulfide (H₂S) on cancer are controversial. Our group previously demonstrated that exogenous H₂S promotes the development of cancer via amplifying the activation of the nuclear factor-κB signaling pathway in hepatocellular carcinoma (HCC) cells (PLC/PRF/5). The present study aimed to further investigate the hypothesis that exogenous H₂S promotes PLC/PRF/5 cell proliferation and migration, and inhibits apoptosis by activating the signal transducer and activator of transcription 3 (STAT3)-cyclooxygenase-2 (COX-2) signaling pathway. PLC/PRF/5 cells were treated with 500 µmol/l NaHS (a donor of H₂S) for 24 h. The expression levels of phosphorylated (p)-STAT3, STAT3, cleaved caspase-3 and COX-2 were measured by western blot assay. Cell viability was detected by Cell Counting kit-8 assay. Apoptotic cells were observed by Hoechst 33258 staining. The expression of STAT3 and COX-2 messenger RNA (mRNA) was detected by semiquantitative reverse transcription-polymerase chain reaction. The production of vascular endothelial growth factor (VEGF) was evaluated by ELISA. The results indicated that treatment of PLC/PRF/5 cells with 500 µmol/l NaHS for 24 h markedly increased the expression levels of p-STAT3 and STAT3 mRNA, leading to COX-2 and COX-2 mRNA overexpression, VEGF induction, decreased cleaved caspase-3 production, increased cell viability and migration, and decreased number of apoptotic cells. However, co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 (an inhibitor of STAT3) or 20 µmol/l NS-398 (an inhibitor of COX-2) for 24 h significantly reverted the effects induced by NaHS. Furthermore, co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 markedly decreased the NaHS-induced increase in the expression level of COX-2. By contrast, co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 20 µmol/l NS-398 inhibited the NaHS-induced increase in the expression level of p-STAT3. In conclusion, the findings of the present study provide evidence that the STAT3-COX-2 signaling pathway is involved in NaHS-induced cell proliferation, migration, angiogenesis and anti-apoptosis in PLC/PRF/5 cells, and suggest that the positive feedback between STAT3 and COX-2 may serve a crucial role in hepatocellular carcinoma carcinogenesis.

Keywords: COX-2; PLC/PRF/5 cells; STAT3; growth; hydrogen sulfide.

Figure 1.

NaHS activates the STAT3-COX-2 signaling pathway in PLC/PRF/5 cells. (A) Exposure of PLC/PRF/5 cells for the indicated times (3, 6, 9, 12 and 24 h) to 500 µmol/l NaHS; (B) co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 for 24 h; (C) the rate of p-STAT3/STAT3; (D) the rate of COX-2/GAPDH; (E) the expression of STAT3 mRNA; (F) the expression of COX-2 mRNA; (G) exposure of PLC/PRF/5 cells for the indicated times (3, 6, 9, 12 and 24 h) to 500 µmol/l NaHS; (H) PLC/PRF/5 cells were co-treated with 500 µmol/l NaHS and 20 µmol/l NS-398 for 24h; (I) the rate of COX-2/GAPDH; (J) the rate of p-STAT3/STAT3. The expression levels of p-STAT3 (A, C, H and J) and COX-2 (B, D, G and I) were semiquantified by western blot assay. (C, D, I and J) Densitometric analysis of the p-STAT3 expression levels shown in panels A, B, G and H, respectively. The mRNA expression levels of (E) STAT3 and (F) COX-2 in PLC/PRF/5 cells were examined by semiquantitative reverse transcription-polymerase chain reaction. GAPDH mRNA was used as a loading control. Data are presented as means ± standard error of the mean (n=3). **P<0.01 vs. the control group. ⁺⁺P<0.01 vs. the group treated with NaHS, a donor of H₂S. Con, control; STAT3, signal transducer and activator of transcription 3; COX-2, cyclooxygenase-2; p-, phosphorylated.

Figure 2.

The signal transducer and activator of transcription 3-cyclooxygenase-2 signaling pathway serves a function in NaHS-induced increase in cell viability in PLC/PRF/5 cells. (A) PLC/PRF/5 cells were treated with different concentration (100, 200, 300, 400 and 500 µmol/l) of NaHS. (B) Treatment of PLC/PRF/5 cells with 500 µmol/l NaHS for the indicated times (12, 24, 36 and 48 h). (C) co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and different doses of AG490 (1, 10, 20 and 30 µmol/l) for 24 h. (D) PLC/PRF/5 cells were co-treated with 500 µmol/l NaHS and NS-398 (0.01–0.5 µmol/l) for 24 h. Cell viability was detected by Cell Counting Kit-8 assay. Data are presented as the mean ± standard error of the mean (n=5). *P<0.05, **P<0.01 vs. the control group. ⁺P<0.05, ⁺⁺P<0.01 vs. with the NaHS-treated group. Con, control.

Figure 3.

The signal transducer and activator of transcription 3-cyclooxygenase-2 signaling pathway serves a function in NaHS-induced anti-apoptosis in PLC/PRF/5 cells. (A) Following the indicated treatments: (Aa) PLC/PRF/5 cells were treated with RPMI 1640 medium for 24 h; (Ab) Treatment of PLC/PRF/5 cells with 500 µmol/l NaHS for 24 h; (Ac) co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 for 24 h; (Ad) PLC/PRF/5 cells were co-treated with 500 µmol/l NaHS and 20 µmol/l NS-398 for 24h; (Ae) Treatment of PLC/PRF/5 cells with 30 µmol/l AG490 for 24 h; (Af) treatment of PLC/PRF/5 cells with 20 µmol/l NS-398 for 24 h; (Ba and Bc) Treatment of PLC/PRF/5 cells with 500 µmol/l NaHS for the indicated times (1, 3, 6, 9, 12 and 24 h); (B-b and d) co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 or 20 µmol/l NS-398 for 24 h. Cellular apoptosis was assessed by Hoechst 33258 staining, followed by photofluorography at ×100 magnification. (B) Western blotting demonstrated varying expression of cleaved caspase-3 associated with (Ba) the times treated with NaHS and (Bb) the different treatment groups. (Bc) Densitometric analysis identified that differences due to time of treatment were significant. (Bd) A number of the differences in cleaved caspase-3 expression as a result of different treatment groups were significant. Data are presented as the mean ± standard error of the mean (n=3). **P<0.01 vs. the control group. ⁺⁺P<0.01 vs. the NaHS-treated group. Con, control.

Figure 4.

The STAT3-COX2 signaling pathway participates in NaHS-induced migration in PLC/PRF/5 cells. (A) Exposure of PLC/PRF/5 cells for the indicated times (1, 3, 6, 9, 12 and 24 h) to 500 µmol/l NaHS; (B) the rate of MMP-2/GAPDH; (C) co-treatment of PLC/PRF/5 cells with 500 µmol/l NaHS and 30 µmol/l AG490 for 24 h; (D) the rate of MMP-2/GAPDH; (E) PLC/PRF/5 cells were co-treated with 500 µmol/l NaHS and 20 µmol/l NS-398 for 24h; (F) the rate of MMP-2/GAPDH; (G) PLC/PRF/5 cells were co-treated with 500 µmol/l NaHS and 30 µmol/l AG490, or 20 µmol/l NS-398 for 24 h. (A-F) The expression levels of MMP-2 were semiquantified by western blot assay. (C, D and F) Densitometric analysis of the p-STAT3 expression levels shown in panels A-C, respectively. (G) Cell migration was evaluated by Transwell migration assay, and the migration rate was calculated under a fully automated inverted microscope. Data are presented as the mean ± standard error of the mean (n=3). **P<0.01 vs. the control group. ⁺⁺P<0.01 vs. the NaHS-treated group. MMP-2, matrix metalloproteinase-2; Con, control.

Figure 5.

The signal transducer and activator of transcription 3-cyclooxygenase-2 signaling pathway serves a function in the NaHS-induced production of VEGF in PLC/PRF/5 cells. PLC/PRF/5 cells were co-conditioned with 500 µmol/l NaHS and 30 µmol/l AG490 or 20 µmol/l NS-398 for 24 h. ELISA was used to measure the production of VEGF. Data are presented as the mean ± standard error (n=3). **P<0.01 vs. the control group; ⁺⁺P<0.01 vs. the NaHS-treated group. VEGF, vascular endothelial growth factor; Con, control; PDTC, pyrrolidine dithiocarbamate.