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Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

Wu YC, Wang XJ, Yu L, Chan FK, Cheng AS, Yu J, Sung JJ, Wu WK, Cho CH - PLoS ONE (2012)

Bottom Line: The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip).Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S.Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.

ABSTRACT
Hydrogen sulfide (H(2)S) is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2)S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC) and a panel of colon cancer cell lines (HT-29, SW1116, HCT116) were exposed to H(2)S at concentrations similar to those found in the human colon. H(2)S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip). Moreover, exposure to H(2)S led to features characteristic of autophagy, including increased formation of LC3B(+) autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S. Further mechanistic investigation revealed that H(2)S stimulated the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2)S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

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Involvement of AMPK/mTOR pathway.(A) Treating colon epithelial cells with NaHS (1 mmol/L; 24 h) resulted in hyperphosphorylation of AMPK and hypophosphorylation of mTOR and p70 S6K. NaHS (1 mmol/L) also increased phosphorylation of acetyl-CoA-carboxylase (ACC) but did not affect Akt phosphorylation at Ser473. (B) NaHS dose- and time-dependently enhanced ACC phosphorylation in HT-29. (C) The AMPK inhibitor compound c (CC) significantly reduced the number of LC3B+ autophagic vacuoles in H2S-treated cells. HT-29 cells were treated with compound C (20 mmol/L) for 8 h followed by 1.0 mmol/L NaHS treatment for another 48 h. (D) Compound C (CC) reversed the inhibitory effect of H2S on HT-29 cell proliferation. (E) Knockdown of AMPK abolished the inhibitory effect of NaHS (1 mmol/L) on cell proliferation in HCT116. **P<0.01, significantly different from respective control group. † P<0.05, significantly different from the NaHS group.
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pone-0037572-g005: Involvement of AMPK/mTOR pathway.(A) Treating colon epithelial cells with NaHS (1 mmol/L; 24 h) resulted in hyperphosphorylation of AMPK and hypophosphorylation of mTOR and p70 S6K. NaHS (1 mmol/L) also increased phosphorylation of acetyl-CoA-carboxylase (ACC) but did not affect Akt phosphorylation at Ser473. (B) NaHS dose- and time-dependently enhanced ACC phosphorylation in HT-29. (C) The AMPK inhibitor compound c (CC) significantly reduced the number of LC3B+ autophagic vacuoles in H2S-treated cells. HT-29 cells were treated with compound C (20 mmol/L) for 8 h followed by 1.0 mmol/L NaHS treatment for another 48 h. (D) Compound C (CC) reversed the inhibitory effect of H2S on HT-29 cell proliferation. (E) Knockdown of AMPK abolished the inhibitory effect of NaHS (1 mmol/L) on cell proliferation in HCT116. **P<0.01, significantly different from respective control group. † P<0.05, significantly different from the NaHS group.

Mentions: As activation of AMPK is known to play an important role in the induction of autophagy, we measured the phosphorylation of AMPK in HT-29 and SW1116. Results show that H2S treatment caused a significant increase of AMPK phosphorylation at Thr-172 in both cell lines. The phosphorylation of AMPK downstrem target acetyl-CoA-carboxylase was also increased by H2S in a time- and dose-dependent manner (Fig. 5A & 5B). Phospho-AMPKThr-172 is known to phosphorylate and activate TSC2, which inhibits the activation of the downstream targets, such as mTOR and S6 [12]. In this regard, phosphorylation of mTOR and p70 S6 kinase was substantially decreased in H2S-treated cells. The phosphorylation of Akt, which has been reported to mediate mTOR phosphorylation, was not affected by H2S (Fig. 5A). Treating the cells with a specific AMPK inhibitor (compound C) significantly reduced the number of LC3+ autophagosomes induced by H2S (Fig. 5C). Moreover, compound C reversed the anti-proliferative effect of H2S in HT-29 (Fig. 5D). The importance of AMPK in the regulation of colon epithelial cell proliferation by H2S was confirmed by RNA interference. Abolition of AMPK rendered HCT1116 cells insensitive to the anti-proliferative effect of NaHS (Fig. 5E).


Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

Wu YC, Wang XJ, Yu L, Chan FK, Cheng AS, Yu J, Sung JJ, Wu WK, Cho CH - PLoS ONE (2012)

Involvement of AMPK/mTOR pathway.(A) Treating colon epithelial cells with NaHS (1 mmol/L; 24 h) resulted in hyperphosphorylation of AMPK and hypophosphorylation of mTOR and p70 S6K. NaHS (1 mmol/L) also increased phosphorylation of acetyl-CoA-carboxylase (ACC) but did not affect Akt phosphorylation at Ser473. (B) NaHS dose- and time-dependently enhanced ACC phosphorylation in HT-29. (C) The AMPK inhibitor compound c (CC) significantly reduced the number of LC3B+ autophagic vacuoles in H2S-treated cells. HT-29 cells were treated with compound C (20 mmol/L) for 8 h followed by 1.0 mmol/L NaHS treatment for another 48 h. (D) Compound C (CC) reversed the inhibitory effect of H2S on HT-29 cell proliferation. (E) Knockdown of AMPK abolished the inhibitory effect of NaHS (1 mmol/L) on cell proliferation in HCT116. **P<0.01, significantly different from respective control group. † P<0.05, significantly different from the NaHS group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362591&req=5

pone-0037572-g005: Involvement of AMPK/mTOR pathway.(A) Treating colon epithelial cells with NaHS (1 mmol/L; 24 h) resulted in hyperphosphorylation of AMPK and hypophosphorylation of mTOR and p70 S6K. NaHS (1 mmol/L) also increased phosphorylation of acetyl-CoA-carboxylase (ACC) but did not affect Akt phosphorylation at Ser473. (B) NaHS dose- and time-dependently enhanced ACC phosphorylation in HT-29. (C) The AMPK inhibitor compound c (CC) significantly reduced the number of LC3B+ autophagic vacuoles in H2S-treated cells. HT-29 cells were treated with compound C (20 mmol/L) for 8 h followed by 1.0 mmol/L NaHS treatment for another 48 h. (D) Compound C (CC) reversed the inhibitory effect of H2S on HT-29 cell proliferation. (E) Knockdown of AMPK abolished the inhibitory effect of NaHS (1 mmol/L) on cell proliferation in HCT116. **P<0.01, significantly different from respective control group. † P<0.05, significantly different from the NaHS group.
Mentions: As activation of AMPK is known to play an important role in the induction of autophagy, we measured the phosphorylation of AMPK in HT-29 and SW1116. Results show that H2S treatment caused a significant increase of AMPK phosphorylation at Thr-172 in both cell lines. The phosphorylation of AMPK downstrem target acetyl-CoA-carboxylase was also increased by H2S in a time- and dose-dependent manner (Fig. 5A & 5B). Phospho-AMPKThr-172 is known to phosphorylate and activate TSC2, which inhibits the activation of the downstream targets, such as mTOR and S6 [12]. In this regard, phosphorylation of mTOR and p70 S6 kinase was substantially decreased in H2S-treated cells. The phosphorylation of Akt, which has been reported to mediate mTOR phosphorylation, was not affected by H2S (Fig. 5A). Treating the cells with a specific AMPK inhibitor (compound C) significantly reduced the number of LC3+ autophagosomes induced by H2S (Fig. 5C). Moreover, compound C reversed the anti-proliferative effect of H2S in HT-29 (Fig. 5D). The importance of AMPK in the regulation of colon epithelial cell proliferation by H2S was confirmed by RNA interference. Abolition of AMPK rendered HCT1116 cells insensitive to the anti-proliferative effect of NaHS (Fig. 5E).

Bottom Line: The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip).Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S.Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.

ABSTRACT
Hydrogen sulfide (H(2)S) is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2)S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC) and a panel of colon cancer cell lines (HT-29, SW1116, HCT116) were exposed to H(2)S at concentrations similar to those found in the human colon. H(2)S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip). Moreover, exposure to H(2)S led to features characteristic of autophagy, including increased formation of LC3B(+) autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S. Further mechanistic investigation revealed that H(2)S stimulated the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2)S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

Show MeSH
Related in: MedlinePlus