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Cited by 109Sulfur Compounds in BiologyAutophagy in Disease and TherapyCatalysis and Hydrodesulfurization Studies

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Hydrogen Sulfide Lowers Proliferation and Induces Protective Autophagy in Colon Epithelial Cells

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Wu, Ya C., Wang, Xiao J., Yu, Le, Chan, Francis K. L., Cheng, Alfred S. L., Yu, Jun, Sung, Joseph J. Y., Wu, William K. K., Cho, Chi H.·2012·PLoS ONE
DOI 10.1371/journal.pone.0037572PMC PMC3362591
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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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Figure 1 Inhibition of colon epithelial cell proliferation and migration by H2S. Incubation of NaHS reduced, in a dose- and time-dependent manner, cell proliferation of (A) YMAC, (B) HCT116, (C) SW1116 and (D) HT-29 as determined by MTT assay. (E) NaHS did not affect necrotic cell death in HT-29 as determined by lactate dehydrogenase release assay. 1% (v/v) Triton X-100 was used as a positive control. (F) The anti-proliferative effect of NaHS (24 h) on colon epithelial cells was further confirmed by direct cell counting. (G) NaHS (1 umol L−1) significantly reduced the migration of SW1116 cells as measured by wound healing assay. * P<0.05; ** P<0.01, significantly different from respective control group.
Figure 1 Inhibition of colon epithelial cell proliferation and migration by H2S. Incubation of NaHS reduced, in a dose- and time-dependent manner, cell proliferation of (A) YMAC, (B) HCT116, (C) SW1116 and (D) HT-29 as determined by MTT assay. (E) NaHS did not affect necrotic cell death in HT-29 as determined by lactate dehydrogenase release assay. 1% (v/v) Triton X-100 was used as a positive control. (F) The anti-proliferative effect of NaHS (24 h) on colon epithelial cells was further confirmed by direct cell counting. (G) NaHS (1 umol L−1) significantly reduced the migration of SW1116 cells as measured by wound healing assay. * P<0.05; ** P<0.01, significantly different from respective control group.CC BY · PubMed Central
Figure 2 Inhibition of cell cycle progression of colon epithelial cells by H2S. (A) DNA histogram shows the accumulation of G0/G1-phase cells induced by NaHS in HT-29. Cells were treated without or with 1 mmol/L NaHS, and their DNA contents were determined by flow cytometry analysis. (B) Quantitative analysis of cell cycle distribution of SW1116 cells treated with or without 1 mmol/L NaHS at various time points. (C) NaHS (1 mmol/L) time-dependently up-regulated p21Cip1 protein expression. The induction was maximal 6 h after NaHS treatment. (D) The effect of H2S on p21Cip1 induction was confirmed in SW1116 and YAMC cells. The results are the representative of three independent experiments. * P<0.05, significantly different from respective control group at 0 h.
Figure 2 Inhibition of cell cycle progression of colon epithelial cells by H2S. (A) DNA histogram shows the accumulation of G0/G1-phase cells induced by NaHS in HT-29. Cells were treated without or with 1 mmol/L NaHS, and their DNA contents were determined by flow cytometry analysis. (B) Quantitative analysis of cell cycle distribution of SW1116 cells treated with or without 1 mmol/L NaHS at various time points. (C) NaHS (1 mmol/L) time-dependently up-regulated p21Cip1 protein expression. The induction was maximal 6 h after NaHS treatment. (D) The effect of H2S on p21Cip1 induction was confirmed in SW1116 and YAMC cells. The results are the representative of three independent experiments. * P<0.05, significantly different from respective control group at 0 h.CC BY · PubMed Central
Figure 3 Induction of autophagosomes in colon epithelial cells by H2S. (A) Treating the cells with NaHS for 24 h or 48 h prominently enhanced the formation of autophagic vacuoles as determined by immunofluorescent staining for LC3B. (B) Ultrastructural analysis by electron microscopy revealed the formation of autophagosome or secondary lysosomes with the residual digested material in NaHS-treated HT-29 (1 mmol/L; 48 h). (C) NaHS increased LC3-II levels in HT-29, SW1116 and YAMC cells after treatment with NaHS (1 mmol/L) for 48 h. (D) NaHS dose-dependently increased LC3B-II level in HT-29 cells at 48 h. The expression of Beclin-1 was not altered. (E) Increased autophagic flux was confirmed by co-treating SW1116 cells with NaHS and bafilomycin A1. Treatment with bafilomycin A1 (10 nmol/L) did not prevent the upregulation of LC3B-II in cells incubated with NaHS (1 mmol/L; 48 h).
Figure 3 Induction of autophagosomes in colon epithelial cells by H2S. (A) Treating the cells with NaHS for 24 h or 48 h prominently enhanced the formation of autophagic vacuoles as determined by immunofluorescent staining for LC3B. (B) Ultrastructural analysis by electron microscopy revealed the formation of autophagosome or secondary lysosomes with the residual digested material in NaHS-treated HT-29 (1 mmol/L; 48 h). (C) NaHS increased LC3-II levels in HT-29, SW1116 and YAMC cells after treatment with NaHS (1 mmol/L) for 48 h. (D) NaHS dose-dependently increased LC3B-II level in HT-29 cells at 48 h. The expression of Beclin-1 was not altered. (E) Increased autophagic flux was confirmed by co-treating SW1116 cells with NaHS and bafilomycin A1. Treatment with bafilomycin A1 (10 nmol/L) did not prevent the upregulation of LC3B-II in cells incubated with NaHS (1 mmol/L; 48 h).CC BY · PubMed Central
Figure 4 Induction and functional role of autophagy in H2S-treated colon epithelial cells. (A) The accumulation of acidic vesicular organelles, which emitted bright red fluorescence (white arrows), induced by 1 mmol/L NaHS was visualized by acridine orange staining. (B) knockdown of Vps34 or Atg7 enhanced the anti-mitogenic effect of 48-h treatment of NaHS (1 mmol/L) in SW1116 cells. (C) The knockdown efficacies of Vps34- and Atg7-siRNAs were confirmed by downregulation of respective targets. (D) Knockdown of Vps34 and Atg7 prevented the upregulation of LC3B-II caused by NaHS (1 mmol/L) but did not alter PARP cleavage. ** P<0.01, significantly different from respective control group; † P<0.05, significantly different from NaHS group transfected with control siRNA.
Figure 4 Induction and functional role of autophagy in H2S-treated colon epithelial cells. (A) The accumulation of acidic vesicular organelles, which emitted bright red fluorescence (white arrows), induced by 1 mmol/L NaHS was visualized by acridine orange staining. (B) knockdown of Vps34 or Atg7 enhanced the anti-mitogenic effect of 48-h treatment of NaHS (1 mmol/L) in SW1116 cells. (C) The knockdown efficacies of Vps34- and Atg7-siRNAs were confirmed by downregulation of respective targets. (D) Knockdown of Vps34 and Atg7 prevented the upregulation of LC3B-II caused by NaHS (1 mmol/L) but did not alter PARP cleavage. ** P<0.01, significantly different from respective control group; † P<0.05, significantly different from NaHS group transfected with control siRNA.CC BY · PubMed Central
Figure 5 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.
Figure 5 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.CC BY · PubMed Central
Figure 6 Schematic diagram showing the effects of H2S on cell proliferation and autophagy in relation to the AMPK signaling pathway.
Figure 6 Schematic diagram showing the effects of H2S on cell proliferation and autophagy in relation to the AMPK signaling pathway.CC BY · PubMed Central

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Authors
Wu, Y. C., Wang, X. J., Yu, L., Chan, F. K. L., Cheng, A. S. L., Yu, J., Sung, J. J. Y., Wu, W. K. K., & Cho, C. H.
Affiliations
Chinese University of Hong Kong (HK)
Corresponding author
Wu, William K. K.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-05-29
Volume
7
Issue
5
Pages
e37572
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0037572
PMC
PMC3362591
OpenAlex
W2088784248
Cited by
109
References
26
Citations by year
2026: 3 · 2025: 7 · 2024: 5 · 2023: 7 · 2022: 9 · 2021: 13 · 2020: 11 · 2019: 8 · 2018: 11 · 2017: 6 · 2016: 7 · 2015: 8 · 2014: 8 · 2013: 6
FWCI
3.01
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Sulfur Compounds in Biology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Physical Sciences › Chemical Engineering › Catalysis › Catalysis and Hydrodesulfurization Studies
Keywords
hydrogen sulfide, colonic epithelial cells, colon cancer, cell proliferation, autophagy, AMPK signaling, mTOR, S6 kinase, p21, G1 cell cycle arrest, Vps34, ATG7, LC3B
MeSH terms
Autophagy-Related Protein 7, Animals, Autophagy, Cell Cycle, Cell Line, Cell Movement, Colon, Epithelial Cells, Humans, Hydrogen Sulfide, Intestinal Mucosa, Microtubule-Associated Proteins, Phagosomes, Signal Transduction, Ubiquitin-Activating Enzymes, Cell Proliferation, Cyclin-Dependent Kinase Inhibitor p21, Mice, AMP-Activated Protein Kinases, Class III Phosphatidylinositol 3-Kinases, TOR Serine-Threonine Kinases

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