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. 2017 Mar 23;8(3):e2688.
doi: 10.1038/cddis.2017.18.

Hydrogen sulfide promotes autophagy of hepatocellular carcinoma cells through the PI3K/Akt/mTOR signaling pathway (V体育平台登录)

Shanshan S Wang  1   2 , Yuhan H Chen  1 , Ning Chen  3 , Lijun J Wang  4 , Dexi X Chen  2 , Honglei L Weng  5 , Steven Dooley  5 , Huiguo G Ding  1
Affiliations

VSports手机版 - Hydrogen sulfide promotes autophagy of hepatocellular carcinoma cells through the PI3K/Akt/mTOR signaling pathway

Shanshan S Wang et al. Cell Death Dis. .

Abstract

Hydrogen sulfide (H2S), in its gaseous form, plays an important role in tumor carcinogenesis. This study investigated the effects of H2S on the cell biological functions of hepatocellular carcinoma (HCC). HCC cell lines, HepG2 and HLE, were treated with NaHS, a donor of H2S, and rapamycin, a classic autophagy inducer, for different lengths of time VSports手机版. Western blotting, immunofluorescence, transmission electron microscopy (TEM), scratch assay, CCK-8 and flow cytometric analysis were carried out to examine the effects of H2S on HCC autophagy, cell behavior and PI3K/Akt/mTOR signaling. Treatment with NaHS upregulated expression of LC3-II and Atg5, two autophagy-related proteins, in HepG2 and HLE cells. TEM revealed increased numbers of intracellular double-membrane vesicles in those cells treated with NaHS. Like rapamycin, NaHS also significantly inhibited expression of p-PI3K, p-Akt and mTOR proteins in HCC cells. Interestingly, the expression of LC3-II was further increased when the cells were treated with NaHS together with rapamycin. In addition, NaHS inhibited HCC cell migration, proliferation and cell division. These findings show that H2S can induce HCC cell apoptosis. The biological function of the gasotransmitter H2S in HCC cells was enhanced by the addition of rapamycin. Hydrogen sulfide influences multiple biological functions of HCC cells through inhibiting the PI3K/Akt/mTOR signaling pathway. .

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Conflict of interest statement

The authors declare no conflict of interest.

"VSports在线直播" Figures

Figure 1
Figure 1
Hydrogen sulfide promotes autophagy of HepG2 and HLE cells. (a) Western blotting detected increased expression of LC3-II, Atg5 and P62 autophagy-related protein in the presence of 10−3 M NaHS in HepG2 and HLE cells. β-actin was used as internal control. (b) Densitometric analysis of LC3-II/LC3-I is shown in the histogram. (c) HepG2 and HLE cells transfected with GFP-LC3 plasmid after 24 h, LC3 puncta dots (green) were observed under a fluorescence microscope; the nuclei (blue) were stained with DAPI. (Scale bar: 20 μm) (d) The percentage of cells presenting typical LC3 puncta dots. (e) Expression of P62 as analyzed by fluorescence microscopy in HepG2 and HLE cells treated with NaHS for 24 h (Scale bar: 100 μm). (f) Intracellular double-membrane vesicles (arrows), the ultrastructural feature of autophagy, were observed by TEM (Scale bars 2 and 1 μm). Each figure is representative of an experiment that was repeated at least three times. The data represent the mean±S.D. of three samples. *P<0.05 compared with control
Figure 2
Figure 2
Hydrogen sulfide inhibits cell migration, proliferation and cell cycle progression, but accelerates apoptosis. Scratch assay was used to evaluate cell migration. HepG2 (a) and HLE (e) cells were treated with NaHS for 48 h then viewed, and images were captured under a light microscope at 0, 12, 24, 36 and 48 h separately (Scale bar: 20 μm). CCK-8 assay was used to verify HepG2 (b) and HLE (f) cell proliferation. Immunofluorescence was used to evaluate M30 immunoreactivity in HepG2 (c) and HLE (g) cells. M30 staining was red and nuclei were blue (Scale bar: 100 μm). Flow cytometry was used to analyze the cell cycle of HepG2 (d) and HLE (h) cells after NaHS treatment for 24 h. The data represents the mean±S.D. of three samples. All the data are representative of an experiment that was repeated at least three times
Figure 3
Figure 3
H2S promotes cell autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway. (a) Western blotting was used to detect the expression of PI3K/ p-PI3K, Akt/p-Akt and mTOR in HepG2 and HLE cells. (b) Immunofluorescence staining revealed that p-PI3K/p-Akt/mTOR decreased after NaHS treatment, with the green indicating positive staining for p-PI3K/p-Akt/mTOR (Scale bar: 40 μm). (c) Western blotting was performed to detect the expression of LC3-II in HepG2 and HLE cells treated with NaHS alone, rapamycin alone or NaHS in combination with rapamycin. β-actin was used as internal control. (d) Densitometric analysis of LC3-II was shown as a histogram. The data represent the mean±S.D. of three samples.*P<0.05 compared with control cells; #P<0.05 versus cells treated with NaHS alone; &P<0.05 versus cells treated with rapamycin alone. (e) Transmission electron microscope observation of cell autophagosomes (Scale bar: 2 μm)
Figure 4
Figure 4
Rapamycin and H2S additively inhibit the migration, proliferation and cell cycle of liver cancer cells. Scratch assay was performed to detect HepG2 (a) and HLE (d) cell migration under different treatments (Scale bar: 200 μm). HepG2 (b) and HLE (e) cell proliferation was calculated by CCK-8 assay. The data represents the mean±S.D. of three samples. HepG2 (c) and HLE (f) cells were incubated under different conditions for 24 h then analyzed by flow cytometry to investigate the cell cycle. The data are representative of an experiment that was repeated at least three times
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