1.Exogenous H2S induces apoptosis and promotes stemness in HCC cell lines
The effect of 10− 3M NaHS (an H2S donor) on apoptosis in HepG2 and Hep3B cell lines was investigated by flow cytometry. Based on the results, Apoptosis rate increased from 6.1–13.4% (P < 0.01) of HepG2(Fig. 1A) and from 4.9–10.8% (P < 0.01) of Hep3B (Fig. 1C) after 10− 3M NaHS treatment. Some studies suggest that CSCs are derived from mutations in adult stem/progenitor cells from the corresponding organ, as they exhibit a high expression of stemness-related genes and specific markers for adult stem/progenitor cells[18]. Therefore, the LCSCs surface markers CD133 and CD44 were examined by flow cytometry. We measured the levels of expression of stemness markers after 24 hours, 48 hours, 72 hours, and 96 hours and we discovered that at 72 hours, CD133 and CD44 were more significantly enriched in 10− 3M NaHS-treated (1.44-fold and 1.54-fold, respectively) (P < 0.01) HepG2(Fig. 1B) and (2,51-fold and 2.11-fold, respectively) (P < 0.01) Hep3B (Fig. 1D) compared with control group, implying that NaHS treatment will confer HCC cell lines to increase CSCs properties.
2. Exogenous H2S regulate multiple different genes and pathways in the LCSCs
To further explain the molecular mechanisms that exogenous H2S promotes LCSCs stemness, flow cytometry was used to sort out LCSCs expressing the surface marker CD133. Next, the LCSCs and HepG2 cell lines were treated with 10− 3M NaHS, using RNA sequencing to explore the differences in gene expression between the NaHS-treated and control groups. As a screening criterion for differentially expressed genes, the absolute value of log2s (fold change > 2 and p < 0.05) was used. The LCSCs contained 393 different genes compared to HepG2 cell lines (Fig. 2A). This finding demonstrates the dramatic change in gene expression following 10− 3M NaHS treatment. To elucidate the molecular mechanism by which NaHS promotes stemness in LCSCs, we retrieved 425 cancer-related stem cell genes from a publicly available stem cell gene database named StemTextSearch from http://bio.yungyun.com.tw/StemTextSearch.aspx, and used Wayne analysis to intersect them with 393 differential genes in the NaHS-treated LCSCs, yielding the identification of 23 differential genes associated with stemness (Fig. 2B). the expression of 7 genes were increased, while the expression of 16 genes were decreased (Fig. 2C). GO functional enrichment results showed that 245 BP terms, 10 CC terms, and 30 MF terms were statistically significantly enriched in DEGs (Fig. 2D). Regulation of epithelial cell proliferation, epithelial cell proliferation, and regulation of endopeptidase activity were the most enriched BP terms. 7 down-regulated genes, including F3, MYC, TGFBR3, JUN, NR4A1, NR4A3, and KLF9, significantly activated epithelial cell proliferation regulation. The most enriched CC terms were transcription factor complexes. Transcriptional activator activity, proximal promoter sequence specific DNA binding, is the most enriched MF term. Furthermore, KEGG pathway enrichment analysis was performed to investigate the pathways that were significantly enriched of DEGs (Fig. 2E). A total of 15 genes were found to be enriched in signaling pathways regulating stem cell pluripotency, the Wnt signaling pathway, the TGF signaling pathway, and the MAPK signaling pathway. Several genes related to Wnt pathways (MYC, JUN, NFATC2) were down-regulated, while ID4 gene was significantly up-regulated. This suggests that the Wnt pathways may be significantly regulated in the process (Fig. 2F).
2. Exogenous H2S upregulates β-catenin expression in HCC cells
With the increasing research on tumour tissue heterogeneity, precise targeting of LCSCs has attracted interest and attention in tumour therapy. Given the critical role of the Wnt signaling pathway in maintaining stemness properties, modulation of the Wnt signaling pathway is considered to be a breakthrough in targeting LCSCs. According to the differential gene enrichment analysis, exogenous H2S may influence the stemness of LCSCs by regulating the Wnt signaling pathway.
HCC cell lines were treated with 10− 3M NaHS in order to examine the changes in stemness properties after its use. Interestingly, the flow cytometry results demonstrated that the expression of the surface markers CD133, CD44 and β-catenin, a downstream molecule of the Wnt signaling pathway, were all significantly increased (1.22-fold 1.25-fold and 1.30-fold, respectively) (p < 0.001) in HepG2 (Fig. 3A, B) and (1.30-fold 1.26-fold and 1.31-fold, respectively) (p < 0.01) in Hep3B (Fig. 4F, G). Then the β-catenin and the nuclear dye Hochest were labeled in both cell lines, the flow cytometric results illustrated that the ability of β-catenin to enter the nucleus were remarkably increased from 0.54–10.55% (p < 0.01) in HepG2 (Fig. 3C, D, E) and from 0.52–17% (p < 0.01) in Hep3B (Fig. 4H, I, J), indicating that the Wnt signaling pathway was significantly activated.
3. H2S regulates the stemness of HCC through Wnt/β-catenin signalling
To further explain the relationship between Wnt/β-catenin signalling and the stemness of HCC cell lines, we treated two HCC cell lines with ICRT3 and compared the changes in stemness properties. ICRT3 is a commonly used responsive transcription inhibitor of Wnt/β-catenin signalling passway, which is a key protein regulating β-catenin. HCC cell lines were divided into four groups: one was treated with NaHS, the second was treated with NaHS and ICRT3, the third was treated with ICRT3 and the last was treated with complete medium for 72 hours. Flow cytometry was used to complete the co-localization analysis. Interestingly, the results demonstrated that the ability of β-catenin to enter the nucleus was changed diversely. In the NaHS treatment group, the extent of β-catenin nucleation ranged from 2.21–14.76%(p < 0.01) in HepG2(Fig. 4A, B, C) and from 2.92–15.2%(p < 0.01) in Hep3B(Fig. 4F, G, H), while when NaHS and ICRT3 were treated simultaneously, the degree of nucleation of β-catenin decreased from 14.76–2.5%(p < 0.05) in HepG2(Fig. 4A, B, C) and from 15.2–0.55%(p < 0.05) in Hep3B(Fig. 4F, G, H), when ICRT3 was treated separately, the degree of nucleation of β-catenin decreased to 0.13%(p < 0.05) in HepG2(Fig. 4A, B, C) and to 0.16%(p < 0.05) in Hep3B(Fig. 4F, G, H). These results implied that β-catenin entry into the nucleus was significantly inhibited in HCC cell lines after treatment with ICRT3. Furthermore, the stemness properties of HCC cell lines were examined. The expression of the surface markers CD133, CD44 and β-catenin in HCC cell lines was significantly downregulated after treatment with ICRT3. The inhibition efficiency of ICRT3 was 35%, 32% and 29%, respectively (p < 0.001) in HepG2(Fig. 4D, E) and was 38%, 34% and 30% respectively (p < 0.05) in Hep3B (Fig. 4I, J). These findings support previous findings that exogenous H2S can promote stemness in HCC cell lines by activating the Wnt signaling pathway, which can be significantly inhibited by the Wnt signaling pathway inhibitor ICRT3.
The proportion of CSCs in tumour tissue or tumour cells is quite low, typically only 0.01-2%. Further studies on CSCs require us to obtain "purified" CSCs in continuous culture without losing the stem cell properties. Specifically, in this study we used DMEM/F12 medium to serially generate sphere-forming cells (SFCs) from HepG2 cells. SFCs were inoculated at a density of 1000 cell/well in 6-well plates and tumour subspheres were evident after 7 days. Figure 4K shows the sphere formation process of SFCs after the treatment of DMEM/F12 medium, NaHS, NaHS with ICRT3 and ICRT3 alone by confocal microscopy. The number of sphere-forming cells was significantly increased in NaHS treated group (> 2-fold) (P < 0.05) compared to that in control group, in the co-treatment group of NAHS and ICRT3, the number of sphere-forming cells was inhibited (> 60%) (P < 0.05) and was more inhibited (> 80%) (P < 0.05) in ICRT3 treatment group (Fig. 4L).