| Hydrogen sulfide (H2S) has been found to play an important role as a novel gasotransmitter, followed with nitric oxide and carbon monoxide, involved in many physiological and patho-physiological processes, such as neuromodulation, hypertension, angiogenesis and inflammation. Cystathionine-y-lyase (CSE) is a pyridoxal-5’-phosphate (PLP)-dependent enzyme that catalyzes the last step in trans-sulfuration pathway, with the production of H2S. CSE is expressed prevalently in the liver, kidney, heart, vasculature, and placenta, CSE gene locates on human chromosome1p31.1. CSE/H2S system is ubiquitously distributed and implicated in various cellular functions, such as cell growth, differentiation, migration, apoptosis and cell cycle progression. Abnormal H2S production in the trans-sulfuration pathway is involved in human cellular dysfunction and diseases, such as atherosclerosis and carcinogenesis. And emerging studies show that CSE/H2S may be involved in the regulation of tumor cells directly. Although in recent years, there has been an explosion of interest in H2S as a biological mediator. But the effect of H2S on cancer cell survival has not been explored in depth. Especially it is unclear whether HiS-producing enzymes can be regulated to control the endogenous H2S level contributing to the tumor biology. PI3K/Akt pathway is classical and important to tumorigenesis with the frequently actived Akt. PI3K/Akt is an important signal pathway frequently constitutively activated in many human cancers, and associated with carcinogenesis including cell survival, metabolism, angiogenesis and malignant transformation. According to the data that the CSE gene was up-regulated in Akt stably transformed mouse embryonic fibroblast cells, the mechanisms that elevated CSE expression by PI3K/Akt signaling pathway and its biological functions in cell survival were further discussed.In the first section of our research, to clarify the relative amount of H2S-producing enzymes in tumor, firstly, we have found that CSE strongly expressed in human hepatocellular carcinoma (HCC) cell lines. And the results showed that PI3K/Akt positively correlated with CSE expression levels in HCC cell lines with Akt phosphorylation. And the positively correlation between Akt2and CSE mRNA levels was also identified. And we observed the significantly enhanced CSE mRNA and protein levels resulted from the high pAkt-Ser473protein in Akt over-expression MEF cells. Here, we present a novel mechanism attributed to the potential role of PI3K/Akt in regulating the CSE enzyme and H2S level on the tumorigenesis. Next, we observed that CSE protein and mRNA levels were inhibited in HCC cell lines by treated with LY294002, a small molecule specific inhibitor of PI3K, in a dose-and time-dependent manner. A significantly decrease of CSE mRNA was found by Akt2siRNA in HCC cell lines, and CSE protein levels decreased significantly in BEL-7404cells of Akt1/2deletion using lentivirus mediated RNAi stably. PTEN knocking down or treatment with insulin-like growth factor-1(IGF-1) or insulin, leaded to the up-regulation of CSE protein with the enhanced Akt phosphorylation. Additionally, after the cells treated with LY294002, the stability of CSE protein was almost not affected, compared to the LY294002-untreated ones by CHX (a protein synthesis inhibitor) decay assay. Taken together, it indicated that PI3K/Akt pathway involved in the regulation of CSE gene expression on the transcriptional level in HCC cell lines.In order to deeply demonstrate the mechanism of regulating CSE gene expression by PI3K/Akt pathway on the transcriptional level, in the second section of our study, a series of deleted CSE promoter luciferase plasmids were constructed. With dual-luciferase reporter assay, the data showed that the promoter pCSE-731(-592/+139) showed the strongest activity, compared with the other deleted ones, represented the core promoter. Furthermore we evaluated the ratio of relative luciferase activity of CSE core promoter and pGL3-Basic by LY294002treated or untreated. It was found that after LY294002treated, the ratio of CSE core promoter was decreased compared with the pGL3-Basic. These results certified that PI3K/Akt increase the expression of CSE at transcription control. The DNA binding activity of transcription factors in different cells is an important determinant to the promoter activity. According to the prediction of transcriptional factor binding sites on CSE promoter by TFSEARCH, ConTra and ALGGEN softwares, the mutation of potential binding sites in the core promoter region mainly included the positive regulators on the CSE gene promoter. We found that four Sp1binding sites mutation decreased the activity of CSE core promoter significantly. It implied that the transcriptional factor Spl have the crucial potential to regulate the CSE gene expression. Transfection of Spl siRNA together with the pCSE-731(-592/+139) construct led to a stronger activity decrease in reporter activity as compared with the control. And also we found that CSE mRNA and protein were down-regulated by the Spl knocking down. By ChIP assay, the direct evidence supported that more sequence-specific DNA-binding factor Spl were recruited into the proximal region of CSE promoter regulated by PI3K/Akt pathway. Furthermore we have excluded the other regulatory sites, predicted by computer software, such as AP1, Oct-1, V-Myb, FoxD3, HNF-3β, c-Ets and E2F by mutation, which were not crucial to the CSE promoter activity. And furthermore, we observed that LY294002stimulation did not induce the increase of CSE mRNA degradation using mRNA decay analysis. So we could draw a conclusion that inhibition of PI3K/Akt down-regulated CSE promoter activity on the transcription control, in which the transcription factor Spl played a crucial role.Understanding the upstream signaling cascades that regulated the CSE expression will help to clarify the biological functions of the gene, such as cell proliferation, cell cycle and apoptosis are associated with tumor development and progression. So in the third sections of our study, we observed that CSE may promote HCC cell growth due to the proliferation inhibited by knocking down CSE. G1/G0phase arrest and S phase decreasing in cells by CSE down-regulation may be the reason why endogenous H2S could promote cellular proliferation and cell viability, with the altered expression of cell cycle regulatory associated protein p21Waf1/Cip1. Apoptosis, migration and EMT analysis showed that there was no significant change in cells with endogenous or exogenous H2S comparing with their parent ones. These indicate that CSE/H2S system may not involve in apoptosis, migration and EMT process, with the treatment of exogenous H2S or CSE-overexpression or knocking down.In summary, in this article, we have, for the first evidence, demonstrated that PI3K/Akt regulates the CSE expression through Spl on transcriptional level in HCC cell lines. This indicated that CSE may play an important role in carcinogenesis and in which the complicated molecular mechanisms need further research. These data may provide the support for the development of specific CSE inhibitors as efficient anti-cancer drugs in HCC, which is particularly important to understand the effects of PI3K/Akt and CSE on hepatocarcinogenesis. |
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