title: Endogenous cystathionine-gamma-lyase/hydrogen sulfide pathway regulates apoptosis of HepG2 cells.
abstract: This study is to investigate the role of endogenous CSE/H2S in regulating apoptosis of HepG2 cells. MTT and Trypan blue assay were performed to determine the effect of CSE inhibitor PAG and CSE siRNA on proliferation of HepG2. Production of H2S from HepG2 cells was assessed spectrophotometrically using N, N-dimethyl-p-phenylenediamine-dihydrochloride. Cells apoptosis was detected by means of double staining of Hoechst 33342 and PI with Array Scan V(TI)HCS600 High-Contents. Dihydroethidine (DHE) and 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to determine intracellular superoxide anion and ROS level. Reduced glutathione (GSH) was determined by OxiSelect Total Glutathione Assay Kit. Recombinant plasmid pcDNA 3.1/myc-His(-)-CSE was constructed and transfected into 293T cells to rescue the ROS and GSH level to further investigate the effect of CSE/H2S on ROS and GSH. Western blotting was performed to test the effect of CSE siRNA on expression of activated caspase 3 and p-AKT and Nrf2 protein. The results showed that PAG and CSE siRNA could significantly decrease the production of H2S in HepG2 cells and inhibit the proliferation of HepG2 cells at a dose-dependent and time-dependent manner, respectively. PAG and CSE siRNA could promote the cell apoptosis of HepG2 cells. Moreover, PAG and CSE siRNA induced increased ROS generation and depletion of the critical antioxidant GSH and recombinant plasmid pcDNA 3.1/myc-His(-)-CSE rescued the level of ROS and GSH. Meanwhile, CSE siRNA increased the expression of activated caspase 3, but CSE siRNA did not affect the expression of p-AKT and Nrf2. These results suggested that the CSE/H2S pathway was involved in suppression of HepG2 cell growth and promoted apoptosis of HepG2 cells in an oxidative stress-dependent manner.
authors.affiliation: Institute of Chinese Materia Medica, College of Pharmacy, Henan University, Kaifeng 475004, China. wtx1975@126.com
authors.forename: Tian-Xiao
authors.initials: TX
authors.lastname: Wang
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Shi
authors.forename: Ying-Hua
authors.initials: YH
authors.lastname: Liu
mesh_terms: D000480:Alkynes / Q000494:pharmacology*; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation; D003542:Cystathionine gamma-Lyase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D006862:Hydrogen Sulfide / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D010957:Plasmids; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D000480:Alkynes; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C009055:propargylglycine; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; D053148:Caspase 3; D003542:Cystathionine gamma-Lyase; D005978:Glutathione; D005998:Glycine; D006862:Hydrogen Sulfide