Glucosylceramide synthase regulates the proliferation and apoptosis of liver cells in vitro by Bcl‑2/Bax pathway

Li,Jun‑Feng; Zheng,Su‑Jun; Wang,Li‑Li; Liu,Shuang; Ren,Feng; Chen,Yu; Bai,Li; Liu,Mei; Duan,Zhong‑Ping

doi:10.3892/mmr.2017.7580

Glucosylceramide synthase regulates the proliferation and apoptosis of liver cells in vitro by Bcl‑2/Bax pathway

Authors:
- Jun‑Feng Li
- Su‑Jun Zheng
- Li‑Li Wang
- Shuang Liu
- Feng Ren
- Yu Chen
- Li Bai
- Mei Liu
- Zhong‑Ping Duan
View Affiliations

Affiliations: The First Clinical Medical School of Lanzhou University, Lanzhou, Gansu 730000, P.R. China, Artificial Liver Center, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, P.R. China, Institute of Liver Diseases, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, P.R. China
Published online on: September 21, 2017 https://doi.org/10.3892/mmr.2017.7580
Pages: 7355-7360
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Our previous study found that glucosylceramide, a type of sphingolipids, was associated with liver inflammation and fibrosis. Glucosylceramide is generated by glucosylceramide synthase (GCS), which is encoded by the UDP‑glucose ceramide glucosyltransferase (UGCG) gene. GCS is a key enzyme to regulate the physiological activity of cells. However, the role of GCS in hepatic cells remains unclear. The aim of the present study was to explore the mechanism of GCS in the proliferation and apoptosis of liver cells. Following the interference of expression of GCS in vitro by UGCG small interfering (si)RNA, the MTT method was performed to detect the proliferation of HL‑7702 hepatocytes, and ELISA was used to determine the concentration of tumor necrosis factor (TNF) α and cytochrome c in the supernatant of culture system. Fluorescence microscopy was used to observe the apoptosis of liver cells stained by Annexin V‑fluorescein isothiocyanate/propidium iodide. Reverse transcription‑quantitative polymerase chain reaction was used to detect the gene expression apoptosis regulator Bcl‑2 (Bcl‑2), apoptosis regulator Bax (Bax) and caspase-3. Western blot analysis was used to detect the expression of caspase-3 protein in the liver cells. Following treatment with UGCG siRNA for 24 h, the proliferation of HL‑7702 hepatocytes was significantly inhibited when compared with the transfection reagent group. Furthermore, the early and advanced apoptosis of liver cells showed an increasing trend. Additionally, concentrations of TNF α and cytochrome c showed no significant difference between the UGCG siRNA and transfection reagent groups. Compared with the transfection reagent group, Bcl‑2 mRNA expression decreased, and Bax and caspase-3 mRNA expression increased in the UGCG siRNA transfection group. The protein expression level of caspase-3 showed increased in hepatocytes following the treatment with UGCG siRNA. In conclusion, the metabolic changes of sphingolipids caused by the lack of GCS may be involved in the proliferation and apoptosis of liver cells through the Bcl‑2/Bax signaling pathway.

View Figures

View References

1	Luedde T and Schwabe RF: NF-κB in the liver-linking injury, fibrosis and hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 8:108–118. 2011. View Article : Google Scholar : PubMed/NCBI
2	Lee YA, Wallace MC and Friedman SL: Pathobiology of liver fibrosis: A translational success story. Gut. 64:830–841. 2015. View Article : Google Scholar : PubMed/NCBI
3	Heymann F and Tacke F: Immunology in the liver-from homeostasis to disease. Nat Rev Gastroenterol Hepatol. 13:88–110. 2016. View Article : Google Scholar : PubMed/NCBI
4	Hannun YA and Obeid LM: Principles of bioactive lipid signalling: Lessons from sphingolipids. Nat Rev Mol Cell Biol. 9:139–150. 2008. View Article : Google Scholar : PubMed/NCBI
5	Nojima H, Freeman CM, Gulbins E and Lentsch AB: Sphingolipids in liver injury, repair and regeneration. Biol Chem. 396:633–643. 2015. View Article : Google Scholar : PubMed/NCBI
6	Li JF, Qu F, Zheng SJ, Ren JY, Wu HL, Liu M, Liu H, Ren F, Chen Y, Zhang JL and Duan ZP: Plasma sphingolipids as potential indicators of hepatic necroinflammation in patients with chronic hepatitis C and normal alanine aminotransferase level. PLoS One. 9:e950952014. View Article : Google Scholar : PubMed/NCBI
7	Li JF, Qu F, Zheng SJ, Wu HL, Liu M, Liu S, Ren Y, Ren F, Chen Y, Duan ZP and Zhang JL: Elevated plasma sphingomyelin (d18:1/22:0) is closely related to hepatic steatosis in patients with chronic hepatitis C virus infection. Eur J Clin Microbiol Infect Dis. 33:1725–1732. 2014. View Article : Google Scholar : PubMed/NCBI
8	Li JF, Qu F, Zheng SJ, Ren F, Wu HL, Liu M, Ren JY, Chen Y, Duan ZP and Zhang JL: Plasma sphingolipids: Potential biomarkers for severe hepatic fibrosis in chronic hepatitis C. Mol Med Rep. 12:323–330. 2015. View Article : Google Scholar : PubMed/NCBI
9	Zheng SJ, Qu F, Li JF, Zhao J, Zhang JY, Liu M, Ren F, Chen Y, Zhang JL and Duan ZP: Serum sphingomyelin has potential to reflect hepatic injury in chronic hepatitis B virus infection. Int J Infect Dis. 33:149–155. 2015. View Article : Google Scholar : PubMed/NCBI
10	Zhang JY, Qu F, Li JF, Liu M, Ren F, Zhang JY, Bian DD, Chen Y, Duan ZP, Zhang JL and Zheng SJ: Up-regulation of plasma hexosylceramide (d18:1/18:1) contributes to genotype 2 virus replication in chronic hepatitis C: A 20-year cohort study. Medicine (Baltimore). 95:e37732016. View Article : Google Scholar : PubMed/NCBI
11	Garcia-Ruiz C, Morales A and Fernández-Checa JC: Glycosphingolipids and cell death: One aim, many ways. Apoptosis. 20:607–620. 2015. View Article : Google Scholar : PubMed/NCBI
12	Merrill AH Jr: Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem Rev. 111:6387–6422. 2011. View Article : Google Scholar : PubMed/NCBI
13	Liu YY, Hill RA and Li YT: Ceramide glycosylation catalyzed by glucosylceramide synthase and cancer drug resistance. Adv Cancer Res. 117:59–89. 2013. View Article : Google Scholar : PubMed/NCBI
14	Haynes CA, Allegood JC, Park H and Sullards MC: Sphingolipidomics: Methods for the comprehensive analysis of sphingolipids. J Chromatogr B Analyt Technol Biomed Life Sci. 877:2696–2708. 2009. View Article : Google Scholar : PubMed/NCBI
15	Morales A, Lee H, Goñi FM, Kolesnick R and Fernandez-Checa JC: Sphingolipids and cell death. Apoptosis. 12:923–939. 2007. View Article : Google Scholar : PubMed/NCBI
16	Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, et al: Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 380:75–79. 1996. View Article : Google Scholar : PubMed/NCBI
17	Brooks C and Dong Z: Regulation of mitochondrial morphological dynamics during apoptosis by Bcl-2 family proteins: A key in Bak? Cell Cycle. 6:3043–3047. 2007. View Article : Google Scholar : PubMed/NCBI
18	Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479–489. 1997. View Article : Google Scholar : PubMed/NCBI
19	Pan G, O'Rourke K and Dixit VM: Caspase-9, Bcl-XL, and Apaf-1 form a ternary complex. J Biol Chem. 273:5841–5845. 1998. View Article : Google Scholar : PubMed/NCBI
20	Mari M and Fernández-Checa JC: Sphingolipid signalling and liver diseases. Liver Int. 27:440–450. 2007. View Article : Google Scholar : PubMed/NCBI
21	García-Ruiz C, Colell A, Marí M, Morales A, Calvo M, Enrich C and Fernández-Checa JC: Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice. J Clin Invest. 111:197–208. 2003. View Article : Google Scholar : PubMed/NCBI
22	Elmore S: Apoptosis: A review of programmed cell death. Toxicol Pathol. 35:495–516. 2007. View Article : Google Scholar : PubMed/NCBI
23	Armstrong JS: Mitochondrial membrane permeabilization: The sine qua non for cell death. Bioessays. 28:253–260. 2006. View Article : Google Scholar : PubMed/NCBI
24	Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, Inohara H, Kubo T and Tsujimoto Y: Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature. 434:652–658. 2005. View Article : Google Scholar : PubMed/NCBI
25	Gogvadze V, Orrenius S and Zhivotovsky B: Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta. 1757:639–647. 2006. View Article : Google Scholar : PubMed/NCBI