RAC1 is a small-molecule G protein that regulates multiple cell cycle, cytoskeletal reorganization, cell migration, and apoptosis. FADD-dependent TRAIL can promote tumor metastasis through RAC1 and PI3K, and down-regulating RAC1 expression can reduce FasL-induced apoptosis. In addition, RIP1 bound to GTP acts as an activating protein for RAC1 and is involved in cytoskeletal reorganization. TRAF6 promotes migration and metastasis by regulating the RAS pathway in tumors. Thus, it is necessary to understand the interaction between RAC1 and TRAF6 as well as FADD and RIP1. In this study, we cultured hepatoma SK-Hep1 cells in vitro , specifically blocked the necroptosis pathway with Nec-1, and silenced FADD, RIP1 and TRAF6 gene expression using RNAi technology. At the same time, the expression of RAC1 was evaluated separately using RT-PCR and Western blot. The hepatoma SK-Hep1 cells survival rate was highest when the concentration of Nec-1 was 60 μM and the concentration of Z-vad-fmk was 20 μM. And the apoptosis rate of the transfected RAC1 siRNA cells was 3.59% compared with transfected siRNA cells 10.01% which was significantly decreased (P < 0.01). RAC1 could promote the occurrence of apoptosis in SK-Hep1 cells. RAC1 expression was suppressed in both protein and gene level in SK-Hep1 cells when the TRAF6 gene was silenced, but there was no significant change in RAC1 gene and protein expression when FADD and RIP1 genes were silenced. TRAF6 affects RAC1 expression and apoptosis in SK-Hep1 cells, while the FADD and RIP1 genes do not affect the role of RAC1. The TRAF6 gene is an important target in liver cancer cells.
KeywordsApoptosisRAC1TRAF6FADDRIP1SK-Hep1 Cells
Liu, L., Li, L., Zhou, S., et al. (2014) Familial Correlations of Onset Age of Hepatocellular Carcinoma a Population-Based Case-Control Family Study. PLoS One, 9, e108391. https://doi.org/10.1371/journal.pone.0108391
Wang, R.C., Huang, C.Y., Pan, T.L., et al. (2015) Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells. PLoS One, 10, e0139232. https://doi.org/10.1371/journal.pone.0139232
Liu, Y., Ren, F., Rong, M., Luo, Y., Dang, Y. and Chen, G. (2015) Association between Underexpression of Microrna-203 and Clinicopathological Significance in Hepatocellular Carcinoma Tissues. Cancer Cell International, 15, 62. https://doi.org/10.1186/s12935-015-0214-0
Hai, H., Tamori, A. and Kawada, N. (2014) Role of Hepatitis B Virus DNA Integration in Human Hepatocarcinogenesis. World Journal of Gastroenterology, 20, 6236-6243. https://doi.org/10.3748/wjg.v20.i20.6236
Papatheodoridis, G.V., Dalekos, G.N., Yurdaydin, C., et al. (2015) Incidence and Predictors of Hepatocellular Carcinoma in Caucasian Chronic Hepatitis B Patients Receiving Entecavir or Tenofovir. Journal of Hepatology, 62, 363-670. https://doi.org/10.1016/j.jhep.2014.08.045
Ma, L., Ji, L., Yu, Y. and Wang, J. (2015) Novel Molecular Targets for Diagnosis and Treatment of Hepatocellular Carcinoma. Discovery Medicine, 19, 7-14.
Fang, P., Hu, J.H., Cheng, Z.G., Liu, Z.F., Wang, J.L. and Jiao, S.C. (2012) Efficacy and Safety of Bevacizumab for the Treatment of Advanced Hepatocellular Carcinoma: A Systematic Review of Phase II Trials. PLoS One, 7, e49717. https://doi.org/10.1371/journal.pone.0049717
Wang, J.Y., Yu, P., Chen, S., et al. (2013) Activation of Rac1 GTPase Promotes Leukemia Cell Chemotherapy Resistance, Quiescence and Niche Interaction. Molecular Oncology, 7, 907-916. https://doi.org/10.1016/j.molonc.2013.05.001
Marei, H. and Malliri, A. (2016) Rac1 in Human Diseases: The Therapeutic Potential of Targeting Rac1 Signaling Regulatory Mechanisms. Small GTPases, 8, 1-25.
Murga, C., Zohar, M., Teramoto, H. and Gutkind, J.S. (2002) Rac1 and RhoG Promote Cell Survival by the Activation of PI3K and Akt, Independently of Their Ability to Stimulate JNK and NF-kappaB. Oncogene, 21, 207-216. https://doi.org/10.1038/sj.onc.1205036
Gastonguay, A., Berg, T., Hauser, A.D., Schuld, N., Lorimer, E. and Williams, C.L. (2012) The Role of Rac1 in the Regulation of NF-kappa B Activity, Cell Proliferation, and Cell Migration in Non-Small Cell Lung Carcinoma. Cancer Biology & Therapy, 13, 647-56. https://doi.org/10.4161/cbt.20082
Tong, L. and Tergaonkar, V. (2014) Rho Protein GTPases and Their Interactions with NFκB: Crossroads of Inflammation and Matrix Biology. Bioscience Reports, 34, e00115. https://doi.org/10.1042/BSR20140021
Chang, F., Lemmon, C., Lietha, D., Eck, M. and Romer, L. (2011) Tyrosine Phosphorylation of RAC1: A Role in Regulation of Cell Spreading. PLoS ONE, 6, e28587. https://doi.org/10.1371/journal.pone.0028587
Huang, L., Liu, Q., Zhang, L., et al. (2015) Encephalomyocarditis Virus 3C Protease Relieves TANK Inhibitory Effect on TRAF6-Mediated NF-kappa B Signaling through Cleavage of TANK. The Journal of Biological Chemistry, 290, 27618-27632. https://doi.org/10.1074/jbc.M115.660761
Kanzaki, H., Shinohara, F., Kanako, I., et al. (2016) Molecular Regulatory Mechanisms of Osteoclastogenesis through Cytoprotective Enzymes. Redox Biology, 8, 186-191. https://doi.org/10.1016/j.redox.2016.01.006
Han, Q., Yao, F., Zhong, C. and Zhao, H. (2014) TRAF6 Promoted the Metastasis of Esophageal Squamous Cell Carcinoma. Tumour Biology, 35, 715-721. https://doi.org/10.1007/s13277-013-1098-z
Festjens, N., Vanden Berghe, T., Cornelis, S. and Vandenabeele, P. (2007) RIP1, a Kinase on the Crossroads of a Cell’s Decision to Live or Die. Cell Death & Differentiation, 14, 400-410. https://doi.org/10.1038/sj.cdd.4402085
Vandenabeele, P., Declercq, W., van Herreweghe, F. and Vanden Berghe, T. (2010) The Role of the Kinases RIP1 and RIP3 in TNF-Induced Necrosis. Science Signaling, 3, re4. https://doi.org/10.1126/scisignal.3115re4
Galluzzi, L., Aaronson, S.A., Abrams, J., et al. (2009) Guidelines for the Use and Interpretation of Assays for Monitoring Cell Death in Higher Eukaryotes. Cell Death & Differentiation, 16, 1093-1107. https://doi.org/10.1038/cdd.2009.44
Hartwig, T., Montinaro, A., von Karstedt, S., et al. (2017) The Trail-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2. Molecular Cell, 65, 730-742. https://doi.org/10.1016/j.molcel.2017.01.021
Jilkina, O. and Bhullar, R.P. (2006) A Serine Kinase Associates with the RAL GTPase and Phosphorylates RAL-Interacting Protein 1. Biochimica et Biophysica Acta, 1763, 948-957. https://doi.org/10.1016/j.bbamcr.2006.07.007
Li, N., Jiang, J., Fu, J., et al. (2016) Targeting Interleukin-1 Receptor-Associated Kinase 1 for Human Hepatocellular Carcinoma. Journal of Experimental & Clinical Cancer Research, 35, 140. https://doi.org/10.1186/s13046-016-0413-0
Nie, F., Zhao, S.Y., Song, F.X. and Li, P.W. (2014) Changes of Cytoskeleton and Cell Cycle in Lovo Cells via Deletion of RAC1. Cancer Biomarkers, 14, 335-342. https://doi.org/10.3233/CBM-140408
Han, J., Weisbrod, R.M., Shao, D., et al. (2016) The Redox Mechanism for Vascular Barrier Dysfunction Associated with Metabolic Disorders: Glutathionylation of RAC1 in Endothelial Cells. Redox Biology, 9, 306-319. https://doi.org/10.1016/j.redox.2016.09.003
Wang, J., Rao, Q., Wang, M., et al. (2009) Overexpression of RAC1 in Leukemia Patients and Its Role in Leukemia Cell Migration and Growth. Biochemical and Biophysical Research Communications, 386, 769-774. https://doi.org/10.1016/j.bbrc.2009.06.125
Mizukawa, B., Wei, J., Shrestha, M., et al. (2011) Inhibition of Rac GTPase Signaling and Downstream Prosurvival Bcl-2 Proteins as Combination Targeted Therapy in MLL-AF9 Leukemia. Blood, 118, 5235-5245. https://doi.org/10.1182/blood-2011-04-351817
Cullen, S.P., Henry, C.M., Kearney, C.J., et al. (2013) Fas/CD95-Induced Chemokines Can Serve as “Find-Me” Signals for Apoptotic Cells. Molecular Cell, 49, 1034-1038. https://doi.org/10.1016/j.molcel.2013.01.025
Tenev, T., Bianchi, K., Darding, M., et al. (2011) The Ripoptosome, a Signaling Platform That Assembles in Response to Genotoxic Stress and Loss of IAPs. Molecular Cell, 43, 432-448. https://doi.org/10.1016/j.molcel.2011.06.006
Bonnet, M.C., Preukschat, D., Welz, P.S., et al. (2011) The Adaptor Protein Fadd Protects Epidermal Keratinocytes from Necroptosis in Vivo and Prevents Skin Inflammation. Immunity, 35, 572-582. https://doi.org/10.1016/j.immuni.2011.08.014
Lu, J.V., Weist, B.M., van Raam, B.J., et al. (2011) Complementary Roles of Fas-Associated Death Domain (FADD) and Receptor Interacting Protein Kinase-3 (RIPK3) in T-Cell Homeostasis and Antiviral Immunity. Proceedings of the National Academy of Sciences of the United States, 108, 15312-15317. https://doi.org/10.1073/pnas.1102779108
Lee, E., Seo, J., Jeong, M., Lee, S. and Song, J. (2012) The Roles of FADD in Extrinsic Apoptosis and Necroptosis. BMB Reports, 45, 496-508. https://doi.org/10.5483/BMBRep.2012.45.9.186
Meng, Q., Zheng, M., Liu, H., et al. (2012) TRAF6 Regulates Proliferation, Apoptosis, and Invasion of Osteosarcoma Cell. Molecular and Cellular Biochemistry, 371, 177-186. https://doi.org/10.1007/s11010-012-1434-4
Chiu, H.W., Lin, S.W., Lin, L.C., et al. (2015) Synergistic Antitumor Effects of Radiation and Proteasome Inhibitor Treatment in Pancreatic Cancer through the Induction of Autophagy and the Down-Regulation of TRAF6. Cancer Letters, 365, 229-239. https://doi.org/10.1016/j.canlet.2015.05.025
Li, J.J., Luo, J., Lu, J.N., et al. (2016) Relationship between TRAF6 and Deterioration of HCC: An Immunohistochemical and in Vitro Study. Cancer Cell International, 16, 76. https://doi.org/10.1186/s12935-016-0352-z
Zhang, P., Yue, S., Jiang, Y., Zhang, X. and Liu, Y. (2013) TNF Receptor-Associated Factor 6 Regulates Proliferation, Apoptosis, and Invasion of Glioma Cells. Molecular and Cellular Biochemistry, 377, 87-96. https://doi.org/10.1007/s11010-013-1573-2
Sun, H., Li, X., Fan, L., Wu, G., Li, M. and Fang, J. (2014) TRAF6 Is Upregulated in Colon Cancer and Promotes Proliferation of Colon Cancer Cells. The International Journal of Biochemistry & Cell Biology, 53, 195-201. https://doi.org/10.1016/j.biocel.2014.04.010