Abstract
Ras homology GTPase activation protein 6 (Arhgap6), as a member of the rhoGAP family of proteins, performs vital functions on the regulation of actin polymerization at the plasma membrane during several cellular processes. The role of Arhgap6 in the progression and development of cancer remains nearly unknown. This study aimed at exploring the effects of Arhgap6 on cervical carcinoma. Human cervical cancer cells HeLa and SiHa were transduced with a lentivirus targeting Arhgap6 (Arhgap6+), while CaSki and C4-1 cells were transfected with miRNA. Cell proliferation was identified by Cell Counting Kit-8 (CCK-8). Cell cycle distribution and cell apoptosis were identified by flow cytometry. The capacity of cell migration, invasion, and adhesion were detected by Transwell assay. Further, quantitative real-time PCR (qRT-PCR) and western blot were used to analyze the expression levels of Arhgap6 and several tumor-related genes. Co-immunoprecipitation assay was performed to validate the interaction between Arhgap6 and Rac3 (Ras-related C3 botulinum toxin substrate 3). Results showed that Arhgap6 inhibited cell proliferation, migration, invasion, and adhesion of cervical carcinoma, induced cell apoptosis, and caused cell cycle arrest in the G0/G1 phase (n = 3, p < 0.05). Expression of the tumor suppressor genes and oncogenes were up- and down-regulated respectively by Arhgap6, and Rac3 was proved to be the target of Arhgap6. Besides, in in vivo assays, tumor size and weight were destructed in Arhgap6+ athymic nude mouse. This study indicated that Arhgap6 may play a role in the treatment of cervical cancer as a tumor supressor.
Similar content being viewed by others
References
Siegel R, Naishadham D, Jemal A. Cancer statistics. 2012. CA Cancer J Clin. 2012;62:10–29.
Jiménez-Wences H, Peralta-Zaragoza O, Fernández-Tilapa G. Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review). Oncol Rep. 2014;31:2467–76.
World Cancer Report 2014. World Health Organization. 2014;Chapter 5.12, pp.
Newmann SJ, Garner EO. Social inequities along the cervical cancercontinuum: a structured review. Cancer Causes Control. 2005;16:63–70.
Egile C, Rouiller I, Xu XP, et al. Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions. PLoS Biol. 2005;3(11), e383.
Ridley AJ. Rho-related proteins: actin cytoskeleton and cell cycle. Curr Opin Genet Dev. 1995;5:24–30.
Schaefer L, Prakash S, Zoghbi HY. Cloning and characterization of an rho-type GTPase-activating protein gene (ARHGAP6) from the critical region for microphthalmia with linear skin defects. Genomics. 1997;46(2):268–77.
Tribioli C, Droetto S, Bione S, Cesareni G, Torrisi MR, Lotti LV, et al. An X chromosome-linked gene encoding a protein with characteristics of a rhoGAP predominantly expressed in hematopoietic cells. Proc Natl Acad Sci U S A. 1996;93(2):695–9.
Prakash SK, Paylor R, Jenna S, et al. Functional analysis of ARHGAP6, a novel GTPase-activating protein for RhoA. Hum Mol Genet. 2000;9(4):477–88.
Guo F, Liu Y, Huang J, et al. Identification of Rho GTP ase activating protein 6 isoform 1 variant as a new molecular marker in human colorectal tumors. Pathol Oncol Res. 2010;6(3):319–26.
Ochocka AM, Grden M, Sakowicz-Burkiewicz M, et al. Regulation of phospholipase C-delta1 by ARGHAP6, a GTPase-activating protein for RhoA: possible role for enhanced activity of phospholipase C in hypertension. Int J Biochem Cell Biol. 2008;40(10):2264–73.
Davies S, Dai D, Pickett G, et al. Effects of bevacizumab in mouse model of endometrial cancer: defining the molecular basis for resistance. Oncol Rep. 2011;25(3):855–62.
Hu JC, Chan HC, Simmer SG, et al. Amelogenesis imperfecta in two families with defined AMELX delections in ARHGAP6. PLoS ONE. 2012;7(12), e52052.
Etienne MS, Hall A. Rho GTPases in cell biology. Nature. 2002;420(6916):629–35.
Cameron NJ, Sergi CB, Laura MC, et al. ARHGAP8 is a novel member of the RHOGAP family related to ARHGAP1/CAA42GAP/p50RHOGAP: mutation and expression analyses in colorectal and breast cancers. Gene. 2004;336:59–71.
Ching YP, Wong CM, Chan SF, et al. Deleted in liver cancer (DLC) 2 encodes a RhoGAP protein with growth suppressor function and is under expressed in hepatocellular carcinoma. J Biol Chem. 2003;278:10824–30.
Liao YC, Lo SH. Deleted in liver cancer-1 (DLC-1): a tumor suppressor not just for liver. Int J Biochem Cell Biol. 2008;40:843–7.
Furukawa Y, Kawasoe T, Daigo Y, et al. Isolation of a novel human gene, ARHGAP9, encoding a rho-GTPase activating protein. Biochem Biophys Res Commun. 2001;284(3):643–9.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2[−Delta DeltaC(T)] method. Methods. 2001;25:402–8.
Healy KD, Hodgson L, Kim T, et al. DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms. Mol Carcinog. 2008;47(5):326–37.
Kandpal RP. Rho GTPase activating proteins in cancer phenotypes. Curr Protein Pept Sci. 2006;7:355–65.
Leve F, Morgado-Díaz JA. Rho GTPase signaling in the development of colorectal cancer. J Cell Biochem. 2012;113(8):2549–59.
Xu J, Zhou X, Wang J, et al. RhoGAPs attenuate cell proliferation by direct interaction with p53 Tetramerization domain. Cell Rep. 2013;3:1526–38.
Nagaraja GM, Kandpal RP. Chromosome 13q12 encoded Rho GTPase activating protein suppresses growth of breast carcinoma cells, and yeast two-hybrid screen shows its interaction with several proteins. Biochem Biopys Res Commun. 2004;313(3):654–65.
Wong CM, Yam JW, Ching YP, et al. Rho GTPase-activating protein deleted in liver cancer suppressor cell proliferation and invasion in hepatocellular carcinoma. Cancer Res. 2005;65(19):8861–8.
Vega FM, Ridley AJ. Rho GTPases in cancer cell biology. FEBS Lett. 2008;582:2093–101.
Jaffe AB, Hall A. RHO GTPases: biochemistry and biology. Annu Rev Cell Dev Biol. 2005;21:247–69.
Riento K, Totty N, Villalonga P, et al. RhoE function is regulated by ROCK I mediated phosphorylation. EMBO J. 2005;24:1170–80.
Xie J, Bai J, Sheng X et al.: Inhibition of proliferation of human cervical cancer HeLa cells by casticin in vitro. China Oncol.
Yuan J, Yan R, Krämer A, et al. Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene. 2004;23(24):5843–52.
Yuan J, Krämer A, Matthess Y, et al. Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo. Oncogene. 2006;25(12):1753–62.
Tiwari N. EMT as the ultimate survival mechanism of cancer cells. Semin Cancer Biol. 2012;22:194–207.
Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev. 2009;28(1–2):15–33.
Ansieau S. Failsafe program escape and EMT: a deleterious partnership. Semin Cancer Biol. 2011;21:392–6.
Julien S, Puig I, Caretti E, et al. Activation of NF-κB by Akt upregulates Snail expression and induces epithelium mesenchyme transition. Oncogene. 2007;26(53):7445–56.
Kyo S, Sakaguchi J, Ohno S, et al. High Twist expression is involved in infiltrative endometrial cancer and affects patient survival. Hum Pathol. 2006;37(4):43–438.
Peña C, García JM, Larriba MJ, et al. SNAI1 expression in colon cancer related with CDH1 and VDR downregulation in normal adjacent tissue. Oncogene. 2009;28:4375–85.
Olmeda D, Jorda M, Peinado H, et al. Snail silencing effectively suppresses tumour growth and invasiveness. Oncogene. 2007;26(13):1862–74.
Stodden GR, Lindberg ME, King ML, et al. Loss of Cdh1 and Trp53 in the uterus induces chronic inflammation with modification of tumor microenvironment. Oncogene. 2015;34:2471–82.
Van Aken E, De Wever O, Correiada Rocha AS, et al. Defective E-cadherin/catenin complexes in human cancer. Virchows Arch. 2001;439(6):725–51.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Li, J., Liu, Y. & Yin, Y. Inhibitory effects of Arhgap6 on cervical carcinoma cells. Tumor Biol. 37, 1411–1425 (2016). https://doi.org/10.1007/s13277-015-4502-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13277-015-4502-z