Effect of CD59-targeted multi-directional intervention on transmembrane apoptotic signal transduction of cervical cancer HeLa cells

Objective: With GPI-anchored protein CD59 as the target, the effect of the abnormal expression of the CD59 gene on the proliferation and apoptosis of cervical cancer HeLa cells was studied to investigate the mechanism of the malignant proliferation of cervical cancer cells. Materials and Methods: Recombinant plasmid pSUPER-siCD59 (CD59 RNA interference) was transfected into HeLa cells via liposome transfection. The plasmid expressed green fluorescent proteins that enabled the observation of the transfection efficiency and was separately transfected. The co-transfection of a pIRES-WTCD59 (wild-type CD59) plasmid, pIRES-MCD59 (with CD59W40 active site mutation) plasmid, and pLeGFP plasmid helped the direct observation of the transfection efficiency, which was examined via an inverted fluorescence microscope at 24, 48, and 72 hours after transfection. Then, the stable transfected cell lines were screened by G418. Western blot, RT-PCR, and immunofluorescence were used to detect the gene and protein expression of CD59 in the stably transfected HeLa cell lines, and the differences were evaluated. The short peptide seal specific to CD59 was exerted on the HeLa cells. Cell proliferation was detected by MTT colorimetric assay, and apoptosis was detected by TUNEL and flow cytometry. Results: The proliferation activity of the HeLa cells with CD59RNAi, CD59W40 active site mutations, and the short peptide seal specific to CD59 significantly decreased. In particular, the proliferation activity of the HeLa cells treated with a high dose of the short peptide seal specific to CD59 was significantly reduced, while the proliferation activity of the HeLa cells with a high expression of CD59 was significantly enhanced. Conclusion: As an inhibitory regulatory protein in the terminal stage of complement regulation, CD59 can promote the proliferation of cervical cancer HeLa cells via inhibiting the dissolution effect of the complement on the cells, promoting transmembrane activation signal transduction, and inhibiting apoptotic signal transduction. Thus, this study provides a new avenue for the clinical treatment of cervical cancer.

CD59 knockout; Short-peptide seal; RNAi; Hela cell; Proliferation; Apoptosis.

[1] Müller-Eberhard H.J.: “Molecular Organization and Function of the Complement System”. Annu. Rev. Biochem., 1988, 57, 321-347.

[2] Esser A.F.: “The membrane attack complex of complement. Assembly, structure and cytotoxic activity”. Toxicology, 1994, 87, 229-247.

[3] Meri S., Morgan B.P., Davies A., Daniels R.H., Olavesen M.G., Waldmann H., et al.: “Human protectin (CD59), an 18,000-20,000 MW complement lysis restricting factor, inhibits C5b-8 catalysed insertion of C9 into lipid bilayers”. Immunology, 1990, 711-9.

[4] Watts M.J., Dankert J.R., Morgan B.P.: “Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids”. Biochem. J., 1990, 265,471- 477.

[5] Farkas I., Baranyi L., Ishikawa Y., Okada N., Bohata C., Budai D., et al.: “CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9”. the Journal of Physiology, 2002, 539, 537-545.

[6] Davies A., Simmons D.L., Hale G., Harrison R.A., Tighe H., Lach-mann P.J., et al.: “CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.”. the Journal of Experimental Medicine, 1989, 170, 637-654.

[7] Sugita Y., Tobe T., Oda E., Tomita M., Yasukawa K., Yamaji N., et al.: “Molecular cloning and characterization of MACIF, an inhibitor of membrane channel formation of complement”. J. Biochem. (Tokyo), 1990, 106, 555-557.

[8] Fletcher C.M., Harrison R.A., Lachmann P.J., Neuhaus D.: “Struc-ture of a soluble, glycosylated form of the human complement regulatory protein CD59”. Structure, 1994, 2, 185-199.

[9] Kieffer B., Driscoll P. C., Campbell I. D., Willis A. C., van der Merwe P. A., Davis S. J.: “Three-Dimensional Solution Structure of the Extracellular Region of the Complement Regulatory Protein CD59, a New Cell-Surface Protein Domain Related to Snake Venom Neurotoxins”. Biochemistry (Mosc.), 1994, 33, 4471-4482.

[10] Petranka J., Zhao J., Norris J., Tweedy N.B., Ware R.E., Sims P.J., et al.: “Structure-Function Relationships of the Complement Regulatory Protein, CD59”. Blood Cells. Mol. Dis., 1996, 22, 281-296.

[11] Smith J.S., Brewer N.T., Saslow D., Alexander K., Chernofsky M. R., Crosby R., et al.: “Recommendations for a national agenda to substantially reduce cervical cancer”. Cancer Causes & Con-trol : CCC, 2014, 24, 1583-1593.

[12] Lammers T., Hennink W.E., Storm G.: “Tumour-targeted nanomedicines: principles and practice”. Br. J. Cancer, 2008, 99, 392- 397.

[13] Penate Medina O., Haikola M., Tahtinen M., Simpura I., Kaukinen S., Valtanen H., et al.: “Liposomal Tumor Targeting in Drug Delivery Utilizing MMP-2- and MMP-9-Binding Ligands”. Journal of Drug Delivery, 2011, 2011, 1-9.

[14] Molek P., Strukelj B., Bratkovic T.: “Peptide Phage Display as a Tool for Drug Discovery: Targeting Membrane Receptors”. Molecules, 2011, 16, 857-887.

[15] Wittrup A., Lieberman J.: “Knocking down disease: a progress report on siRNA therapeutics”. Nature Reviews Genetics, 2015, 16, 543- 552.

[16] Sharei A., Zoldan J., Adamo A., Sim W. Y., Cho N., Jackson E., et al.: “A vector-free microfluidic platform for intracellular delivery”. Proc. Natl. Acad. Sci. U. S. a., 2013, 110, 2082-2087.

[17] Chen S., Caragine T., Cheung N.K., Tomlinson S.: “CD59 ex-pressed on a tumor cell surface modulates decay-accelerating factor expression and enhances tumor growth in a rat model of human neuroblastoma”. Cancer Res, 2017, 60, 3013-3118.

[18] Gelderman K.A., Tomlinson S., Ross G.D., Gorter A.: “Comple-ment function in mAb-mediated cancer immunotherapy”. Trends Immunol., 2004, 25, 158-164.

[19] Huang Y., Smith C.A., Song H., Morgan B.P., Abagyan R., Tomlinson S.: “Insights into the human CD59 complement binding interface toward engineering new therapeutics”. the Journal of Biological Chemistry, 2005, 280, 34073-34079.

[20] Nakano Y., Tozaki T., Kikuta N., Tobe T., Oda E., Miura N., et al.: “Determination of the active site of CD59 with synthetic piptides”. Mol Immunol, 1995, 32, 241-247.

[21] Bodian D.L., Davis S.J., Morgan B.P., Rushmere N.K.: “Mutational Analysis of the Active Site and Antibody Epitopes of the Complement-inhibitory Glycoprotein, CD59”. J. Exp. Med., 1997, 185, 507-516.

[22] Yu J., Abagyan R., Dong S., Gilbert A., Nussenzweig V., Tomlinson S.: “Mapping the active site of CD59”. the Journal of Experimental Medicine, 1997, 185, 745-753.

[23] BRANNEN C., SODETZ J.: “Incorporation of human complement C8 into the membrane attack complex is mediated by a binding site located within the C8β MACPF domain”. Mol. Immunol., 2007, 44, 960- 965.

[24] De Groot A.S., Martin W.: “Reducing risk, improving outcomes: Bioengineering less immunogenic protein therapeutics”. Clin. Immunol., 2009, 131, 189-201.

[25] Liu J., Liu J., Chu L., Wang Y., Duan Y., Feng L., et al.: “Novel peptide-dendrimer conjugates as drug carriers for targeting nonsmall cell lung cancer”. Int. J. Nanomedicine, 2011, 659-69.

[26] Lee T., Lin C., Kuo S., Chang D., Wu H.: “Peptide-Mediated Targeting to Tumor Blood Vessels of Lung Cancer for Drug Delivery”. Cancer Res., 2007, 67, 10958-10965.

[27] Yang W., Luo D., Wang S., Wang R., Chen R., Liu Y., et al.: “TMTP1, a Novel Tumor-Homing Peptide Specifically Targeting Metastasis”. Clin. Cancer Res., 2008, 14, 5494-5502.

[28] Chen B., Cao S., Zhang Y., Wang X., Liu J., Hui X., et al.: “A novel peptide (GX1) homing to gastric cancer vasculature inhibits angiogenesis and cooperates with TNF alpha in anti-tumor therapy”. Bmc Cell Biol., 2009, 1063.

[29] Wu X., Yan Q., Huang Y., Huang H., Su Z., Xiao J., et al.: “Isolation of a novel basic FGF-binding peptide with potent antiangiogenetic activity”. J. Cell. Mol. Med., 2010, 14, 351-356.

[30] Negi S.S., Braun W.: “Automated Detection of Conformational Epi-topes Using Phage Display Peptide Sequences”. Bioinformatics and Biology Insights, 2009, 3, BBI.S2745.

[31] Waisberg J., De Souza Viana L., Affonso Junior R.J., Silva S.R.M., Denadai M.V.A., Margeotto F.B., et al.: “Overexpression of the IT-GAV gene is associated with progression and spread of colorectal cancer”. Anticancer Res., 2014, 34, 5599-5607.

[32] Howell R.C., Revskaya E., Pazo V., Nosanchuk J.D., Casadevall A., Dadachova E.: “Phage Display Library Derived Peptides that Bind to Human Tumor Melanin as Potential Vehicles for Targeted Radionuclide Therapy of Metastatic Melanoma”. Bioconjug. Chem., 2007, 18, 1739-1748.