Anticarcinogenic activity of Cl-Amidine on non-small cell lung cancer

Year 2024, Volume: 41 Issue: 1, 80 - 85, 29.03.2024

Pınar Naile Öğüten , Selin Engür Öztürk Miriş Dikmen

Abstract

Peptidyl arginine deiminases (PADs) are enzymes that convert arginine to citrulline. They play a role in embryogenesis and cell signal transduction activities. However, it has been determined that excess or dysregulated PAD levels increase in many diseases and may be associated with diseases. Cl-amidine, used as a PAD inhibitor, has been shown to suppress PAD activity and reduce the severity of developing clinical pictures in animal experimental models such as rheumatoid arthritis and ulcerative colitis. Anti-proliferative activities have also been reported due to their cytotoxic effects on various cancer cell lines. In our study, the anti-carcinogenic activity of Cl-amidine was investigated in A549 human non-small cell lung cancer cells. The anti-proliferative effects of Cl-amide on A549 cells were determined by the WST-1 method. Apoptotic effects were analyzed on the flow cytometry device with Annexin V-PI, caspase-3 activation and mitochondrial membrane depolarization (JC-1) methods. It was determined by the WST-1 method that Cl-amidine had a significant anti-proliferative effect on A549, depending on time and concentration. The apoptotic effects analyzed by Annexin V-PI and JC-1 methods were found to be especially significant at the 48th hour. It has been determined that Cl-amidine, in particular, causes cells to undergo apoptosis by increasing mitochondrial depolarization. In addition, it was observed that Cl-amidine had a less cytotoxic effect on CCD-19Lu healthy lung cells than cisplatin, one of the existing treatment agents. This study shows that Cl-amidine has the potential to be an important chemotherapy agent in the lung cancer treatment, as it has a significant anti-proliferative and apoptotic effect on A549 cells, as well as being less cytotoxic when compared to existing cisplatin treatment on healthy lung cells. We believe that Cl-amidine will contribute to the discovery of new anti-cancer drugs by investigating it with single or combined agents.

Keywords

Peptidyl arginine deiminase inhibitor, Cl-amidine, lung cancer, A549 cell line

Supporting Institution

Scientific Research Projects Coordination Unit of Ordu University

Project Number

AP-1409

References

• Herbst RS, Heymach JV, Lippman SM. Lung cancer. The New England journal of medicine 2008;359(13): 1367-1380.
• Jones JE, Causey CP, Knuckley B, Slack-Noyes JL, Thompson PR. Protein arginine deiminase 4 (PAD4): Current understanding and future therapeutic potential. Curr Opin Drug Discov Devel. 2009; 12(5): 616-27.
• Chang X, Han J, Pang L, Zhao Y, Yang Y, Shen Z. Increased PADI4 expression in blood and tissues of patients with malignant tumors. BMC Cancer. 2009; 9: 40.
• Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays. 2003; 25(11): 1106-18.
• Chang X, Han J. Expression of peptidylarginine deiminase type 4 (PAD4) in various tumors. Mol Carcinog. 2006; 45(3): 183-96.
• Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 1998; 101: 273–281.
• Moscarello MA, Mastronardi FG, Wood DD. The role of citrullinated proteins suggests a novel mechanism in the pathogenesis of multiple sclerosis. Neurochem Res. 2007; 32: 251–256.
• Ishigami A, Ohsawa T, Hiratsuka M, Taguchi H, Kobayashi S, Saito Y, Murayama S, Asaga H, Toda T, Kimura N, Maruyama N. Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease. J Neurosci Res 2005; 80: 120–128.
• Chen CC, Isomoto H, Narumi Y, Sato K, Oishi Y, Kobayashi T, Yanagihara K, Mizuta Y, Kohno S, Tsukamoto K. Haplotypes of PADI4 susceptible to rheumatoid arthritis are also associated with ulcerative colitis in the Japanese population. Clin Immunol. 2008; 126: 165–171.
• Carmicheal J, DeGrafit WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of tetrazolium-based semiautomated colorimetric assay: assasment of chemosensivity testing. Cancer Research. 1987; 47: 936-942.
• Lian Z, Niwa K, Gao J, Tagami K, Mori H, Tamaya T. Association of cellular apoptosis with anti-tumor effects of the Chinese herbal complex in endocrine-resistant cancer cell line. Cancer Detection and prevention. 2003; 27: 147-154.
• Boğa C. Akım sitometri ile apoptozis tayini. Sözer O, Ed. Klinik ve pratikte akım sitometri. 1. baskı, Haberal Eğitim Vakfı. 2009; 155-158.
• Gatti R, Belletti S, Orlandini G, Bussolati O, Dall’asta V, Gazzola, G.C. Comparison of Annexin V and calcein-AM as early vital markers of apoptosis in adherent cells by confocal laser microscopy. J. Histochem. Cytochem. 1998; 46: 895–900.
• Kopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers, NH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994; 84: 1415–1420.
• Overbeeke R, Steffens-Nakken H, Vermes I, Reutelingsperger C, Hanen C. Early features of apoptosis detected by four different flow cytometry assays. Apoptosis. 1998; 3: 115.
• Zhang G, Gurtu V, Kain S.R, Yan G. Early detection of apoptosis using a fluorescent conjugate of Annexin V. Biotechniques. 1997; 23: 525–531.
• Tesarik J, Greco E, Cohen-Bacrie P, Mendoza C. Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol. Hum. Reprod. 1998; 4: 757.
• Dispersyn G, Nuydens R, Connors R, Borgers M, Geerts H. Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane poteintial depolarization in PC12 cells. Biochimica et Biophysica Acta. 1999; 1428: 357-371.
• Chen TK, Ming JH, Bing CC, Chien CC, Che MT, Shiow LP, Chien HL. Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction. Toxicology Letters. 2008; 177: 48-58.
• Iijima T. Mitochondrial membrane potential and ischemic neuronal death. Neuroscience Research. 2006; 55: 234-243.
• Yao G, Ling L, Luan J, Ye D, Zhu P. Nonylphenol induces apoptosis of Jurkat cells by a caspase-8 dependent mechanism. International Immunopharmacology. 2007; 7: 444-453.
• Kaufmann SH, Hengartner MO. Programmed cell death: alive and well in the new millennium. Trends Cell Biol. 2001;11(12): 526-534.
• Slee EA, Adrain C, Martın SJ. Executioner caspases-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. The Journal of Biological Chemistry. 2001; 276(10): 7320-7326.
• Jones JE, Slack JL, Fang P, Zhang X, Subramanian V, Causey CP, Coonrod SA, Guo M, Thompson PR. Synthesis and screening of a haloacetamidine containing library to identify PAD4 selective inhibitors. ACS Chem Biol. 2012; 20;7(1): 160-165.
• Slack JL, Causey CP, Thompson PR. Protein arginine deiminase 4: a target for an epigenetic cancer therapy. Cell Mol Life Sci. 2011; 68(4): 709-20.
• Cui X, Witalison EE, Chumanevich AP, Chumanevich AA, Poudyal D, Subramanian V, Schetter AJ, Harris CC, Thompson PR, Hofseth LJ. The induction of microRNA-16 in colon cancer cells by protein arginine deiminase inhibition causes a p53-dependent cell cycle arrest. PLoS One. 2013; 8(1):e53791.
• Cherrington BD, Zhang X, McElwee JL, Morency E, Anguish LJ, Coonrod SA. Potential role for PAD2 in gene regulation in breast cancer cells. PLoS One. 2012; 7(7):e41242.
• Wang Y, Li P, Wang S, Hu J, Chen XA, Wu J, Fisher M, Oshaben K, Zhao N, Gu Y, Wang D, Chen G, Wang Y. Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity. J Biol Chem. 2012; 27;287(31):25941-53.