Author(s): Wiwit Nurwidyaningtyas, Djanggan Sargowo, Ferry Sandra, Titin Andri Wihastuti


DOI: 10.52711/0974-360X.2022.00117   

Address: Wiwit Nurwidyaningtyas1, Djanggan Sargowo2, Ferry Sandra3, Titin Andri Wihastuti4*
1Doctoral Program of Medical Science, Faculty of Medicine, Brawijaya University, Malang, Indonesia.
1Department Biochemistry and Molecular Biology, STIKES Kendedes Malang, East Java, Indonesia.
2Department of Cardiology, Faculty of Medicine, Brawijaya University, Malang, Indonesia.
3Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia.
4Department of Biomedical Nursing Science, Faculty of Medicine, Brawijaya University, Malang, Indonesia.
*Corresponding Author

Published In:   Volume - 15,      Issue - 2,     Year - 2022

p16INK4a (CDKN2A) represent as primary cell-cycle regulation, which arranges the moment of continued or interrupt cell proliferation. Cells expressing p16INK4a accumulate in aging tissue and age-related accumulation DNA damage, yet their physiologic compensated effects in human peripheral blood mononuclear cells (PBMNCs) after different stress inducer are poorly understood. Blood samples were obtained from healthy and non-sedentary lifestyle volunteers. Human mononuclear cells (MNCs) were purified from peripheral blood with Ficoll-density gradient centrifugation subsequently seeded into a medium culture. 80% confluence cells were divided into untreated cells and four treated cells with Asymmetric dimethylarginine (ADMA) and H2O2 in different doses for 24 h. Quantification p16INK4a positive cells were analyzed by FACS. The difference of p16INK4a positive cells after ADMA treated cells, H2O2 treated cells and untreated cells were also analyzed with a statistical test. We found that ADMA and H2O2 treatment in human MNCs induce elevation of p16INK4a (p=0.001), continuous p16INK4a expression incline to increase attendant extension dose of cellular stress inducer (p=0.000). Expression of p16INK4a has been proven higher in ADMA treated cells (1.43% ± 0.21) than control cells (0.34% ± 0.125) after 24h, the number of p16INK4a positive cells tended to increase when the ADMA exposure dose is added at 500µM (2.76% ± 1.39) compare with H2O2 treated cells (1.22% ± 0.33). These findings showed that p16INK4a positive cells are a part of the cellular stress response that results in temporary adaptation to some stressors, and may promote inhibition of inappropriate cell division.

Cite this article:
Wiwit Nurwidyaningtyas, Djanggan Sargowo, Ferry Sandra, Titin Andri Wihastuti. Differentiation of Intracellular p16INK4a Expression in the Circulating Human Mononuclear Isolated Cells after ADMA and H2O2 Exposure. Research Journal of Pharmacy and Technology. 2022; 15(2):707-2. doi: 10.52711/0974-360X.2022.00117

Wiwit Nurwidyaningtyas, Djanggan Sargowo, Ferry Sandra, Titin Andri Wihastuti. Differentiation of Intracellular p16INK4a Expression in the Circulating Human Mononuclear Isolated Cells after ADMA and H2O2 Exposure. Research Journal of Pharmacy and Technology. 2022; 15(2):707-2. doi: 10.52711/0974-360X.2022.00117   Available on:

1.    Dahlöf B. Cardiovascular Disease Risk Factors: Epidemiology and Risk Assessment. Am J Cardiol. 2010; 105(1):3A-9A.
2.    Zoccali C. Traditional and emerging cardiovascular and renal risk factors: an epidemiologic perspective. Kidney Int. 2006; 70:26–33
3.    Zoccali C. ADMA: a critical cardio-renal link in heart failure? Eur J Clin Invest. 2003;33:361–2.
4.    Titin Andri Wihastuti, Fibe Yulinda Cesa, Reyhan Amiruddin, Meddy Setiawan, Danisa Namira Wijayanti, Teuku Heriansyah. Polysaccharide Peptide (PsP) of Ganoderma Lucidum as vasa vasorum anti-Angiogenesis agent in Dyslipidemic state by Measuring Lp-PLA2 and H2O2 Levels: In Vivo Study using Wistar strain Rattus novergicus model of Atherosclerosis with Dyslipidemia. Research J. Pharm. and Tech. 2020; 13(7): 3241-3245.
5.    Said M.Y., Bollenbach A., Minovic I., van Londen M., Frenay A.R., de Borst M.H., van den Berg E., Kayacelebi A.A., Tsikas D., van Goor H., et al. Plasma ADMA, urinary ADMA excretion, and late mortality in renal transplant recipients. Amino Acids. 2019; 51(6):913-927
6.    Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, Blaschke TF, Cooke JP. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation.1998; 98:1842–1847
7.    Fulton M. Brown T. Zheng Y. The Biological Axis of Protein Arginine Methylation and Asymmetric Dimethylarginine, Int J Mol Sci. 2019; 20(13):3322.
8.    Teerlink, T.; Luo, Z.; Palm, F.; Wilcox, C.S. Cellular ADMA: Regulation and action. Pharmacol. Res. 2009, 60, 448–460.
9.    Shin S, Thapa SK, Fung H-L. Cellular interactions between L-arginine and asymmetric dimethylarginine: Transport and metabolism. PLoS ONE 2017; 12(5): e0178710
10.    Palacin M. Estevez R. Bertran J. Zorzano A. Molecular biology of mammalian plasma membrane amino acid transporters. Physiological Reviews. 1998, 78(4), 970-1054
11.    Ohno, Y. and J. I. Gallin. Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide. J Biol Chem. 1985; 260(14): 8438-46.
12.    Lennicke C. Rahn J. Lichtenfels R. Wessjohans L. Seliger B. Hydrogen peroxide – production, fate and role in redox signaling of tumor cells. Cell Commun Signal. 2015; 366:150–9
13.    Bienert G. P., Møller A. L. B., Kristiansen K. A., et al. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. The Journal of Biological Chemistry. 2007; 282(2):1183–1192.
14.    Rhee, S. G., Y. S. Bae, S. R. Lee and J. Kwon (2000). "Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation." Sci STKE 2000; 53: PE1.
15.    Lassegue, B. and R. E. Clempus. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol. 2003; 285(2): R277-97.
16.    Marzo N. Chisci E. Geovannoni R. The Role of Hydrogen Peroxide in Redox-Dependent Signaling: Homeostatic and Pathological Responses in Mammalian Cells. Cells 2018; 7(10), 156
17.    Poljsak B. & Milisav I. Clinical implications of cellular stress responses. Bosn J Basic Med Sci. 2012; 12(2): 122–126.
18.    Fulda S. Gorman A. Hori O. Samali A. Cellular stress responses: cell survival and cell death, International Journal of Cell Biology 2010, Article ID 214074, 1-23
19.    Negrini S., Gorgoulis V. G., Halazonetis T. D. Genomic instability an evolving hallmark of cancer. Nature Reviews Molecular Cell Biology. 2010;11(3):220–228.
20.    Simmons SO, Fan CY, and Ramabhadran R (2009) Cellular stress response pathway system as a sentinel ensemble in toxicological screening. Toxicol Sci 2009; 111:202–225.
21.    Jiang F. Zhang Y. Dusting G. NADPH Oxidase-Mediated Redox Signaling: Roles in Cellular Stress Response, Stress Tolerance, and Tissue Repair, Pharmacol Rev 2011; 63:218–242.
22.    Enriquez G. Arroyo A. Grijalva M. Israel R. Zafra A. Camacho J. Molecular and Cellular Effects of Hydrogen Peroxide on Human Lung Cancer Cells: Potential Therapeutic Implications, Oxid Med Cell Longev. 2016; 2016: 1908164.
23.    Jenkins N. Liu T. Cassidy P. Leachman S. Boucher K. Goodson A. Samadashwily G. Grossman D. The p16INK4A tumor suppressor regulates cellular oxidative stress. Oncogene. 2011; 30(3): 265–274.
24.    Lukas J, Parry D, Aagaard L, Mann DJ, Bartkova J, Strauss M, et al. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumor suppressor p16. Nature. 1995; 375:503–506.
25.    Sharpless NE and DePinho RA. The INK4A/ARF locus and its two gene products. Current Opinion Genet. Dev. 1999; (9): 22–30.
26.    Alcorta DA, Xiong Y, Phelps D, Hannon G, Beach D, Barrett JC. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci U S A. 1996; 93:13742–13747.
27.    Shapiro GI, Edwards CD, Ewen ME, Rollins BJ. p16INK4A participates in a G1 arrest checkpoint in response to DNA damage. Mol Cell Biol. 1998; 18:378–387.
28.    Ku¨ ltz D. Molecular and evolutionary basis of the cellular stress response. Annu Rev Physiol 2005; 67:225–257.
29.    Welch WJ. The mammalian heat shock (or stress) response: a cellular defense mechanism. Adv Exp Med Biol 1987; 225:287–304.
30.    Huizer K, Mustafa DAM, Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol. PLoS ONE 2017; 12(9): e0184895.
31.    Long Ma W. Wang L. Liu L. Wang X. Effect of phosphorylation and methylation on the function of the p16INK4a protein in non-small cell lung cancer A549 cells. Oncol Lett. 2015; 10(4): 2277–2282.
32.    Chen Y.H., Xu X., Sheng M.J., Zhang X.Y., Gu Q., Zheng Z. PRMT-1 and DDAHs-induced ADMA upregulation is involved in ROS- and RAS-mediated diabetic retinopathy. Exp. Eye Res. 2009; 89:1028–1034.
33.    Yokoro M., Suzuki M., Murota K., Otsuka C., Yamashita H., Takahashi Y., Tsuji H., Kimoto M. Asymmetric dimethylarginine, an endogenous NOS inhibitor, is actively metabolized in rat erythrocytes. Biosci. Biotechnol. Biochem. 2012; 76:1334–1342.
34.    Leiper J.M., Vallance P. The synthesis and metabolism of asymmetric dimethylarginine (ADMA) Eur. J. Clin. Pharmacol. 2006;62:33–38.
35.    Vallance P., Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway. Arterioscler. Thromb. Vasc. Biol. 2004; 24:1023–1030
36.    Cooke P. Does ADMA Cause Endothelial Dysfunction? Arteriosclerosis, Thrombosis, and Vascular Biology. 2000; 20(9):2032-2037
37.    Boger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the "L-arginine paradox" and acts as a novel cardiovascular risk factor. J Nutr. 2004;134(10):2842S–7S
38.    Bode-Boger SM, Scalera F, Ignarro LJ. The l-arginine paradox: Importance of the l-arginine/asymmetrical dimethylarginine ratio. Pharmacol Ther. 2007;114(3):295–306.
39.    Closs E. Boissel J. Habermeier A. Rotmaan A. Structure and Function of Cationic Amino Acid Transporters (CATs), The Journal of Membrane Biology 2006; 213(2):67-77
40.    Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabloized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol. 2003; 23:1455–9.
41.    Siroen MP, Teerlink T, Nijveldt RJ, Prins HA, Richir MC, Van Leeuwen PA. The clinical significance of asymmetric dimethylarginine. Annu Rev Nutr. 2006; 26:203–28
42.    Richir MC, Bouwman RH, Teerlink T, Siroen MP, de Vries TP, Van Leeuwen PA. The prominent role of the liver in the elimination of asymmetric dimethylarginine (ADMA) and the consequences of impaired hepatic function. JPEN J Parenter Enteral Nutr. 2008;32:613–21
43.    Cardounel, A.J.; Cui, H.; Samouilov, A.; Johnson, W.; Kearns, P.; Tsai, A.L.; Berka, V.; Zweier, J.L. Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function. J. Biol. Chem. 2007, 282, 879–887.
44.    Villabo A. Nitric oxide and cells proliferation, FEBS Journal 2006; 273(11):2329-2344
45.    Sessa WC, Hecker M, Mitchell JA, Vane JR. The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: L-glutamine inhibits the generation of L-arginine by cultured endothelial cells. Proc Natl Acad Sci U S A. 1990;87:8607–11.
46.    Arnal JF, Munzel T, Venema RC, James NL, Bai C, Mitch WE. Interactions between L-arginine and L-glutamine change endothelial NO production. An effect independent of NO synthase substrate availability. J Clin Invest. 1995; 95:2565–72.
47.    Marino SM, Gladyshev VN, Marino SM, Gladyshev VN. Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces. J Mol Biol. 2010; 404:902–16.
48.    Zhao H., Traganos F., Albino A. P., Darzynkiewicz Z. Oxidative stress induces cell cycle-dependent Mre11 recruitment, ATM and Chk2 activation and histone H2AX phosphorylation. Cell Cycle. 2008; 7(10):1490–1495
49.    Sfikas A., Batsi C., Tselikou E., et al. The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage. Cellular Signalling. 2012; 24(11):2007–2023.
50.    K. Gowthami Balashri, S. Kanmani. Decolorisation of Reactive Orange using Coupled Oxidation Process. Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 231-234.
51.    Hardik Joshi, Manoj Pagare, Leena Patil, Vilasrao Kadam. In–Vitro Antioxidant Activity of Ethanolic Extract of Leaves of Buchanania Lanzan Spreng. Research J. Pharm. and Tech. 4(6): June 2011; Page 920-924.
52.    Manoj S. Pagare, Hardik Joshi, Leena Patil, Vilasrao J. Kadam. In Vitro Antioxidant Activity of Fruit of Benincasa hispida Cogn. Research J. Pharm. and Tech. 4(7): July 2011; Page 1082-1085.
53.    Serrano M, Lee H, Chin L, Cordon-Cardo C, Beach D, DePinho RA. Role of the INK4a locus in tumor suppression and cell mortality. Cell. 1996; 85:27–37.
54.    Chung JS, Lee SB, Park SH, Kang ST, Na AR, Chang TS, et al. Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27(Kip1) expression. Free Radic Res. 2009; 43:729–737.
55.    Didi Chinnu Raju, T Diana Victoria, Nancy Biji, Gandra Nikitha. Evaluation of Antioxidant Potential of Ethanolic Extract of Centella asiatica L. Research J. Pharm. and Tech. 8(9): Sept, 2015; Page 1289-1293.
56.    Ganesh Choudhari, Vishnu Choudhari, Akshay Baheti, Manini Mantri, Savita Matapurkar, Chandrakant More. Synergistic Antioxidant activity of a Polyherbal Preparation. Research J. Pharm. and Tech 2020; 13(3):1193-1197.
57.    Suprava Sahoo, Subrat Kumar Kar, Bhaskar Chandra Sahoo, Sanghamitra Nayak, Basudeba Kar. Free radical scavenging potential of Alpinia calcarata Roscoe leaves. Research J. Pharm. and Tech. 2020; 13(7): 3356-3360.
58.    Mary Thomas. Entrepreneurial Orientation and the Management Grid: A roadmap for the entrepreneurial journey. Asian J. Management. 2016; 7(4): 293-296.
59.    N. Naidu, G. Sudheer Kumar, K. Sivakrishna, K. Anjinaik, L. Praveen Kumar, G. Sneha. Anti microbial and antioxidant evolution of aqueous extract of Terminalia chebula using disc diffusion, H2O2 scavenging methods. Asian J. Res. Pharm. Sci. 2017; 7(2): 112-114
60.    Seenaa Kadhum Ali, Mahmoud Hussein Hadwan. Precise Spectrophotometric Method for measurement of Peroxiredoxin activity in Biological Samples. Research J. Pharm. and Tech. 2019; 12(5):2254-2260.

Recomonded Articles:

Research Journal of Pharmacy and Technology (RJPT) is an international, peer-reviewed, multidisciplinary journal.... Read more >>>

RNI: CHHENG00387/33/1/2008-TC                     
DOI: 10.5958/0974-360X 

56th percentile
Powered by  Scopus

SCImago Journal & Country Rank

Recent Articles


Not Available