Modulation of Vascular Endothelial Growth Factor and Annexin A2 in Response to 4-(Methylnitrosamino)-1-(3-pyridyl)-1-Butanone -Induced Inflammation via Swimming Training

  • Ali Barzegari Department of Physical Education and Sport, Payame Noor University, Tehran, Iran
  • Shadmehr Mirdar Department of Sport Sciences, University of Mazandaran, Babolsar, Iran
  • Mohammad Ranayi Department of Pathology, Babol University of Medical Sciences, Babol, Iran
Keywords: Annexin A2, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, Swimming, Vascular endothelial growth factor


The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; nicotine derived nitrosamine ketone) is one of the strongest carcinogens in tobacco which is involved in induction of lung cancer by changing the stimulation of vascular endothelial growth factor (VEGF) and annexin A2 expression. The aim of this study was to investigate the changes in resting levels of annexin A2 and VEGF in lung tissues of rats exposed NNK after 12 weeks of aerobic submaximal swimming training. For this purpose, 46 Wistar rats were randomly divided into five groups consist of training, training + NNK, NNK, saline and control. NNK-induced groups received NNK subcutaneously one day per week at a rate of 12/5 mg per kg body weight and the training groups performed submaximal swimming training for 12 weeks. The levels of VEGF and annexin A2 in lung tissue were measured respectively by ELISA and immunohistochemistry. To analyze the data; ANOVA and Tukey's test were used at a significance level of p<0.05. Findings indicated that 12 weeks submaximal swimming training decreased the levels of VEGF and annexin A2 in lung tissue significantly when compared to NNK group (p<0.001). There was no significant correlation between VEGF and annexin A2 levels in all study groups (p≥0.05). Generally, it could be confirmed that regular submaximal aerobic training plays an important role in inhibition of the effects of lung inflammation induced by NNK via decreased levels of VEGF and annexin A2.


1. Keohavong P, Kahkonen B, Kinchington E, Yin J, Jin J, Liu X, et al. K-ras Mutations in Lung Tumors from NNK-treated Mice with Lipopolysaccharide-elicited Lung Inflammation. Anticancer Res 2011; 31(9):2877-82.
2. Huang RY, Li MY, Hsin MK, Underwood MJ, Ma LT, Mok TS, et al. 4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A2 and its receptor. Oncogene 2011; 30(1):106-16.
3. Peterson LA, Thomson NM, Crankshaw DL, Donaldson EE and Kenney PJ. Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis. Cancer Res 2001; 61(15):5757-63.
4. Hecht SS. Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst 1999; 91(14):1194-210.
5. Lin RK, Hsieh YS, Lin P, Hsu HS, Chen CY, Tang YA, et al. The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients. J Clin Invest 2010; 120(2):521-32.
6. Barta P, Van Pelt C, Men T, Dickey B, Lotan R, Moghaddam SJ. Enhancement of lung tumorigenesis in a Gprc5a knockout mouse by chronic extrinsic airway inflammation. Mol Cancer 2012; 11(4):1-11.
7. Adcock IM, Caramori G, Barnes PJ. Chronic obstructive pulmonary disease and lung cancer: new molecular insights. Respiration 2011; 81(4):265-84.
8. Swisher JFA, Khatri U, Feldman GM. Annexin A2 is a soluble mediator of macrophage activation. J Leukoc Biol 2007; 82 (5):1174-84.
9. Morel E, Gruenberg J. The p11/S100A10 light chain of annexin A2 is dispensable for annexin A2 association to endosomes and functions in endosomal transport. PLoS One 2007; 82(5):1174-84.
10. Yao H, Zhang Z, Xiao Z, Chen Y, Li C, Zhang P, et al. Identification of metastasis associated proteins in human lung squamous carcinoma using two-dimensional difference gel electrophoresis and laser capture microdissection. Lung Cancer 2009; 65(1):41-8.
11. Yang SF, Hsu HL2, Chao TK3, Hsiao CJ4, Lin YF5, Cheng CW6. Annexin A2 in renal cell carcinoma: Expression, function, and prognostic significance. Urol Oncol 2014; 33(1):22.
12. Luo CH, Liu QQ, Zhang PF, and Li MY, Chen ZC, Liu YF. Prognostic significance of annexin II expression in non-small cell lung cancer. Clin Transl Oncol 2013; 15(11):938-46.
13. Zhao S, Huang L, Wu J, Zhang Y, Pan D, Liu X. Vascular endothelial growth factor upregulates expression of annexin A2 in vitro and in a mouse model of ischemic retinopathy. Mol Vis 2009; 15:1231–42.
14. Jarnicki AG, Lysaght J, Todryk S, Mills KH. Suppression of antitumor immunity by IL-10 and TGF-beta-producing T-cells infiltrating the growing tumor: influence of tumor environment on the induction of CD4+ and CD8+ regulatory T-cells. J Immunol 2006; 177(2):896-904.
15. Heist RS, Zhai R, Liu G, Zhou W, Lin X, Su L, et al. VEGF polymorphisms and survival in early-stage non-small cell lung cancer. J Clin Oncol 2008; 26(6):856-62.
16. Wada J, Suzuki H, Fuchino R, Yamasaki A, Nagai S, Yanai K, Koga K, Nakamura M, Tanaka M, Morisaki T, Katano M. The Contribution of Vascular Endothelial Growth Factor to the Induction of Regulatory T-Cells in Malignant Effusions. Anticancer Res 2009; 29(3):881-8.
17. Zhang N, Sun X, Sun M, Zhu S, Wang L, Ma D, et al. 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Promotes Esophageal Squamous Cell Carcinoma Growth via Beta-Adrenoceptors In Vitro and In Vivo. PLoS One 2015; 10(3):1-16.
18. Cui JW, Wang YL. Expression and function of Annexin栻 in lung cancer tissue. Asian Pac J Trop Med 2013; 6(2):150-2.
19. Xu XH, Pan W, Kang LH, Feng H, Song YQ. Association of Annexin A2 with Cancer Development (Review). Oncol Rep 2015; 33(5):2121-8.
20. Koutsokera A, Kiagia, M, Saif MW, Souliotis K, Syrigos, KN. Nutrition Habits, Physical Activity, and Lung Cancer: An Authoritative Review. Clin Lung Cancer 2013; 14(4):342-50.
21. Ávila LCM, Bruggemann TR, Bobinski F, Silva MD, Oliveira RC, Martins DF, et al. Effects of High-Intensity Swimming on Lung Inflammation and Oxidative Stress in a Murine Model of DEP-Induced Injury. PLoS One 2015; 10(9):e137273.
22. Vieira RP, Toledo AC, Silva LB, Almeida FM, Damaceno-Rodrigues NR, Caldini EG, et al. Anti-inflammatory effects of aerobic exercise in mice exposed to air pollution. Med Sci Sports Exerc 2012; 44(7):1227-34.
23. Lee HH, Kim H, Lee JW, Kim YS, Yang HY, Chang HK, Lee TH, Shin MC, Lee MH, Shin MS, Park S, Baek S, Kim CJ. Maternal swimming during pregnancy enhances short-term memory and neurogenesis in the hippocampus of rat pups. Brain Dev 2006; 28(3):147-54.
24. Sinner P, Folsom Ar, Harnack L, Eberly Le, Schmitz Kh. The Association of Physical Activity with Lung Cancer Incidence in a Cohort of Older Woman: The Lowa Woman's Health Study. Cancer Epidemiol Biomarkers Prev 2006; 15(2):2359-63.
25. Colombo R, Siqueira R, Conzatti A, de Lima Seolin BG, Fernandes TR, Godoy AE, et al. Exercise training contributes to H2O2/VEGF signaling in the lung of rats with monocrotaline-induced pulmonary hypertension. Vascul Pharmacol 2016; 87:49-59.
26. Krenc Z, Mazurowski W, Wosik-Erenbek M. Changes in VEGF and bFGF serum concentration after long-term sports training in young athletes – The significance of adaptive angiogenesis in arterial blood pressure adjustment. Pediatria Polska 2016; 91(6): 552-558.
27. Olfert ED, Cross BM, McWilliam AA. Guide to the care and use of experimental animals. Canadian Council on Animal Care Ottawa. 1993; 1: 1-209.
28. Belinsky SA, Foley JF, White CM, Anderson MW, Maronpot RR. Dose-response relationship between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res 1990; 50(12):3772-80.
29. Barzegari A, Mirdar S. Effect of a 12-week submaximal swimming training in rats exposed to tobacco- derived nitrosamine ketone. Caspian J Intern Med 2018; 9(2):158-163.
30. ianbin C, Liyuan H, Shuli S, Zhonghui H, Haitao J. Expression of vascular endothelial growth factor and hypoxia-inducible factor 1α in rat traumatic lung injury. Int J Clin Exp Pathol 2017; 10(1):80-7.
31. Jin L, Shen Q, Ding S, Jiang W, Jiang L, Zhu X. Immunohistochemical expression of Annexin A2 and S100A proteins in patients with bulky stage IB-IIA cervical cancer treated with neoadjuvant chemotherapy. Gynecol Oncol 2012; 126(1):140-6.
32. Ribatti D, Conconi MT, Nussdorfer GG. Nonclassic endogenous novel regulators of angiogenesis. Pharmacol Rev 2007; 59(2):185-205.
33. Czarkowska-Paczek B, Bartlomiejczyk I, PrzybylskiJ. The serum levels of growth factors: PDGF, TGF-betaand VEGF are increased after strenuous physical exercise. J Physiol Pharmacol 2006; 57(2):189-97.
34. Mirdar SH, Mehdinia E Bayani T. The effect of swimming endurance training on the level of lung VEGF in pregnant mice that exposed to cadmium. Journal of sport biosciences 2016; 8(2): 177-191.
35. Mirdar Sh, Jarrahi M, Hedayati M, Hajizade A, Hamidian Gh. Effect of swimming during pregnancy on vascular endothelial growth factor level of neonatal rat kidney tissue. J Gorgan Uni Med Sci 2014; 16(4):106-10.
36. Leggate M, Carter WG, Evans MJ, Vennard RA, Sribala-Sundaram S, Nimmo MA. Determination of inflammatory and prominent proteomic changes in plasma and adipose tissue after high-intensity intermittent training in overweight and obese males. J Appl Physiol 2012; 112(8):1353-60.
37. Hord JM, Botchlett R, Lawle JM. Age-related alterations in the sarcolemmal environment are attenuated by lifelong caloric restriction and voluntary exercise. Exp Gerontol 2016; 83:148–57.
38. Hussey SE, Sharoff CG, Garnham A, Yi Z, Bowen BP, Mandarino LJ, et al. Effect of Exercise on the Skeletal Muscle Proteome in Patients with Type 2 Diabetes. Med Sci Sports Exerc 2013; 45(6):1069–76.
39. Zhang D, Lei Jianjun, Ma J, Chen X, Sheng L, Jiang Z, et al. β2-Adrenogenic signaling regulates NNK-induced pancreatic cancer progression via upregulation of HIF-1α. Oncotarget 2016; 7(14):17760–72.
40. Akopyan G, Bonavida B. Understanding tobacco smoke carcinogen NNK and lung tumorigenesis (Review). Int J Oncol 2006; 29(4):745-52.
41. Wang CY, Chen CL, Tseng YL, Fang YT, Lin YS, Su WC, et al. Annexin A2 Silencing Induces G2 Arrest of Non-small Cell Lung Cancer Cells through p53-dependent and –independent Mechanisms. J Biol Chem 2012; 287(39):32512–4.
42. Wang CY, Lin CF. Annexin A2: Its Molecular Regulation and Cellular Expression in Cancer Development. Dis Markers 2014; 2014:308976.
43. Lokman NA, Ween MP, Oehler MK, Ricciardelli C. The Role of Annexin A2 in Tumorigenesis and Cancer Progression. Cancer Microenviron 2011; 4(2):199–208.
44. Amani SS, Agha-Alinejad H, Alizadeh Sh, Shahbazi Sh, Kashani Khatib Z, Kazemi AR, et al. The effect of exercise training on the level of tissue IL-6 and vascular endothelial growth factor in breast cancer bearing mice. Iran J Basic Med Sci 2014; 17(4):231–58.
45. Suhr F, Brixius K, de Marées M, Bölck B, Kleinöder H, Achtzehn S, Bloch W, Mester J. Effects of short-term vibration and hypoxia during high-intensity cycling exercise on circulating levels of angiogenic regulators in humans. J Appl Physiol 2007; 103(2):474-83.
How to Cite
Barzegari A, Mirdar S, Ranayi M. Modulation of Vascular Endothelial Growth Factor and Annexin A2 in Response to 4-(Methylnitrosamino)-1-(3-pyridyl)-1-Butanone -Induced Inflammation via Swimming Training. Iran J Allergy Asthma Immunol. 17(5):418-427.
Original Article(s)