İnsan servikal karsinoma hücre dizisinde nitrik oksit ile yönlendirilen hücre ölümü üzerinde hidrojen peroksidin etkisi

Amaç: Nitrik oksit (NO) ve reaktif oksijen türleri (ROT) gibi organ mikroçevre faktörleri veonların apoptoz ile etkileşimleri, normal hücrelerin ve kanser hücrelerinin davranışlarınıetkileyebilir . Bu olasılık sağaltım stratejileri için yeni hedefler sağlayabilir . NO'nın tümörhücresinin yaşamını sürdürmesi, redoks homeostazı; tümör hücrelerinin genetik ve epigenetikyapılanması gibi olaylarda rolü bulunmaktadır . Bu çalışmada NO'nın yönlendirdiği hücreölümünde ROT'un [Hidrojen peroksit (H O ) ile taklit edilmiştir] etkileri araştırılmıştır . 2 2 Yöntem: İnsan servikal karsinoma hücre dizisi (HeLa); Dulbecco's Minimum Essential Medium(DMEM) ile üretilmiştir . Hücrenin yaşamı ve proliferasyonları, niceleyici koloritmetrik bir 3 (4,5dimetiltiyazol- 2 il)-2,5- difenil tetrazolyum bromid (MTT) deneyi ile taranmıştır . DNA'yabağlanan boya Hoechst 33342 (HOE 33342) ve propidyum iyodid (PI), nükleer morfoloji vemembran bütünlüğünün kantifikasyonu ve mikroskopik identifikasyonu için ayırt edici boyaçalışması olarak kullanılmıştır .Bulgular: HeLa hücreleri yalnızca L-arjinin (NO uyarıcısı) ile uyarıldığında (NO grubu) % 66± 9.2 hücre ölmüş ve ölen bu hücrelerin % 51±7.3'ü “apoptotik yolak”tan geçerek olmuştur . NO,H O varlığında (peroksi nitrit grubu) uyarıldığında daha fazla hücre ölmüş (% 98.3±1.7) ve bu 2 2 ölümler “nekrotik yolak”tan geçerek olmuştur (% 69.3±5.7). Hücreler H Oile uyarıldığında 2 2 (peroksit grubu) bütün hücreler ölmüş (% 99.3±4.1) ve hücresel ölüm “nekrotik yolak” üzerindenolmuştur (% 85±6.9).Sonuç: HeLa hücrelerinde L-arjinin (L-arg) ve H O 'nin hücresel ölümü belirgin olarak, fakat 2 2 farklı yolaklardan giderek arttırdıkları görülmüştür . Ancak birlikte kullanıldıklarında, H O 'nin, L- 2 2 arg'in yarattığı hücresel ölümü “apoptotik yolak”tan, “nekrotik yolak”a çevirdiğigözlemlenmiştir .

Effects of hydrogen peroxide on nitric oxide mediated cellular death in human cervical carcinoma cell line

Objective: Organ microenvironment factors such as nitric oxide (NO) and reactive oxygenpecies (ROS), and their interactions with apoptosis may influence the behavior of normal andcancer cells. This possibility may provide new targets for therapy strategies. The effect of NO onumor cell survival is influenced by multiple factors, including the levels of redox homeostasis,and genetic and epigenetic make up of tumor cells. The effects of ROS [mimicked by hydrogenperoxide(H O )] on NO-mediated cellular death were examined in this study. 2 2 Method: Human cervical carcinoma cell line (HeLa) was grown using Dulbecco's MinimumEssential Medium (DMEM). Cell survival and proliferations were determined by a quantitativecolorimetric MTT assay. The DNA binding dye Hoechst 33342 and propidium iodide (HOE/PI) were Results: Conclusion: used together in a differential dye uptake assay for microscopic identification and quantification of membrane integrity andnuclear morphology.Results: When HeLa cells were induced by L-arginine (NO inducer) alone (nitric oxide group), 66 + 9,2 % of cells died, andof these cells 51 + 7,3 % died through an “apoptotic pathway”. We found that when NO was induced in the presence of H O2 2 (peroxynitrite group), more cells died (98, 3 + 1, 7 %) through a “necrotic pathway” (69, 3 + 5, 7 %). When cells were induced byhydrogen peroxide (peroxide group) all cells died (99, 3 + 4, 1 %) and cellular death was through a “necrotic pathway” (85 + 6, 9%).Conclusion: In HeLa cells L-arginine (L-arg) and H O significantly increased cellular death but in distinct pathways. But 2 2 when treated together the presence of H Oshifted cellular death caused by L-arg from apoptotic pathway to necrotic 2 2 pathway.

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1. Sen S, D'Incalci M. Apoptosis: Biochemical events and relevance to cancer chemotherapy. FEBS Letters, 1992; 307:122-129.
2. Mohr S, McCormick TS, Lapetina EG. Macrophages resistant to endogenously generated nitric oxide mediated apoptosis are hypersensitive to exogenously added nitric oxide donors: Dichotomous apoptotic response independent of caspase 3 and reversal by the mitogen-activated protein kinase (MEK) inhibitor PD 098059. Proc Natl Acad Sci, 1998; 95: 5045-5050.
3. Dröge W. Free radicals in the physiological control of cell function. Physiol Rev , 2002; 82: 47-95.
4. Brüne B, Von Knethen A, Sandau KB. Nitric oxide and its role in apoptosis. Eur J Pharmacology, 1998; 351: 261 272.
5. Cookson MR, Ince PG, Shaw PJ. Peroxynitrite and hydrogen peroxide induced cell death in the NSC34 neuroblastoma X spinal cord cell line: role of poly (ADP Ribose) polymerase. Journal of Neurochemistry, 1998; 70: 501-508.
6. Melkova Z, Lee SB, Rodriquez D, Esteban M. Bcl-2 prevents nitric oxide-mediated apoptosis and poly (ADP ribose) polymerase cleavage. FEBS Letters, 1997; 403: 273-278.
7. Brüne B: Nitric oxide. NO apoptosis or turning it ON? Cell Death and Differentiation, 2003; 10: 864-869
8. Oh-Hashi K, Maruyama W, Isobe K. Peroxynitrite induced GADD34, 45, and 153 via p38 MAPK in human neuroblastoma SH-SY5Y cells. Free Radical Biology and Medicine, 2001; 30: 213-221.
9. Xie K, Huang S. Contribution of nitric oxide-mediated apoptosis to cancer metastasis inefficiency. Free Radical Biology and Medicine, 2003; 34: 969-986.
10.Shacter E, Williams JA, Hinson RM, Senturker S, Lee YJ. Oxidative stress interferes with cancer chemotherapy: inhibition of lymphoma cell apoptosis and phagocytosis. Blood, 2000; 96: 308-313.
11.Baskin H, Ellerman-Eriksen S, Lovmand J, Mogensen CS. Herpes simplex virus type 2 synergizes with interferon gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumor necrosis factor-alfa. J Gen Virol, 1997; 78: 195 203.
12.Brune B. The intimate relation between nitric oxide in apoptosis and cell survival. Antioxid Redox Signal, 2005; 3-4: 497-507.
13.Steinmann M, Moosmann N, Schimmel M, Gerhardus C, Bauer G. Differential role of extra-and intracellular superoxide anions for nitric oxide-mediated apoptosis induction. In Vivo, 2004; 18: 293-309.
14.Mc Keague AL, Wilson DJ, Nelson J. Staurosporine induced apoptosis and Hydrogen peroxide-induced necrosis in two human breast cell lines. British Journal of Cancer , 2003; 88: 125-131.
15.Rocha M, Kruger A, Van Rooijen N, Schirrmacher V, Umansky V. Liver endothelial cells participate in T-cell dependent host resistance to lymphoma metastasis by production of nitric oxide in vivo. Int J Cancer , 1995; 63: 405-411.
16.Sade H and Sarin A. Reactive oxygen species regulate quiescent T-cell apoptosis via the BH3-only proapoptotic protein BIM. Cel l Death and Differentiation, 2004; 11:416-23.
17.Park IC, Woo SH, Park MJ et al. Ionizing radiation and nitric oxide donor sensitize Fas-induced apoptosis via up regulation of Fas in human cervical cancer cells. Oncol Rep, 2003; 10: 629-633.
18.Chung HT , Pae HO, Choi BM, Billiar TR. Nitric oxide as a bioregulator of apoptosis. Biochem Biophys Res Commun, 2001; 282: 1075-1079.
19.Heigold S, Sers C, Bechtel W, Ivanovas B, Schafer R, Bauer G. Nitric oxide mediates apoptosis induction selectively in transformed fibroblasts compared to non transformed fibroblasts. Carcinogenesis, 2002; 23: 929 941.
20.Baskin Y , Baskin H, Guner G, Tuzun E, Oto O. The inversely proportional relation between nitric oxide and lipid peroxidation in atherosclerotic plaque formation in human. Int J Cardiol, 2003; 9: 53-57.
21.Tews DS. Cell death and oxidative stress in gliomas. Neuropathology and Applied Neurobiology, 1999; 25: 272-284.
22.Toomey D, Condron C, Wu QD et al. TGF-beta1 is elevated in breast cancer tissue and regulates nitric oxide production from a number of cellular sources during hypoxia reoxygenation injury. Br J Biomed Sci, 2001; 58: 177-183.
23.Bauer G. Reactive oxygen and nitrogen species: efficient, selective, and interactive signals during intercellular induction of apoptosis. Anticancer Res, 2000; 20: 4115-39.
24.Engelmann I, Bauer G. How can tumor cells escape intercellular induction of apoptosis? Anticancer Res, 2000; 20: 2297-306.