Nadir KİRAZ, Aysun ÖZKAN, Ödül ÖZKAN, Ayşe ERDOĞAN

Comparison of cellular responses of parental and epirubicin-resistant non-small cell lung cancer cells against stabilized-ag ion solution induced injury

Amaç: Bu çalışmanın amacı, stabilize iyon solüsyonu ile muamele edildikten sonra bu çözeltinin uyardığı oksidatif DNA ve membran hasarına karşı parental ve epirubisin dirençli küçük hücreli dışı akciğer kanseri (NSCLC) hücrelerinin cevaplarını karşılaştırmaktır. Gereç ve Yöntem: Parental ve ilaca dirençli NSCLC hücreleri için IC50 değerleri 3-(4,5-dimetiltiazol-2-yl) -2,5difeniltetrazolyum bromür (MTT) ve resazurin-temelli deneyler ile ortaya kondu. Hücrelerdeki malondialdehit (MDA) seviyeleri florometrik yöntemi kullanılarak analiz edilmiştir. Genomik DNA örnekleri rekabetçi ELISA kiti ile 8-hidroksi-2'-deoksiguanozin (8-OHdG) miktarını belirlemek için kullanılmıştır. Bulgular: MTT deneyi ile IC50 değeri parental hücreleri için 139 ?g/mL ve dirençli hücreler için 224 ?g/mL olarak hesaplanmıştır. Resazurin-temelli deney ile IC50 değeri sırasıyla 96 ?g/ mL ve 189 ?g/mL olarak hesaplanmıştır. Hücreler St-Ag iyon çözeltisinin IC50 konsantrasyonu ile muamele edildiğinde oksidatif stresin net işaretlerinden, glutatyon (GSH) (1.5 kat parentalde, 2 kat dirençlide) ve glutatyon peroksidaz (GSH-Px) (2 kat parentalde) azaldığı bunların yanı sıra lipid peroksidasyonun (1.6 kat parentalde, 2 kat dirençlide) ve 8-OHdG oluşumunun (1.5 kat parentalde, 2 kat dirençlide) arttığı görülmüştür. Her iki hücre seviyelerindeki ve dirençli hücrelerdeki GSH-Px seviyesindeki değişimler istatistiksel olarak önemli değildir. Dirençli hücrelerin yüksek GST ve GSH-Px aktivitesine sahip olmasına rağmen St-Ag iyon çözeltisi dirençli hücrelerde parental hücrelerden daha yüksek membran ve DNA hasar etkisi vardır. Sonuç: Böylece verilerimiz St-Ag iyon solüsyonunun oksidatif stresle ilişkili konsantrasyona parental ve associated with oxidative stress. Anticancer potential of anticancer drugs for especially drug resistant cells can be increased by combining therapy with stabilized-silver ion solution. epirubisin dirençli kanser hücreleri üzerinde farklı potansiyel toksisiteye sahip olduğunu göstermektedir. Özellikle ilaca dirençli hücreler için anti-kanser ilaçların antikanser potansiyeli stabilize gümüş iyonu çözeltisi ile tedavinin birleştirilmesiyle arttırılabilir.

Stabilize-ag iyon çözeltisinin uyardığı hasara karşı parental ve epirubicin dirençli küçük hücreli dışı akciğer kanseri hücrelerinin hücresel yanıtların karşılaştırılması

Purpose: The aim of this study was to compare the responses of parental and epirubicin-resistant non-small cell lung cancer (NSCLC) cells against stabilized-silver (StAg) ion induced oxidative DNA and membrane injury after the cells treated with this solution. Material and Methods: IC50 values for parental and drug-resistant NSCLC cells as revealed by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and resazurin-based assay. Malondialdehyde (MDA) levels in the cells were assayed by using fluorometric method. The genomic DNA samples were used to determine the amount of 8-hydroxy-2'- deoxyguanosine (8-OHdG) with a competitive ELISA kit. Results: 139 ?g/mL for parental cells and 224 ?g/mL for resistant cells were calculated as IC50 values by MTT assay. 96 ?g/mL and 189 ?g/mL respectively were calculated as IC50 values by resazurin-based assay. When the cells were treated with IC50 concentration of St-Ag ion solution, clear signs of oxidative stress, i.e. decreased is glutathione (GSH) (1.5-folds in parental, 2-folds in resistant) and glutathione peroxidase (GPx) (2-folds in parental), as well as increased lipid peroxidation (1.6-folds in parental, 2-folds in resistant) and 8-OHdG formation (1.5-folds in parental, 2-folds in resistant) were seen. Changes in the levels of glutathione-S-transferase (GST) in both cell type and levels GPx in resistant cells were statistically insignificant. The St-Ag ion solution has higher membrane and DNA damaging effect on resistant cells than parental cells even though resistant-cells have high GST and GPx activity. Conclusion: Thus our data suggest St-Ag ion solution has different potential toxicity on parental and epirubicinresistant cancer cells depend on concentration that is associated with oxidative stress. Anticancer potential of anticancer drugs for especially drug resistant cells can be increased by combining therapy with stabilized-silver ion solution.

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Rescifina A, Zagni C, Romeo G, Sortino S. Synthesis and biological activity of novel bifunctional isoxazolidinyl polycyclic aromatic hydrocarbons. Bioorg Med Chem. 2012;20:4978-984.
Martinez R, Chacon-Garcia L. The search of DNA- intercalators as antitumoral drugs: what it worked and what did not work. Curr Med Chem. 2005;12:127-51.
Ono A, Cao S, Togashi H, Tashiro M, Fujimoto T, Machinami T, Oda S, Miyake Y, Okamoto I, Tanaka Y. Specific interactions between silver (I) ions and cytosine-cytosine pairs in DNA duplexes. Chem Commun. 2008;39:4825-827.
Shukla S, Mishra AP. Synthesis, structure, and anticancerous properties of silver complexes. Journal of Chemistry 2013; Article number: 527123.
Pedahzur R, Shuval HI, Ulitzur S. Silver and hydrogen peroxide as potential drinking water disinfectants: their bactericidal effects and possible modes of action. Water Sci Tech. 1997;35:87-93.
Ozkan A, Fışkın K. Epirubicin-HCl cytotoxicity in non-small cell lung cancer (NSCLC) cells. Turkish Journal of Hematology Oncology. 2003;13:125-33.
Rihova B, Strohalm J, Kobackova K. Acquired and specific immunological mechanisms co-responsible for efficacy of polymer-bound drugs. J Cont Release. 2002;17:97-114.
Tatar P, Kiraz N, Asiltürk M, Sayılkan F, Sayılkan H, Arpaç E. Antibacterial thin films on glass substrate by sol gel process. J Inorg Organomet Polym Mater. 2007;3:127-32.
Ozkan A. Lymphokine-Activated Killer cell susceptibility in epirubicin resistant and parental human non-small cell lung cancer (NSCLC). Biologia. 2007;62:232-37.
Gloeckner H, Jonuleit T, Lemke HD. Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue (TM). J Immunol Methods. 2001;252:131-38.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55- 63.
Wasowicz W, Neve J, Petretz A. Optimized steps in fluorometric determination of thiobarbitiric acid- reactive substances in serum; importance of extraction pH and influence of sample preservation and storage. Clin Chem. 1993;39:2522-526.
Bradford MM. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72:248-54.
Garcon G, Dagher Z, Zerimech F, Ledoux F, Courcot D, Aboukais A et al. Dunkerque City air pollution particulate matter-induced cytotoxicity, oxidative stress and inflammation in human epithelial lung cells (L132) in culture. Toxicol in Vitro. 2006;20:519-28.
Habig WH, Jakoby WB. Assay for differentiation of glutathione 1974;77:389-405. Meth Enzymol.
Flohe L, Gunzler WA. Glutathione peroxidase. Meth Enzymol. 1984;105:115-21.
Beutler E. Glutathione peroxidase. In Red Cell Metabolism. A Manual of Biochemical Methods. Grune & Stratton, New York. 1975.
Kirkman TW. Statistics to use [Online]. Available:http://www.physics.csbsju.edu/stats/1996 [17 August 2008].
Mukherjee SG, O'Claonadh N, Casey A, Chambers G. Comparative in vitro cytotoxicity study of silver nanoparticle on two mammalian cell lines. Toxicol In Vitro. 2012;26:238-51.
Erdogan A, Ozkan A. A comparative study of cytotoxic, membrane and DNA damaging effects of Origanum majorana's essential oil and its oxygenated monoterpene component linalool on parental and epirubicin-resistant H1299 cells. Biologia.2013;8:754-61.
Tirkey N, Pilkhwal S, Kuhad A, Chopra K. Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbontetrachloride in rat liver and kidney. BMC Pharmacol. 2005;5:2.
Harhaji L, Isakovic A, Raicevic N, Markovic Z, Todorovic-Markovic B, Nikolic N, Vranjes-Djuric S, Markovic I, Trajkovic V. Multiple mechanisms underlying the anticancer action of nanocrystalline fullerene. Eur J Pharmacol. 2007;568:89-98.
Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress. Environ Sci Technol. 2007;41:4158-163.
Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ. The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett. 2008;179:130-39.
Kim S, Choi JE, Choi J, Chung KH, Park K, Yi J, Ryu DY. Oxidative stress dependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol In Vitro. 2009;23:1076-84.
Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro. 2005;19:975-83.
Oberdörster E. Manufactured nanomaterials (Fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass. Environ Health Perspect. 2004;112:1058-62.
Arora S, Jain J, Rajwade JM, Paknikar KM. Cellular responses induced by silver nanoparticles: In vitro studies. Toxicol Lett. 2008;179:93-100.
Piao MJ, Kang KA, Leeb IK, Kim HS, Kim S, Choi JY et al. Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria- involved apoptosis. Toxicol Lett. 2011;201:92-100.
www.invitrogen.com
Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 3rd ed. Oxford: Oxford Univ. Press. 1999.
Cooke MS, Evans MD, Burd RM, Patel K, Barnard A, Lunec J et al. Induction and excretion of ultraviolet-induced 8-oxo- 2V-deoxyguanosine and thymine dimers in vivo: implications for PUVA. J Invest Dermatol. 2001;116:281 - 85.