Biologia, Bratislava, 62/2: 232—237, 2007 Section Cellular and Molecular Biology DOI: 10.2478/s11756-007-0040-5 Lymphokine-activated killer cell susceptibility in epirubicin-resistant and parental human non-small cell lung cancer (NSCLC) Aysun Ozkan Akdeniz University, Art-Science Faculty, Biology Department, TR-07058 Antalya Turkey; e-mail: aozkan@akdeniz.edu.tr Abstract: The aim of this study was to evaluate that: (i) epirubicin-HCl (EPI) and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radical generation; and (ii) resistant H1299 cells may be more sensitive to combined treatment of LAK cells plus EPI than the LAK or EPI treatment alone. Viability of H1299 cells treated with EPI, LAK and LAK plus EPI was measured using the MTT test. Amount of glutathione (GSH), protein content and enzymatic activity were measured by spectrophotometer. Glutathione S-transferase (GST)-pi expression in the cells was determined by western blot analysis. LAK plus EPI combined treatment increased susceptibility of H1299 WT and H1299 EPI(R) (300–fold EPI resistant) cells to LAK cell cytotoxicity. The resistance of H1299 EPI(R) cells to EPI appears to be associated with a developed tolerance to free radicals, most likely because of a 2-fold increase in NADPH-dependent- cytochrome-P450 reductase (NADPH-CYP reductase) activity, 11-fold GST activity and 11– and 7-fold augmented selenium dependent and independent glutathione peroxidase (GSH-Px) activity, respectively. Amount of GST-pi in H1299 EPI(R) cells is statistically different from negative control and H1299 WT (p< 0.01). It is proposed that production of reactive oxygen species and hydrogen peroxide by the treatment of EPI and LAK cells can cause cytotoxicity of H1299 WT and H1299 EPI(R) cells. Superoxide dismutase, catalase, GSH-Px, GST, NADPH-CYP reductase and GSH must be considered as part of the intracellular antioxidant defense mechanism of H1299 WT and H1299 EPI(R) cells against reactive oxygen species. Combined treatment of EPI plus LAK cells caused the increasing cytotoxicity on the H1299 EPI(R) cells. Key words: non-small cell lung cancer; lymphokine-activated killer cells; epirubicin-HCl; antioxidants. Abbreviations: CYP, cytochrome P450; EPI, epirubicin-HCl; EPI(R), 300–fold EPI resistant; GSH, glutathione; GSH- Px, glutathione peroxidase; GST, glutathione S-transferase; IL, interleukin; LAK, lymphokine-activated killer; NK, natural killer; NSCLC, non-small cell lung cancer; PBS, phosphate-buffered saline; SOD, superoxide dismutase. Introduction The antracycline analogue epirubicin-HCl (EPI) is an intensely potent cytotoxic compound. It causes less car- diac injury than doxorubicin derivatives at doses pro- ducing equal antitumor activity. The proposed mecha- nism for their cytotoxic effects involves the formation of intracellular free radicals caused by the quinone group of antracycline (Dobbs et al. 1994). Glutathione peroxidase (GSH-Px), catalase and superoxide dismutase (SOD) protect the cells from the effects of reactive oxygen species generated dur- ing the one-electron reduction of quinines (Ozkan & Fiskin 2004). Antioxidants and detoxification mech- anisms that can be involved in the drug resistance of various tumors include glutathione (GSH), GSH- Px and glutathione S-transferase (GST) (Ozkan et al. 2004). Natural killer (NK) cells exhibit cytotoxic ac- tivity against virus-infected cells and tumor cells. NK cells that were stimulated with interleukin 2 (IL-2) con- tribute to the lymphokine-activated killer (LAK) cell phenomenon (Fibeiro-Dias et al. 2000). In the human non-small cell lung cancer (NSCLC), GSH played a vital role in the protection of trichloroethylene- and perchloroethylene-induced oxidative stress (Chen et al. 2002). We tested free radical-mediated cytotoxicity of EPI, LAK and LAK plus EPI (1/10 of the IC50 con- centration) in H1299 WT and H1299 EPI(R) cells. The addition of a low concentration of EPI to LAK cells exposed to H1299 cells caused a significant increase in the susceptibility of H1299 WT and H1299 300-fold EPI resistant (EPI(R)) cells to LAK cells. Human lung can- cer is probably a good candidate for the combination of immunotherapy and chemotherapy. Material and methods Drug and chemicals EPI was purchased from Pharmacia (Italy). Reducing sam- ple buffer (BL 46056) was from Perbio (USA). ECLTM 1059243 batch 25 detection reagent I and 1059250 batch 25 detection reagent II were from Amersham (USA). The anti- bodies for western blotting were from Santa Cruz Biotech- nology (CA, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The water was deionized and distilled in glass. c 2007 Institute of Molecular Biology, Slovak Academy of Sciences Unauthenticated Download Date | 7/27/18 2:04 PM