IP: 5.10.31.211 On: Tue, 25 Jun 2019 18:13:15 Delivered by Ingenta Article(s) and/or figure(s) cannot be used for resale. Please use proper citation format when citing this article including the DOI, publisher reference, volume number and page location. Oncology Research, Vol. 14, pp. 381–386 0965-0407/04 $20.00 + .00 Printed in the USA. All rights reserved. Copyright  2004 Cognizant Comm. Corp. www.cognizantcommunication.com Differential Effects of Doxorubicin and Docetaxel on Nitric Oxide Production and Inducible Nitric Oxide Synthase Expression in MCF-7 Human Breast Cancer Cells Gulperi Oktem,* Bulent Karabulut,† Nur Selvi,† Canfeza Sezgin,† Ulus Ali Sanli,† Ruchan Uslu,† Mine Ertem Yurtseven,* and Serdar Bedii Omay† 1 *Histology and Embryology and †Hematology-Oncology, Ege University Medical Faculty, Bornova 35100, Izmir, Turkey (Submitted December, 2003; revision received April 2, 2002; accepted April 30, 2004) Anthracyclines and docetaxel are frequently used agents in the chemotherapy of breast cancer. In this study we examined the role of inducible nitric oxide synthase (iNOS) expression during the cytotoxicity of these drugs in the MCF-7 breast cancer cell line. Cell viability and cytotoxicity were evaluated by the trypan blue dye exclusion method. Apoptosis and necrosis were determined by the acridine orange/ethidium bromide dye method. The percentage of apoptotic cells was significantly higher with doxorubicin. However, total cytotoxic cell numbers were higher in the docetaxel group compared with doxorubicin, with respect to the control. Most of the cells were seen to be necrotic with the dye method. Cell extracts during the apoptotic process were applied to immunoblot by anti-iNOS monoclonal antibodies. While there was an increase in iNOS expression during docetaxel induced-cytotoxicity, a significant decrease in iNOS expression was de- tected during doxorubicin-induced cytotoxicity. The Griess method was used for detection of nitrate levels. It was compatible with immunoblot results. These data open a window for further studies to understand the mechanism underlining the cytotoxicity of docetaxel and doxorubicin. Key words: MCF-7; Inducible nitric oxide synthase (iNOS); Nitric oxide; Cytotoxicity; Doxorubicin; Docetaxel Breast cancer is frequently diagnosed in women and a increased production of NO in murine melanoma cells is associated with apoptosis (8). This small molecule is number of problems occur during its treatment planning and prognosis prediction (1). Unfortunately, a consider- the product of the conversion of L-arginine to L-citrul- line by nitric oxide synthase (NOS), with three distinc- able number of patients develop emerging resistance, which causes serious problems such as metastasis, re- tive isoenzymes that are constitutively expressed in a variety of cells: the calcium-dependent endothelial (eNOS), fractory state to conventional treatment modalities, and relapses (2). Anthracyclines and docetaxel are frequently neuronal (nNOS), and calcium-independent inducible NOS (iNOS) (9). Inducible calcium-independent form used agents in the treatment of this cancer (3). However, because of the growing resistance to these drugs, new of NO synthase (iNOS) can be found in macrophages, hepatocytes, neutrophils, endothelial cells, and astro- molecular targets and new agents are essential for fur- ther treatment modalities. cytes. NO derived from iNOS activity is extremely im- portant in the immunogenic response of T lymphocytes, Nitric oxide (NO 2 ) is a short-lived inorganic free radi- cal gas (4). The roles of NO can be described in three macrophages, and reticuloendothelial cells. The iNOS isoform is normally associated with immune effector categories: as an intracellular signal, as a transcellular messenger, and as a cytotoxic agent. NO acts as an inter- cells such as macrophages, which can be induced to ex- press iNOS as a part of their cytotoxic repertoire (10). cellular secondary messenger in various phenomena such as vascular homeostasis, neurotransmission, and antimi- Therefore, the finding that iNOS expression is present in stromal elements of breast and gastric cancer tissue is crobial activity (5,6). Many studies suggest that NO can play a significant role in tumor growth (7). It has been important. There are in vivo studies suggesting that iNOS has a role and potential as a therapeutic target in shown that the generation of NO by tumor cells corre- lates inversely with their propensity for metastasis. Also, human cancers (11,12). Overall, these studies, together 1 Address correspondence to Serdar Bedii Omay, M.D., Ph.D., Ege University School of Medicine Department of Hematology-Oncology, 35100 Bornova, Izmir, Turkey. Tel/Fax: +90 232 374 73 21; E-mail: omay@med.ege.edu.tr 2 Abbreviations used: NO, nitric oxide; iNOS, inducible nitric oxide synthase. 381