The Influence of Phenothiazine Derivatives on Doxorubicin Treatment in Sensitive and Resistant Human Breast Adenocarcinoma Cells (phenothiazine derivatives / doxorubicin / breast adenocarcinoma / multidrug resistance)

Breast cancer is commonly treated by various combinations of surgery, radiation therapy, chemotherapy and hormone therapy. Most cancers either are increasingly resistant to any initial treatment or acquire resistance to a broad spectrum of anticancer drugs over time. Combination of more than one drug or combination with multidrug resistance (MDR) modifiers will possibly support the efficiency of the applied therapy. Understanding the MDR mechanisms in malignancies is crucial for developing novel strategies for treatment. The main goal of our study was to determine the cytostatic effect of doxorubicin in combination with phenothiazine derivatives (PD; promazine and triflupromazine) in doxorubicin-sensitive (MCF-7/WT) and -resistant (MCF-7/DOX) human breast adenocarcinoma cell lines. We determined cytotoxicity of the investigated compounds (MTT assay) after 24 and 48 h. The effect of phenothiazine derivatives was evaluated and doxorubicin localization was performed using confocal microscopy. The mode of the cell death was examined by the comet assay. We also determined the expression of P-glycoprotein (P-gp), which is a membrane-associated protein responsible for the multidrug resistance. Introduction Chemotherapy is applied in all stages of breast cancer. In early stages, as well as in all advanced stages, its application depends on the risk and hormone receptor status. The efficiency of chemotherapy in cancer treatment is decreased by the resistance to cytostatics called multidrug resistance (MDR). Resistance to chemotherapy is a common clinical problem in patients with all types of cancer. The medicine targets of malignant cells often show a cross-refractoriness to a variety of drugs that have different structures and functions (Hait, 1990; Bartosz, 1997; Lenart, 2005). Multidrug resistance is a consequence of complex molecular events, which consolidated in malignant cells during evolution. MDR is still one of the reasons of the adversity in anticancer treatment. The active cytostatics transport out of the cells is also responsible for MDR occurrence. The main role in this process is played by proteins called multidrug transporters (Michalak, 2002). The expression of adenosine-5’-triphosphate (ATP)-binding cassette (ABC) proteins is increased in the membranes of resistant cells. Irregular, increased expression of transporter proteins is a factor involved in cancer resistance to cytostatics. The ABC protein family includes membrane proteins. They are constructed of several or dozen domains built in the cell membrane. Their common feature is a specific domain responsible for binding and hydrolysis of ATP (Bartosz, 1997). The energy from ATP decomposition is exploited by these proteins for transport of different substances. Transporters in normal cells play important physiological functions involved in inner and outer transport and participate in detoxification processes. The main transporters of MDR are P-glycoprotein (MDR1) and MDR-related proteins (MRP). They possess the ability to pump out a wide spectrum of compounds differing in structure. The mechanism of action of these MDR transReceived January 5, 2011. Accepted August 5, 2011 This research was supported by Wroclaw Medical University grants Nos. 1709, 1903 and Pbmn/2. Corresponding author: Julita Kulbacka, Department of Biophysics, Wroclaw Medical University, Chalubinskiego 10, 50-367 Wroclaw, Poland. e-mail: [email protected] Abbreviations: ABC – ATP-binding cassette, ATP – adenosine5’-triphosphate, DOX – doxorubicin, MDR – multidrug resistance, MRP – MDR-related proteins, MTT – 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, PD – phenothiazine derivatives, P-gp – P-glycoprotein.