1. The Effects of Drug-Resistant MCF7 Sublines on Cellular Proliferation and Gene Expression Pattern of Drug- Sensitive MCF7 Cells Esin Gülce SEZA*, Laçin YAPINDI*, Çağrı URFALI MAMATOĞLU, Ufuk GÜNDÜZ Middle East Technical University, Department of Biological Sciences, 06800 Ankara-Turkey Middle East Technical University, Department of Biological Sciences, 06800 Ankara-Turkey *Authors contributed equally INTRODUCTIONINTRODUCTION MATERIALS & METHODS The resistance of tumor cells to structurally and functionally unrelated drugs is named as multidrug resistance (MDR) [1]. Tumors consist of cells with different genetic make-ups. Due to the tumor heterogeneity, some of the cells in a tumor could be drug-sensitive whereas the others could be intrinsicly resistant or acquire resistance during the course of chemotherapy. This heterogenous nature of tumors could cause the failure in chemotherapy. The aim of the current study is to investigate the effects of drug- resistant MCF7 sublines on drug-sensitive MCF7 cells. Chemicals Zoledronic acid was kindly donated by Novartis Pharma AG, Switzerland. Doxorubicin and docetaxel,were provided by Gülhane Military Medical School, Ankara,Turkey. Cell culture Parental MCF7 cells were obtained from ŞAP Institute, Ankara, Turkey. Docetaxel, doxorubicin and zoledronic acid resistant MCF7 cells were developed previously in our laboratory by stepwise selection of parental MCF7 cells in increasing drug concentrations [2,3,4]. Parental and drug resistant MCF7 cells were maintained in RPMI1640 medium supplemented with 10% FBS in an humidified atmosphere with 5% CO2 at 37 o. Drug-resistant cells were treated with corresponding anticancer agents to maintain drug resistance. Growth curve analyses The effect of drug-resistant MCF7 cells on the proliferation of drug-sensitive MCF7 cells was investigated by mimicking co-culture conditions. To this end, 2x10 5 drug-resistant and drug- sensitive MCF7 cells were seeded onto 6-well plates, separately. At each 24h interval, the growth medium of drug-resistant cells (conditioned medium) was collected, centrifuged at 1000 rpm for 5 min and the supernatant was added onto drug-sensitive cells. Drug-sensitive cells were harvested by trypsinization and counted every 24h to plot a growth curve. Results were analyzed by 2 -DDCt method [5]. Gene expression analyses 2x10 5 parental and doxorubicin resistant MCF7 (MCF7/Dox) cells were seeded onto 6-well plates, separately. At each 24h intervalconditioned medium obtained from MCF7/Dox cells was collected, centrifuged at 1000 rpm for 5 min and the supernatant was added onto drug-sensitive cells. Total RNA was isolated at 24, 48 and 72h by using TRI Reagent according to the manufacturer’sinstructions. cDNA was synthesized by using 1μg RNA and random hexamer primers. MDR1 expression was examined by FastStart Universal SYBR Green Master mix in qRT-PCR analysis. Results were analyzed by 2 -DDCt method [5]. Statistical analysis All experiments were performed in duplicates.Data were analyzed by one-way ANOVA and post-hoc Tukey’s tests onGraphPad Prism 5.0 software. The results were significant when p<0.05. RESULTS & DISCUSSION References [1] Simon, SM; Schindler, M. Cell biological mechanisms of multidrug resistance in tumors. Proc Natl Acad Sci USA. 1994, 91, 3497-3504. [2] İşeri ÖD. Investigation of docetaxel and doxorubicin resistance in MCF-7 breast carcinoma cell line. PhD Thesis, February 2009, Middle East Technical University, Ankara, Turkey. [3] İşeri, OD; Kars, MD; Gündüz, U. Two different docetaxel resistant MCF7 sublines exhibited different gene expression pattern. Mol Biol Rep. 2012, 39(4), 3505-16. [4] Kars, MD; İşeri, OD; Ural, AU; Gündüz, U. In vitro evaluation of zoledronic acid resistance developed in MCF7 cells. Anticancer Res. 2007, 27(6B), 4031-7. [5] Livak, KJ; Schmittgen, TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001, 25(4), 402-408. The major problem in chemotherapy is the development of multidrug resistance. The heterogenous nature of tumor cells decreases the efficiency of anticancer agents, thus causes failure in chemotherapy. a) b) Figure 1. Proliferation profiles of MCF7 cells in conditioned medium obtained from a) MCF7/Zol, b) MCF7/Dox and c) MCF7/Doc cells. The growth curve analyses showed that the proliferation rate of drug- sensitive MCF7 cells significantly increased after 120h when they were treated with conditioned medium obtained from doxorubicin docetaxel resistant MCF7 cells. cellular proliferation was found to be significantly higher even in the absence of FBS, which is essential for cellular growth. However, the growth rate of drug-sensitive MCF7 cells was not significantly affected when they were treated with conditioned medium obtained from zoledronic acid resistant MCF7 cells. c) Drug-resistant MCF7 cells could secrete various growth stimulators such as growth factors, cytokines and chemokines, into the medium which may stimulate the growth of drug-sensitive cells. further studies are required to identify the exact nature of biomolecules in the secretions of resistant cells which can potentially enhance cellular proliferation of others Gene expression analyses revealed that MDR1 expression in MCF7 cells was not significantly affected by the treatment with conditioned medium obtained from doxorubicin resistant MCF7 cells. It could be a result of short treatment period or the molecular composition of conditioned medium obtained from MCF7/Dox cells. Figure 2. MDR1 expression of drug-sensitive MCF7 cells after treatment with conditioned medium obtained from MCF7/Dox cells for 24, 48 and 72h