Investigation of the effect of Thioridazine in breast cancer cell cultures

The aim of this study was to investigate cytotoxic effects of antipsychotic Thioridazine and to reveal type of cell death in breast cancer (MDA-MB-231, MCF-7) and breast cancer stem (CD44+/CD24-) cells. The second aim is, to reval role of THZ in epithelial mesenchymal transition. The percentage viability of THZ (1-30 μM, 24 hours) treated cells was measured with XTT test. IC50 values were calculated by Calcusyn 2.1 software. To validate uptake of THZ by cells, specific inhibitor of dopamine activator transporters, Threo-methylphenidate hydrochloride (TMH), was used. ELISA kit and Annexin V/7-AAD were used to investigate apoptosis, measurement of protein expressions of LC-3 I/II and Beclin-1 were done by performing western blot analysis. Monodansylcadaverine dye (stain) was used to show autophagosomes in the fluorescence microscope. The effect of THZ on epithelial mesenchymal transition was inveastigated by using cell migration, invasion and Western blot methods. IC50 values of THZ were found as 9±1.2 µM (MDA-MB-231, 24 hours), 16.4±1.8 µM (MCF-7, 24 hours), and 18.1±2.3 µM (CD44+/CD24-, 24 hours). The significant correlation between basal expressions of DR2 [(MDA-MB-231, 111.6±2 ng/mL), (MCF-7, 86.5±1.4 ng/mL), (CD44+/CD24-, 52±2.3 ng/mL)] and IC50 values was detected. In TMH inhibited cell cultures, THZ mediated cytotoxic effect was observed to be decreased (p<0.05). THZ mediated apoptotic cell percentages were calculated as 10.8% for MDA-MB-231, 30% for MCF-7, and 46.8% for CD44+/CD24-. Apoptosis was detected in MCF-7 and CD44+/CD24- while MDA-MB-231 cells were autophagic. Invasion potency of MCF-7, CD44+/CD24- and MDA-MB-231 cells was determined as 29%, 24%, 16.5%, respectively. Migration potency of of MCF-7, CD44+/CD24- and MDA-MB-231 cells was determined as 28.5%, 63.2%, 61%, respectively. Among the three cell lines, MDA-MB-231 cells have highest invasive and migration ability than other cell lines. We demonstrated that THZ significantly increases E-cadherin, Cytokeratin-18, β-Catenin, while inhibiting N-cadherin, Vimentin, Fibronectin at protein level. We also confirmed the expressions of E-cadherin and Vimentin by using immunocytochemistry. E-cadherin expression increased while Vimentin decreasing all three tested cell lines. In this study, the cytotoxic effect of THZ is mediated by two different types of cell death in breast cancer and breast cancer stem cells. Inhibition of epithelial mesenchymal transition was confirmed by using independent methods. In conclusion, these data suggest that THZ might be a novel cytotoxic and anti-metastatic agent for breast cancer at in vitro cell culture.