THE ANTICANCER POTENTIAL OF THYMOQUINONE IS ENHANCED IN BREAST CANCER CELLS ENRICHED WITH COPPER

Abstract

Cellular and serum levels of copper are seen to be upregulated in many cancer types including aggressive breast cancer. High copper levels aid aggressive breast cancer in multiple facets including angiogenesis and proliferation signalling. Thymoquinone (TQ) the bioactive constituent of Black Seed has displayed promising antioxidant and antiproliferative effects in many cancer types both in vitro and in vivo. Further, TQ was demonstrated to be a copper redox modulator able to induce oxidative DNA breakage in cells via the mobilization of endogenous copper ions. However, the effect of TQ on breast cancer displaying increased copper levels is unknown. Here, we investigated the anticancer potential of TQ on two breast cancer cell lines MDA-MB-231 and MCF-7 cells enriched with copper to mimic the in vivo conditions. The effect of TQ on the viability and cell cycle progression of both cell lines cultured in normal and increased copper conditions was evaluated by MTT assay and flow cytometry, respectively. In addition, reactive oxygen species generation after TQ treatment was assessed by DCFDH assay and PARP expression was evaluated by Western blot. This study is the first to report increased cytotoxic effects of TQ in breast cancer cells enriched with copper. TQ alone inhibited cell viability in a dose-dependent manner in both MDA-MB-231 and MCF-7 cell lines. The IC50 values of TQ were 32 µM and 46 µM in MDA- MB-231 and MCF-7 cell lines respectively at 24 hours post treatment with TQ in low copper conditions. IC50 values dropped to 10 µM and 38 µM for MDA_MB-231 and MCF-7 cells respectively when in high copper conditions. TQ caused greater inhibition of cell viability, increased sub-G1 cells and elevated ROS generation when both cell lines were enriched with copper compared to those cultured in normal copper conditions. Pre-treatment with N-acetyl cysteine (NAC) abrogated the inhibitory effect of TQ on cell viability and cell cycle distribution suggesting that cell death is ROS dependent. In conclusion, our results suggest that the anticancer efficacy of TQ was further enhanced in breast cancer cells containing high copper. Further studies are needed to elucidate the role of copper in the anticancer mechanism of TQ and explore TQ ability to selectively induce cell death in cancer cells while sparing normal cells having normal copper level.