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.