Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin

Doxorubicin is a mainstay of cancer chemotherapy despite its clinical limitations that arise from its cardiotoxicity and the high incidence of multi-drug resistance. Recent studies revealed a protective effect of thymoquinone, a non-toxic constituent of the essential oil of Nigella sativa, against doxorubicin-induced cardiotoxicity. We now investigated the influence of thymoquinone on various other effects exerted by doxorubicin in human cancer cells. Doxorubicin, thymoquinone and equimolar mixtures of both were tested for cytotoxicity on human cells of HL-60 leukaemia, 518A2 melanoma, HT-29 colon, KB-V1 cervix, and MCF-7 breast carcinomas as well as multi-drug-resistant variants thereof and on non-malignant human fibroblasts (HF). Apoptosis induction was analysed via DNA fragmentation, activity studies of the caspases-3, -8 and -9, determination of changes in the mitochondrial membrane potential and in the ratio of the mRNA expressions of pro- and anti-apoptotic proteins bax and bcl-2. The generation of reactive oxygen species (ROS) was assessed by the NBT assay. Thymoquinone improved the anti-cancer properties of doxorubicin in a cell line-specific manner. We found a significant rise of the growth inhibition by doxorubicin in HL-60 and multi-drug-resistant MCF-7/TOPO cells when thymoquinone had been added. The mode of action of both drugs and of their mixture was mainly apoptotic. In HL-60 cells, the drug mixture caused an additional concentration maximum of effector caspase-3 not observed for either of the pure drugs. The impact of the drug mixture on the mitochondria of HL-60 cells was also greater than those of the individual quinones alone. In addition, the drug mixture led to a higher concentration of reactive oxygen species in HL-60 cells. In summary, thymoquinone is a booster for the anti-cancer effect of doxorubicin in certain cancer cell lines. Distinct improvements on efficacy, selectivity, and even breaches of multi-drug resistance were observed for equimolar mixtures of doxorubicin and thymoquinone.