(M1130-10-55) Combination of Thymoquinone and Curcumin Induces Cell Death by Alteration of Redox Homeostasis in A375 Melanoma Cell Line

Purpose: Thymoquinone (TQ) is a potent antioxidant and anti-inflammatory extracted from Nigella Sativa (N. Sativa). Thymoquinone carries out its anticancer activity by activating molecular signaling pathways, leading to apoptosis and cell cycle arrest in various cancer cell lines. Curcumin (CU) is an antioxidant with a broad spectrum of anticarcinogenic activity. In this study, we investigated the combination of thymoquinone and curcumin on a melanoma cell line (A375). The redox rewiring, apoptotic and metabolomic reprogramming effects of thymoquinone and the combination of these two potent antioxidants.

Methods: Human melanoma A375 cell line was treated with different concentrations of thymoquinone, curcumin and the combination of both for 24 and 48 hours. Cell viability studies were performed using AlamarBlue assay. Cytoplasmic level of 116 metabolites were investigated using LC/MS. Reactive oxygen species (ROS) and Annexin v related apoptosis were measured by flow cytometry.

Results: Cell viability studies showed the increased killing effects of thymoquinone and curcumin in a dose and time- dependent manner. Curcumin showed protective effects with high dose TQ (40 uM), where the viability increased from 37% to 46%, 57% and 61% when TQ combined with 5uM, 10uM and 15uM of CU, respectively (Figure 1.). While ROS level was decreased at low concentrations of TQ ( 20 uM), it has significantly increased at a high dose (40 uM). These findings suggest a dual effect of TQ (i.e. pro- and anti-oxidant) (Figure 3.). Pairing ROS levels with the viability results proves that TQ exerts its killing effect by the production of ROS. To take a further step in understanding the mechanisms and effects of TQ on melanoma cell lines, we studied the mitochondrial metabolites, which emphasized these findings. At high doses, TQ and CU significantly increased the level of glutathione, a potent biological scavenger (Figure 3.) Nonetheless, TQ and CU increased the pool level of TCA cycle metabolites at a high dose.
These findings were consistent with ROS levels, where their levels decreased with low dose, and increased with high dose thymoquinone and curcumin. The combination also increased the cells in the early apoptosis stage (Figure 2.). While each treatment alone increased the cells number in the late apoptosis stage which proved by the protective effect in the cell viability assay.

Conclusion: In conclusion, combination of TQ and CU showed a promising in vitro result which may be useful for the treatment of melanoma. High dose of TQ and CU increased ROS levels and induced programmed cell death. TQ and low concentration of curcumin had a protective effect on cell line. The combination of TQ and CU increase the pool size of glutathione and decrease the ROS production.


Dose dependent effect of curcumin and thymoquinone for 24 hours on the proliferation of human (A375) was determined using AlamarBlue assay (N=3).


Apoptosis effect of TQ and CU in melanoma cell line A375 for 24hr. TQ increases early and late apoptosis in A375 cells and the combination with CU increase the early apoptosis. sample were analyzed in flow cytometry using Annexin-V-FIT dye. All concentrations were made in three independent study (N=3)


(A) Effect of CU and TQ on cellular ROS production. After indicated treatment for 24hr, cells were incubated with 10uM DCFH-DA for 30 min and then immediately subjected to flow-cytometric analysis. Results are expressed as a ratio of relative fluorescent intensity compared to control group (N=3)
(B) The effect of TQ and CU on the pool size of glutathione (N=3).