Study of the Chemo‐sensitizing Effects of Thymoquinone, the Active Constituent of Negilla Sativa (Black Seed), to Nitrogen Mustards, Chlorambucil and Cyclophosphamide, in Human Melanoma Cell Lines

Abstract

Black seed is often used as a spice in food and as a preservative in many products. The main active constituent of black seed oil is thymoquinone (TQ) which constitutes 30–57% of the oil. Thymoquinone has shown varying therapeutic benefits in various disease states, including cancer. The aim of this study was to investigate the cytotoxic and chemo‐sensitizing potential of TQ and compare it to ethacrynic acid (EA), a known cytotoxic agent in sensitizing melanoma cells to the effects of the N‐mustards, chlorambucil (CHL) and cyclophosphamide (CP). Both TQ and EA have an α, β unsaturated carbonyl system which could act as potent Michael Acceptors to mediate the cytotoxicity. Studies also assessed the cytotoxic potential of cyclophosphamide(CP) and chlorambucil (CHL) alone and in combination with TQ. Approximately 1×104 cells/100μL/well of two melanoma cell lines, lightly pigmented SK‐MEL19 and pigmented SK‐MEL23 were seeded in 96‐well plates and incubated for 24h at 37C° and 5% CO2. The cells were then treated with TQ (25–500 μM), EA (25–200 μM), CHL (25–500 μM) and CP (25–500 μM), for 24h and 48h. Combination treatment included pretreatment of the cells with TQ (100 μM) for 24 h followed by further exposure to CHL for an additional 24 h. Similarly, cells were pretreated with EA (100 μM) for 24h, followed by further exposure to CHL for 24h. Colorimetric MTS (Abcam) cytotoxicity assays, per manufacturer's instructions, was then carried out to determine cell viability. The results indicated that TQ exerts its toxic effect in a concentration‐dependent manner in SKMEL19 cell lines at concentrations ≥75μM, while in SK‐MEL23 at concentrations ≥ 100 μM, relative to the control. In addition, EA reduced cells viability significantly in both cell lines at concentration of 100 μM and above. However, CP did not show any cytotoxic effect in both cell lines at 24h or 48h up to concentrations of 500 μM. In contrast, CHL reduced cell viability in both cell lines at high concentrations (250 and 500μM) within 24h. When the incubation time was increased to 48h, the cytotoxic effect of CHL in SK‐MEL19 was observed at concentrations ≥ 100 μM, while in SK‐MEL23 at concentrations ≥ 150 μM. Pre‐treatment with TQ (100 μM), as well as EA (100 μM) greatly enhanced the cytotoxic effect of a non‐toxic dose of CHL (100 μM) in both cell lines after 24h as compared to the effect of CHL alone. These studies demonstrate the potentiating effect of TQ on CHL cytotoxicity possibly by sensitizing the human melanoma cell lines to the chemotherapeutic agent.Support or Funding InformationMA acknowledges SACM for financial supportThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.