Concurrent Reactive Oxygen Species Generation and Aneuploidy Induction Contribute to Thymoquinone Anticancer Activity

Abstract

Thymoquinone (TQ) is the main biologically active constituent of Nigella sativa. Many studies have confirmed its anticancer actions. Herein, we investigated the different anticancer activities of, and considered resistance mechanisms to, TQ. MTT and clonogenic data showed TQ’s ability to suppress breast MDA-MB-468 and T-47D proliferation at lower concentrations compared to other cancer and non-transformed cell lines tested (GI50 values ≤ 1.5 µM). Flow-cytometric analyses revealed that TQ consistently induced MDA-MB-468 and T-47D cell-cycle perturbation, specifically inducing pre-G1 populations. In comparison, less sensitive breast MCF-7 and colon HCT-116 cells exhibited only transient increases in pre-G1 events. Annexin V/PI staining confirmed apoptosis induction in MDA-MB-468 and HCT-116 cells, which was continuous in the former and transient in the latter. Experiments revealed the role of reactive oxygen species (ROS) generation and aneuploidy induction in MDA-MB-468 cells within the first 24 h of treatment. The ROS-scavenger NAD(P)H dehydrogenase (quinone 1) (NQO1; DT-diaphorase) and glutathione (GSH) were implicated in resistance to TQ. Indeed, western blot analyses showed that NQO1 is expressed in all cell lines in this study, except those most sensitive to TQ-MDA-MB-468 and T-47D. Moreover, TQ treatment increased NQO1 expression in HCT-116 in a concentration-dependent fashion. Measurement of GSH activity in MDA-MB-468 and HCT-116 cells found that GSH is similarly active in both cell lines. Furthermore, GSH depletion rendered these cells more sensitive to TQ’s antiproliferative actions. Therefore, to bypass putative inactivation of the TQ semiquinone metabolite, the benzylamine analogue was designed and synthesised following modification of TQ’s carbon-3 atom. However, the structural modification negatively impacted potency against MDA-MB-468 cells. In conclusion, we disclose the following: (i) The anticancer activity of TQ may be a consequence of ROS generation and aneuploidy; (ii) Early GSH depletion could substantially enhance TQ’s anticancer activity; (iii) Benzylamine substitution at TQ’s carbon-3 failed to enhance anticancer activity.

Thymoquinone effect on the Dictyostelium discoideum model correlates with functional roles for glutathione S-transferases in eukaryotic proliferation, chemotaxis, and development

“…A notable, two-fold increase in ROS was observed in cells that were treated with 7.5 μM thymoquinone for 5hr ( Fig 8A and 8B ). Whereas GSH is known to control ROS production and is implicated in thymoquinone resistance [ 34 ], recent findings indicate that sulfur/cysteine sequestration is a rate limiting variable to initiate development during starvation [ 33 ]. Moreover, because GSH is an essential to the conjugation of multiple substrates in GST enzyme activity, we measured the levels of oxidized and reduced glutathione (GSSG, GSH).…”

Metformin and thymoquinone co‐treatment enhance 5‐fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells

“…28,42,43 Furthermore, TQ induced apoptosis in cancer cells by promoting glucose oxidative phosphorylation following inhibition of LDHA enzyme, enhancing ROS production with declines in antioxidants, and downregulating the PI3K/mTOR/HIF1α network. [24][25][26][27][28][29] Further studies have also reported that combining 5-FU with TQ 28,32 or Met 23,33 resulted in boosted in vitro and in vivo anti-cancer actions against CRC. Although more recent reports have also shown enhanced anti-cancer effects for TQ and Met co-therapy in human leukemic cell lines 30 and breast cancer in mice, 31 there is no report regarding their combination against CRC.…”

“…[17][18][19][20][21][22][23] Similarly, the main active compound of the black seed, thymoquinone (TQ), has demonstrated efficient tumoricidal actions against CRC by inhibiting the PI3K/mTOR/HIF1α network, inducing oxidative glycolysis, and increasing oxidative stressmediated apoptosis. [24][25][26][27][28][29] Moreover, others have reported boosted anti-cancer effects against leukemia and breast cancer following co-treatment with metformin and TQ, 30,31 as well as by adding each of them with 5-FU for the treatment of CRC. 23,28,32,33 However, none of the prior reports investigated the potential beneficial effects of combining metformin and TQ, with and without 5-FU, against colon neoplasia.…”

Nutraceutical Prevention of Diabetic Complications—Focus on Dicarbonyl and Oxidative Stress

“…The ability of thymoquinone (a key component of Nigella sativa —black cumin seed—oil) and of pyrroloquinoline quinone (PQQ)—a vitamin-like compound in the diet that binds with high affinity to lactate dehydrogenase—to boost Sirt1 activity rests in their ability to promote oxidation of NADH to NAD+. The antioxidant enzyme NAD(P)H quinone oxidoreductase 1 (NQO1) can reduce thymoquinone to thymohydroquinone, converting NADH to NAD+ in the process; thymohydroquinone can then act as a scavenging antioxidant [ 160 ]. This mechanism can explain thymoquinone’s ability to activated Sirt1 [ 161 , 162 , 163 ].…”