Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage and consequent cell death: putative anticancer mechanism of antioxidants

Abstract

Plant-derived dietary antioxidants have attracted considerable interest in recent past for their chemopreventive and cancer therapeutic abilities in animal models. Thymoquinone (TQ) is the major bioactive constituent of volatile oil of Nigella sativa and has been shown to exert various pharmacological properties, such as anti-inflammatory, cardiovascular, analgesic, anti-neoplastic, anticancer and chemopreventive. Although several mechanisms have been suggested for the chemopreventive and anticancer activity of TQ, a clear mechanism of action of TQ has not been elucidated. TQ is a known antioxidant at lower concentrations and most of the studies elucidating the mechanism have centered on the antioxidant property. However, recent publications have shown that TQ may act as a prooxidant at higher concentrations. It is well known that plant-derived antioxidants can switch to prooxidants even at low concentrations in the presence of transition metal ions such as copper. It is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Copper is an important metal ion present in the chromatin and is closely associated with DNA bases, particularly guanine. Using human peripheral lymphocytes and comet assay, we first show that TQ is able to cause oxidative cellular DNA breakage. Such a DNA breakage can be inhibited by copper-chelating agents, neocuproine and bathocuproine, and scavengers of reactive oxygen species. Further, it is seen that TQ targets cellular copper in prostate cancer cell lines leading to a prooxidant cell death. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of plant-derived antioxidants.

Figure 1

Effect of increasing (a) native DNA base pair molar ratios and (b) copper concentrations on the fluorescence emission spectra of TQ. TQ (in 10 mM Tris-HCl, pH 7.5) was excited at 255 nm in the presence of (a) increasing native DNA base pair molar ratios and (b) increasing concentration of Cu(II) and the emission spectra were recorded between 500 and 540 nm

Figure 2

Reduction of Cu(II) to Cu(I) by TQ. Detection of TQ-induced Cu(I) production by bathocuproine. Reaction mixture contained 10 mM Tris-HCl (pH 7.5) along with 300 μM bathocuproine and indicated concentrations of the following: bathocuproine+100 μM Cu(II); bathocuproine+50 μM TQ; bathocuproine+50 μM TQ+100 μM Cu(II)

Figure 3

Generation of hydroxyl radical generation by TQ. Formation of hydroxyl radicals as a function of increasing concentration of TQ in the presence of Cu(II). The reaction mixture (0.5 ml) contained 100 μg calf thymus DNA as substrate, 50 μM Cu(II) and indicated concentrations of TQ and incubated at 37 °C for 30 min. Hydroxyl radical formation was measured by determining the TBA-reactive material. All values reported are mean±S.E.M. of three independent experiments

Figure 4

Agarose gel electrophoretic pattern of ethidium bromide-stained pBR322 plasmid DNA after treatment with TQ in the absence and presence of copper. Agarose gel electrophoretic pattern of ethidium bromide-stained pBR322 DNA after treatment with TQ and Cu(II). Reaction mixtures were incubated at 37 °C for 1 h in dark. Lane D: pBR322 DNA alone; lane C: pBR322 DNA+Cu (II) 30 μM; lanes 1, 2, 3: pBR322 DNA+TQ (50, 100, 150 μM, respectively); lanes 4, 5, 6: pBR322 DNA+Cu(II) 30 μM+TQ (50, 100, 150 μM, respectively)

Figure 5

DNA breakage by TQ in human peripheral lymphocytes. (a) Whole lymphocyte cells were incubated in microfuge tubes with reaction mixture at 4 °C for 1 h in dark. Reaction mixture contained RPMI (400 μl), Ca²⁺- and Mg²⁺-free PBS, increasing concentrations of TQ (0–40 μM), alone (◊) and with fixed concentration of Cu(II) (25 μM) (□) and processed further for comet assay. (b) Comparison of DNA breakage by TQ in intact lymphocytes and lymphocyte nuclei as measured by comet assay. Whole lymphocyte cells (⧫)/lymphocyte nuclei (▪) embedded in agarose were incubated with the reaction mixture containing indicated concentrations of TQ (0–40 μM) at 4 °C for 1 h and processed further for comet assay. Whole lymphocyte (c)/lymphocyte nuclei (d) embedded in agarose were incubated with the reaction mixture (2.0 ml) containing TQ (40 μM) and indicated concentrations of membrane permeable copper chelator (Neo, neocuproine); membrane impermeable copper chelator (Batho, bathocuproine) at 4 °C for 1 h and processed further for comet assay. Values reported are mean±S.E.M. of three independent experiments. Error bars denote ±S.E.M. **P<0.05 when compared with *(untreated control cells)

Figure 6

Effect of preincubation of lymphocyte with neocuproine and thiourea on TBARS generated by increasing concentrations of TQ. Effect of preincubation of lymphocyte with neocuproine and thiourea on TBARS generated by increasing concentrations of TQ. Lymphocyte cells were preincubated with fixed concentration of neocuproine and thiourea for 30 min at 37 °C after which it was further incubated for 1 h in the presence of increasing concentrations of TQ. Values reported are mean of three independent experiments

Figure 7

Effect of TQ on cell proliferation and induction of apoptosis in prostate cancer cells. (a) The effect of TQ on cell growth in prostate cancer cell lines as detected by MTT assay. The cells were incubated with indicated concentrations of TQ for 72 h. (b) The effect of copper chelator neocuproine (Neo), iron chelator desferrioxamine mesylate (DM) and zinc chelator histidine (His) on TQ-induced cell growth inhibition in PC3 and LNCaP prostate cancer cells as detected by MTT assay. The cells were incubated with 5 μM TQ in the presence of 50 μM metal-specific chelator for 72 h and MTT assay performed. (c) The Cell Death Detection ELISA Kit (Roche) was used to detect apoptosis in PC3 and LNCaP prostate cancer cells treated with increasing concentrations of TQ as indicated and the effect of 50 μM metal chelators on TQ-induced apoptosis on PC3 and LNCaP cells. Cells were incubated with 5 μM TQ alone or in the presence of copper chelator neocuproine (Neo), iron chelator desferioxamine mesylate (DM) and zinc chelator histidine (His). All results are expressed as percentage of control±S.E. of triplicate determinations from three independent experiments. *P<0.01 when compared with untreated (control) cells