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

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.

Effect of vitamins C and E on antioxidant status of breast-cancer patients undergoing chemotherapy.

WHAT IS KNOWN AND OBJECTIVE: Reactive oxygen/nitrogen species generated by antineoplastic agents are prime suspects for the toxic side-effects of acute or chronic chemotherapy. The present study was undertaken to test whether vitamins C and E (VCE) supplementation protect against some of the harmful effects of commonly used anticancer drugs in breast-cancer patients. METHODS: In a randomized 5-month study, the activity of various antioxidant enzymes (superoxide dismutase, catalase, glutathione-S-transferase and glutathione reductase) and the levels of malondialdehyde and reduced glutathione were measured in forty untreated breast-cancer patients (stage II) and compared with those of healthy controls. The degree of DNA damage was also assessed in the peripheral lymphocytes of the patients by alkaline single cell gel electrophoresis. The untreated patients were then randomly assigned to either treatment with chemotherapy alone (5-fluorouracil 500 mg/m(2) i.v. day 1, doxorubicin 50 mg/m(2) i.v. day 1 and cyclophosphamide 500 mg/m(2) i.v. day 1, every 3 weeks for six cycles) or to the same chemotherapy regimen supplemented with VCE (vitamin C 500 mg tablet and vitamin E 400 mg gelatin capsule). On completion of the treatments, both the groups were studied again for the levels of the markers measured prior to treatment. RESULTS AND DISCUSSION: The untreated group showed significantly lower levels of antioxidant enzymes (P < 0·001) and reduced glutathione (P < 0·001), and more extensive lipid peroxidation (P < 0·001) and DNA damage than healthy controls. Similar but less pronounced patterns were observed in the patients receiving chemotherapy alone. The group of patients receiving VCE supplementation had all the marker levels moving towards normal values. Activities of superoxide dismutase, catalase, glutathione-S-transferase and glutathione reductase, and the levels of reduced glutathione were significantly increased (P < 0·01) while, the levels of malondialdehyde and DNA damage were significantly (P < 0·01) reduced in the VCE supplemented group relative to those of patients receiving chemotherapy alone as well as relative to the pretreatment levels. WHAT IS NEW AND CONCLUSION: Co-administration of VCE restored antioxidant status, lowered by the presence of breast-cancer and chemotherapy. DNA damage was also reduced by VCE. The results suggest that VCE should be useful in protecting against chemotherapy-related side-effects and a randomized control trial to evaluate the effectiveness of VCE in breast-cancer patients using clinical outcomes would be appropriate.

The antioxidant ascorbic acid mobilizes nuclear copper leading to a prooxidant breakage of cellular DNA: implications for chemotherapeutic action against cancer.

PURPOSE: Ascorbic acid is an essential micronutrient and is considered to have an antioxidant function in living systems. For the past several decades, ascorbic acid has been the subject of considerable interest as an anticancer agent. Several studies have shown that ascorbic acid is cytotoxic to a variety of cancer cells, whereas normal cells are relatively resistant to such cytotoxic action. In this study, we propose a putative molecular mechanism that accounts for the preferential cytotoxicity of ascorbic acid against cancer cells. METHODS: Standard and lysed version of alkaline single-cell gel electrophoresis (Comet assay); ferrous oxidation-xylenol orange (FOX) assay. RESULTS: We show that ascorbic acid acts as a prooxidant and leads to oxidative DNA breakage in lymphocytes and lymphocyte nuclei. Scavengers of reactive oxygen species were able to inhibit ascorbic acid-induced DNA breakage, suggesting the involvement of reactive oxygen species in this reaction. We further show that such DNA breakage is inhibited by both iron and copper chelators in cells, whereas in nuclei, similar inhibition was achieved only by copper chelators, indicating an important role of chromatin-bound copper in the prooxidant cellular DNA breakage by ascorbic acid. CONCLUSION: We propose that the copper-dependent cellular redox status is an important element in the cytotoxic action of ascorbic acid against cancer cells. It is well established that cellular copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to electron transfer between copper and ascorbate to generate reactive oxygen species. In light of these observations and those in literature, in this paper we explain that the preferential cytotoxicity of ascorbic acid against cancer cells is the result of elevated copper levels in such cells. Further, this study identifies nuclear copper as a novel molecular target for cytotoxic action of ascorbic acid, which has implications for its chemotherapeutic properties against cancer.

Resveratrol mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for chemoprevention of cancer.

Plant polyphenols are important components of human diet, and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring anti-oxidants but also act as pro-oxidants catalyzing DNA degradation in the presence of metal ions such as copper. The plant polyphenol resveratrol confers resistance to plants against fungal agents and has been implicated as a cancer chemopreventive agent. Of particular interest is the observation that resveratrol has been found to induce apoptosis in cancer cell lines but not in normal cells. Over the last few years, we have shown that resveratrol is capable of causing DNA breakage in cells such as human lymphocytes. Such cellular DNA breakage is inhibited by copper specific chelators but not by iron and zinc chelating agents. Similar results are obtained by using permeabilized cells or with isolated nuclei, indicating that chromatin-bound copper is mobilized in this reaction. It is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to electron transfer between copper ions and resveratrol to generate reactive oxygen species responsible for DNA cleavage. The results are in support of our hypothesis that anti-cancer mechanism of plant polyphenols involves mobilization of endogenous copper and the consequent pro-oxidant action. Such a mechanism better explains the anti-cancer effects of resveratrol, as it accounts for the preferential cytotoxicity towards cancer cells.