Showket H. Bhat

Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties.

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 antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. Using human peripheral lymphocytes and Comet assay we have previously confirmed that resveratrol–Cu(II) is indeed capable of causing DNA degradation in cells. In this paper we show that the polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that polyphenols generate oxidative stress in lymphocytes which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.

Resveratrol–Cu(II) induced DNA breakage in human peripheral lymphocytes: Implications for anticancer properties

Resveratrol (3,4′,5‐trihydroxy stilbene), a plant derived polyphenol found in mulberries, grapes and red wine is considered to possess chemopreventive properties against cancer. It is recognized as a naturally occurring antioxidant but also catalyzes oxidative DNA degradation in vitro in the presence of transition metal ions such as copper. Using a cellular system of lymphocytes isolated from human peripheral blood and Comet assay, we have confirmed that resveratrol–Cu(II) system is indeed capable of causing DNA degradation in cells such as lymphocytes. Also, trans‐stilbene, which does not have any hydroxyl groups, is inactive in the lymphocyte system. Pre‐incubation of lymphocytes with resveratrol indicates that it is capable of either traversing the cell membrane or binding to it. Our results are in partial support of our hypothesis that anticancer properties of various plant derived polyphenols may involve mobilization of endogenous copper and the consequent prooxidant action.

Plant polyphenols mobilize nuclear copper in human peripheral lymphocytes leading to oxidatively generated DNA breakage: Implications for an anticancer mechanism

It was earlier proposed that an important anti-cancer mechanism of plant polyphenols may involve mobilization of endogenous copper ions, possibly chromatin-bound copper and the consequent pro-oxidant action. This paper shows that plant polyphenols are able to mobilize nuclear copper in human lymphocytes, leading to degradation of cellular DNA. A cellular system of lymphocytes isolated from human peripheral blood and comet assay was used for this purpose. Incubation of lymphocytes with neocuproine (a cell membrane permeable copper chelator) inhibited DNA degradation in intact lymphocytes. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. This study has further shown that polyphenols are able to degrade DNA in cell nuclei and that such DNA degradation is inhibited by neocuproine as well as bathocuproine (both of which are able to permeate the nuclear pore complex), suggesting that nuclear copper is mobilized in this reaction. Pre-incubation of lymphocyte nuclei with polyphenols indicates that it is capable of traversing the nuclear membrane. This study has also shown that polyphenols generate oxidative stress in lymphocyte nuclei which is inhibited by scavengers of reactive oxygen species (ROS) and neocuproine. These results indicate that the generation of ROS occurs through mobilization of nuclear copper resulting in oxidatively generated DNA breakage.

The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA.

Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence of transition metal ions such as copper. In this paper we show that similar to various other classes of polyphenols, delphinidin is also capable of causing oxidative degradation of cellular DNA. Lymphocytes were exposed to various concentrations of delphinidin (10, 20, 50 microM) for 1h and the DNA breakage was assessed using single cell alkaline gel electrophoresis (Comet assay). Inhibition of DNA breakage by several scavengers of reactive oxygen species (ROS) indicated that it is caused by the formation of ROS. Incubation of lymphocytes with neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation in intact lymphocytes in a dose dependent manner. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. We have further shown that delphinidin is able to degrade DNA in cell nuclei and that such DNA degradation is also inhibited by neocuproine suggesting that nuclear copper is mobilized in this reaction. These results indicate that the generation of ROS possibly occurs through mobilization of endogenous copper ions. The results are in support of our hypothesis that the prooxidant activity of plant polyphenols may be an important mechanism for their anticancer properties.

A new liquid chromatography/mass spectrometry method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine produced upon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the lungs. NNAL undergoes further uridine diphosphate glucuronosyltransferase (UGT)-mediated metabolism to give N- and O-glucuronide metabolites, which together with free (non-conjugated) NNAL are then excreted in the urine. The ability to conduct validated analyses of free and conjugated NNAL in human urine is important in order to assess inter-individual differences in lung cancer risk from exposure to cigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography/multiple reaction monitoring/mass spectrometry (LC/MRM-MS) provides the highest bioanalytical specificity possible for such analyses. We describe a novel derivatization procedure, which results in the formation of a pre-ionized N-propyl-NNAL derivative. The increased LC/MS sensitivity arising from this derivative then makes it possible to analyze free NNAL in only 0.25 mL urine. This substantial reduction in urine volume when compared with other methods that have been developed will help preserve the limited amounts of stored urine samples that are available from on-going longitudinal biomarker studies. The new high sensitivity SID LC/MRM-MS assay was employed to determine free and conjugated NNAL concentrations in urine samples from 60 individual disease-free smokers. Effects of inter-individual differences in urinary creatinine clearance on NNAL concentrations were then assessed and three metabolizer phenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/free NNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2 (mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean = 3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1).

Pharmacological Intervention through Dietary Nutraceuticals in Gastrointestinal Neoplasia

Neoplastic conditions associated with gastrointestinal (GI) tract are common worldwide with colorectal cancer alone accounting for the third leading rate of cancer incidence. Other GI malignancies such as esophageal carcinoma have shown an increasing trend in the last few years. The poor survival statistics of these fatal cancer diseases highlight the need for multiple alternative treatment options along with effective prophylactic strategies. Worldwide geographical variation in cancer incidence indicates a correlation between dietary habits and cancer risk. Epidemiological studies have suggested that populations with high intake of certain dietary agents in their regular meals have lower cancer rates. Thus, an impressive embodiment of evidence supports the concept that dietary factors are key modulators of cancer including those of GI origin. Preclinical studies on animal models of carcinogenesis have reflected the pharmacological significance of certain dietary agents called as nutraceuticals in the chemoprevention of GI neoplasia. These include stilbenes (from red grapes and red wine), isoflavones (from soy), carotenoids (from tomatoes), curcuminoids (from spice turmeric), catechins (from green tea), and various other small plant metabolites (from fruits, vegetables, and cereals). Pleiotropic action mechanisms have been reported for these diet-derived chemopreventive agents to retard, block, or reverse carcinogenesis. This review presents a prophylactic approach to primary prevention of GI cancers by highlighting the translational potential of plant-derived nutraceuticals from epidemiological, laboratory, and clinical studies, for the better management of these cancers through consumption of nutraceutical rich diets and their intervention in cancer therapeutics.

Simulating hypoxia-induced acidic environment in cancer cells facilitates mobilization and redox-cycling of genomic copper by daidzein leading to pro-oxidant cell death: implications for the sensitization of resistant hypoxic cancer cells to therapeutic challenges

Abstract This study was conducted to investigate the mechanism of action involved in the anti-cancer activity of daidzein and identification of cancer specific micro-environment as therapeutic target of this secondary metabolite derived from soy. Our data indicated that daidzein induces cellular DNA breakage, anti-proliferative effects and apoptosis in a concentration-dependent manner. We demonstrated that such a daidzein-induced anti-cancer action involves a copper-dependant pathway in which endogenous copper is mobilized by daidzein and redox-cycled to generate reactive oxygen species which act as an upstream signal leading to pro-oxidant cell death. Further in the context of hypoxia being a resistant factor against standard therapies and that an effect secondary to hypoxia is the intracellular acidification, we show that the anticancer activity of daidzein is modulated positively in acidic pH but copper-specific chelator is still able to inhibit daidzein activity. Moreover, an experimental setup of hypoxia mimic (cobalt chloride) revealed an enhanced sensitivity of cancer cells to the cytotoxic effects of daidzein which was neutralized in the presence of neocuproine. The findings support a paradigm shift from the conventional antioxidant property of dietary isoflavones to molecules capable of initiating a pro-oxidant signaling mediated by reactive oxygen species. Further, the clinical relevance of such an action mechanism in cancer chemoprevention is also proposed. This study identified endogenous copper as a molecular target and acidic pH as a modulating factor for the therapeutic activity of daidzein against cancer. The evidence presented highlights the potential of dietary agents as adjuvants to standard therapeutic regimens.

Dietary Factors May Influence the Clinical Outcome of Chemotherapy in Cancer Multidrug Resistance

A substantive burden of cancer mortality results from poor prognosis of the disease due to the failure of chemotherapeutic regimen under the influence of Multidrug Resistance (MDR). The outcome of chemotherapy which is the most effective treatment for patients with cancer is impeded by the development of drug resistance. Anticancer drugs can fail to kill cancer cells for various reasons that include variations in the absorption, metabolism, and delivery of drug to target tissues and tumor location in parts of the body into which the drugs do not easily penetrate. In addition, certain cancer cells develop resistance by micro-evolutionary means through mutations occurring in the drug target, thus rendering the drugs ineffective. However, the most common of these mechanisms is the efflux of hydrophobic drugs mediated by energy driven ATP-binding cassette (ABC) family of transporters such as P-glycoprotein (P-gp), an integral membrane protein over-expressed in several malignancies. Various generations of MDR modulators have presented novel and improved interventions, though not to the perfection. Studies have shown that natural compounds found in vegetables, fruits, plant-derived beverages, and herbal dietary supplements not only have anticancer properties but may also modulate P-gp activity. P-gp inhibitors found in natural products, especially those found in plants of dietary origin and traditional medicine, have the potential to be developed as MDR reversing agents as adjuvant to chemotherapy leading to better clinical prognosis.