Estefanía Burgos-Morón

A Review on the Dietary Flavonoid Kaempferol

Epidemiological studies have revealed that a diet rich in plant-derived foods has a protective effect on human health. Identifying bioactive dietary constituents is an active area of scientific investigation that may lead to new drug discovery. Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a flavonoid found in many edible plants (e.g. tea, broccoli, cabbage, kale, beans, endive, leek, tomato, strawberries and grapes) and in plants or botanical products commonly used in traditional medicine (e.g. Ginkgo biloba, Tilia spp, Equisetum spp, Moringa oleifera, Sophora japonica and propolis). Some epidemiological studies have found a positive association between the consumption of foods containing kaempferol and a reduced risk of developing several disorders such as cancer and cardiovascular diseases. Numerous preclinical studies have shown that kaempferol and some glycosides of kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, anticancer, cardioprotective, neuroprotective, antidiabetic, anti-osteoporotic, estrogenic/antiestrogenic, anxiolytic, analgesic and antiallergic activities. In this article, the distribution of kaempferol in the plant kingdom and its pharmacological properties are reviewed. The pharmacokinetics (e.g. oral bioavailability, metabolism, plasma levels) and safety of kaempferol are also analyzed. This information may help understand the health benefits of kaempferol-containing plants and may contribute to develop this flavonoid as a possible agent for the prevention and treatment of some diseases.

The dark side of curcumin.

Dear Editor, Curcumin is a yellow–orange pigment obtained from the plant Curcuma longa. The powdered rhizome of this plant, called turmeric, is a common ingredient in curry powders and has a long history of use in traditional Asian medicine for a wide variety of disorders. In the last decade a large number of reports have been published on the beneficial effects of curcumin, and it has repeatedly been claimed that this natural product is efficient and safe for the prevention and treatment of several diseases including cancer. It is not surprising, therefore, that curcumin is currently sold as a dietary supplement and that numerous clinical trials are ongoing or recruiting participants to evaluate curcumin activity. But there is accumulating evidence that curcumin may not be so effective and safe. Because such evidence is not generally acknowledged, the purpose of this letter is to briefly review the negative properties of curcumin so that they can be balanced against its beneficial effects. Most of the evidence that supports the therapeutic potential of curcumin is mainly based on in vitro studies in which curcumin was tested at concentrations in the micromolar range. Several reports have demonstrated, however, that the plasma concentrations of curcumin in people taking relatively high oral doses of this compound are very low, typically in the nanomolar range (reviewed in Ref. 4). For instance, a recent study examined the pharmacokinetics of a curcumin preparation in 12 healthy human volunteers 0.25–72 hr after an oral dose of 10 or 12 g. Using a high-performance liquid chromatography assay with a limit of detection of 50 ng mL , only 1 subject had detectable free curcumin at any of the time points assayed. The fact that curcumin also undergoes extensive metabolism in intestine and liver means that high concentrations of curcumin cannot be achieved and maintained in plasma and tissues after oral ingestion. This is a major obstacle for the clinical development of this agent and suggests that the therapeutic potential of oral curcumin is limited. The low clinical efficiency of curcumin in the treatment of several chronic diseases such as Alzheimer’s disease and cardiovascular diseases has been discussed recently. As far as cancer is concerned, in vitro studies have demonstrated that cancer cells do not die unless they are exposed to curcumin concentrations of 5–50 lM for several hours. Because of its poor bioavailability, these concentrations are not achieved outside the gastrointestinal tract when curcumin is taken orally. Because of its extensive metabolism in intestine and liver, these concentrations cannot be maintained for several hours in the gastrointestinal tract. This suggests that the chemotherapeutic potential of oral curcumin is limited even for the treatment of cancers of the gastrointestinal tract. Accordingly, when 15 patients with advanced colorectal cancer were treated with curcumin at daily doses of 3.6 g for up to 4 months, no partial responses to treatment or decreases in tumor markers were observed. A search of the website www.clinicaltrials.gov in July 2009 showed 34 clinical trials using curcumin in a wide variety of diseases, particularly in cancer. In some of these trials, patients with several types of cancer are receiving or will receive curcumin through the oral route. For instance, in an ongoing Phase II clinical trial (NCT00094445), participants with pancreatic cancer are receiving 8 g of curcumin by mouth every day for several 8-week-periods. As discussed before, the plasma concentrations of curcumin in people taking relatively high oral doses of curcumin are very low, typically in the nanomolar range. This means that the oral administration of curcumin does not lead to cytotoxic concentrations outside the gastrointestinal tract. If one assumes that tumor cell death is necessary to achieve an efficient therapeutic response, one should not expect a very positive outcome from this trial. A Phase II Trial is also recruiting participants to test if a daily oral dose of 8 g of curcumin can improve the efficacy of the standard chemotherapy gemcitabine in patients with locally advanced or metastatic adenocarcinoma of the pancreas (NCT00192842). The rationale for this trial is based on in vitro and in vivo data that suggest that noncytotoxic concentrations of curcumin may sensitize cancer cells to the effects of anticancer drugs such as gemcitabine. Although a daily dose of 1 g kg 1 of curcumin increased the antitumor effects of gemcitabine in an orthotopic model of pancreatic cancer, this dose of curcumin (e.g. 70 g in a 70-kg person) is almost 10 times higher than that used in the clinical trial testing the combination of curcumin and gemcitabine (8 g). This makes the outcome of this trial uncertain, as curcumin can either increase or reduce the efficiency of chemotherapy depending on the concentration at which it is used. Several strategies have been proposed to overcome the low oral bioavailability of curcumin. One of these strategies has entered clinical trials and consists of using the black pepper alkaloid piperine (bioperine) to increase the bioavailability of curcumin. This strategy, however, should be used cautiously, as piperine is a potent inhibitor of drug Le tt er s to th e E di to r

Pro-Oxidant Natural Products as Anticancer Agents

Cancer cells produce high levels of reactive oxygen species (ROS) that lead to a state of increased basal oxidative stress. Since this state of oxidative stress makes cancer cells vulnerable to agents that further augment ROS levels, the use of pro-oxidant agents is emerging as an exciting strategy to selectively target tumor cells. Natural products have provided a significant contribution to the development of several drugs currently used in cancer chemotherapy. Although many natural products are known to affect the redox state of the cell, most studies on these compounds have focused on their antioxidant activity instead of on their pro-oxidant properties. This article provides an overview of natural products with pro-oxidant and anticancer activities, with special focus on plant secondary metabolites, and discusses their possible use as cancer chemotherapeutic agents.

Green tea constituents (−)-epigallocatechin-3-gallate (EGCG) and gallic acid induce topoisomerase I– and topoisomerase II–DNA complexes in cells mediated by pyrogallol-induced hydrogen peroxide

Green tea and its major active constituent, (-)-epigallocatechin-3-gallate (EGCG), are in clinical trials for the prevention and treatment of several diseases such as cancer. DNA topoisomerase (topo) poisons are commonly prescribed anticancer drugs that kill cancer cells by inducing topo-DNA complexes. Using purified topoisomerases, previous in vitro studies have shown that EGCG induces the formation of topo-DNA complexes. Because the activity of a drug on purified topoisomerases does not always represent the activity in a cell, we have used an immunofluorescence technique that allows the visualisation of topo I- and topo II-DNA complexes produced in individual cells to evaluate the activity of EGCG on both enzymes. High levels of topo I- and topo II-DNA complexes were observed in K562 leukaemia cells exposed to EGCG. Similar levels of topo I- and topo II-DNA complexes were visualised in cells treated with gallic acid (GA) (the acid part of the EGCG ester). Pyrogallol (PG) also induced topo-DNA complexes with both enzymes, therefore suggesting that the activity of EGCG and GA is mediated by their PG moieties. Catalase prevented both the cytotoxicity and the formation of topo I- and topo II-DNA complexes induced by EGCG, GA, PG and myricetin (a PG-containing flavonoid recently shown to induce topo I- and topo II-DNA complexes in cells), indicating that hydrogen peroxide mediates these activities. Hydrogen peroxide induced topo I- and topo II (α and β)-DNA complexes in a time- and dose-dependent manner. The formation of topo I- and topo II-DNA complexes in cells exposed to hydrogen peroxide correlated well with the induction of apoptosis, suggesting that the topo-DNA complexes induced at long exposure times by the compounds tested in our study may be apoptotic topo-DNA complexes. Finally, we report results suggesting that PG-containing drugs may selectively kill tumour cells by generating hydrogen peroxide.

Evaluating the cancer therapeutic potential of cardiac glycosides.

Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an unsaturated lactone ring and the ability to induce cardiotonic effects mediated by a selective inhibition of the Na+/K+-ATPase. Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias. Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. These compounds typically inhibit cancer cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identified cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models, which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in mice xenografted with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells versus rodent cells. This paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer therapeutic potential of cardiac glycosides in preclinical studies.

The Coffee Constituent Chlorogenic Acid Induces Cellular DNA Damage and Formation of Topoisomerase I– and II–DNA Complexes in Cells

Chlorogenic acid (CGA) is a plant polyphenol with known antioxidant properties. Although some studies suggest that CGA has anticancer properties, others indicate that this dietary constituent may cause DNA damage and induce carcinogenic effects. Because CGA is widely consumed in the form of coffee, it is important to further evaluate the putative DNA-damaging activity of CGA. Here we have employed two standard techniques commonly used for DNA damage detection (the comet assay and the γ- H2AX focus assay) and observed that CGA (0.5-5 mM) induces DNA damage in normal and cancer cells. We report for the first time that CGA induces high levels of topoisomerase I- and topoisomerase II-DNA complexes in cells (TARDIS assay). Catalase pretreatment abolished the formation of these topoisomerase-DNA complexes and reduced the cytotoxic activity of CGA, therefore indicating that hydrogen peroxide plays an important role in these activities. Lung cancer cells (A549) were more sensitive than normal lung fibroblasts (MRC5) to the cytotoxic activity of CGA, supporting previous findings that CGA may induce selective killing of cancer cells. Taking into consideration our results and the pharmacokinetic profile of CGA, the possible cancer preventive, carcinogenic and therapeutic potential of this dietary agent are discussed.

The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions.

Decitabine (5-aza-2′-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1–DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML.

5-Aza-2′-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair

5-Aza-2′-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.

Isoprenyl-thiourea and urea derivatives as new farnesyl diphosphate analogues: synthesis and in vitro antimicrobial and cytotoxic activities.

A series of new isoprenyl-thiourea and urea derivatives were synthesized by the reaction of alkyl or aryl isothiocyanate or isocyanate and primary amines. The structures of the compounds were established by (1)H NMR, (13)C NMR, MS, HRMS and elemental analysis. The new compounds were screened for in vitro antimicrobial activity against seven strains representing different types of gram-positive and gram-negative bacteria. More than a third of the synthesized compounds showed variable inhibition activities against the tested strains. Best antimicrobial activities were found for those thiourea analogues with 3-methyl-2-butenyl, isobutyl or isopentyl groups and aromatic rings possessing electron withdrawing substituents. The new compounds were also subjected to a preliminary screening for antitumoral activity. The presence of a highly lipophilic group and an electron withdrawing group in the aromatic rings enhanced anticancer activity of the synthesized compounds, showing in most cases more activity than that of the controls.

Selective cytotoxic activity of new lipophilic hydroxytyrosol alkyl ether derivatives.

Recent data suggest that hydroxytyrosol, a phenolic compound of virgin olive oils, has anticancer activity. This communication reports the synthesis of decyl and hexadecyl hydroxytyrosyl ethers, as well as the cytotoxic activity of hydroxytyrosol and a series of seven hydroxytyrosol alkyl ether derivatives against A549 lung cancer cells and MRC5 non-malignant lung fibroblasts. Hydroxytyrosyl dodecyl ether (HTDE) showed the highest selective cytotoxicity, and possible mechanisms of action were investigated; results suggest that HTDE can moderately inhibit glycolysis, induce oxidative stress, and cause DNA damage in A549 cells. The combination of HTDE with the anticancer drug 5-fluorouracil induced a synergistic cytotoxicity in A549 cancer cells but not in non-malignant MRC5 cells. HTDE also displayed selective cytotoxicity against MCF7 breast cancer cells versus MCF10 normal breast epithelial cells in the 1-30 μM range. These results suggest that the cytotoxicity of HTDE is more potent and selective than that of parent compound hydroxytyrosol.