Differential Apoptotic Effects of Bee Product Mixtures on Normal and Cancer Hepatic Cells.

Most effective anticancer drugs normally generate considerable cytotoxicity in normal cells; therefore, the preferential activation of apoptosis in cancer cells and the reduction of toxicity in normal cells is a great challenge in cancer research. Natural products with selective anticancer properties used as complementary medicine can help to achieve this goal. The aim of the present study was to analyze the effect of the addition of bee products [propolis (PR) or royal jelly (RJ) or propolis and royal jelly (PR+RJ), 2-10%] to thyme (TH) and chestnut honeys (CH) on the differential anticancer properties, mainly the cytotoxic and pro-apoptotic effects, in normal and cancer hepatic cells. The cytotoxic effects of samples were analyzed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (0-250 mg/mL) and the effects on apoptosis were analyzed using cell cycle analysis, TdT-dUTP terminal nick-end labeling (TUNEL) assay, DR5 (Death Receptor 5) and BAX (BCL-2-Associated X) activation, and caspases 8, 9, and 3 activities. Both honey samples alone and honey mixtures had no or very little apoptotic effect on normal cells. Antioxidant honey mixtures enhanced the apoptotic capacity of the corresponding honey alone via both extrinsic and intrinsic pathways. Of all the samples, chestnut honey enriched with 10% royal jelly and 10% propolis (sample 14, CH+10RJ+10PR) showed the highest apoptotic effect on tumor liver cells. The enrichment of monofloral honey with bee products could be used together with conventional anticancer treatments as a dietary supplement without side effects. On the other hand, it could be included in the diet as a natural sweetener with high added value.

Protective Effect of Thyme and Chestnut Honeys Enriched with Bee Products against Benzo(a)pyrene-Induced DNA Damage.

The aim of the present study was to validate the cytotoxicity, genotoxicity, and preventive potential against benzo(a)pyrene (BaP)-induced DNA damage of nine samples of thyme and chestnut honeys enriched with bee products (royal jelly and propolis, 2-10%). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (0-250 mg/mL) to select nontoxic concentrations, and DNA damage (0.1-10 µg/mL) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Treatment with honey samples or royal jelly and propolis did not affect the viability of HepG2 cells up to 100 and 50 mg/mL, respectively. Treatment with 100 µM BaP significantly increased (p ≤ 0.001) the levels of the DNA strand breaks. None of the tested concentrations (0.1-10 µg/mL) of the honey samples (thyme and chestnut), royal jelly, and propolis caused DNA damage per se. All tested samples at all the concentrations used decreased the genotoxic effect of BaP. In addition, all mixtures of thyme or chestnut honeys with royal jelly or propolis showed a greater protective effect against BaP than the samples alone, being the thyme and chestnut honey samples enriched with 10% royal jelly and 10% propolis the most effective (70.4% and 69.4%, respectively). The observed protective effect may be associated with the phenolic content and antioxidant capacity of the studied samples. In conclusion, the thyme and chestnut honey samples enriched with bee products present potential as natural chemoprotective agents against the chemical carcinogen BaP.

Enhancement of the Antioxidant Capacity of Thyme and Chestnut Honey by Addition of Bee Products.

Honey consumption and imports have increased in recent years, and it is considered by consumers to be a healthy alternative to more commonly used sweeteners. Honey contains a mixture of polyphenols and antioxidant compounds, and the botanical origin and geographical area of collection play an important role on its chemical composition. The present study investigated the physicochemical properties, total phenolic content and antioxidant capacity of Spanish thyme honey and chestnut honey, and their mixtures with royal jelly (2% and 10%) and propolis (2% and 10%). The analysis of the physicochemical parameters of both honey samples showed values within the established limits. Propolis showed the highest value of total phenolic content (17.21-266.83 mg GAE/100 g) and antioxidant capacity (DPPH, ORAC and ABTS assays; 0.63-24.10 µg eq. Tx/g, 1.61-40.82 µg eq. Tx/g and 1.89-68.54 µg eq. Tx/g, respectively), and significantly reduced ROS production in human hepatoma cells. In addition, mixtures of honey with 10% of propolis improved the results obtained with natural honey, increasing the value of total phenolic content and antioxidant capacity. A significant positive correlation was observed between total phenolic compounds and antioxidant capacity. Therefore, the antioxidant capacity could be attributed to the phenolic compounds present in the samples, at least partially. In conclusion, our results indicated that thyme and chestnut honey supplemented with propolis can be an excellent natural source of antioxidants and could be incorporated as a potential food ingredient with biological properties of technological interest, added as a preservative. Moreover, these mixtures could be used as natural sweeteners enriched in antioxidants and other bioactive compounds.

New Insights into Bacillus-Primed Plant Responses to a Necrotrophic Pathogen Derived from the Tomato-Botrytis Pathosystem.

Induced systemic resistance (ISR) is one of the most studied mechanisms of plant−microbe interaction and is considered a very promising alternative for integrated pest management programs. In our study, we explored the plant defense response induced by Bacillus velezensis BBC047 in relation to its application before or after Botrytis cinerea infection of tomato plants. The inoculation of BBC047 did not considerably alter the gene expression of the tomato tissues, whereas infection with B. cinerea in BBC047-primed plants induced expression of LRR and NBS-LRR receptors, which are highly related to the ISR response. As expected, B. cinerea infection generated molecular patterns typical of a defense response to pathogen infection as the overexpression of pathogenesis-related proteins (PRs) in leaflets distant to the point of infection. The curative treatment (P + F + B) allowed us to gain insights into plant response to an inverted priming. In this treatment, B. cinerea caused the m tissue damage, extending nearly entirely across the entire infected leaves. Additionally, genes generally associated with early SAR response (<16 h) were overexpressed, and apparently, the beneficial strain was not perceived as such. Therefore, we infer that the plant defense to the curative treatment represents a higher degree of biological stress triggered by the incorporation of strain BBC047 as second arriving microorganism. We highlight the importance the phytosanitary status of plants prior to inoculation of beneficial microorganism for the biocontrol of pathogens.

Resistance to MAPK Inhibitors in Melanoma Involves Activation of the IGF1R-MEK5-Erk5 Pathway.

Combined treatment of metastatic melanoma with BRAF and MEK inhibitors has improved survival, but the emergence of resistance represents an important clinical challenge. Targeting ERK is a suitable strategy currently being investigated in melanoma and other cancers. To anticipate possible resistance to ERK inhibitors (ERKi), we used SCH772984 (SCH) as a model ERKi to characterize resistance mechanisms in two BRAF V600E melanoma cell lines. The ERKi-resistant cells were also resistant to vemurafenib (VMF), trametinib (TMT), and combined treatment with either VMF and SCH or TMT and SCH. Resistance to SCH involved stimulation of the IGF1R-MEK5-Erk5 signaling pathway, which counteracted inhibition of Erk1/2 activation and cell growth. Inhibition of IGF1R with linsitinib blocked Erk5 activation in SCH-resistant cells and decreased their growth in 3D spheroid growth assays as well as in NOD scid gamma (NSG) mice. Cells doubly resistant to VMF and TMT or to VMF and SCH also exhibited downregulated Erk1/2 activation linked to stimulation of the IGF1R-MEK5-Erk5 pathway, which accounted for resistance. In addition, we found that the decreased Erk1/2 activation in SCH-resistant cells involved reduced expression and function of TGFα. These data reveal an escape signaling route that melanoma cells use to bypass Erk1/2 blockade during targeted melanoma treatment and offer several possible targets whose disruption may circumvent resistance. SIGNIFICANCE: Activation of the IGF1R-MEK5-Erk5 signaling pathway opposes pharmacologic inhibition of Erk1/2 in melanoma, leading to the reactivation of cell proliferation and acquired resistance.

Validation of coffee silverskin extract as a food ingredient by the analysis of cytotoxicity and genotoxicity.

The aim of the present study was to validate the food safety of CSE, by studying its effect on cytotoxicity (100-20000µg/ml) and genotoxicity (10, 100 and 1000µg/ml) and also to investigate its preventive potential (1, 10 and 100µg/ml) against B(a)P induced DNA damage. Prior to analyses, the antioxidant capacity and the microbiological quality of CSE were tested. DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. HepG2 cells were pre-treated with CSE (1, 10 and 100µg/ml) for 24h followed by the addition of 100µM B(a)P in presence of CSE for other 24h. Detection of oxidized purines and pyrimidines was carried out using Formamidopyrimidine DNA glycosylase or Endonuclease III enzymes, respectively. Chlorogenic acid (CGA), the major antioxidant present in coffee, was used as a control. Treatment with 100 µM B(a)P significantly increased (p<0.05) levels of DNA strand breaks and oxidized purine and pyrimidine bases. Treatment of HepG2 cells with CSE did not induce either cytotoxicity or genotoxicity. CSE significantly inhibited (p<0.05) genotoxicity induced by B(a)P and the observed effect may be associated to its antioxidant capacity. CGA alone at the concentration present in CSE was effective against B(a)P. Thus, CGA seems to be a contributor to the preventive effect of CSE against B(a)P induced DNA damage in HepG2 cells. In conclusion, CSE presents potential as a natural sustainable chemoprotective agent against the chemical carcinogen B(a)P.

Interactions of manufactured silver nanoparticles of different sizes with normal human dermal fibroblasts.

Silver compounds have been used for their medicinal properties for centuries. At present, silver nanoparticles (AgNPs) are reemerging as a viable topical treatment option for infections encountered in burns, open wounds and chronic ulcers. This study evaluated the in vitro mechanisms of two different sizes of AgNPs (4·7 and 42 nm) toxicity in normal human dermal fibroblasts. The toxicity was evaluated by observing cell viability and oxidative stress parameters. In all toxicity endpoints studied (MTT and lactate dehydrogenase assays), AgNPs of 4·7 nm were much more toxic than the large AgNPs (42 nm). The cytotoxicity of both AgNPs was greatly decreased by pre-treatment with the antioxidant N-acetyl-L-cysteine. The oxidative stress parameters showed significant increase in reactive oxygen species levels, depletion of glutathione level and slight, but not statistically significant inactivation of superoxide dismutase, suggesting generation of oxidative stress. Thus, AgNPs should be used with caution for the topical treatment of burns and wounds, medical devices etc, because their toxicity depends on the size, the smaller NPs being much more cytotoxic than the large.

In vivo genotoxicity assesment of silver nanoparticles of different sizes by the Somatic Mutation and Recombination Test (SMART) on Drosophila.

Silver nanoparticles (AgNPs) with antimicrobial activity are by far the most commercialized nano-compound. They are commonly used in medical products and devices, food storage materials, cosmetics and industrial products. Despite the increasing human exposure to AgNPs, they remain a controversial research area with regard to their toxic and genotoxic effects to biological systems. Although previous data have suggested that AgNPs induce toxicity in vitro, the in vivo studies on this topic are very limited. In the present study, the potential genotoxic activity of AgNPs of different sizes (4.7 and 42 nm) was evaluated using the in vivo Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Larvae were treated with 25, 30 and 50 µg/ml of AgNPs 4.7 nm, and 250, 500 and 1000 µg/ml of AgNPs 42 nm. Data showed that AgNPs at the applied concentrations did not modify the spontaneous frequencies of spots indicating lack of mutagenic and recombinogenic activity. However, both AgNPs induced pigmentation defects and reduction in locomotor ability in adult flies. Therefore, further experiments must be carried out to gain a better understanding of the mechanism of action of AgNPs to ensure their safe use.

Effects of silver and gold nanoparticles of different sizes in human pulmonary fibroblasts.

Silver and gold nanoparticles (Ag-AuNPs) are currently some of the most manufactured nanomaterials. Accordingly, the hazards associated with human exposure to Ag-AuNPs should be investigated to facilitate the risk assessment process. In particular, because pulmonary exposure to Ag-AuNPs occurs during handling of these nanoparticles, it is necessary to evaluate the toxic response in pulmonary cells. The aim of this study was to evaluate the in vitro mechanisms of toxicity of different sizes of silver (4.7 and 42 nm) and gold nanoparticles (30, 50 and 90 nm) in human pulmonary fibroblasts (HPF). The toxicity was evaluated by observing cell viability and oxidative stress parameters. Data showed that AgNPs-induced cytotoxicity was size-dependent, whereas the AuNPs of the three sizes showed similar cytotoxicity. Silver nanoparticles of 4.7 nm were much more toxic than the large silver nanoparticles and the AuNPs. However, the pre-treatment with the antioxidant, N-acetyl-L-cysteine, protected HPF cells against treatment with Ag-AuNPs. The oxidative stress parameters revealed significant increase in reactive oxygen species levels, depletion of glutathione level and slight, but not statistically significant inactivation of superoxide dismutase, suggesting generation of oxidative stress. Hence, care has to be taken while processing and formulating the Ag-AuNPs till their final finished product.

Cytotoxicity and ROS production of manufactured silver nanoparticles of different sizes in hepatoma and leukemia cells.

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. In spite of the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs. The aim of this study was investigate how AgNPs of different sizes (4.7 and 42 nm) interact with two different tumoral human cell lines (hepatoma [HepG2] and leukemia [HL-60]). In addition, glutathione depletion, inhibition of superoxide dismutase (SOD) and reactive oxygen species (ROS) generation were used to evaluate feasible mechanisms by which AgNPs exerted its toxicity. AgNPs of 4.7 nm and 42 nm exhibited a dramatic difference in cytotoxicity. Small AgNPs were much more cytotoxic than large AgNPs. A difference in the cellular response to AgNPs was found. HepG2 cells showed a higher sensitivity to the AgNPs than HL-60. However, the cytotoxicity induced by AgNPs was efficiently prevented by NAC treatment, which suggests that oxidative stress is primarily responsible for the cytotoxicity of AgNPs. Furthermore, cellular antioxidant status was disturbed: AgNPs exposure caused ROS production, glutathione depletion and slight, but not statistically significant inactivation of SOD.

Oxidative stress contributes to gold nanoparticle-induced cytotoxicity in human tumor cells.

Due to their exceptional properties, gold nanoparticles (AuNPs) have shown promising medical and technological applications in the treatment of cancer and the development of antimicrobial packaging and time-temperature indicators in the food sector. However, little is known about their cytotoxicity when they come into contact with biological systems. The aim of this work was to compare the effects of three commercially available AuNPs of different sizes (30, 50 and 90 nm) on human leukemia (HL-60) and hepatoma (HepG2) cell lines. AuNP-induced cytotoxicity was dose and time-dependent, with IC50 values higher than 15 µg/mL. Nanoparticle (NP) size and cell line slightly influenced on the cytotoxicity of AuNPs, although HL-60 cells proved to be more sensitive to the cytotoxic response than HepG2. N-Acetyl-L-cysteine (NAC) protected HL-60 and HepG2 cells only against treatment with 30 nm AuNPs. In both cell types, glutathione (GSH) content was drastically depleted after 72 h of incubation with the three AuNPs (less than 30% in all cases), while the reduction of superoxide dismutase activity (SOD) activity depended on cell line. HepG2, but not HL-60 cells, exhibited a decrease of SOD activity (∼ 45% of activity). The three AuNPs also caused a two-fold elevation of reactive oxygen species (ROS) production in both cell lines. Thus, protective effect of NAC, depletion of GSH and increase of ROS appear to be determined by NP size and indicate that oxidative stress contributes to cytotoxicity of AuNPs.

Nanopartículas de oro: aplicaciones y citotoxicidad in vitro / Gold nanoparticles: Applications and in vitro cytotoxicity

En los últimos años, la evolución en el desarrollo de productos elaborados a partir de nanotecnología ha experimentado un espectacular crecimiento. En particular, las nanopartículas de oro han despertado gran interés en los sectores biomédico y alimentario, donde se ha descrito su utilización en el tratamiento frente al cáncer o como parte integrante de envases resistentes a la abrasión, con propiedades antimicrobianas. Por tanto, se cree que la exposición humana a las nanopartículas de oro aumentará considerablemente en los próximos años, pudiendo tener esto repercusiones sobre la salud. En este marco, el estudio de la toxicología de las nanopartículas ha revelado que su toxicidad depende de multitud de factores. Además, en la bibliografía hay cierta controversia en torno a los posibles efectos citotóxicos inducidos por las nanopartículas de oro. Diversos estudios de exposición in vitro han destacado su inocuidad en algunas líneas celulares, mientras que otros trabajos demostraron respuesta citotóxica. La siguiente revisión tiene por objeto describir las propiedades más relevantes de las nanopartículas de oro considerando sus potenciales aplicaciones en medicina y en la industria de los alimentos, así como examinar su posible toxicidad, con especial énfasis en los estudios de citotoxicidad in vitro disponibles hasta el momento.(AU)

In the recent years, the development of nanotechnology-based products has experienced a spectacular growth. Especially, gold nanoparticles have awoken a great interest in the biomedical and food sector, where their applications in cancer treatment as well as their incorporation in abrasion resistant and antimicrobial packaging have been described. Therefore, it is believed that human exposure to gold nanoparticles will increase considerably in the next few years, which may arise possible human health hazards. Hence, toxicology studies on nanoparticles revealed that their toxicity depends on various factors. Furthermore, there is some controversy regarding to gold nanoparticle-induced cytotoxicity. Several in vitro studies have reported that gold nanoparticles are innocuous, while some investigations have demonstrated a cytotoxic response after the exposure to these. The aim of this review is to describe the most relevant properties of gold nanoparticles according to their possible applications in medicine and in food industry, as well as to provide information about their possible toxic effects, taking into account the cytotoxic in vitro studies published at present.(AU)

Nanopartículas de oro: aplicaciones y citotoxicidad in vitro / Gold nanoparticles: Applications and in vitro cytotoxicity

En los últimos años, la evolución en el desarrollo de productos elaborados a partir de nanotecnología ha experimentado un espectacular crecimiento. En particular, las nanopartículas de oro han despertado gran interés en los sectores biomédico y alimentario, donde se ha descrito su utilización en el tratamiento frente al cáncer o como parte integrante de envases resistentes a la abrasión, con propiedades antimicrobianas. Por tanto, se cree que la exposición humana a las nanopartículas de oro aumentará considerablemente en los próximos años, pudiendo tener esto repercusiones sobre la salud. En este marco, el estudio de la toxicología de las nanopartículas ha revelado que su toxicidad depende de multitud de factores. Además, en la bibliografía hay cierta controversia en torno a los posibles efectos citotóxicos inducidos por las nanopartículas de oro. Diversos estudios de exposición in vitro han destacado su inocuidad en algunas líneas celulares, mientras que otros trabajos demostraron respuesta citotóxica. La siguiente revisión tiene por objeto describir las propiedades más relevantes de las nanopartículas de oro considerando sus potenciales aplicaciones en medicina y en la industria de los alimentos, así como examinar su posible toxicidad, con especial énfasis en los estudios de citotoxicidad in vitro disponibles hasta el momento.

In the recent years, the development of nanotechnology-based products has experienced a spectacular growth. Especially, gold nanoparticles have awoken a great interest in the biomedical and food sector, where their applications in cancer treatment as well as their incorporation in abrasion resistant and antimicrobial packaging have been described. Therefore, it is believed that human exposure to gold nanoparticles will increase considerably in the next few years, which may arise possible human health hazards. Hence, toxicology studies on nanoparticles revealed that their toxicity depends on various factors. Furthermore, there is some controversy regarding to gold nanoparticle-induced cytotoxicity. Several in vitro studies have reported that gold nanoparticles are innocuous, while some investigations have demonstrated a cytotoxic response after the exposure to these. The aim of this review is to describe the most relevant properties of gold nanoparticles according to their possible applications in medicine and in food industry, as well as to provide information about their possible toxic effects, taking into account the cytotoxic in vitro studies published at present.

Antiproliferative and apoptotic effects of spanish honeys.

BACKGROUND: Current evidence supports that consumption of polyphenols has beneficial effects against numerous diseases mostly associated with their antioxidant activity. Honey is a good source of antioxidants since it contains a great variety of phenolic compounds. OBJECTIVE: The main objective of this work was to investigate the antiproliferative and apoptotic effects of three crude commercial honeys of different floral origin (heather, rosemary and polyfloral honey) from Madrid Autonomic Community (Spain) as well as of an artificial honey in human peripheral blood promyelocytic leukemia cells (HL-60). MATERIAL AND METHODS: HL-60 cells were cultured in the presence of honeys at various concentrations for up to 72 hours and the percentage of cell viability was evaluated by MTT assay. Apoptotic cells were identified by chromatin condensation and flow cytometry analysis. ROS production was determined using 2´,7´-dichlorodihydrofluorescein diacetate (H2DCFDA). RESULTS: The three types of crude commercial honey induced apoptosis in a concentration and time dependent-manner. In addition, honeys with the higher phenolic content, heather and polyfloral, were the most effective to induce apoptosis in HL-60 cells. However, honeys did not generate reactive oxygen species (ROS) and N-acetyl-L-cysteine (NAC) could not block honeys-induced apoptosis in HL-60 cells. CONCLUSION: These data support that honeys induced apoptosis in HL-60 cells through a ROS-independent cell death pathway. Moreover, our findings indicate that the antiproliferative and apoptotic effects of honey varied according to the floral origin and the phenolic content.

Spanish honeys protect against food mutagen-induced DNA damage.

BACKGROUND: Honey contains a variety of polyphenols and represents a good source of antioxidants, while the human diet often contains compounds that can cause DNA damage. The present study investigated the protective effect of three commercial honey samples of different floral origin (rosemary, heather and heterofloral) from Madrid Autonomic Community (Spain) as well as an artificial honey on DNA damage induced by dietary mutagens, using a human hepatoma cell line (HepG2) as in vitro model system and evaluation by the alkaline single-cell gel electrophoresis or comet assay. RESULTS: Rosemary, heather and heterofloral honeys protected against DNA strand breaks induced by N-nitrosopyrrolidine (NPYR), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but none of the honey samples tested prevented DNA strand breaks induced by N-nitrosodimethylamine (NDMA). Heterofloral and heather (unifloral) honeys with higher phenolic content were most effective in protecting HepG2 cells against DNA damage induced by food mutagens. Heterofloral honey was more protective against NPYR and BaP, while heather honey was more protective against PhIP. Artificial honey did not show a protective effect against DNA damage induced by any of the food mutagens tested, indicating that the protective effects of honeys could not be due to their sugar components. CONCLUSION: The results suggest that the protective effect of three kinds of Spanish honey of different floral origin could be attributed in part to the phenolics present in the samples. Honeys with higher phenolic content, i.e. heather and heterofloral honeys, were most effective in protecting against food mutagen-induced DNA damage in HepG2 cells. In addition, a possible synergistic effect between other minor honey components could also be involved.

In vitro evaluation of silver nanoparticles on human tumoral and normal cells.

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. Despite the widespread use of AgNPs, relatively few studies have been undertaken to determine the toxicity effects of AgNPs exposure. The aim of the present work was to study how AgNPs interact with four different human cell lines (hepatoma, leukemia, dermal and pulmonary fibroblast) in order to understand the impact of such nanomaterials on cellular biological functions. For toxicity evaluations, mitochondrial function (MTT assay) and membrane leakage of lactate dehydrogenase (LDH assay) were assessed under control and exposed conditions (24, 48 and 72 h of exposure). Furthermore, we evaluated the protective effect of N-acetyl-l-cysteine (NAC) against AgNP-induced cytotoxicity. Results showed that mitochondrial function decreased in all cell lines exposed to AgNPs at 6.72-13.45 µg/ml. LDH leakage also increased in all cell lines exposed to AgNPs (6.72-13.45 µg/ml). However, the cytotoxic effect of AgNPs (13.45 µg/ml) was prevented by pretreatment of different concentrations of NAC (1-20 mM). Our findings indicate that AgNPs are cytotoxic on human tumor and normal cells, the tumor cells being more sensitive to the cytotoxic effect of AgNPs. In addition, NAC protects human cells from cytotoxicity of AgNPs, suggesting that oxidative stress is in part responsible of this effect.

Selective apoptotic effects of piceatannol and myricetin in human cancer cells.

Numerous studies have shown the potential of dietary polyphenols as anticarcinogenic agents. The aim of the present study was to evaluate the apoptotic effects of piceatannol and myricetin, naturally occurring polyphenols in red wine, alone or in combination, in two human cell lines: HL-60 (leukemia) and HepG2 (hepatoma). Apoptotic cells were identified by chromatin condensation, poly(ADP-ribose) polymerase cleavage and flow cytometry analysis. Results from TUNEL assay showed that piceatannol or myricetin alone induced apoptotic cell death in a concentration- and time-dependent manners in HL-60 cells. Furthermore, in combined treatment the percentage of apoptotic HL-60 cells was significantly higher. Nevertheless, the percentage of TUNEL positive HepG2 cells only was significant after piceatannol treatment and in combined treatment was even lower than in cells treated with piceatannol alone. Moreover, we also studied the relative reactive oxygen species (ROS) production. Our results indicate that apoptosis induced by piceatannol or myricetin occurs through an ROS-independent cell death pathway. In conclusion, piceatannol and myricetin synergistically induced apoptosis in HL-60 cells but not in HepG2 cells. These findings suggest that the potential anticarcinogenic properties of dietary polyphenols depend largely on the cell line used. The relevance of these data needs to be verified in human epidemiological studies.

Effects of (+)-catechin and (-)-epicatechin on heterocyclic amines-induced oxidative DNA damage.

The aim of the present study was to evaluate the protective effect of (+)-catechin and (-)-epicatechin against 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline (4,8-diMeIQx) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Increasing concentrations of 8-MeIQx, 4,8-diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose-dependent manner. Among those, PhIP (300 µm) exerted the highest genotoxicity. (+)-Catechin exerted protection against oxidized purines induced by 8-MeIQx, 4,8-diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)-catechin. Otherwise, (-)-epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8-MeIQx and 4,8-diMeIQx. One feasible mechanism by which (+)-catechin and (-)-epicatechin exert their protective effect towards heterocyclic amines-induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O-deethylation (CYP1A1) activity was moderately inhibited by (+)-catechin, while little effect was observed by (-)-epicatechin. However, (+)-catechin showed the greatest increase in UDP-glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)-catechin was more efficient than (-)-epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8-MeIQx and 4,8-diMeIQx.

N-Nitrosopiperidine and N-Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway.

The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.