Pro-Apoptotic Activity of Artichoke Leaf Extracts in Human HT-29 and RKO Colon Cancer Cells.

(1) Background: Cynara cardunculus L. subsp. scolymus (L.) Hegi, popularly known as artichoke, is an herbaceous plant belonging to the Asteraceae family. Artichoke leaf extracts (ALEs) have been widely used in traditional medicine because of their hepatoprotective, cholagogic, hypoglycaemic, hypolipemic and antibacterial properties. ALEs are also recognized for their antioxidative and anti-inflammatory activities. In this study, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as effect on cell growth of ALEs on human colon cancer HT-29 and RKO cells. HT-29 and RKO cells exhibit a different p53 status: RKO cells express the wild-type protein, whereas HT-29 cells express a p53-R273H contact mutant. (2) Methods: Four different ALEs were obtained by sequential extraction of dried artichoke leaves; ALEs were characterized for their content in chlorogenic acid, cynaropicrin, and caffeoylquinic acids. HT-29 and RKO cells were used for in vitro testing (i.e., cytotoxicity and genotoxicity assessment, cell cycle analysis, apoptosis induction). (3) Results: Two out of the four tested ALEs showed marked effects on cell vitality toward HT-29 and RKO tumour cells. The effect was accompanied by a genotoxic activity exerted at a non-cytotoxic concentrations, by a significant perturbation of cell cycle (i.e., with increase of cells in the sub-G1 phase), and by the induction of apoptosis. (4) Conclusions: ALEs rich in cynaropicrin, caffeoylquinic acids, and chlorogenic acid showed to be capable of affecting HT-29 and RKO colon cancer cells by inducing favourable biological effects: cell cycle perturbation, activation of mitochondrial dependent pathway of apoptosis, and the induction of genotoxic effects probably mediated by the induction of apoptosis. Taken together, these results weigh in favour of a potential cancer chemotherapeutic activity of ALEs.

Imbalance in the antioxidant defence system and pro-genotoxic status induced by high glucose concentrations: In vitro testing in human liver cells.

It has been hypothesized that high glucose concentrations might contribute to the overall intracellular oxidative stress either by the direct generation of reactive oxygen species (ROS) or by altering the redox balance. Moreover, it has also been suggested that high glucose concentration can increase the susceptibility of DNA to genotoxic effects of xenobiotics. The aim of this approach was to test high glucose concentrations for pro-genotoxicity in human liver cells by setting up an in vitro model for hyperglycaemia. The experimental design included performing of tests on both human HepG2 tumour cells and HepaRG immortalized cells. Increased cell susceptibility to genotoxic xenobiotics was tested by challenging cell cultures with 4-nitroquinoline-N-oxide (4NQO) and evaluating the extent of primary DNA damage by comet assay. Moreover, we evaluated the relationship between glucose concentration and intracellular ROS, as well as the effects of glucose concentration on the induction of Nrf2-dependent genes such as Glutathione S-transferases, Heme­oxygenase-1, and Glutathione peroxidase-4. To investigate the involvement of ROS in the induced pro-genotoxic activity, parallel experimental sets were set up by considering co-treatment of cells with the model mutagen 4NQO and the antioxidant, glutathione precursor N-acetyl-L-cysteine. High glucose concentrations caused a significant increase in the levels of primary DNA damage, with a pro-genotoxic condition closely related to the concentration of glucose in the culture medium when cells were exposed to 4NQO. High glucose concentrations also stimulated the production of ROS and down-regulated genes involved in contrasting of the effects of oxidative stress. In conclusion, in the presence of high concentrations of glucose, the cells are in unfavourable conditions for the maintenance of genome integrity.

Mutagenic and genotoxic effects induced by PM0.5 of different Italian towns in human cells and bacteria: The MAPEC_LIFE study.

Particulate matter (PM) is considered an atmospheric pollutant that mostly affects human health. The finest fractions of PM (PM2.5 or less) play a major role in causing chronic diseases. The aim of this study was to investigate the genotoxic effects of PM0.5 collected in five Italian towns using different bioassays. The role of chemical composition on the genotoxicity induced was also evaluated. The present study was included in the multicentre MAPEC_LIFE project, which aimed to evaluate the associations between air pollution exposure and early biological effects in Italian children. PM10 samples were collected in 2 seasons (winter and spring) using a high-volume multistage cascade impactor. The results showed that PM0.5 represents a very high proportion of PM10 (range 10-63%). PM0.5 organic extracts were chemically analysed (PAHs, nitro-PAHs) and tested by the comet assay (A549 and BEAS-2B cells), MN test (A549 cells) and Ames test on Salmonella strains (TA100, TA98, TA98NR and YG1021). The highest concentrations of PAHs and nitro-PAHs in PM0.5 were observed in the Torino, Brescia and Pisa samples in winter. The Ames test showed low mutagenic activity. The highest net revertants/m3 were observed in the Torino and Brescia samples (winter), and the mutagenic effect was associated with PM0.5 (p < 0.01), PAH and nitro-PAH (p < 0.05) concentrations. The YG1021 strain showed the highest sensitivity to PM0.5 samples. No genotoxic effect of PM0.5 extracts was observed using A549 cells except for some samples in winter (comet assay), while BEAS-2B cells showed light DNA damage in the Torino, Brescia and Pisa samples in winter, highlighting the higher sensitivity of BEAS-2B cells, which was consistent with the Ames test (p < 0.01). The results obtained showed that it is important to further investigate the finest fractions of PM, which represent a relevant percentage of PM10, taking into account the chemical composition and the biological effects induced.

Buccal micronucleus cytome assay in primary school children: A descriptive analysis of the MAPEC_LIFE multicenter cohort study.

BACKGROUND: Recent data support the hypothesis that genetic damage occurring early in life during childhood can play an important role in the development of chronic diseases in adulthood, including cancer. OBJECTIVES: The objective of this paper, part of the MAPEC_LIFE project, is to describe the frequency of micronuclei and meta-nuclear alterations in exfoliated buccal cells of 6-8year-old Italian children recruited in five Italian towns (i.e., Brescia, Torino, Pisa, Perugia and Lecce) with different air pollution levels. METHODS: About 200 children per town were recruited from primary schools. Biological samples were collected twice from the same children, in two different seasons (winter 2014-15 and late spring 2015). Cytogenetic damage was evaluated by the buccal micronucleus cytome assay. RESULTS: Overall,n = 1046 children represent the final cohort of the MAPEC_LIFE study. On the whole, the results showed a higher mean MN frequency in winter (0.42 ±â€¯0.54‰) than late-spring (0.22 ±â€¯0.34‰). MN frequency observed among the five Italian towns showed a trend that follows broadly the levels of air pollution in Italy: the highest MN frequency was observed in Brescia during both seasons, the lowest in Lecce (winter) and Perugia (late-spring). CONCLUSIONS: To the best of our knowledge, the number of recruited children included in the analysis (n = 1046) is the highest compared to previous studies evaluating the frequency of MN in exfoliated buccal cells so far. MN frequency was associated with winter season and living in towns at various levels of air pollution, suggesting an important role of this exposure in determining early cytogenetic effects.

Sulforaphane and Epigallocatechin Gallate Restore Estrogen Receptor Expression by Modulating Epigenetic Events in the Breast Cancer Cell Line MDA-MB-231: A Systematic Review and Meta-Analysis.

BACKGROUND/AIMS: Epigenetics refers to modifications in gene activity and expression without alteration at the DNA sequence. Environment and diet could influence gene expression. Diet modifications may be meaningful in preventing and treating chronic diseases, cancer included. Dietary bioactive compounds, such as polyphenols (e.g., curcumin, resveratrol, or epigallocatechin gallate [EGCG]) or isothiocyanate (e.g., sulforaphane [SFN]), can regulate histone acetylation. The aim of this systematic review and meta-analysis was to evaluate the effect of SFN and EGCG on breast cancer (BC) cells cultured in vitro. METHODS: Due to the enormous variability observed in study protocols and the innumerable genes involved, only studies analyzing the number of apoptotic cells in the MDA-MB-231 cell line were evaluated. The effect size (ES) was computed as the ratio of means. RESULTS: We identified 7 studies, 4 regarding the effect of 10 µM SFN on MDA-MB-231 cells (ES = 4.59, 95% confidence interval 4.05-5.20) and 3 focusing on the impact of 20 µM EGCG (ES = 2.84, 95% confidence interval 2.60-3.10). CONCLUSION: The findings suggest beneficial effects of dietary bioactive compounds such as SFN and EGCG and their effect on BC cells by restoring estrogen receptor gene expression, modulating epigenetic changes and events, and interfering with tumor growth rate. Publication bias limits the generalizability of the conclusions. High-quality studies are needed.

In vitro Biological Effects of Sulforaphane (SFN), Epigallocatechin-3-gallate (EGCG), and Curcumin on Breast Cancer Cells: A Systematic Review of the Literature.

Much of the recent research in neoplasia has been focusing on the epigenetics of cancer cells, particularly as regards the search for potential molecular biomarkers that could be used for early diagnosis, effective treatment, and prognosis of several types of cancer. Carcinogenesis often starts with mutations in oncogenes and tumor suppressor genes, and it leads to anomalies in cellular processes as vital as cell cycle regulation and apoptosis. Because malignant changes arise as a result of genetic as well as epigenetic mechanisms, one possible means of intervention involves reprogramming gene expression, so as to-at least in part-revert the molecular alterations. DNA methylation and demethylation, acetylation and deacetylation of histones, and microRNAs are a few examples of the epigenetic mechanisms responsible for tumor development and progression. Many biologically active compounds present in food-including sulforaphane, curcumin, and epigallocatechin-have been found to modulate those processes. We here systematically review information on the effects of such bioactive dietary compounds on human breast cancer cell lines, and explore the mechanisms underlying those effects with a view to their potential therapeutic application.

Acid sphingomyelinase as target of Lycium Chinense: promising new action for cell health.

BACKGROUND: Sphingomyelin plays very important roles in cell function under physiological and pathological conditions. Physical and chemical stimuli produce reactive oxygen species that stimulate acid sphingomyelinase to induce apoptosis. Antioxidant plants of the traditional Chinese Pharmacopoeia, such as Lycium Barbarum and Lycium Chinense, have become increasingly popular in Western countries. We investigated the effects of Lycium Chinense on acid sphingomyelinase and sphingomyelin species in relation to gene expression. METHODS: We prepared Lycium Chinense berry extracts and evaluated their antioxidant properties. Increasing amount of extracts was used to test cytotoxic and genotoxic effect on HepG2 cells. Gene expression, protein amount and enzyme activity of acid sphingomyelinase were tested by RT-PCR, immunoblotting and enzymatic activity assay, respectively. Sphingomyelin species were analyzed by UFLC MS/MS. A panel of 96 genes involved in oxidative stress, proliferation, apoptosis and cancer was used to test the effect of LC on gene expression. GLRX2, RNF7, and PTGS1 proteins were analyzed by immunoblotting. RESULTS: We showed that Lycium Chinense berries have high antioxidant properties, have an IC50value of 9.55 mg/mL, do not induce genotoxic effect and maintain high level of cell viability. The berry extracts inhibit acid sphingomyelinase activity and increase both very long fatty acid sphingomyelin species and unsaturated fatty acid sphingomyelin species. Among 96 genes, Lycium Chinense berries up-regulate Glutaredoxin 2 and Ring Finger Protein 7 genes and proteins, able to protect cells from apoptosis. Intrigantly, Lycium Chinense berries down-regulates Prostaglandin H synthase 1 gene but the protein is not expressed in HepG2 cells. CONCLUSION: The results identify acid sphingomyelinase as a novel target of Lycium Chinense berries to decrease saturated/unsaturated fatty acid sphingomyelin ratio, known to be useful for cell health. Consistent with these data, the berries regulate specifically gene expression to protect cells from apoptosis.

In Vitro Safety/Protection Assessment of Resveratrol and Pterostilbene in a Human Hepatoma Cell Line (HepG2).

The aim of this work was to evaluate in vitro the genotoxic and/or antigenotoxic effects of resveratrol (RESV) and pterostilbene (PTER) on HepG2 cells. Moreover, additional tests were performed to evaluate early and late apoptosis events induced by the tested stilbenes. RESV and PTER did not show any genotoxic activity. As regards antigenotoxicity testing, RESV and PTER showed a typical, U-shaped hormetic dose-response relationship characterized by a biphasic trend with small quantities having opposite effects to large ones. HepG2 cells treated with PTER exhibited a marked increase in early apoptosis (40.1%) at 250 microM; whereas, the highest concentration tested for both RESV and PTER significantly increased the proportion of HepG2 cells undergoing late apoptosis (32.5 and 51.2%, respectively). The observed pro-apoptotic activity could, at least in part, explain the hormetic response observed when the compounds were tested for antigenotoxicity (i.e., in the presence of induced DNA damage).

Primary DNA damage in welders occupationally exposed to extremely-low-frequency magnetic fields (ELF-MF).

BACKGROUND: Electric arc welding is known to involve considerable exposure to extremely-low-frequency magnetic fields (ELF-MF; 50 Hz). The aim of the present study was to evaluate individual exposure to ELF-MF during arc welding and to assess the eventually associated genotoxic hazard by evaluating primary DNA damage. METHODS: The study group comprised 21 electric arc welders (exposed) and 21 non-exposed control subjects (healthy blood donors). Occupational exposure to ELF-MF was measured using personal dosimeters worn during one complete work-shift (7 am to 5 pm). The extent of primary DNA damage was measured in peripheral blood leukocytes with the standard procedure of the alkaline comet assay. RESULTS: Tail length showed to have similar values in welders and controls. Whereas, the data showed a significant decrease for tail intensity (p = 0.01) and tail moment (p = 0.02) counts in exposed subjects compared to controls. CONCLUSIONS: The different results of our present study and published investigations from other research groups reporting positive results in the comet assay might be a result of different chromium and/or nickel (or other metals) exposure levels, which lead to DNA-protein cross-links at lower concentrations and DNA single-strand breakages at higher concentrations. Since these results are derived from a small-scale pilot study, a larger scale study should be undertaken.

Effects of the "PreveDi" lifestyle modification trial on metabolic syndrome.

BACKGROUND: Metabolic syndrome (MetS) is a cluster of metabolic disorders that includes central obesity, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia and high blood pressure (BP). Statistical reports suggest that the prevalence of MetS has dramatically increased during the recent years and is considered a worldwide epidemic. MetS has been found to be associated with increased risk of all-cause mortality, cardiovascular diseases (CVD), type 2 diabetes mellitus, chronic kidney disease and some types of cancer. MetS has a high socioeconomic cost and it is therefore extremely important that MetS is prevented and treated by simple and feasible methods. METHODS: The PreveDi study is a pilot before/after preventive trial aimed at the evaluation of the impact of a brief lifestyle intervention on changes in metabolic syndrome (MetS) risk factors. Recruitment was carried out in two community (council-run) pharmacies in the province of Perugia, Italy and the sample population consisted of 186 adults aged 45 or more who volunteered to participate. At enrolment the participants received a booklet illustrating general recommendations for MetS. During the 6-months follow-up period, participants were invited (by brochures and text messaging on cellular phones) to attend five conferences, five cooking classes, and twelve physical activity sessions. The conferences and the kitchen course were aimed to disseminate a healthy diet strategy focused primarily on reducing glycemic and insulinemic response. At baseline and follow-up MetS parameters were evaluated using medical equipments available in community pharmacies. RESULTS: At baseline, MetS was observed approximately in 52.2% of the PreveDi population, the MetS prevalence decreased with a higher education level in women, but not in men. Attendance to intervention programs was low and there was no significant difference between physical activity recorded at baseline and at follow-up. A slightly increased adherence to the healthy diet recommendations was observed for males. Waist circumference, systolic and diastolic blood pressure, fasting glucose and triglycerides did not change significantly, whereas weight, BMI and total cholesterol did. At follow-up the prevalence of MetS decreased, though not significantly, only in women (from 54,1 to 45.2%). Moreover, in women the negative correlation with education level was lost. CONCLUSIONS: This pilot study, even if with some limitations, suggests that MetS can be prevented and/or treated by simple and sustainable methods.

Investigation of the cytotoxic, genotoxic, and apoptosis-inducing effects of estragole isolated from fennel (Foeniculum vulgare).

The present study was undertaken to evaluate, in the HepG2 human hepatoma cell line, the in vitro cytotoxic, genotoxic, and apoptotic activities of estragole (1), contained in the essential oil of Foeniculum vulgare (fennel) and suspected to induce hepatic tumors in susceptible strains of mice. Toward this end, an MTT cytotoxicity assay, a trypan blue dye exclusion test, a double-staining (acridine orange and DAPI) fluorescence viability assay, a single-cell microgel-electrophoresis (comet) assay, a mitochondrial membrane potential (Δψm) assay, and a DNA fragmentation analysis were conducted. In terms of potential genotoxic effects, the comet assay indicated that estragole (1) was not able to induce DNA damage nor apoptosis under the experimental conditions used.