Francine Carla Cadoná

Tucumã fruit extracts (Astrocaryum aculeatum Meyer) decrease cytotoxic effects of hydrogen peroxide on human lymphocytes.

This study quantifies the bioactive molecules in and determines the in vitro protective effect of ethanolic extracts isolated from the peel and pulp of tucumã (Astrocaryum aculeatum, Mart.), an Amazonian fruit rich in carotenoids. The cytoprotective effect of tucumã was evaluated in lymphocyte cultures exposed to H2O2 using spectrophotometric, fluorimetric, and immunoassay assays. The results confirmed that tucumã pulp extract is rich in β-carotene and quercetin, as previously described in the literature. However, high levels of these compounds were also found in tucumã peel extract. The extracts also contained significant amounts rutin, gallic acid, caffeic acid, and chlorogenic acid. Despite quantitative differences in the concentration of these bioactive molecules, both extracts increased the viability of cells exposed to H2O2 in concentrations ranging from 300 to 900 μg/mL. Caspases 1, 3, and 8 decreased significantly in cells concomitantly exposed to H2O2 and these extracts, indicating that tucumã cryoprotection involves apoptosis modulation.

Superoxide-hydrogen peroxide imbalance interferes with colorectal cancer cells viability, proliferation and oxaliplatin response

The role of superoxide dismutase manganese dependent enzyme (SOD2) in colorectal cancer is presently insufficiently understood. Some studies suggest that high SOD2 levels found in cancer tissues are associated with cancer progression. However, thus far, the role of colorectal cancer superoxide-hydrogen peroxide imbalance has not yet been studied. Thus, in order to address this gap in extant literature, we performed an in vitro analysis using HT-29 colorectal cell line exposed to paraquat, which generates high superoxide levels, and porphyrin, a SOD2 mimic molecule. The effect of these drugs on colorectal cancer cell response to oxaliplatin was evaluated. At 0.1 μM concentration, both drugs exhibited cytotoxic and antiproliferative effect on colorectal cancer cells. However, this effect was more pronounced in cells exposed to paraquat. Paraquat also augmented the oxaliplatin cytotoxic and antiproliferative effects by increasing the number of apoptosis events, thus causing the cell cycle arrest in the S and M/G2 phases. The treatments were also able to differentially modulate genes related to apoptosis, cell proliferation and antioxidant enzyme system. However, the effects were highly variable and the results obtained were inconclusive. Nonetheless, our findings support the hypothesis that imbalance caused by increased hydrogen peroxide levels could be beneficial to cancer cell biology. Therefore, the use of therapeutic strategies to decrease hydrogen peroxide levels mainly during oxaliplatin chemotherapy could be clinically important to the outcomes of colorectal cancer treatment.

The in vitro genotoxic effect of Tucuma (Astrocaryum aculeatum), an Amazonian fruit rich in carotenoids.

Tucuma (Astrocaryum aculeatum) is an Amazonian fruit that presents high levels of carotenoids and other bioactive compounds such as quercetin. The extracts of tucuma peel and pulp present strong antioxidant activity which illustrate an elevated concentration that causes cytotoxic effects in human peripheral blood mononuclear cells (PBMCs). This study performed additional investigations to analyze the potential genotoxic effects of the tucuma extracts on PBMCs. The genotoxicity was evaluated by DNA fragmentation, Comet assay, and chromosomal instability G-band assays. The acute tucuma extract treatment showed genoprotective effects against DNA denaturation when compared with untreated PBMC cells. However, in the experiments with 24 and 72 h treatments to tucuma treatments, we observed low genotoxicity through a concentration of 100 μg/mL, some genotoxic effects related to intermediary concentrations (100-500 μg/mL), and more pronounced genotoxic effects on higher tucuma extract concentrations. After 24 h of treatment, the reactive oxygen species were similar among treatments and PBMC control groups. However, the caspase-1 activity related to the apoptosis and pyroptosis process increased significantly in higher tucuma concentrations. In summary, tucuma extracts, despite their higher antioxidant content and antioxidant activity, would present PBMCs genotoxic effects that are dependent on concentration and time exposition. These results need to be considered in future in vitro and in vivo studies of tucuma effects.

Cytoprotective and genoprotective effects of β-glucans against aflatoxin B1-induced DNA damage in broiler chicken lymphocytes

The polysaccharide β-glucan presents beneficial effects on the immune system, although the mechanisms of the immunomodulatory effect remain poorly understood. The potential cytoprotective and genoprotective effects of β-glucans were evaluated in broiler chicken lymphocytes exposed to increasing concentrations of aflatoxin B₁ (AFB₁) and/or β-glucans. AFB₁ significantly decreased cell viability at the concentrations of 10 and 20 μg/ml at 72 h of incubation (p<0.01 and p<0.001, respectively). Moreover, the AFB₁ concentrations of 1, 10 and 20 μg/ml increased DNA fragmentation levels at 24 h (p<0.001). Conversely, lymphocyte death was prevented by β-glucans at the concentrations of 1% and 10%, indicating a cytoprotective effect. Reactive oxygen species levels were increased in the cells treated with 20 μg/ml AFB₁ at 24 h (p<0.05) and 10% β-glucans with or without AFB₁ at 24, 48 and 72 h of incubation (p<0.001). DNA damage increased by more than 100% in AFB₁-treated lymphocytes when compared to control group. β-glucans at 1% was able to fully revert the AFB₁-induced lymphocyte DNA damage, indicating a genoprotective effect and maintaining DNA integrity. In conclusion, β-glucans showed in vitro dose-dependent cytoprotective and genoprotective effects in broiler chicken lymphocytes exposed to AFB₁.

Neuroprotective Effects of Açaí (Euterpe oleracea Mart.) against Rotenone In Vitro Exposure

Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5 μg/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy.

Effect of Uncaria tomentosa Extract on Apoptosis Triggered by Oxaliplatin Exposure on HT29 Cells

Background/Aim. The use of herbal products as a supplement to minimize the effects of chemotherapy for cancer treatment requires further attention with respect to the activity and toxicity of chemotherapy. Uncaria tomentosa extract, which contains oxindole alkaloids, is one of these herbal products. The objective of this study was to evaluate whether Uncaria tomentosa extract modulates apoptosis induced by chemotherapy exposure. Materials and Methods. Colorectal adenocarcinoma cells (HT29 cells) were grown in the presence of oxaliplatin and/or Uncaria tomentosa extract. Results. The hydroalcoholic extract of Uncaria tomentosa enhanced chemotherapy-induced apoptosis, with an increase in the percentage of Annexin positive cells, an increase in caspase activities, and an increase of DNA fragments in culture of the neoplastic cells. Moreover, antioxidant activity may be related to apoptosis. Conclusion. Uncaria tomentosa extract has a role for cancer patients as a complementary therapy. Further studies evaluating these beneficial effects with other chemotherapy drugs are recommended.

Genomodifier capacity assay: a non-cell test using dsDNA molecules to evaluate the genotoxic/genoprotective properties of chemical compounds

We describe here an ultrasensitive and fast protocol called a GEMO assay (genomodifier capacity assay). This non-cell method was developed to identify chemicals with genomodifier (genotoxic and/or genoprotective) capacity. The assay is performed in a black 96-well plate using calf thymus dsDNA exposed to different concentrations of chemicals tested (CT) for 30 minutes with and without the addition of a prooxidant substance that causes dsDNA damage (H2O2, 3 M). Furthermore, PicoGreen®, a highly sensitive dsDNA dye is added and so the fluorescence is emitted according to the concentration of intact dsDNA. Chemicals that cause a break in dsDNA are identified by a decrease in fluorescence in comparison with the fluorescence observed in an untreated dsDNA (control group) indicating genotoxic capacity. In contrast, attenuation of dsDNA degradation caused by H2O2 exposition indicates CT genoprotective capacity. The GEMO assay was validated by comparing peripheral blood mononuclear cells (PBMCs) and an HT29 colorectal cell line exposed to similar conditions where the effect on dsDNA was also evaluated by a DNA alkaline comet assay. Vitamin C was used as CT and other variables were also evaluated to confirm the cytotoxic action of H2O2. The results showed a strong negative correlation between the GEMO assay and the comet assay performed in PBMCs (r2 = −0.828; p < 0.0001) since higher dsDNA fluorescence measured by the GEMO assay was associated with lower index damage measured by the DNA alkaline comet assay. Therefore, the GEMO assay could be useful for early screening of genoprotective and genotoxic effects of chemicals and plant extracts without interfering cell biological variables.

Guaraná a Caffeine-Rich Food Increases Oxaliplatin Sensitivity of Colorectal HT-29 Cells by Apoptosis Pathway Modulation

We investigated the in vitro effects of guaraná and its main metabolites (caffeine, theobromine and catechin) on cytotoxicity and cell proliferation on colorectal cancer (CRC) line HT-29 cells and on oxaliplatin sensitivity. The cells were exposed to different concentrations of guaraná extract with and without oxaliplatin. The concentrations of bioactive molecules were also estimated considering their potential proportion on guaraná hydro-alcoholic extract. Apoptosis effect was analyzed by annexin V quantification using flow cytometry, while apoptosis pathway gene modulation (p53, Bax/Bcl-2 genes ratio, caspases 8 and 3) was determined by qRT-PCR analysis. Cells exposed to guaraná at a concentration of 100 μg/mL presented a similar cytotoxic effect as HT-29 cells treated with oxaliplatin and did not affect the sensitivity of the drug. Guaraná presented cell anti-proliferative effect and increased anti-proliferative oxaliplatin sensitivity at all concentrations tested here. Guaraná was able to induce apoptosis and up-regulate the p53 and Bax/Bcl-2 genes.

Guaraná, a Highly Caffeinated Food, Presents in vitro Antitumor Activity in Colorectal and Breast Cancer Cell Lines by Inhibiting AKT/mTOR/S6K and MAPKs Pathways

ABSTRACT The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinases (MAPKs) pathways are frequently upregulated in cancer. Some authors have reported that some antioxidant molecules could be potential inhibitors of these pathways. Therefore, we investigated the in vitro antitumor effect of guaraná by inhibiting the AKT/mTOR/S6K and MAPKs pathways. Colorectal and breast cancer cell lineages, HT-29 and MCF-7 cells, respectively, were exposed to different guaraná concentrations (0.1, 1, 10, and 100 µg/mL) as well as its main bioactive molecule, caffeine, in proportional concentrations to those found in the extract. Western blot, clonogenic assay, and growth curve were performed. Moreover, we investigated the potential cytotoxic effect of guaraná in normal cells. The results revealed that guaraná and caffeine inhibited some MAPKs proteins (p-p38 and p-HSP27) in MCF-7 cells. However, they did not affect this pathway in HT-29 cells. Furthermore, guaraná inhibited mTORC1 (p-S6K) and mTORC2 (p-AKT) in MCF-7 cells, but only mTORC1 in HT-29 cells. Caffeine only inhibited the mTOR pathway in MCF-7 cells. Guaraná decreased the colony formation and cell growth in MCF-7 and HT-29 cells. Guaraná did not affect normal cells. In conclusion, guaraná could be an important agent in antitumor pharmacologic therapies by inhibiting the mTOR and MAPKs pathways.

The Pavonia xanthogloea (Ekman, Malvaceae): Phenolic compounds quantification, anti-oxidant and cytotoxic effect on human lymphocytes cells

Introduction: Pavonia xanthogloea is traditionally used as an antimicrobial and anti-tumour medicine in Southern Brazilian region. However, investigations about this species are still incipient. Hypothesis Tested: The study postulated that P. xanthologea specie present some phenolic compound and present some biological properties as anti-oxidant and cytoprotective effect against oxidative stress. Materials and Methods: The content of eight phenolic molecules in the crude ethanolic extract of the aerial part of P. xanthogloea and its five fractions (hexane, dichloromethane, ethyl-acetate, n-butanol, and water) was determined by heterotrophic plate count method. The anti-oxidant capacity of the extract and the fractions was determined by 1,1-diphenyl-2-picryl-hydrazyl assay. The potential anti-oxidant and cytoprotective effect was also analyzed in human lymphocyte culture treated with extract/fractions at different concentrations with and without oxidative stress generated by hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) exposition. Results: Tiliroside was the molecule detected in all extract. Water and ethyl-acetate fractions showed the highest radical-scavenging activity. The crude extract, hexane, water, and n-butanol reversed the higher reactive oxygen specie levels generated by H2O2 and SNP to levels similar to those observed in the control group. In addition, crude extract, hexane, ethyl-acetate and n-butanol did not caused cytotoxicity, whereas water fraction was cytotoxic at higher concentration tested here (300 μg/mL). The cytotoxicity reversion caused by SNP exposition was concentration-dependent of the extract and fractions. However, dichloromethane fraction increased cell mortality in all concentrations investigated and was not able to decrease cell death in the lymphocytes exposed to SNP. Conclusion: The results suggest potential medicine use of this species.