Oxidative Stress and Annexin A2 Differential Expression in Free Fatty Acids-Induced Non-Alcoholic Fatty Liver Disease in HepG2 Cells.

Non-alcoholic fatty liver disease (NAFLD) is a rising global burden, affecting one in four adults. Despite the increasing prevalence of NAFLD, the exact cellular and molecular mechanisms remain unclear, and effective therapeutic strategies are still limited. In vitro models of NAFLD are critical to understanding the pathogenesis and searching for effective therapies; thus, we evaluated the effects of free fatty acids (FFAs) on NAFLD hallmarks and their association with the modulation of Annexin A2 (ANXA2) and Keratin 17 (KRT17) in HepG2 cells. Our results show that oleic and palmitic acids can differentially induce intracellular lipid accumulation, cell death, and promote oxidative stress by increasing lipid peroxidation, protein carbonylation, and antioxidant defense depletion. Moreover, a markedly increased expression of inflammatory cytokines demonstrated the activation of inflammation pathways associated with lipotoxicity and oxidative stress. ANXA2 overexpression and KRT17 nuclear translocation were also observed, supporting the role of both molecules in the progression of liver disease. Taken together, these data provide insights into the interplay between ANXA2 and KRT17 in NAFLD, paving the way for understanding molecular mechanisms involved with the disease and developing new therapeutic strategies.

A Novel Compound from the Phenylsulfonylpiperazine Class: Evaluation of In Vitro Activity on Luminal Breast Cancer Cells.

Breast cancer (BC) is the most common cancer in women, and is characterized by its histological and molecular heterogeneity. Luminal BC is an estrogen receptor-positive subtype, with varied clinical courses. Although BC patients are eligible for hormone therapy, both early and late relapses still occur, and thus there is a demand for new cytotoxic and selective treatment strategies for these patients. In the present study, inspired by the structure of phenylsulfonylpiperazine, a series of 20 derivatives were tested in bioassays against MCF7, MDA-MB-231 and MDA-MB-453 BC cells to discover new hit compounds. After 48 h of treatment, 12 derivatives impaired cell viability and presented significant IC50 values against at least one of the tumor lineages. Overall, the luminal BC cell line MCF7 was more sensitive to treatments. Compound 3, (4-(1H-tetrazol-1-yl)phenyl)(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)methanone, was the most promising, with IC50 = 4.48 µM and selective index (SI) = 35.6 in MCF7 cells. Compound 3 also presented significant antimigratory and antiproliferative activities against luminal BC cells, possibly by affecting the expression of genes involved in the epithelial-mesenchymal transition mechanism, upregulating E-Cadherin transcripts (CDH1). Our findings suggest that phenylsulfonylpiperazine derivatives are potential candidates for the development of new therapies, especially those targeting luminal BC.

Glyphosate and Aminomethylphosphonic Acid (AMPA) Modulate Glutathione S-Transferase in Non-Tumorigenic Prostate Cells.

Glyphosate (GLY) was developed in the early 1970s and has become the most used broad-spectrum herbicide in the world so far. Its main metabolite is aminomethylphosphonic acid (AMPA), and the accumulation of GLY and its derivative compounds raises some concerns regarding possible health outcomes. In this study, we aimed to evaluate the effects of GLY and AMPA on prostate cell lines by evaluating cell viability, proliferation, gene and protein expression, and cellular pathways involved in the response to oxidative stress. Our results indicated that GLY and AMPA reduced the cell viability of tumorigenic and non-tumorigenic prostate cell lines only at higher concentrations (10 mM GLY and 20 mM AMPA). In contrast, both compounds increased the clonogenicity of non-tumorigenic PNT2 cells, mainly at concentrations below the IC50 (5 mM GLY and 10 mM AMPA). Moreover, treatment of non-tumorigenic cells with low concentrations of GLY or AMPA for 48 h increased GSTM3 expression at both mRNA and protein levels. In contrast, the treatments decrease the GST activity and induced an increase in oxidative stress, mainly at lower concentrations. Therefore, both compounds can cause cellular damage even at lower concentrations in non-tumorigenic PNT2 cells, mainly affecting cell proliferation and oxidative stress.

Endothelial dysfunction due to the inhibition of the synthesis of nitric oxide: Proposal and characterization of an in vitro cellular model.

The vascular endothelium plays a pivotal role in the maintenance of vascular homeostasis, mediated by vasoactive molecules produced by endothelial cells. The balance between vasoconstrictor and vasodilator biomolecules is what guarantees this equilibrium. Therefore, an increase in the bioavailability of vasoconstrictors along with a reduction in vasodilators may indicate a condition known as endothelial dysfunction. Endothelial dysfunction is marked by an inflammatory process and reduced activity of vasoprotective enzymes, being characterized by some factors like the reduction of the bioavailability of nitric oxide (NO) and increase in the production of reactive oxygen species (ROS), pro-inflammatory and vasoconstrictor molecules. This condition is a predictive marker of several cardiovascular diseases (e.g., atherosclerosis, hypertension, and diabetes). Research is affected by the scarcity of suitable in vitro models that simulate endothelial dysfunction. The goal of this study was to induce an in vitro condition to mimic endothelial dysfunction by inhibiting NO synthesis in cells. Thymus-derived endothelial cells (tEnd.1) were treated with different concentrations of L-NAME (from 1 to 1,000 µM) for different times (12, 24, 48, 72, 96, and 120 h without and with retreatment every 24 h). Cell viability, nitrite concentration, p22phox, NOX2, NOX4, IL-6, and ACE genes expression and lipid peroxidation were evaluated. The results indicate that the treatment with 100 µM L-NAME for 72 h without retreatment reduced NO concentration and NOX4 gene expression while increasing ACE expression, thus mimicking reduced vascular protection and possibly increased vasoconstriction. On the other hand, treatment with 100 µM L-NAME for 96 h with retreatment reduced the concentration of NO and the expression of the p22phox gene while increasing the expression of the IL-6 and ACE genes, mimicking the increase in inflammation and vasoconstriction parameters. Based on these results, we thus propose that both 100 µM L-NAME for 72 h without retreatment and 100 µM L-NAME for 96 h with retreatment may be used as models for in vitro endothelial dysfunction according to the purpose of the study to be conducted.

Jaboticaba (Myrciaria cauliflora) Peel Supplementation Prevents Hepatic Steatosis Through Hypolipidemic Effects and Cholesterol Metabolism Modulation in Diet-Induced Nonalcoholic Fatty Liver Disease Rat Model.

Jaboticaba (Myrciaria cauliflora), a Brazilian fruit, is a good source of dietary fiber and phenolic compounds, which are concentrated mainly in the peel. These compounds have been considered promising in prevention and treatment of hypercholesterolemia and hepatic steatosis. In this study, we investigated the effects of 4% jaboticaba peel powder (JPP) supplementation on cholesterol metabolism and hepatic steatosis in livers of rats fed a high-fat (HF) diet. The rats were fed a standard AIN-93M (control) diet or an HF diet containing 32% lard and 1% cholesterol, both with and without 4% JPP. The M. cauliflora peel composition revealed a low-lipid high-fiber content and phenolic compounds. The phenolic compounds in JPP, tentatively identified by high-performance liquid chromatography and mass spectrometry (HPLC-MS/MS) analysis, were confirmed to contain phenolic acids, flavonoids, and anthocyanins. Moreover, JPP presented significant antioxidant activity in vitro and was not cytotoxic to HepG2 cells, as determined by the lactate dehydrogenase (LDH) assay. After 6 weeks of treatment, our results showed that JPP supplementation increased lipid excretion in feces, reduced serum levels of total cholesterol and nonhigh-density lipoprotein cholesterol, decreased serum aspartate aminotransferase (AST) activity, and attenuated hepatic steatosis severity in rats fed the HF diet. Furthermore, JPP treatment downregulated expression of ACAT-1, LXR-α, CYP7A1, and ABCG5 genes. Therefore, jaboticaba peel may represent a viable dietary strategy to prevent nonalcoholic fatty liver disease as the JPP treatment alleviated hepatic steatosis through improvement of serum lipid profiles and modulation of mRNA expression of genes involved in cholesterol metabolism.