Characterization of the Interaction Between SARS-CoV-2 Membrane Protein (M) and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target.

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M-PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and PLA (Proximity Ligation Assay). In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase in PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

Increased mTOR Signaling and Impaired Autophagic Flux Are Hallmarks of SARS-CoV-2 Infection.

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling.

Anticancer effects of root and beet leaf extracts (Beta vulgaris L.) in cervical cancer cells (HeLa).

Cervical cancer is the fourth leading cause of cancer mortality in women worldwide. Beetroot (Beta vulgaris L.) has bioactive compounds that can inhibit the progression of different types of cancer. To analyze the antiproliferative effects of beet leaf and root extracts, we performed MTT, clonogenic survival, cell cycle analysis, Annexin/PI labeling, and western blotting. Here, we report that 10 and 100 µg/ml of root and leaf extracts decreased cell viability and potentiated rapamycin and cisplatin effects while decreased the number of large colonies, especially at 10 µg/ml (293.6 of control vs. 200.0 of leaf extract, p = .0059; 138.6 of root extract, p = .0002). After 48 hr, 100 µg/ml of both extracts led to increased sub-G1 and G0/G1 populations. In accordance, 100 µg/ml of root extract induced early apoptosis (mean = 0.64 control vs. 1.56 root; p = .048) and decreased cell size (p < .0001). Both extracts decreased phosphorylation and expression of mechanistic Target of Rapamycin (mTOR) signaling, especially by inhibiting ribosomal protein S6 (S6) phosphorylation, increasing cleaved poly(ADP-ribose) polysomerase 1 (PARP1) and Bcl-2-like protein 11 (BIM), and decreasing cyclin D1 expression, which regulates cell cycle progression. Here, we demonstrate that beetroot and leaf extracts could be an efficient strategy against cervical cancer.

STAT3 contributes to cisplatin resistance, modulating EMT markers, and the mTOR signaling in lung adenocarcinoma.

Lung cancer is the second leading cause of cancer death worldwide and is strongly associated with cisplatin resistance. The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells and coordinates critical cellular processes as survival, self-renewal, and inflammation. In several types of cancer, STAT3 controls the development, immunogenicity, and malignant behavior of tumor cells while it dictates the responsiveness to radio- and chemotherapy. It is known that STAT3 phosphorylation at Ser727 by mechanistic target of rapamycin (mTOR) is necessary for its maximal activation, but the crosstalk between STAT3 and mTOR signaling in cisplatin resistance remains elusive. In this study, using a proteomic approach, we revealed important targets and signaling pathways altered in cisplatin-resistant A549 lung adenocarcinoma cells. STAT3 had increased expression in a resistance context, which can be associated with a poor prognosis. STAT3 knockout (SKO) resulted in a decreased mesenchymal phenotype in A549 cells, observed by clonogenic potential and by the expression of epithelial-mesenchymal transition markers. Importantly, SKO cells did not acquire the mTOR pathway overactivation induced by cisplatin resistance. Consistently, SKO cells were more responsive to mTOR inhibition by rapamycin and presented impairment of the feedback activation loop in Akt. Therefore, rapamycin was even more potent in inhibiting the clonogenic potential in SKO cells and sensitized to cisplatin treatment. Mechanistically, STAT3 partially coordinated the cisplatin resistance phenotype via the mTOR pathway in non-small cell lung cancer. Thus, our findings reveal important targets and highlight the significance of the crosstalk between STAT3 and mTOR signaling in cisplatin resistance. The synergic inhibition of STAT3 and mTOR potentially unveil a potential mechanism of synthetic lethality to be explored for human lung cancer treatment.

Short-Term Strength Exercise Reduces Hepatic Insulin Resistance in Obese Mice by Reducing PTP1B Content, Regardless of Changes in Body Weight.

Obesity is closely related to insulin resistance and type 2 diabetes genesis. The liver is a key organ to glucose homeostasis since insulin resistance in this organ increases hepatic glucose production (HGP) and fasting hyperglycemia. The protein-tyrosine phosphatase 1B (PTP1B) may dephosphorylate the IR and IRS, contributing to insulin resistance in this organ. Aerobic exercise is a great strategy to increase insulin action in the liver by reducing the PTP1B content. In contrast, no study has shown the direct effects of strength training on the hepatic metabolism of PTP1B. Therefore, this study aims to investigate the effects of short-term strength exercise (STSE) on hepatic insulin sensitivity and PTP1B content in obese mice, regardless of body weight change. To achieve this goal, obese Swiss mice were submitted to a strength exercise protocol lasting 15 days. The results showed that STSE increased Akt phosphorylation in the liver and enhanced the control of HGP during the pyruvate tolerance test. Furthermore, sedentary obese animals increased PTP1B content and decreased IRS-1/2 tyrosine phosphorylation; however, STSE was able to reverse this scenario. Therefore, we conclude that STSE is an important strategy to improve the hepatic insulin sensitivity and HGP by reducing the PTP1B content in the liver of obese mice, regardless of changes in body weight.

Beetroot and leaf extracts present protective effects against prostate cancer cells, inhibiting cell proliferation, migration, and growth signaling pathways.

Beet (Beta vulgaris L.) has high nutritional value, containing bioactive compounds such as betalains and flavonoids. Scientific evidence points to the use of these natural compounds in the treatment of several types of cancer, such as prostate cancer, one of the main causes of morbidity and mortality in men. Here, we compared beet roots and leaves extracts, and their main compounds, apigenin, and betanin, respectively, in DU-145 and PC-3 prostate cancer cell lines. Both cells presented the proliferation decreased for beetroot and beet leaves extracts. The apigenin treatment also reduced the proliferation of both cell lines. Regarding cell migration, beet leaves extract was able to decrease the scratch area in both cell lines, whereas apigenin affected only PC-3 cells' migration. In colony formation assay, both extracts were effective in reducing the number of colonies formed. Besides, the beet leaves extracts and apigenin presented strong inhibition of growth-related signaling pathways in both cell lines, and the beetroot extract and betanin presented effects only in DU-145 cells. Furthermore, the extracts and isolated compounds were able to reduce the levels of apoptotic and cell cycle proteins. This study reveals that beet extracts have important anti-cancer effects against prostate cancer cells.

Metformin impairs cisplatin resistance effects in A549 lung cancer cells through mTOR signaling and other metabolic pathways.

Lung cancer is the leading cause of cancer­associated death worldwide and exhibits intrinsic and acquired therapeutic resistance to cisplatin (CIS). The present study investigated the role of mTOR signaling and other signaling pathways after metformin (MET) treatment in control and cisplatin­resistant A549 cells, mapping pathways and possible targets involved in CIS sensitivity. MTT, flow cytometry, clonogenic assay, western blotting, proteomic analysis using the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) approach and reverse transcription­quantitative PCR were performed. The results revealed that CIS treatment induced mTOR signaling pathway overactivation, and the mTOR status was restored by MET. MET and the mTOR inhibitor rapamycin (RAPA) decreased the viability in control and resistant cells, and decreased the cell size increase induced by CIS. In control cells, MET and RAPA decreased colony formation after 72 h and decreased IC50 values, potentiating the effects of CIS. Proteomics analysis revealed important pathways regulated by MET, including transcription, RNA processing and IL­12­mediated signaling. In CIS­resistant cells, MET regulated the apoptotic process, oxidative stress and G2/M transition. Annexin 4 (ANXA4) and superoxide dismutase 2 (SOD2), involved in apoptosis and oxidative stress, respectively, were chosen to validate the SILAC analysis and may represent potential therapeutic targets for lung cancer treatment. In conclusion, the chemosensitizing and antiproliferative effects of MET were associated with mTOR signaling and with potential novel targets, such as ANXA4 and SOD2, in human lung cancer cells.

The Hallmarks of Flavonoids in Cancer.

Flavonoids represent an important group of bioactive compounds derived from plant-based foods and beverages with known biological activity in cells. From the modulation of inflammation to the inhibition of cell proliferation, flavonoids have been described as important therapeutic adjuvants against several diseases, including diabetes, arteriosclerosis, neurological disorders, and cancer. Cancer is a complex and multifactor disease that has been studied for years however, its prevention is still one of the best known and efficient factors impacting the epidemiology of the disease. In the molecular and cellular context, some of the mechanisms underlying the oncogenesis and the progression of the disease are understood, known as the hallmarks of cancer. In this text, we review important molecular signaling pathways, including inflammation, immunity, redox metabolism, cell growth, autophagy, apoptosis, and cell cycle, and analyze the known mechanisms of action of flavonoids in cancer. The current literature provides enough evidence supporting that flavonoids may be important adjuvants in cancer therapy, highlighting the importance of healthy and balanced diets to prevent the onset and progression of the disease.

Strength exercise reduces hepatic pyruvate carboxylase and gluconeogenesis in DIO mice.

Obesity is linked to a reduction in the control of hepatic glucose production, which is the primary mechanism related to fasting hyperglycemia and the development of type 2 diabetes mellitus (T2DM). The main system involved in hepatic gluconeogenesis synthesis is controlled by pyruvate carboxylase (PC), which increases in obesity conditions. Recently, we showed that short-term strength training is an important tool against obesity-induced hyperglycemia. As aerobic exercise can reduce the hepatic PC content of obese animals, we hypothesized that strength exercise can also decrease this gluconeogenic enzyme. Therefore, this study investigated whether the metabolic benefits promoted by short-term strength training are related to changes in hepatic PC content. Swiss mice were divided into three groups: lean control (Ctl), obese sedentary (ObS), and obese short-term strength training (STST). The STST protocol was performed through one session/day for 15 days. The obese exercised animals had reduced hyperglycemia and insulin resistance. These results were related to better control of hepatic glucose production and hepatic insulin sensitivity. Our bioinformatics analysis showed that hepatic PC mRNA levels have positive correlations with glucose levels and adiposity, and negative correlations with locomotor activity and muscle mass. We also found that hepatic mRNA levels are related to lipogenic markers in the liver. Finally, we observed that the obese animals had an increased hepatic PC level; however, STST was efficient in reducing its amount. In conclusion, we provide insights into new biomolecular mechanisms by showing how STST is an efficient tool against obesity-related hyperglycemia and T2DM, even without body weight changes.

Short-Term Combined Exercise Improves Inflammatory Profile in the Retina of Obese Mice.

Excess of adipose tissue increases the concentration of proinflammatory cytokines, triggering a subclinical inflammatory condition. This inflammatory profile contributes to retina damage, which can lead to retinal dysfunction and reduced vision. Regularly practicing both aerobic and strength exercises is well known for promoting anti-inflammatory effects on different organs in the peripheral and central regions. However, the effects of combined physical exercise (CPE; strength + aerobic) on the inflammatory process in the retina tissue are not yet known. This study aimed to investigate the effects of CPE on the inflammatory profile of the retina in obese mice. Swiss mice were distributed into control, sedentary obese, and trained obese groups. The trained obese group was subjected to short-term CPE, 1 h/day, for 7 days. The CPE was composed of aerobic and strength exercises in the same exercise session. The strength exercise protocol consisted of 10 climbing series, with 12 ± 1 dynamic climbing movements at 70% of the maximum voluntary carrying capacity (MVCC), and the aerobic exercise protocol consisted of 30 min of treadmill running, with an intensity of 75% of the exhaust velocity. Subsequently, the retina was excised and analyzed by Western blot. Obese animals presented impairment on glucose homeostasis and elevated levels of proinflammatory proteins in the serum and retina; however, CPE was effective in reversing these parameters, independently of changes in body adiposity. Therefore, for the first time, we have shown that short-term CPE can be an important strategy to treat an inflammatory profile in the retina.

Protective effects of beet (Beta vulgaris) leaves extract against oxidative stress in endothelial cells in vitro.

Beetroot is an herb used worldwide as a food product, raw material for food industry, ethanol production and source of food coloring. Beet leaves are an unconventional food with antioxidant properties, which might neutralize reactive oxygen species (ROS) induced by oxidized Low-Density Lipoprotein (LDL) present in dyslipidemias. This study aimed to elucidate the effects of beet leaves on the suppression of LDL oxidative processes. Beet leaves extract was produced, characterized, and tested for its antioxidant capacity using endothelial cells in vitro. A model of human umbilical vein endothelial cells was used in various tests, including viability assay, molecular analysis of antioxidant genes, ROS labeling, and macrophage adhesion assay. The extract improved the antioxidative protection of endothelial cells against different agents including oxidized LDL-cholesterol and H2 O2 . It acted on ROS directly due to its high content of natural antioxidants, but also due to the activation and improvement of cellular defenses such as Superoxide dismutase 1, Superoxide dismutase 2, and catalase. The inhibition of LDL-mediated oxidative effects on endothelial cells may turn this unconventional food a functional food with great potential for phytotherapy of atherosclerosis as an adjuvant for medicinal treatments.

Short-term high-fat diet modulates several inflammatory, ER stress, and apoptosis markers in the hippocampus of young mice.

The consumption of saturated fatty acids is one of the leading risk factors for Alzheimer's Disease (AD) development. Indeed, the short-term consumption of a high-fat diet (HFD) is related to increased inflammatory signals in the hippocampus; however, the potential molecular mechanisms linking it to AD pathogenesis are not fully elucidated. In our study, we investigated the effects of short-term HFD feeding (within 3, 7 and 10 days) in AD markers and neuroinflammation in the hippocampus of mice. The short period of HFD increased fasting glucose and HOMA-IR. Also, mice fed HFD increased the protein content of ß-Amyloid, pTau, TNFα, IL1ß, pJNK, PTP1B, peIF2α, CHOP, Caspase3, Cleaved-Caspase3 and Alzheimer-related genes (Bax, PS1, PEN2, Aph1b). At 10 days, both neuronal (N2a) and microglial (BV2) cells presented higher expression of inflammatory and apoptotic genes when stimulated with palmitate. These findings suggest that a short period of consumption of a diet rich in saturated fat is associated with activation of inflammatory, ER stress and apoptotic signals in the hippocampus of young mice.