Rosmarinic acid modulates purinergic signaling and induces apoptosis in melanoma cells.

Cancer cases have increased worldwide. Cutaneous melanoma (CM), a highly metastatic skin cancer, largely contributes to global statistical cancer death data. Research has shown that rosmarinic acid (RA) is a promising phenolic compound with antineoplastic properties. Thus, we investigated the effects of RA on apoptosis-inducing in melanoma cells, purinergic signaling modulation, and cytokine levels. We treated SK-MEL-28 cells for 24 h with different concentrations of RA and assessed the apoptosis, CD39, CD73, and A2A expression, and cytokine levels. We found RA-induced apoptosis in melanoma cells. Regarding the purinergic system, we verified that RA downregulated the expression of CD73 and A2A, specially at high concentrations of treatment. Additionally, RA increased IL-6, IL-4, IL-10, IFN-γ, and TNF-α levels. Our in vitro results confirm RA's potential to be used to induce melanoma cell apoptosis, having CD73 and A2A as targets when reversion of immune suppression is desired. Further studies in animal models and clinical trials focusing on RA's modulation of purinergic signaling in melanoma are required.

Curcumin modulates purinergic signaling and inflammatory response in cutaneous metastatic melanoma cells.

Cutaneous melanoma (CM) poses a therapeutic challenge due to its aggressive nature and often limited response to conventional treatments. Exploring novel therapeutic targets is essential, and natural compounds have emerged as potential candidates. This study aimed to elucidate the impact of curcumin, a natural compound known for its anti-inflammatory, antioxidant, and anti-tumor properties, on metastatic melanoma cells, focusing on the purinergic system and immune responses. Human melanoma cell line SK-Mel-28 were exposed to different curcumin concentrations for either 6 or 24 h, after which we assessed components related to the purinergic system and the inflammatory cascade. Using RT-qPCR, we assessed the gene expression of CD39 and CD73 ectonucleotidases, as well as adenosine deaminase (ADA). Curcumin effectively downregulated CD39, CD73, and ADA gene expression. Flow cytometry analysis revealed that curcumin significantly reduced CD39 and CD73 protein expression at specific concentrations. Moreover, the A2A receptor's protein expression decreased across all concentrations. Enzymatic activity assays demonstrated that curcumin modulated CD39, CD73, and ADA activities, with effects dependent on concentration and duration of treatment. Extracellular ATP levels increased after 24 h of curcumin treatment, emphasizing its role in modulating hydrolytic activity. Curcumin also displayed anti-inflammatory properties by reducing NLRP3 gene expression and impacting the levels of key inflammatory cytokines. In conclusion, this study unveils the potential of curcumin as a promising adjuvant in CM treatment. Curcumin modulates the expression and activity of crucial components of the purinergic system and exhibits anti-inflammatory effects, indicating its potential therapeutic role in combating CM. These findings underscore curcumin's promise and warrant further investigation in preclinical and clinical settings for melanoma management.

Effects of calcium supplementation on changes in the IL2, IL4, IL6, IL10 axes and oxidative stress in pregnant women at risk for pre-eclampsia.

BACKGROUND: Pregnant women with hypertensive disorders are at increased risk for inflammatory diseases and oxidative stress. The dilemma raised by the best dosage of calcium supplementation on these factors is evident. The aim of the current study was to examine the effects of calcium on biomarkers of the purinergic system, inflammation and oxidative stress, which are factors contributing to vascular damage in pregnant women at high risk of pre-eclampsia. METHODS: A prospective, double-blind and placebo-controlled study conducted with 101 women at risk of pre-eclampsia were randomized to take 500 mg calcium/day or 1,500 mg calcium/day or placebo for 6 weeks from the 20th gestational week until delivery. Fasting blood samples were collected at the beginning of the study and 6 weeks after the intervention. RESULTS: Taking calcium supplements (500 mg calcium/day) led to a significant increase in ATP hydrolysis (p < 0.05), NTPDase activity with increased hydrolysis of ADP and AMP nucleotides in platelets and lymphocytes. In the intragroup analysis IL-2, IL-6, IL-4 and interferon-É£ presented lower values in the calcium 1,500 mg/day group (p < 0.005). Oxidative stress was assessed by TBARS pro-oxidant marker, with an increase for the calcium groups when compared to the placebo group. The Vitamin C antioxidant marker presented a significant increase (p < 0.005) for the group that received high calcium doses. CONCLUSIONS: Calcium administration for 6 weeks had antioxidant action and positively modulated the purinergic system and inflammatory markers in pregnant women at risk of pre-eclampsia.

Curcumin promotes apoptosis of human melanoma cells by caspase 3.

Cutaneous melanoma (CM) is a malignant neoplasm with a high metastatic rate that shows poor response to systemic treatments in patients with advanced stages. Recently, studies have highlighted the antineoplastic potential of natural compounds, such as polyphenols, in the adjuvant therapy context to treat CM. The objective of the present study was to evaluate the effect of different concentrations of curcumin (0.1-100 µM) on the metastatic CM cell line SK-MEL-28. The cells were treated for 6 and 24 h with different concentrations of curcumin. Cell viability was assessed by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and fluorescence microscopy. The apoptotic-inducing potential was detected by annexin V flow cytometry. The wound healing assay was used to verify cell migration after the curcumin exposition. The redox profile was evaluated by levels of the pro-oxidant markers reactive oxygen species (ROS) and Nitric oxide (NOx) and antioxidants of total thiols (PSH) and nonprotein thiols. The gene expression and enzymatic activity of caspase 3 were evaluated by reverse transcription-quantitative polymerase chain reaction and a sensitive fluorescence assay, respectively. Curcumin significantly decreased the cell viability of SK-MEL-28 cells at both exposure times. It also induced apoptosis at the highest concentration tested (p < .0001). SK-MEL-28 cell migration was inhibited by curcumin after treatment with 10 µM (p < .0001) and 100 µM (p < .0001) for 6 and 24 h (p = .0006 and p < .0001, respectively). Furthermore, curcumin significantly increased levels of ROS and NOx. Finally, curcumin was capable of increasing the gene expression at 10 µM (p = .0344) and 100 µM (p = .0067) and enzymatic activity at 10 µM (p = .0086) and 100 µM (p < .0001) of caspase 3 after 24 h. For the first time, we elucidated in our study that curcumin increases ROS levels, promoting oxidative stress that activates the caspase pathway and culminates in SK-MEL-28 metastatic CM cell death.

Rosmarinic acid decreases viability, inhibits migration and modulates expression of apoptosis-related CASP8/CASP3/NLRP3 genes in human metastatic melanoma cells.

Cutaneous melanoma is the most aggressive type of skin cancer; it is difficult to treat, and has been highlighted in recent years due to increasing numbers of cases worldwide. The use of antitumoral therapeutics for this neoplasm has been associated with severe side effects, low quality of life, and resistance. We aimed in this study to explore the effect of the phenolic compound rosmarinic acid (RA) on human metastatic melanoma cells. SK-MEL-28 melanoma cells were treated for 24 h with different concentrations of RA. In parallel, peripheral blood mononuclear cells (PBMCs) also were treated with RA under the same experimental conditions to verify the cytotoxic effect on non-tumoral cells. Then, we assessed cell viability and migration, levels of intracellular and extracellular reactive oxygen species (ROS), as well as nitric oxide (NOx), non-protein thiols (NPSH), and total thiol (PSH). Gene expression of the caspase 8, caspase 3 and NLRP3 inflammasome was evaluated by RT-qPCR. The enzymatic activity of the caspase 3 protein was assessed by a sensitive fluorescent assay. Fluorescence microscopy was employed to corroborate the effects of RA on melanoma cell viability, mitochondria transmembrane potential and apoptotic bodies formation. We found that RA potently reduces melanoma cell viability and migration after 24 h of treatment. On the other hand, it has no cytotoxic effect on non-tumoral cells. The fluorescence micrographics indicated that RA reduces transmembrane potential of mitochondria and induces apoptotic bodies formation. Moreover, RA significantly decreases intracellular and extracellular ROS levels, and increases the antioxidant defenders NPSH and PSH. A remarkable feature found in our study was that RA strongly upregulates the gene expression of the caspase 8 and caspase 3, and downregulates NLRP3 inflammasome expression. Similar to gene expression, RA greatly increases the enzymatic activity of caspase 3 protein. Taken together, we have shown for the first time that RA reduces cell viability and migration of human metastatic melanoma cells, in addition to modulates apoptosis-related gene expression. We suggest that RA may have the potential to be used in a therapeutic perspective, particularly for CM cell treatment.

Eugenia uniflora L. seed and pulp extracts: phytochemical profile, cytotoxic potential, antitumoral activity, and α-amylase and α-glucosidase inhibition capacity.

In this study, phytochemical profiling, cytotoxic potential, antitumoral activity, and α-amylase and α-glucosidase inhibition capacity of extracts of seed and pulp of Eugenia uniflora L. fruits were investigated. The extracts were obtained using a cellulase complex and the phenolic compounds were quantified. The cytotoxic potential and antitumoral activity were evaluated using peripheral blood mononuclear cells and melanoma-type tumor cells, respectively. The α-amylase and α-glucosidase inhibition capacity was determined. For all extracts, the compounds identified and quantified were salicylic acid, protocatechuic acid, gallic acid and, myricitrin. For extract of pulp, ellagic and p-coumaric acids were also identified and quantified. The extracts do not show cytotoxicity in peripheral blood mononuclear cells. Extract of seed was able to decrease cell viability in melanoma-type tumor cells within 24 h of exposure. The concentration 5 µg mL-1 of extracts inhibited 7.73% of α-amylase and 15.34% of α-glucosidase. The Pitanga extracts presents substances with biological activities.

Phytochemical profiling, antidiabetic, antitumoral and cytotoxic potential of Psidium cattleianum Afzel. ex Sabine leaves of red variety.

In this study, phytochemical profiling, and antidiabetic, antitumoral and cytotoxic potential of aqueous extracts of leaves of red variety of Psidium cattleianum Afzel. ex Sabine were investigated. The extracts were obtained using a cellulase complex. The total phenolic compounds (TPC) were determined, and the individual phenolic compounds were quantified by HPLC-ESI-MS/MS. For the TPC, the amounts varied from 85.91 to 106.33 mg EAG g-1. Eighteen compounds have been identified. The compounds with the highest concentrations were gallic acid, quercetin and protocatechuic acid. Antidiabetic activity was obtained through α-amylase and α-glucosidase inhibition tests. The extract inhibited 17.94% of α-amylase activity and 73.34% of α-glucosidase activity. The antitumoral activity in cells of cutaneous melanoma (SK-MEL-28) and the cytotoxic activity was determined in peripheral blood mononuclear cells (PBMC). The cellular migration was determined for cells SK-MEL-28. Antitumoral effects on cells SK-MEL-28 were observed and the absence of cytotoxicity on the PBMCs.

Deregulation of purinergic ectoenzyme activity in head and neck cancer promotes immunosuppression.

BACKGROUND: Head and neck cancer (HNC) comprises a spectrum of neoplasms that affect the upper aerodigestive tract and are the sixth most common cancers worldwide. Individuals with HNC exhibit various symptoms and metabolic changes, including immune alterations and alterations of the purinergic pathway, which may signal worse outcomes. Therefore, the purpose of this research was to measure the activity of purinergic ectoenzymes and interleukins in patients with HNC, oral cavity cancer, and larynx cancer. METHODS AND RESULTS: We recruited 32 patients and 33 healthy control subjects and performed the laboratory analyses. We identified dysregulation in the purinergic signaling pathway characterized by an increase in adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolysis and a decrease in the deamination of adenosine to inosine in these cancers (p < 0.05). These alterations were likely caused by increased activity of the ectoenzymes E-NTPDase and ecto-5'-nucleotidase and reduced adenosine deaminase activity. This dysregulation was associated with immune alterations, increased levels of IL-10, and decreased myeloperoxidase activity (p < 0.05), suggesting immunosuppression in these patients and suggesting possible accumulation of adenosine in the extracellular environment. CONCLUSIONS: Adenosine is a potent immunosuppressive molecule associated with tumor progression and immune evasion. Our findings suggest a relationship between extracellular purines and the development and progression of the tumor microenvironment and poor outcomes. These findings increase the understanding of biological mechanisms related to HNC and demonstrate that these components are potential diagnostic markers and therapeutic targets for future management strategies and improvement in the quality of life.

High levels of extracellular ATP lead to different inflammatory responses in COVID-19 patients according to the severity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has significantly impacted the world and has driven many researchers into the pathophysiology of COVID-19. In the findings, there is a close association between purinergic signaling and the immune response. Then, this study aimed to evaluate alterations in the purinergic signaling in COVID-19 patients according to range severity. We divided the COVID-19 patients into moderate and severe cases following the guideless of NIH and WHO, together with clinical characteristics. The blood samples were collected to obtain PBMCs and platelets. We analyzed the ectonucleotidase activities through ATP, ADP, AMP, Ado hydrolysis, E-NTPDase1 (CD39), and 5'-NT (CD73) expression by flow cytometry in total leukocytes. The extracellular ATP was measured by bioluminescence, and cytokines were analyzed by flow cytometry. We observed a decrease in ATP hydrolysis and increased AMP hydrolysis in PBMCs for both groups. In severe cases, ATP hydrolysis was raised for the platelets, while ADP and AMP hydrolysis have risen significantly in both groups. Additionally, there was a significant increase in ADP hydrolysis in severe cases compared to moderate cases. In addition, we observed an increase in the ADA activity in platelets of moderate patients. Moderate and severe cases showed increased expression of CD39 and CD73 in total leukocytes. To finalize the purinergic signaling, extracellular ATP was increased in both groups. Furthermore, there was an increase in IL-2, IL-6, IL-10, and IL-17 in moderate and severe groups. Thus, for the first time, our findings confirm the changes in purinergic signaling and immune response in COVID-19, in addition to making it more evident that the severity range directly impacts these changes. Therefore, the therapeutic potential of the purinergic system must be highlighted and studied as a possible target for the treatment of SARS-CoV-2 disease. KEY MESSAGES: COVID-19 patients exhibit alterations in purinergic system and immune response. High levels of extracellular ATP lead to different inflammatory responses. CD39 and CD73 expression were increased in COVID-19 patients. Cytokines IL-2, IL-6, IL-10, and IL-17 also were altered in these patients. The purinergic system may be a possibility target to SARS-CoV-2 treatments.

Supplementation with detox juice added with probiotic improves atherogenic parameters in healthy individuals

Abstract Phytochemicals present in detox juices and probiotics have demonstrated protective effects on cardiovascular risk factors. The consumption of these products alone modulate metabolic mechanisms and biomarkers. However, the effects of the combination of detox juice and probiotics have not yet been evaluated on atherogenic parameters. A randomized controlled study was carried out with 40 healthy volunteers (20 men and 20 women), aged between 18 and 50 years old. The volunteers ingested 200mL of juice for 30 days. Before and after supplementation, the anthropometric and lipid profiles and plasma concentrations of TBARS, Myeloperoxidase, Glutathione, Protein and non-protein Thiols and Vitamin C were analyzed. A reduction in LDL-c (p=0.05), triglycerides (p=0.05) and a significant increase in HDL-c (p=0.002) was observed. There was a significant decrease in the concentrations of TBARS (p=0.01), myeloperoxidases (p=0.02) and a significant increase in the Vitamin C and GSH (p=0.01). There wasn`t improvement in anthropometric parameters and total cholesterol. The findings highlight that supplementation with probiotic detox juice improves the lipid and antioxidant profile, suggesting a possible positive effect in reducing the risk of cardiovascular disease in healthy volunteers. Nevertheless, more robust researches with a prolonged treatment period should be conducted.

Intermittent Fasting before Laparotomy: Effects on Glucose Control and Histopathologic Findings in Diabetic Rats.

(1) Background: Intermittent fasting is a nutrition practice in which individuals fast for several hours in a day, mainly with feeding time during the daylight hours. They seek to improve metabolic performance and cellular resistance to stress. In this study, we tested the fasting protocol to investigate the glycemic effect in a laparotomy perioperative period in diabetic rats and histopathologic findings. (2) Methods: The animals were diabetic-induced with alloxan. Two groups were set according to the feeding protocol: free food and intermittent fasting, whose rats could only eat 8 h in the daylight. Both groups were anesthetized, and a laparotomy was performed. We evaluated the glucose levels during the perioperative period, and we accessed organ histology seeking damage of kidney, bowel and liver after surgical trauma, and we evaluated the wound healing process. (3) Results: Glycemic levels were improved in the intermittent fasting group, especially in the post-operative period after laparotomy. Comparing both groups' tubular damage showed interdependency with mice with worse glycemic level (Z = 2.3; p = 0.0215) and wound-healing parameters showed interdependency with rats with better glycemic status for neovascularization (Z = 2.2; p = 0.0273) and the presence of sebaceous and sweat gland in the healing process (Z = 2.30; p = 0.0215). (4) Conclusions: Intermittent fasting before surgery can be a tool to improve glycemic levels in diabetic rats, with improvement especially in the post-operative period.

Targeting purinergic receptors to suppress the cytokine storm induced by SARS-CoV-2 infection in pulmonary tissue.

The etiological agent of coronavirus disease (COVID-19) is the new member of the Coronaviridae family, a severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2), responsible for the pandemic that is plaguing the world. The single-stranded RNA virus is capable of infecting the respiratory tract, by binding the spike (S) protein on its viral surface to receptors for the angiotensin II-converting enzyme (ACE2), highly expressed in the pulmonary tissue, enabling the interaction of the virus with alveolar epithelial cells promoting endocytosis and replication of viral material. The infection triggers the activation of the immune system, increased purinergic signaling, and the release of cytokines as a defense mechanism, but the response can become exaggerated and prompt the so-called "cytokine storm", developing cases such as severe acute respiratory syndrome (SARS). This is characterized by fever, cough, and difficulty breathing, which can progress to pneumonia, failure of different organs and death. Thus, the present review aims to compile and correlate the mechanisms involved between the immune and purinergic systems with COVID-19, since the modulation of purinergic receptors, such as A2A, A2B, and P2X7 expressed by immune cells, seems to be effective as a promising therapy, to reduce the severity of the disease, as well as aid in the treatment of acute lung diseases and other cases of generalized inflammation.

Viabilidade das células-tronco mesenquimais do tecido adiposo proveniente de lipoaspirado humano / Viability of mesenchymal stem cells of adipose tissue from human liposuction

Introdução: A lipoenxertia é uma alternativa com importante aplicabilidade para reconstrução de mama e/ou correções de assimetrias decorrentes do tratamento oncológico. Esta técnica consiste na transferência de gordura autóloga, cujo estroma contém células-tronco derivadas do tecido adiposo que tem capacidade de diferenciar-se em toda a linhagem mesodermal. Para o preparo do tecido adiposo, Coleman fundamentou a centrifugação, de material aspirado por seringa, em 3000 rotações por minuto (rpm) durante 3 minutos. Contudo, estudos questionam se velocidades menores de centrifugação poderiam ser menos deletérias para viabilidade celular. Métodos: Foi realizado um estudo experimental, onde foram avaliadas as células adiposas de seis pacientes; a partir de 60mL de lipoaspirado de cada um. A amostra coletada foi fracionada em quatro tubos, e submetidos a diferentes protocolos, decantação e centrifugação nas velocidades 500, 1000 e 3000rpm por 3 minutos. Após as amostras foram processadas com colagenase IA por 30 min, submetidas ao cultivo celular por 24 horas e realizado a análise da viabilidade celular. Os resultados foram tabulados e analisados pelo teste ANOVA utilizando os programas Graphpad Prism 6.0® e SAS®. Resultados: A viabilidade celular foi maior na amostra celular centrifugada a 3000rpm e menor na amostra decantada. A coloração com Giemsa indicou manutenção da morfologia celular entre as amostras. Conclusão: As células centrifugadas na velocidade de 3000rpm apresentaram maior viabilidade celular. A centrifugação foi efetiva na compactação do tecido e eliminação de resíduos indesejados (sangue e óleo residual).

Introduction: Lipografting is an alternative with important applicability for breast reconstruction and/or corrections of asymmetries resulting from cancer treatment. This technique consists of autologous fat transfer, whose stroma contains stem cells derived from adipose tissue that can differentiate itself throughout the mesodermal lineage. For adipose tissue preparation, Coleman-based centrifugation of syringe-aspirated material at 3000 revolutions per minute (rpm) for 3 minutes. However, studies question whether lower centrifugation speeds could be less harmful to cell viability. Methods: An experimental study was conducted to evaluate the adipose cells of six patients; from 60mL of liposuction of each one. The sample collected was fractionated into four tubes and submitted to different protocols, decanting and centrifugation at speeds 500, 1000, and 3000rpm for 3 minutes. Afterward, the samples were processed with collagenase IA for 30 min, submitted to cell culture for 24 hours, and a cell viability analysis. The results were tabulated and analyzed by the ANOVA test using the Graphpad Prism 6.0® and SAS®. Results: Cell viability was higher in the cell sample centrifuged at 3000rpm and lower in the decanted sample. Giemsa staining indicated maintenance of cell morphology on the samples. Conclusion: Centrifuged cells at a speed of 3000rpm showed higher cell viability. Centrifugation was effective in compacting tissue and eliminating unwanted waste (blood and residual oil).