Cellular and molecular antiproliferative effects in 2D monolayer and 3D-cultivated HT-29 cells treated with zerumbone.

Zerumbone (ZER) is a phytochemical isolated from plants of the Zingiberaceae family. Numerous studies have demonstrated its diverse pharmacological properties, particularly its potent antitumorigenic activity. This study aimed to assess the antiproliferative effects of ZER on HT-29 cells cultivated in both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture systems. The evaluation of growth (size), cell death, and cell cycle arrest in 3D spheroid HT-29 cells was correlated with mRNA expression data. Treatment of 2D cells revealed that ZER exhibited cytotoxicity at concentrations above 30 µM, and an IC50 of 83.54 µM (24-h post-ZER treatment) effectively suppressed cell migration. In the 3D model, ZER induced an increase in spheroid volume over a 72-h period attributed to disaggregation and reconfiguration of characteristic zones. Analysis of cell death demonstrated a significant rise in apoptotic cells after 24 h of ZER treatment, along with cell cycle arrest in the G1 phase. Furthermore, ZER treatment resulted in alterations in mRNA expression, affecting key signaling pathways involved in cell death (BCL2 and BBC3), endoplasmic reticulum stress (ERN1), DNA damage (GADD45A), cell cycle regulation (CDKN1A, NFKB1, MYC, and TP53), and autophagy (BECN1 and SQSTM1). These findings suggested that ZER holds promise as a potential candidate for the development of novel anticancer agents that can modulate crucial cell signaling pathways. Additionally, the use of the 3D culture system proved to be a valuable tool in our investigation.

The prevalence of hyperpalatable food intake among individuals with food addiction seeking bariatric surgery.

INTRODUCTION: The prevalence of food addiction among patients seeking bariatric surgery is approximately 30 %. While hyper-palatable foods (HPF) have been identified as the potential 'substance' in food addiction and a contributor to severe obesity, consumption of HPF among individuals with food addiction, including those seeking bariatric surgery, is unknown. Thus, the aim of this study was to evaluate the consumption of HPF among individuals seeking bariatric surgery with food addiction, compared to those without food addiction. METHODS: Participants were N = 54 individuals with severe obesity seeking bariatric surgery. The Yale Food Addiction Scale was used to identify individuals with food addiction (FA) (37 % of sample). Dietary recalls were used to quantify HPF intake. Analyses were conducted to characterize average HPF intake and to determine whether there were significant differences between HPF intake among those with FA compared to those without FA, and whether HFP intake was correlated with FA symptoms. RESULTS: On average, 71 % of participants' daily calorie intake was from HPF. There were no significant differences in HPF items intake among individuals with and without FA (70.46 % vs 71.34; p = 0.85). A positive correlation between number of FA symptoms and the intake of HPF high in fat and sugar ([0.3]; p = 0.03) was observed. CONCLUSION: In this pilot study, HPF consumption among individuals with and without FA seeking bariatric surgery was high overall, however there were no differences across groups. In addition, intake of HPF with fat and sugar was associated with the number of symptoms of food addiction. More studies with a larger sample are needed to confirm these preliminary findings.

Disruption of caspase-independent cell proliferation pathway on spheroids (HeLa cells) treated with curcumin.

Curcumin is an antiproliferative phytochemical extracted from Curcuma longa L and which has been studied in preclinical drug screening using cell monolayers and animal models. However, several limitations of these culture systems may be overcome by performing screening with three-dimensional (3-D) cell culture. The aim of this study was to investigate the effects of curcumin on cytotoxicity and genotoxicity as well as spheroid growth using cervical adenocarcinoma HeLa cell spheroids by performing RT-PCR mRNA expression of genes involved in cell death (CASP3, CASP8, CASP9, PARP1, BBC3, BIRC5, BCL2, TNF), autophagy (BECN1, SQSTM1), cell cycle regulation (TP53, C-MYC, NF-kB, CDKN1A, m-TOR, TRAF-2), DNA damage repair (H2AFX, GADD45A, GADD45G), oxidative stress (GPX1), reticulum stress (EIF2AK3, ERN1), and invasion (MMP1, MMP9) was investigated. Curcumin was cytotoxic in a concentration-dependent manner. Curcumin-treated spheroids exhibited lower proliferative recovery and cell proliferation attenuation, as observed in the clonogenic assay. Further, no marked genotoxicity was detected. Curcumin-treated spheroids displayed reduced expression of BECN1 (2.9×), CASP9 (2.1×), and PARP1 (2.1×) mRNA. PARP1 inhibition suggested disruption of essential pathways of proliferation maintenance. Downregulated expression of CASP9 mRNA and unchanged expression of CASP3/8 mRNA suggested caspase-independent cell death, whereas downregulated expression of BECN1 mRNA indicated autophagic disruption. Therefore, curcumin exhibits the potential for drug development with antiproliferative activity to be considered for use in cancers.

Differential mRNA expression in the induction of DNA damage, G2/M arrest, and cell death by zerumbone in HepG2/C3A cells.

Zerumbone (ZER) is a phytochemical with antioxidant and antiproliferative properties. This study evaluated the cytoxicity of ZER combined with chemotherapeutic agents and the expression of mRNA genes related to cell cycle, cell death, xenobiotic metabolism, DNA damage, and endoplasmic reticulum (ER) stress in HepG2/C3A cells. ZER was cytotoxic (IC50, 44.31 µM). ZER-induced apoptosis was related to BBC3 and ERN1 upregulation (ER stress), and its antiproliferative effects were attributable to MYC, IGF1, and NF-kB mRNA inhibition. ZER-induced G2/M arrest and DNA damage was associated with mRNA expression of cell cycle (CDKN1A) and DNA damage (GADD45A) genes. Increased CYP1A2 and CYP2C19 mRNA expression suggested ZER metabolization, and reduced CYP1A1 and CYP2D6 expression indicated a longer time of action of ZER in the cell, enhancing its pharmacological effect. ZER downregulated TP53, PARP1, BIRC5 (apoptosis), and MAP1LC3A (autophagy). In apoptosis assay, the data of the association treatments with ZER suggested antagonism. In cytotoxicity assay, the data of the association treatments with ZER suggested synergism action to cisplatin and antagonism action to doxorubicin and 5-fluorouracil. Thus, ZER has potential for application in chemotherapy as it modulates mRNA targets; however, it may not have the desired efficiency when combined with other chemotherapeutic agents.

Vitamin D3 and Salinomycin synergy in MCF-7 cells cause cell death via endoplasmic reticulum stress in monolayer and 3D cell culture.

1α, 25, dihydroxyvitamin D3 (1,25D), the active form of vitamin D3, has antitumor properties in several cancer cell lines in vitro. Salinomycin (Sal) has anticancer activity against cancer cell lines. This study aims to examine the cytotoxic and antiproliferative effect of Sal associated with 1,25D on MCF-7 breast carcinoma cell line cultured in monolayer (2D) and three-dimensional models (mammospheres). We also aim to evaluate the molecular mechanism of Sal and 1,25D-mediated effects. We report that Sal and 1,25D act synergistically in MCF-7 mammospheres and monolayer causing G1 cell cycle arrest, reduction of mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) overproduction with a long-lasting cytotoxic response represented by clonogenic and mammosphere assay. We observed the induction of cell death by apoptosis with upregulation in mRNA levels of apoptosis-related genes (CASP7, CASP9, and BBC3). Extensive cytoplasmic vacuolization, a morphological characteristic found in paraptosis, was also seen and could be triggered by endoplasmic reticulum stress (ER) as we found transcriptional upregulation of genes related to ER stress (ATF6, GADD153, GADD45G, EIF2AK3, and HSPA5). Overall, Sal and 1,25D act synergistically, inhibiting cell proliferation by activating simultaneously multiple death pathways and may be a novel and promising luminal A breast cancer therapy strategy.

Diosgenin increases BBC3 expression in HepG2/C3A cells and alters cell communication in a 3D spheroid model.

Preclinical studies have shown that diosgenin, a steroidal sapogenin, is a promising phytochemical for treating different pathological conditions, such as cancer, diabetes, and cardiovascular diseases. However, the toxicological safety of this molecule for therapeutic use in humans needs to be better understood. Thus, this study aimed to evaluate the mechanisms of action of diosgenin in HepG2/C3A human hepatocellular carcinoma cells. Cytotoxicity, genotoxicity, alterations in the cell cycle, and cell death (apoptosis) were investigated and associated with the gene expression profile of pathways involved in these processes. The effects of diosgenin on the growth of spheroids were also tested. Diosgenin induced a dose-dependent reduction in cell viability and cell cycle arrest in S and G2/M phases and apoptosis in response to DNA damage. Apoptosis was associated with an increase in the expression of BBC3, a participant in the intrinsic apoptosis pathway. Diosgenin also promoted an increase in volume and greater cellular breakdown in spheroids. These results allowed a better understanding of the toxicity of diosgenin in human cells and contributed to the development of treatments based on this phytochemical.

Effect of probiotic supplementation on plasma metabolite profile after Roux-Y gastric bypass: a prospective, randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND/OBJECTIVE: There is evidence that metabolic profile changes after Roux-Y gastric bypass (RYGB), especially due to modifications in the gastrointestinal tract. In addition, previous studies have suggested that probiotics can modify the microbiome and produce metabolites important for metabolic health maintenance. In this sense, the aim of this study was to verify the influence of probiotic supplementation on the plasma metabolite profile after RYGB. METHODS: This was a randomized, double-blind, placebo-controlled clinical trial conducted with 31 patients subjected to RYGB surgery, randomized in probiotic group that was supplemented with a probiotic supplement (FloraVantage®) for 3 months after surgery or a placebo group. Plasma metabonomics was performed using nuclear magnetic resonance (NMR) at the preoperative period (T0) and at 45-50 days (T1) and 90-95 days (T2) during the postoperative period/intervention. RESULTS: Reductions in trimethylamine-N-oxide (TMAO) and alanine were observed in both groups, however this reduction was greater in the probiotic group (TMAO 13.82%, p = 0.01 and alanine 14.03%, p = 0.03) at T2. Additionally, ß-hydroxybutyrate (BHB) levels increased 10.77% in the probiotic group (p = 0.03) compared to the placebo group at T2. CONCLUSION: Supplementation with Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 was able to associate with significant differences in relevant plasma metabolites associated with improved metabolic health.

DNA damage and reticular stress in cytotoxicity and oncotic cell death of MCF-7 cells treated with fluopsin C.

Fluopsin C is an antibiotic compound derived from secondary metabolism of different microorganisms, which possesses antitumor, antibacterial, and antifungal activity. Related to fluopsin C antiproliferative activity, the aim of this study was to examine the following parameters: cytotoxicity, genotoxicity, cell cycle arrest, cell death induction (apoptosis), mitochondrial membrane potential (MMP), colony formation, and mRNA expression of genes involved in adaptive stress responses and cellular death utilizing a monolayer. In addition, a three-dimensional cell culture was used to evaluate the effects on growth of tumor spheroids. Fluopsin C was cytotoxic (1) producing cell division arrest in the G1 phase, (2) elevating expression of mRNA of the CDKN1A gene and (3) decrease in expression of mRNA H2AFX gene. Further, fluopsin C enhanced DNA damage as evidenced by increased expression of mRNA of GADD45A and GPX1 genes, indicating that reactive oxygen species (ROS) may be involved in the observed genotoxic response. Reticulum stress was also detected as noted from activation of the ribonuclease inositol-requiring protein 1 (IRE1) pathway, since a rise in mRNA expression of the ERN1 and TRAF2 genes was observed. During the cell death process, an increase in mRNA expression of the BBC3 gene was noted, indicating participation of this antibiotic in oncotic (ischemic) cell death. Data thus demonstrated for the first time that fluopsin C interferes with the volume of tumor spheroids, in order to attenuate their growth. Our findings show that fluopsin C modulates essential molecular processes in response to stress and cell death.

PROBIOTIC SUPPLEMENTATION ATTENUATES BINGE EATING AND FOOD ADDICTION 1 YEAR AFTER ROUX-EN-Y GASTRIC BYPASS: A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED TRIAL.

AIM: The use of probiotics as adjuvants in the treatment of eating disorders, known as psychobiotics, has already been investigated as a means of modulating the microbiota-gut-brain axis. This study aimed to assess the effect of probiotic supplementation on binge eating and food addiction in subjects after Roux-en-Y gastric bypass surgery. METHODS: This is a randomized, double-blind, placebo-controlled trial involving 101 patients who received probiotic (Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07) or placebo supplements for 90 days after bariatric surgery, starting on the seventh postoperative day. They were evaluated preoperatively (T0) and postoperatively at 90 days (T1) and 1 year (T2) after surgery. The Yale Food Addiction Scale (YFAS) and Binge Eating Scale (BES) were applied to assess food addiction and binge eating, respectively. RESULTS: Before surgery, one-third of the patients presented with a food addiction and binge eating diagnosis. The number of symptoms of YFAS and the BES score decreased significantly in both groups at T1 compared to T0. However, a significant effect of treatment with probiotics was observed 1 year after surgery (T2). Both the number of symptoms of food addiction and the binge eating score were lower in the probiotic group than in the placebo group (p=0.037 and p=0.030, respectively). CONCLUSION: The use of probiotic supplementation for 90 days in the immediate postoperative period may decrease food addiction symptoms and binge eating score up to 1 year after surgery compared to controls.

Probiotic supplementation attenuates binge eating and food addiction 1 year after roux-en-y gastric bypass: a randomized, double-blind, placebo-controlled trial / Suplementação de probióticos atenua compulsão e vício alimentar um ano após cirurgia bariátrica por bypass gástrico em y de roux: um estudo randomizado, duplo-cego, placebo controlado

BACKGROUND: The use of probiotics as adjuvants in the treatment of eating disorders, known as psychobiotics, has already been investigated as a means of modulating the microbiota-gut-brain axis. AIM: This study aimed to assess the effect of probiotic supplementation on binge eating and food addiction in subjects after Roux-en-Y gastric bypass surgery. METHODS: This is a randomized, double-blind, placebo-controlled trial involving 101 patients who received probiotic (Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07) or placebo supplements for 90 days after bariatric surgery, starting on the seventh postoperative day. They were evaluated preoperatively (T0) and postoperatively at 90 days (T1) and 1 year (T2) after surgery. The Yale Food Addiction Scale (YFAS) and Binge Eating Scale (BES) were applied to assess food addiction and binge eating, respectively. RESULTS: Before surgery, one-third of the patients presented with a food addiction and binge eating diagnosis. The number of symptoms of YFAS and the BES score decreased significantly in both groups at T1 compared to T0. However, a significant effect of treatment with probiotics was observed 1 year after surgery (T2). Both the number of symptoms of food addiction and the binge eating score were lower in the probiotic group than in the placebo group (p=0.037 and p=0.030, respectively). CONCLUSION: The use of probiotic supplementation for 90 days in the immediate postoperative period may decrease food addiction symptoms and binge eating score up to 1 year after surgery compared to controls.

RESUMO - RACIONAL: O uso de probióticos como coadjuvantes no tratamento de distúrbios alimentares, conhecidos como psicobióticos, já foi investigado na modulação do eixo intestino-microbiota-cérebro. OBJETIVO: Analisar a influência da suplementação com probióticos no vício e compulsão alimentar em indivíduos submetidos à cirurgia de bypass gástrico em Y-de-Roux MÉTODOS: Trata-se de um estudo randomizado, duplo-cego, controlado por placebo, envolvendo 101 pacientes que receberam suplementação de probiótico (Lactobacillus acidophilus NCFM e Bifidobacterium lactis Bi-07) ou placebo, durante 90 dias após a cirurgia bariátrica, com início no sétimo dia de pós-operatório. Os indivíduos foram avaliados no pré-operatório (T0) e no pós-operatório aos 90 dias (T1) e 1 ano (T2) após a cirurgia. A Escala de Dependência Alimentar de Yale (YFAS) e a Escala de Compulsão Alimentar Periódica (ECAP) foram aplicadas para avaliar o vício e compulsão alimentar, respectivamente. RESULTADOS: Antes da cirurgia, um terço dos pacientes apresentou diagnóstico de dependência alimentar e compulsão alimentar. O número de sintomas da YFAS e a pontuação da ECAP diminuiu significativamente em ambos os grupos em T1 em comparação com T0. Entretanto, um ano após a cirurgia (T2), tanto o número de sintomas de vício alimentar como a pontuação de compulsão alimentar foram menores no grupo probiótico do que no grupo placebo (p = 0,037 e p = 0,030, respectivamente). CONCLUSÃO: A utilização de suplemento probiótico durante 90 dias após a cirurgia pode diminuir os sintomas de vício alimentar e a pontuação de compulsão alimentar um ano após a cirurgia em comparação com o grupo controle.

Effects of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 Supplementation on Nutritional and Metabolic Parameters in the Early Postoperative Period after Roux-en-Y Gastric Bypass: a Randomized, Double-Blind, Placebo-Controlled Trial.

Studies have suggested that Roux-en-Y gastric bypass (RYGB) causes changes in the intestinal microbiota composition and function due to anatomical and physiological modifications. The role of probiotic supplementation after bariatric procedures remains to be determined. PURPOSE: The aim of this study was to investigate the effects of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 supplementation on nutritional and metabolic parameters after RYGB. MATERIALS AND METHODS: This is a randomized, double-blind, placebo-controlled clinical trial. Patients were assigned to receive either a probiotic supplement (FloraVantage®) or placebo for three consecutive months, beginning 7 days after surgery. Anthropometric and biochemical indexes were evaluated in the preoperative period and at the end of the study. RESULTS: Following RYGB, serum 25-OH vitamin D increased in both groups compared to baseline; however, this increase was significant only in the probiotic group (p = 0.004). Vitamin B12 levels tended to be higher in the probiotic group compared to the placebo group (p = 0.063), and triglyceride levels showed a significant reduction in the probiotic group only (p < 0.001). In addition, a significant reduction was observed in the anthropometric parameters and glycemic profile (p < 0.05) in both groups. CONCLUSION: Probiotic supplementation after RYGB improves the vitamin and lipid profile.

RNAm expression profile of cancer marker genes in HepG2 cells treated with different concentrations of a new indolin-3-one from Pseudomonas aeruginosa.

The present study tested the effects of a newly identified indolin-3-one compound (compound 1), produced by Pseudomonas aeruginosa, on HepG2 cells. The MTT assays demonstrated decreased metabolic activities in HepG2 cells treated with compound 1, with dose- and time-dependent intensifying effect, starting at a concentration of 40 µM. The IC50 after 24, 48, 72, and 96 h treatments were 41.35, 52.7, 92.79 and 66.65 µM of compound 1, respectively. Below 80 µM, no significative damage on erythrocytes membranes was observed by the hemolytic assays. The RT-qPCR revealed that the compound modulated key genes involved in carcinogenesis process, indicating possible indolin-3-one mechanisms of action. The data showed that gene expression alterations promoted by compound 1, in concentrations up to 60 µM after 48 h, led to a decrease in cellular progression and there was no direct cellular damage. In addition, non-cytotoxic concentrations of compound 1 halved the concentration of the chemotherapeutic doxorubicin, maintaining similar therapeutic effect against HepG2 cells. The novelty of the molecule and the biological activities observed in the present study emphasize the potential of the compound 1 in cancer therapy research.

Vitamin D: Correlation with biochemical and body composition changes in a southern Brazilian population and induction of cytotoxicity in mesenchymal stem cells derived from human adipose tissue.

Studies have shown that metabolic disorders, serum inflammatory markers and weight gain (obesity) are correlated with vitamin D deficiency. Therefore, the present study correlated the serum calcidiol (s25(OH)D3) levels in a sample of individuals from southern Brazil with variables related to metabolic disorders, obesity and lifestyle habits and assessed the cytotoxic effect of calcitriol on adipose tissue-derived mesenchymal stem cells (ADSCs). The results showed a 79.23% prevalence of hypovitaminosis D in the study population and a correlation (p<0.05) between a low serum vitamin D concentration and an elevated low-density lipoprotein cholesterol (LDL-c) level. Univariate linear regression analysis using 25(OH)D3 as a regressor showed a negative association (p<0.05) with an indoor work environment (ß=-2.305), increased body fat (ß=-0.095), age (ß=-0.065) and high-density lipoprotein cholesterol (HDL-c; ß=-0.109). An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay performed with ADSCs using five calcitriol concentrations (15.625, 31.25, 62.5, 125 and 250nM) indicated cytotoxic potential (p<0.05) at the 62.5nM concentration at 48 and 72h and at the 125 and 250nM concentrations at 24, 48 and 72h. The results reported herein corroborate one another and suggest a key association between vitamin D deficiency and the development of obesity because ADSCs are involved in adipose tissue hyperplasia and differentiate into adipocytes that can sequester the bioavailable vitamin D necessary for homeostasis.