Cristina P. R. Xavier

Luteolin, quercetin and ursolic acid are potent inhibitors of proliferation and inducers of apoptosis in both KRAS and BRAF mutated human colorectal cancer cells.

KRAS and BRAF mutations are frequent in colorectal carcinoma (CRC) and have the potential to activate proliferation and survival through MAPK/ERK and/or PI3K signalling pathways. Because diet is one of the most important determinants of CRC incidence and progression, we studied the effects of the dietary phytochemicals quercetin (Q), luteolin (L) and ursolic acid (UA) on cell proliferation and apoptosis in two human CRC derived cell lines, HCT15 and CO115, harboring KRAS and BRAF activating mutations, respectively. In KRAS mutated HCT15 cells, Q and L significantly decreased ERK phosphorylation, whereas in BRAF mutated CO115 cells the three compounds decreased Akt phosphorylation but had no effect on phospho-ERK. Our findings show that these natural compounds have antiproliferative and proapoptotic effects and simultaneously seem to act on KRAS and PI3K but not on BRAF. These results shed light on the molecular mechanisms of action of Q, L and UA and emphasize the potential of dietary choices for the control of CRC progression.

Salvia Fruticosa, Salvia Officinalis, and Rosmarinic Acid Induce Apoptosis and Inhibit Proliferation of Human Colorectal Cell Lines: The Role in MAPK/ERK Pathway

Epidemiological studies have shown that nutrition is a key factor in modulating sporadic colorectal carcinoma (CRC) risk. Aromatic plants of the genus Salvia (sage) have been attributed many medicinal properties, which include anticancer activity. In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signalling pathways. These pathways are commonly altered in CRC, leading to increased proliferation and inhibition of apoptosis. Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.

Quercetin enhances 5-fluorouracil-induced apoptosis in MSI colorectal cancer cells through p53 modulation

PurposeColorectal tumors (CRC) with microsatellite instability (MSI) show resistance to chemotherapy with 5-fluorouracil (5-FU), the most widely used pharmacological drug for CRC treatment. The aims of this study were to test the ability of quercetin (Q) and luteolin (L) to increase the sensitivity of MSI CRC cells to 5-FU and characterize the dependence of the effects on cells’ p53 status.MethodsTwo MSI human CRC-derived cell lines were used: CO115 wild type (wt) for p53 and HCT15 that harbors a p53 mutation. Apoptosis induction in these cells by 5-FU, Q and L alone, and in combinations was evaluated by TUNEL and western blot. The dependence of the effects on p53 was confirmed by small interference RNA (siRNA) in CO115 cells and in MSI HCT116 wt and p53 knockout cells.ResultsCO115 p53-wt cells are more sensitive to 5-FU than the p53-mutated HCT15. The combination treatment of 5-FU with L and Q increased apoptosis with a significant effect for Q in CO115. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The significant apoptotic enhancement in CO115 incubated with Q plus 5-FU involved the activation of the apoptotic mitochondrial pathway. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence of the effect on p53 modulation by Q.ConclusionThis study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in MSI CRC therapy, especially Q in p53 wt tumors.

Ursolic acid induces cell death and modulates autophagy through JNK pathway in apoptosis-resistant colorectal cancer cells

Colorectal carcinomas (CRCs) with P53 mutations have been shown to be resistant to chemotherapy with 5-fluorouracil (5-FU), the most widely used chemotherapeutic drug for CRC treatment. Autophagy is emerging as a promising therapeutic target for drug-resistant tumors. In the present study, we tested the effects of ursolic acid (UA), a natural triterpenoid, on cell death mechanisms and its effects in combination with 5-FU in the HCT15 p53 mutant apoptosis-resistant CRC cell line. The involvement of UA in autophagy and its in vivo efficacy were evaluated. Our data show that UA induces apoptosis independent of caspases in HCT15 cells and enhances 5-FU effects associated with an activation of c-jun N-terminal kinase (JNK). In this cell line, where this compound has a more pronounced effect on the induction of cell death compared to 5-FU, apoptosis corresponds only to a small percentage of the total cell death induced by UA. UA also modulated autophagy by inducing the accumulation of LC3 and p62 levels with involvement of JNK pathway, which indicates a contribution of autophagy on JNK-dependent induction of cell death by UA. By using nude mice xenografted with HCT15 cells, we verified that UA was also active in vivo decreasing tumor growth rate. In conclusion, this study shows UAs anticancer potential both in vitro and in vivo. Induction of cell death and modulation of autophagy in CRC-resistant cells were shown to involve JNK signaling.

Hypericum androsaemum water extract inhibits proliferation in human colorectal cancer cells through effects on MAP kinases and PI3K/Akt pathway

MAP kinase and PI3K/Akt signalling pathways are commonly altered in colorectal carcinoma (CRC) leading to tumor growth due to increased cell proliferation and inhibition of apoptosis. Several species of the genus Hypericum are used in Portugal to prepare herbal teas to which digestive tract effects are attributed. In the present study, the antiproliferative and proapoptotic effects of the water extracts of H. androsaemum (HA) and H. perforatum (HP) were investigated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which harbour activating mutations of KRAS and BRAF, respectively. Contrarily to HP, HA significantly inhibited cell proliferation and induced apoptosis in both cell lines. HA decreased BRAF and phospho-ERK expressions in CO115, but not in HCT15. HA also decreased Akt phosphorylation in CO115 and induced p38 and JNK in both cell lines. HA induced cell cycle arrest at S and G2/M phases as well as caspase-dependent apoptosis in both cell lines. Chlorogenic acid (CA), the main phenolic compound present in the HA extract and less represented in the HP water extract, did, however, not show any of those effects when used individually. In conclusion, water extract of HA, but not of HP, controlled CRC proliferation and specifically acted on mutant and not wild-type BRAF. The effect of HA was, however, not due to CA alone.

The anticancer potential of Mediterranean medicinal plants on colorectal cancer treatment

Scopoletin is a coumarin derivative known for its antioxidant capacity. The present study was undertaken to investigate the blood pressure lowering effect of scopoletin in multiple models of hypertensive rats. To obtain animal models of hypertension, a number of 18 Wistar-Kyoto male rats were divided into two groups receiving a combination of oral prednisone and salt for 14 days (PN group, the endocrine hypertension model) while another half received the same inducers with an additional Nω-nitro-L-arginine methyl ester (L-NAME) for 2 days (PNL group, oxidative stressassociated hypertension model). The rats were anesthetized with propofol and subdivided into 3 groups receiving control, scopoletin 10 mg/kg, and tempol 100 μmol/kg. The systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MAP) and heart rate (HR) were recorded for 120 minutes after a single dose of drug commencement. The serum concentration of nitric oxide (NO) was measured before and at the end of the experiment. The data were analyzed using two-way ANOVA followed by Tukey’s HSD with 95% confidence interval. The results showed that scopoletin decreased the SBP, DBP, and MAP of the rats significantly (p<0.05) without any significant effect on the animal HR (p>0.1). The concentration of NO in animals receiving scopoletin and tempol was greater as compared control, but there was no significant difference in blood NO between both hypertensive models (p>0.1). The study concludes that scopoletin is effective as blood pressure lowering agent on oxidative stress-associated hypertensive rats.

Should the diet of colon cancer patients be personalized based on gene mutation profiles of tumor cells

F many years now, scientists have understood that the onset of breast cancer is a gradual and stepwise process. Chemoprevention researches in recent days are focused on finding substances or components of natural sources that can prevent or inhibit carcinogenesis. Targeted cancer therapies using natural bioactive compounds in combination with chemoprevention drugs are also used to target specific characteristics of cancer cells, such as a gene or protein that allows the cancer cells to grow in a rapid or abnormal way. Therefore, chemoprevention of breast cancer, the attempt to use natural and synthetic compounds to intervene in the early precancerous stages of carcinogenesis before invasion begins, is undertaken as a measure to reduce breast cancer risk for women at high risk. A number of bioactive dietary components are of particular interest in the field of breast cancer. One such compound known as the sub-group of vitamin-E family, the tocotrienols display anticancer properties and may play a role in cancer prevention. To date, there are many studies, which show that tocotrienols can inhibit proliferation of human breast cancer cells in vitro. The inhibitory effects of four isoforms of tocotrienols on the human breast cancer cells appear to be different. The γ and δ-tocotrienols have been shown to have a more potent inhibitory effect on cell growth compared to α and β-tocotrienols. In this study, the effect of tocotrienol isomers (γ and δ) were used to postulate the mechanism of action of these compounds using an in vitro model. The integration of omics methodologies provided insights into functions and the mechanisms of tocotrienols action in breast cancer cellular and molecular environment.Introduction: Type-1 diabetes mellitus patients are at a risk for developing eating disorders because of their intense diet consciousness which is imbibed at an early age. The main objectives of this study were to screen for the risk of developing of eating disorders amongst young adults with and without type-1 diabetes mellitus and study its associations with metabolic control in type-1 diabetes mellitus patients.I recent decades, obesity has become a major health problem. So far, several studies have showed that physical activity is related to creating negative energy balance and changes in appetite directly. However, few of the studies examined the effect of exercise time during the day on these factors. The main aim of the present study was demonstrating the effect of 6 weeks morning and evening aerobic exercise on appetite and anthropometric indices. Forty eight overweight females were recruited in this clinical trial. By the time of exercise, they were divided into two groups (morning or evening) and performed 6 weeks of aerobic exercise with the target heart rate on the ventilatory threshold. Appetite change, calorie intake and anthropometric indices were assessed. All of the variables were compared between two groups and within groups at baseline in the third and sixth week. Some parameters of appetite indices were changed significantly in morning and evening exercise groups in time point’s measurements. However, consistent changes in the appetite scores were not found along the 6 weeks (P>0.05). Calorie consumption of the morning group decreased along the 6 weeks significantly greater than evening group (P=0.02). In addition, significant changes in weight, BMI, abdominal skin fold thickness and abdominal circumference were seen in the morning group (P<0.05). It seems that moderate to high intensity aerobic exercise in the morning along 6 weeks could be considered as a more effective program than evening exercise on appetite control, calorie intake and weight loss.P of chronic health conditions such as diabetes and cardiovascular diseases has not been decreased but rather increased in UK, one of most developed countries in which huge amount of effort have been implemented by government and health care professionals. The statistic possibly indicates that ordinary nutritional instruction such as ‘eat less, move more’ has not worked appropriately, therefore, we may need to turn our perspective toward newly emerging care paradigm such as person centered care in order to provision of quality nutrition care. The person centered care has emerged alongside four themes such as ‘care with dignity, compassion, respect’ ‘coordinated care’ ‘personalized care’ and ‘enable care’. The objective of this review was firstly to elicit gaps alongside those four themes of person centered care from scientific surveys that had conducted inside the UK. Secondly, in order to respond questions such as how to resolve the gaps as well as to clarify health care professional’s roles and responsibilities, recommendations from government and non-government stakeholders were reviewed; as a results of the finding, a few elements including liaison, training and education and awareness should be undertaken by all health care professionals. Doctors should perform specific activities such as detection and solving nutritional problem, whilst nurses should aware their legal limits and consult when required. Dietitians should develop nutritional training packages as well as provide talks and lectures for all health care professionals.G is one of the nutritional factors that involves in developing of obesity and type 2 diabetes in human. The studies indicated that enterocyte cells on intestine might play a role in dietary glucose sensing during obesity. Obese people are consumed high amount of dietary glucose and enterocyte cells consequently are exposed to high glucose. Thus, we aimed to find relevant physiological pathways and genome-wide mRNA expression profiles that can be regulated by glucose in fully differentiated human intestinal epithelial (CaCo-2). The cells were maintained two different glucose levels (5.5 mM for control, 25 mM for high glucose) at least three passages. The cells were grown on transwell system for 21 days to mimic human intestine system. Transepithelial electrical resistances (TEER) were measured to control monolayer formation and polarization. RNA isolation was performed and whole genome mRNA expression profile was determined following gene ontology analysis to find affected molecular pathways. Compared to control relative glucose level was found high in basolateral site of CaCo-2 cells that were under high glucose condition without effecting TEER. GLUT2, SGLT1, GLUT5 mRNA levels were significantly reduced during elevated glucose levels which is consistent with literature. Significant fold change analysis showed that 351 genes up-regulated and 468 genes under high glucose condition. We found high glucose significantly leads changes of molecular pathways (down-regulated; insulin signaling, focal adhesion, inositol phosphate, fructose/mannose, glycolysis and up-regulated; ubiquitin-mediated proteolysis, spliceosome, protein export). These results provide us better understanding and open new window for glucose metabolism of enterocytes during obesity.T close to 1.2 billion people, approximately a fifth of the world’s population continues to live in conditions of abject poverty and almost 800 million people in the developing world are chronically hungry. A basic right to adequate and nutritious food, which most people take for granted, remains a distant dream for those who struggle with food shortages every day of their lives. The presence of extreme poverty and hunger on such a vast scale in a world of apparent opulence is a moral outrage. Evidence-based strategies for nutrition intervention commonly address direct determinants such as food/nutrient intake, whereas community-based intervention tends to take more holistic approach resulting in greater efficacy when combined with other proximal determinants such as women’s economic and social empowerment. We need to address not simply the immediate causes of malnutrition, but also their underlying and basic factors if we are to achieve nutritional well-being and reach full potential for functional and productive capacity in a population. It is thus imperative that food and nutrition programs succeed and that success is sustained. In the case of Bukoba rural, Republic of Tanzania identified direct determinants of undernutrition were lack of availability, accessibility and utilization of food whereas identified proximal determinants were women’s lack of sustainable purchasing power, decision making power, business acumen and horticultural and technical assistance. The proximal determinants imply the lack of women person and social empowerment in their family and community context. These data lead to a cooperative farming project creating a collective way to cultivate soybean crops to improve the quality of the existing diet (direct determinants) and an infrastructure to effect the women’s economic sustainability and empowerment (proximal determinants). The success of ongoing programs suggests when feeding programs and food-based safety nets are supplied from local production, there is a double benefit; not only are the hungry fed but local markets for food expand, opening income-earning and employment opportunities for the poor thereby creating sustainability. The body of evidence clearly suggests that nutrition interventions work best when it is designed with long-term sustainability as an explicit goal.

Ursolic acid, a dietary phytochemical, decreases KRAS signaling and modulates cell death pathways in resistant CRC cells

KRAS mutations are frequent in colorectal cancer (CRC) and have the potential to activate proliferation and inhibit cell death through effects on MAPK/ERK and PI3K/Akt signaling pathways. Because diet is one of the most important determinants of CRC incidence and progression, we studied the effects of the dietary triterpenoid ursolic acid (UA) on proliferation and cell death induction in human CRC derived KRAS mutated cell lines. Our results show that UA decreases cell proliferation and induces cell death while decreasing signaling through KRAS as indicated by a decrease in ERK and Akt phosphorylation (western blot). UA also induced cell death. TP53 mutated cells are known to be resistant to the chemotherapeutic drug 5-FU. Caspase independent apoptosis (Tunel assay), was increased 6 fold by co-incubation of UA with 5-FU. However, apoptosis was only a small percentage of the total cell death induced by UA. In order to explain these observations, we looked into effects on autophagy. Autophagy is emerging as a promising therapeutic target for drug resistant tumors. UA modulated autophagy by inducing the accumulation of LC3 II and p62 levels an effect dependent on JNK activation. In conclusion, this study shows UA’s anticancer potential as a modulator of KRAS signaling and cell death mechanisms increasing sensitivity to the chemotherapeutic drug 5-FU.

Quercetin synergistically induces sensitivity to 5-fluorouracil through p53 modulation in colorectal cancer cells

TUNEL (TdT mediated dUTP Nick End Labelling) staining was performed to estimate the percentage of apoptotic cells of 5-FU alone and 5-FU in combination with Q or L, as well as, z-VAD-fmk (zVAD) alone and zVAD in combination with Q, L and staurosporine (STS). Cells were collected, fixed and cytospined to a polylysine treated slide. Then, cells were permeabilised with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. TUNEL assay was performed following the manufacture’s instructions (Roche). The percentage of apoptotic cells was calculated from the ratio between TUNEL positive cells and total number of cells (nuclei staining with Hoechst) using a fluorescent microscope. • Apoptosis-related-Protein Expression by Western Blotting