Cristovao F. Lima

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.

Evaluation of toxic/protective effects of the essential oil of Salvia officinalis on freshly isolated rat hepatocytes

For this study the essential oil (EO) of sage (Salvia officinalis L.) was isolated from air-dried vegetative aerial parts of the plants by hydrodistillation and analysed by GC and GC-MS. A total yield of 12.07 mg of EO per g of plant dry mass was obtained and more than 50 compounds identified. The major compounds were cis-thujone (17.4%), alpha-humulene (13.3%), 1,8-cineole (12.7%), E-caryophyllene (8.5%) and borneol (8.3%). The EO fraction of sage tea was also isolated by partition with pentane and the respective components identified. The toxic and antioxidant protective effects of S. officinalis EO were evaluated on freshly isolated rat hepatocytes. Cell viability (LDH leakage), lipid peroxidation and glutathione status were measured in experiments undertaken with cells (suspensions of 1 x 10(6) cells per millilitre) exposed to EO alone (toxicity of the EO;t-BHP as positive control); and with cells exposed to EO and an oxidative compound (t-BHP) together (in EO protection evaluation; quercetin as positive control) for 30 min. The results show that the EO is not toxic when present at concentrations below 200 nl/ml; it was only at 2000 nl EO/ml that a significant LDH leakage and GSH decrease were observed indicating cell damage. In the range of concentrations tested, the EO did not show protective effects against t-BHP-induced toxicity.

Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells: Evaluation by the comet assay

In the present study, the chemoprotective effects of quercetin, rutin and ursolic acid on tert-butyl hydroperoxide (t-BHP)-induced DNA damage in a human hepatoma cell line (HepG2) were investigated by the comet assay. To determine whether protection was due to direct chemical interactions alone or to cellular-mediated responses three different types of treatments were used: simultaneous incubation of cells with individual test compounds and the toxicant; pre-treatment with test compound before addition of the toxicant followed or not by a recovery period. The expression of Hsp70 was quantified by Western blotting to test the involvement of heat shock proteins in the cellular responses to the test compounds. In addition, effects on proliferation were evaluated by the MTT assay. The results show that quercetin and ursolic acid prevented DNA damage and had antiproliferative properties in HepG2 cells suggesting an anticarcinogenic potential for these compounds. The protective effects of quercetin against t-BHP-induced DNA damage seem to be due to both direct effects on t-BHP toxicity and to cellularly mediated indirect effects which reflect the potentiation of the cellular antioxidant defenses. Ursolic acid seems to exert effects only through cellularly mediated mechanisms since it was not protective in simultaneous incubation. Quercetin and ursolic acid also showed to increase the rate of DNA repair. Rutin did not have effects at any level. These results, obtained with liver cells, emphasize and confirm the chemopreventive potential of quercetin and ursolic acid, which may help explain the lower cancer incidence in human population with high dietary intakes of fruits and vegetables. These results also demonstrate that Hsp70 is not involved in the observed effects in HepG2.

Heat Stress and Hormetin-Induced Hormesis in Human Cells: Effects on Aging, Wound Healing, Angiogenesis, and Differentiation:

Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of cellular aging. Mild stress-induced hormesis can be an effective way for reducing the accumulation of molecular damage, and thus slowing down aging from within. We have shown that repeated mild heat stress (RMHS) has anti-aging effects on growth and various other cellular and biochemical characteristics of normal human skin fibroblasts and keratinocytes undergoing aging in vitro. RMHS given to human cells increased the basal levels of various chaperones, reduced the accumulation of damaged proteins, stimulated proteasomal activities, increased the cellular resistance to other stresses, enhanced the levels of various antioxidant enzymes, enhanced the activity and amounts of sodium-potassium pump, and increased the phosphorylation-mediated activities of various stress kinases. We have now observed novel hormetic effects of mild heat stress on improving the wound healing capacity of skin fibroblasts and on enhancing the angiogenic ability of endothelial cells. We have also tested potential hormetins, such as curcumin and rosmarinic acid in bringing about their beneficial effects in human cells by inducing stress response pathways involving heat shock proteins and heme-oxygenase HO-1. These data further support the view that mild stress-induced hormesis can be applied for the modulation, intervention and prevention of aging and age-related impairments.

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.

Metformin-like effect of salvia officinalis (common sage) : is it useful in diabetes prevention?

Common sage (Salvia officinalis L.) is among the plants that are claimed to be beneficial to diabetic patients, and previous studies have suggested that some of its extracts have hypoglycaemic effects in normal and diabetic animals. In the present study, we aimed to verify the antidiabetic effects of an infusion (tea) of common sage, which is the most common form of this plant consumed. Replacing water with sage tea for 14 d lowered the fasting plasma glucose level in normal mice but had no effect on glucose clearance in response to an intraperitoneal glucose tolerance test. This indicated effects on gluconeogenesis at the level of the liver. Primary cultures of hepatocytes from healthy, sage-tea-drinking rats showed, after stimulation, a high glucose uptake capacity and decreased gluconeogenesis in response to glucagon. Essential oil from sage further increased hepatocyte sensitivity to insulin and inhibited gluconeogenesis. Overall, these effects resemble those of the pharmaceutical drug metformin, a known inhibitor of gluconeogenesis used in the treatment and prevention of type 2 diabetes mellitus. In primary cultures of rat hepatocytes isolated from streptozotocin (STZ)-induced diabetic rats, none of these activities was observed. The present results seem to indicate that sage tea does not possess antidiabetic effects at this level. However, its effects on fasting glucose levels in normal animals and its metformin-like effects on rat hepatocytes suggest that sage may be useful as a food supplement in the prevention of type 2 diabetes mellitus by lowering the plasma glucose of individuals at risk.

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.

Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

In the present study, two phytochemicals – ursolic acid (UA) and luteolin‐7‐glucoside (L7G) – were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase‐3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low‐density lipoprotein levels, and, in addition, UA increased plasma high‐density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition. Copyright

Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin.

A series of chalcone and flavonol derivatives were synthesized in good yield by an eco-friendly approach. A pharmacological evaluation was performed with the human colorectal carcinoma cell line HCT116 and revealed that the anticancer activity of flavonols was higher when compared with that of the respective chalcone precursors. The antiproliferative activity of halogenated derivatives increases as the substituent in the 3- or 4-positon of the B-ring goes from F to Cl and to Br. In addition, halogens in position 3 enhance anticancer activity in chalcones whereas for flavonol derivatives the best performance was registered for the 4-substituted derivatives. Flow cytometry analysis showed that compounds 3p and 4o induced cell cycle arrest and apoptosis as demonstrated by increased S, G2/M and sub-G1 phases. These data were corroborated by western blot and fluorescence microscopy analysis. In summary, halogenated chalcones and flavonols were successfully prepared and presented high anticancer activity as shown by their cell growth and cell cycle inhibitory potential against HCT116 cells, superior to that of quercetin, used as a positive control.