Can dietary flavonoids be useful in the personalized treatment of colorectal cancer?

Activating mutations in the oncogenes KRAS, BRAF and PI3K define molecular colorectal cancer (CRC) subtypes because they play key roles in promoting CRC development and in determining the efficacy of chemotherapeutic agents such as 5-fluorouracil and anti-epidermal growth factor receptor monoclonal antibodies. Survival of patients with cancers displaying these molecular profiles is low. Given the limited efficacy of therapeutic strategies for CRC presenting mutational activations in mitogen-activated protein kinase and/or PI3K pathways, developing combination therapies with natural flavonoids or other phytochemicals with demonstrated effects on these pathways (and little or no toxic effects) may constitute a valuable path forward. Much has been published on the anticancer effects of dietary phytochemicals. However, even an exhaustive characterization of potential beneficial effects produced by in vitro studies cannot be extrapolated to effects in humans. So far, the available data constitute a good starting point. Published results show quercetin and curcumin as possibly the best candidates to be further explored in the context of adjuvant CRC therapy either as part of dietary prescriptions or as purified compounds in combination regimens with the drugs currently used in CRC treatment. Clinical trial data is still largely missing and is urgently needed to verify relevant effects and for the development of more personalized treatment approaches.Portuguese Foundation for Science and Technology (FCT), No. UIDB/04469/2020; I&D&I AgriFood XXI, No. NORTE-01-0145-FEDER-000041; and Fundo Europeu de Desenvolvimento Regional (FEDER) through the NORTE 2020 program (Programa Operacional Regional do Norte 2014/2020).info:eu-repo/semantics/publishedVersio

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

Colorectal 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 identify compounds that increase sensitivity of MSI CRC cells to 5-FU and characterize their dependence on the p53 status of the cells. Two MSI human CRC derived cell lines were used: CO115 wildtype for p53 and HCT15 that harbors a p53 mutation. The sensitivity of these cells to 5-FU was evaluated by TUNEL assay and the effects on apoptosis induction of co-incubation of the flavonoids, quercetin (Q) or luteolin (L), with 5-FU were characterized. The mechanisms of apoptosis induction were assessed by western blot and p53 mediated effects confirmed by small interference RNA (siRNA) in CO115 and in HCT116 wt and p53 knockout cells. Our results demonstrate that CO115 is more sensitive to 5-FU than the p53 mutated HCT15. Additive effects on apoptosis were shown for L (in both cell lines) and Q (in HCT15). In CO115 Q synergistically induced apoptosis with 5-FU. Apoptosis induction was caspase dependent in CO115 cells but not in HCT15 cells. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The synergistic effect of Q and 5-FU in CO115 involved the activation of the mitochondrial pathway with an increase in the expression of cleaved caspase 9 and 3 and PARP, as well as a decrease in Bcl-2 expression. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence on p53 modulation of apoptosis induction by Q. This study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in CRC therapy, especially Q in p53 wild-type tumors.Fundação para a Ciência e a Tecnologia (FCT

Drinking of Salvia officinalis tea increases CCl4-induced hepatotoxicity in mice

In a previous study, the drinking of a Salvia officinalis tea (prepared as an infusion) for 14 days improved liver antioxidant status in mice and rats where, among other factors, an enhancement of glutathione-S-transferase (GST) activity was observed. Taking in consideration these effects, in the present study the potential protective effects of sage tea drinking against a situation of hepatotoxicity due to free radical formation, such as that caused by carbon tetrachloride (CCl4), were evaluated in mice of both genders. Contrary to what was expected, sage tea drinking significantly increased the CCl4-induced liver injury, as seen by increased plasma transaminase levels and histology liver damage. In accordance with the previous study, sage tea drinking enhanced significantly GST activity. Additionally, glutathione peroxidase was also significantly increased by sage tea drinking. Since CCl4 toxicity results from its bioactivation mainly by cytochrome P450 (CYP) 2E1, the expression level of this protein was measured by Western Blot. An increase in CYP 2E1 protein was observed which may explain, at least in part, the potentiation of CCl4-induced hepatotoxicity conferred by sage tea drinking. The CCl4-induced hepatotoxicity was higher in females than males. In conclusion, our results indicate that, although sage tea did not have toxic effects of its own, herb–drug interactions are possible and may affect the efficacy and safety of concurrent medical therapy with drugs that are metabolized by phase I enzymes.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/6942/2001, POCI/AGR/62040/200

Protective effects of ursolic acid and luteolin against oxidative DNA damage include enhancement of DNA repair in Caco-2 cells

Consumption of fruits and vegetables is associated with a reduced risk of developing a wide range of cancers including colon cancer. In this study, we evaluated the effects of two compounds present in fruits and vegetables, ursolic acid, a triterpenoid, and luteolin, a flavonoid, on DNA protection and DNA repair in Caco-2 cells using the comet assay. Ursolic acid and luteolin showed a protective effect against H2O2-induced DNA damage. Repair rate (rejoining of strand breaks) after treatment with H2O2 was increased by pre-treatment of Caco-2 cells for 24 h with ursolic acid or luteolin. To evaluate effects on induction of base oxidation, we exposed cells to the photosensitizer Ro 19-8022 plus visible light to induce 8-oxoguanine. Luteolin protected against this damage in Caco-2 cells after a short period of incubation. We also measured the incision activity of a cell extract from Caco-2 cells treated for 24 h with test compounds, on a DNA substrate containing specific damage (8-oxoGua), to evaluate effects on base excision repair activity. Preincubation for 24 h with ursolic acid enhanced incision activity in Caco-2 cells. In conclusion, we demonstrated for the first time that ursolic acid and luteolin not only protect DNA from oxidative damage but also increase repair activity in Caco-2 cells. These effects of ursolic acid and luteolin may contribute to their anti-carcinogenic effects.Fundação para a Ciência e a Tecnologia (FCT

Phenolic compounds protect HepG2 cells from oxidative damage : relevance of glutathione levels

Prova tipográfica (In Press)In the present work, the potential hepatoprotective effects of five phenolic compounds were evaluated against oxidative damages induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells in order to relate in vitro antioxidant activity with cytoprotective effects. t-BHP induced considerable cell damages to HepG2 cells as shown by significant LDH leakage, increased lipid peroxidation, DNA damage as well as decreased reduced glutathione (GSH) levels. All tested phenolic compounds significantly decreased cell death induced by t-BHP (when in co-incubation). If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound’s lipophilicity in addition to its antioxidant potential for its biological activity. All tested phenolic compounds were found to significantly decrease lipid peroxidation and prevent GSH depletion induced by t-BHP, but only luteolin and quercetin significantly decreased DNA damage. Therefore, the lipophilicity of the natural antioxidants tested appeared to be of even higher importance for DNA protection than for cell survival. The protective potential against cell death was probably achieved mainly by preventing intracellular GSH depletion. The phenolic compounds studied here showed protective potential against oxidative damages induced in HepG2 cells that could be beneficial against liver diseases where it is known that oxidative stress plays a crucial role.Fundação para a Ciência e Tecnologia -SFRH/BD/6942/2001; POCTI/AGR/62040/2004

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.CPRX was supported by the Foundation for Science and Technology (FCT), Portugal, through the grant SFRH/BD/27524/2006 and the work was supported by the FCT research grants PTDC/AGR-AAM/70418/2006 (HypericumBiotech) and PEst-C/BIA/UI4050/2011. All projects are co-funded by the program COMPETE from QREN with co-participation from the European Community fund FEDER

Salvia fruticosa, salvia officinalis and rosmarinic acid induce apoptosis and inhibit proliferation of human colorectal cell lines: the role in MAPK/ERK pathway

Epidemiologic 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 pro-apoptotic 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.POCI/AGR/62040/2004. CPRX and CFLSFRH/BD/27524/2006 and SFRH/BPD/26316/2006Foundation for Science and Technology (FCT

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

Publicado em "BMC Proceedings 2012, 6(Suppl 3)"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