RESUMO
We have recently demonstrated that the anthocyanidin delphinidin (DEL), one of the most abundant dietary flavonoids, inhibits activation of ErbB and vascular endothelial growth factor receptor family members. These receptors play crucial roles in the context of tumor progression and the outgrowth of blood and lymphatic vessels. Here, we have developed an improved chemical synthesis for DEL in order to study the effects of the aglycon and its degradation product gallic acid (GA) on endothelial and tumor cells in vitro and in vivo. We found that DEL blocked the proliferation in vitro of primary human blood and lymphatic endothelial cells as well as human HT29 colon and rat MT-450 mammary carcinoma cells in a dose-dependent manner. In contrast, its degradation product GA had little effect. At higher concentrations, DEL induced apoptosis of endothelial and tumor cells. Furthermore, DEL potently blocked the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In the MT-450 rat syngeneic breast tumor model, it also significantly reduced angiogenesis and tumor-induced lymphangiogenesis when administered in vivo. These data reveal DEL to be a novel antilymphangiogenesis reagent. Surprisingly, however, the application of DEL unexpectedly promoted tumor growth and metastasis in the MT-450 tumor model, suggesting that the antiproliferative effect of DEL on cultured cells does not necessarily reflect the response of tumors to this anthocyanidin in vivo. Furthermore, while DEL may have utility as a cancer chemopreventative agent, its ability to promote tumor growth once a neoplasm develops also needs to be taken into consideration.
Assuntos
Antocianinas/farmacologia , Linfangiogênese/efeitos dos fármacos , Metástase Linfática/prevenção & controle , Neoplasias Mamárias Animais/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimioprevenção/métodos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células HT29 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
In cell culture were compared the different release rates of anthocyanins from a bilberry pomace extract encapsulated either in food grade whey protein-based matrix capsules (WPC) or in pectin amid-based hollow spherical capsules (PHS). The impact of the formulations on typical anthocyanin-associated biological end points such as inhibition of the epidermal growth factor receptor (EGFR) and suppression of cell growth in HT29 colon carcinoma cells was assessed. The purpose was to find whether the release rates are sufficient to maintain biological activity and whether encapsulation affected EGFR inhibitory and growth suppressive properties of the extract. Even though anthocyanin release from extract-loaded capsules was proven under cell culture conditions, the inhibitory potential toward the EGFR was diminished. However, nonencapsulated extract as well as both extract-loaded encapsulation systems diminished the growth of HT29 cells to a comparable extent. The loss of EGFR inhibitory properties by encapsulation despite anthocyanin release indicates substantial contribution of other further constituents not monitored so far. Taken together, both applied encapsulation strategies allowed anthocyanin release and maintained biological activity with respect to growth inhibitory properties. However, the loss of EGFR inhibitory effects emphasizes the need for biological profiling to estimate process-induced changes of plant constituent's beneficial potencies.
Assuntos
Antocianinas/análise , Frutas/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Vaccinium myrtillus/química , Cápsulas , Composição de Medicamentos/efeitos adversos , Estabilidade de Medicamentos , Receptores ErbB/antagonistas & inibidores , Inibidores do Crescimento/farmacologia , Células HT29/química , Células HT29/efeitos dos fármacos , Células HT29/patologia , Humanos , Proteínas do Leite , Pectinas , Proteínas do Soro do LeiteRESUMO
Complex polyphenol-rich extracts from apples are known to inhibit the activity of the epidermal growth factor receptor (EGFR) in vitro. The aim of the present study was to identify the bioactive constituents of the apple juice extract which contribute substantially to this potentially chemopreventive effect and to address the question whether the effect is specific to the EGFR or whether other members of the ErbB-receptor family might also be affected. Apple-derived dihydrochalcones and their respective glycosides were found to decrease EGFR activity under cell-free conditions with IC50-values ranging from 0.4 ± 0.1 to 267.0 ± 50.0 µM but showed no activity on human cancer cells. The concentration of quercetin or its glycosides in the extract was too low to contribute substantially to the EGFR-inhibitory properties. In contrast, fractions derived from the apple juice extract comprising ≥86% oligomeric procyanidins (OPCs) suppressed the activity of the EGFR in cell culture with an IC50 â¼ 100 µg mL(-1). In addition, the activity of further members of the ErbB-receptor family was potently inhibited, with ErbB3 receptor activity being most potently decreased (IC50 â¼ 10 µg mL(-1)). From the apple polyphenols identified so far OPCs were found to add the highest contribution to the inhibitory effects towards members of the ErbB-receptor family. Considering the crucial role of the ErbB-receptors in carcinogenesis, these results support the hypothesis that apple-derived OPCs as well as OPC-rich apple preparations might be of interest with respect to chemoprevention.
Assuntos
Bebidas/análise , Malus/química , Proantocianidinas/farmacologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Concentração Inibidora 50 , Fosforilação , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Transdução de SinaisRESUMO
This study investigated Nrf2-activating properties of a coffee blend combining raw coffee bean constituents with 5-O-caffeoylquinic acid (CGA) as a lead component with typical roasting products such as N-methylpyridinium (NMP). In cell culture (HT29) the respective coffee extract (CN-CE) increased nuclear Nrf2 translocation and enhanced the transcription of ARE-dependent genes as exemplified for NAD(P)H:quinone oxidoreductase and glutathione-S-transferase (GST)A1, reflected in the protein level by an increase in GST enzyme activity. In a pilot human intervention study (29 healthy volunteers), daily consumption of 750 mL of CN-coffee for 4 weeks increased Nrf2 transcription in peripheral blood lymphocytes on average. However, the transcriptional response pattern of Nrf2/ARE-dependent genes showed substantial interindividual variations. The presence of SNPs in the Nrf2-promoter, reported recently, as well as the detection of GSTT1*0 (null) genotypes in the study collective strengthens the hypothesis that coffee acts as a modulator of Nrf2-dependent gene response in humans, but genetic polymorphisms play an important role in the individual response pattern.
Assuntos
Ácido Clorogênico/análogos & derivados , Café/química , Fator 2 Relacionado a NF-E2/genética , Compostos de Piridínio/farmacologia , Ácido Quínico/análogos & derivados , Ácido Clorogênico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Células HT29/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/metabolismo , Polimorfismo de Nucleotídeo Único , Transporte Proteico/efeitos dos fármacos , Ácido Quínico/farmacologia , Elementos de RespostaRESUMO
The Nrf2/ARE pathway is a major cellular defense mechanism that prevents damage by reactive oxygen species through induction of antioxidative phase II enzymes. However, the activity of the Nrf2/ARE system is not uniform with variability in response presumed to be dependent on the Nrf2 genotype. We recently completed a pilot human coffee intervention trial with healthy humans, where large interindividual differences in the antioxidative response to the study coffee were examined. Here, we address the question whether differences in the modulation of Nrf2 gene transcription, assessed as an induction of Nrf2 gene transcription by Q-PCR, might be correlated with specific Nrf2 genotypes. To date, nine single nucleotide polymorphisms (SNPs) have been identified in the Nrf2 (NFE2L2) gene. Two of these, the -617C/A and -651G/A SNPs are located within the promoter region and have previously been reported to influence the activity of the Nrf2/ARE pathway by reducing Nrf2 transcriptional activity. Sequencing of the critical Nrf2 gene promoter region not only confirmed the existence of these SNPs within the participants of the trial at the expected frequency (33% carrying the -617C/A, 17% the -651G/A and 56% the -653A/G SNP) but also indicated reduced Nrf2 gene transcription associated with a normal diet if the SNPs at position -617, -651 or -653 were present. Of note, the data also indicated the study coffee increased Nrf2 gene transcription even in SNP carriers. This further highlights the relevance of genotype-dependent induction of Nrf2 gene transcription that appears to be largely influenced by dietary factors.
Assuntos
Café , Fator 2 Relacionado a NF-E2/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Antioxidantes/metabolismo , Sequência de Bases , Cafeína/farmacologia , Dano ao DNA , Frequência do Gene , Estudos de Associação Genética , Genótipo , Glutationa/sangue , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 µM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 µM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 µM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.
Assuntos
Antocianinas/farmacologia , Dano ao DNA/efeitos dos fármacos , Extratos Vegetais/farmacologia , Rosaceae/química , Inibidores da Topoisomerase/farmacologia , Antocianinas/análise , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Frutas/química , Glucosídeos/farmacologia , Células HT29 , Humanos , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/farmacologiaRESUMO
Recently, the coffee constituents 5-O-caffeoylquinic acid (CGA) and N-methylpyridinium (NMP) were identified as inducers of the Nrf2/antioxidant-response element (ARE) detoxifying pathway under cell-culture condition. To study the impact of CGA and NMP on the Nrf2-activating properties of a complex coffee beverage, two different model coffees were generated by variation of the roasting conditions: a low-roast coffee rich in CGA and a heavy-roast low in CGA but containing high levels of NMP. Activation of the Nrf2/antioxidant-response element pathway was monitored in vitro and in vivo.
Assuntos
Antioxidantes/farmacologia , Quimioprevenção , Ácido Clorogênico/farmacologia , Café/química , Fator 2 Relacionado a NF-E2/fisiologia , Compostos de Piridínio/farmacologia , Elementos de Resposta/fisiologia , Indução Enzimática/efeitos dos fármacos , Glutamato-Cisteína Ligase/biossíntese , Células HT29 , Heme Oxigenase-1/biossíntese , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
SCOPE: The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined. METHODS AND RESULTS: The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 µg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 µg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 µg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 µg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 µg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 µg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells. CONCLUSION: These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy.
Assuntos
Antocianinas/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Extratos Vegetais/farmacologia , Camptotecina/farmacologia , Sistema Livre de Células , Neoplasias do Colo/tratamento farmacológico , DNA/efeitos dos fármacos , DNA/metabolismo , Doxorrubicina/farmacologia , Células HT29 , Humanos , Vaccinium myrtillus/químicaRESUMO
Oxidative cellular stress initiates Nrf2 translocation into the nucleus, thus inducing antioxidant response element (ARE)-mediated expression of Phase II enzymes involved in detoxification and antioxidant defence. We investigated whether coffee extracts (CEs) of different proveniences and selected constituents have an impact on the Nrf2/ARE pathway in human colon carcinoma cells (HT29). Assessed as increased nuclear Nrf2 protein, Nrf2 nuclear translocation was modulated by different CEs as observed by Western blot analysis. In addition to the known Nrf2 activator 5-O-caffeoylquinic acid (CGA), pyridinium derivatives like the N-methylpyridinium ion (NMP) were identified as potent activators of Nrf2 nuclear translocation and ARE-dependent gene expression of selected antioxidative Phase II enzymes in HT29. Thereby, the substitution pattern at the pyridinium core structure determined the impact on Nrf2-signalling. In contrast, trigonelline was found to interfere with Nrf2 activation, effectively suppressing the NMP-mediated induction of Nrf2/ARE-dependent gene expression. In conclusion, several coffee constituents, partly already present in the raw material as well as those generated during the roasting process, contribute to the Nrf2-translocating properties of consumer-relevant coffee. A fine tuning in the degradation/formation of activating and deactivating constituents of the Nrf2/ARE pathway during the roasting process appears to be critical for the chemopreventive properties of the final coffee product.
Assuntos
Antioxidantes/farmacologia , Café/química , Expressão Gênica/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Extratos Vegetais/farmacologia , Antineoplásicos/farmacologia , Western Blotting , Ácidos Cafeicos/farmacologia , Núcleo Celular , Ácido Clorogênico/análise , Células HT29 , Humanos , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Transporte Proteico , Compostos de Piridínio/metabolismo , Ácido Quínico/análogos & derivados , Ácido Quínico/farmacologia , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais , Transcrição GênicaRESUMO
PURPOSE: Red grape pomace extract (oenocyanin) is a cheap and rich source of anthocyanins, the agents suggested to possess cancer chemopreventive properties. Here the hypothesis was tested that oenocyanin added to the diet can interfere with intestinal adenoma development in the Apc(Min) mouse, a model of intestinal carcinogenesis linked to an Apc mutation. METHODS: Mice received oenocyanin (0.3%) in their diet until week 16, when adenoma number and burden were recorded. Expression of Akt and ERK proteins was studied by Western blot in adenomas to discover effects of anthocyanins on cellular signalling via the PI3 and MAP kinase pathways. Levels of anthocyanins were measured by HPLC with visible spectroscopic or mass spectrometric detection. RESULTS: In mice which had consumed oenocyanin, overall adenoma burden was halved and adenoma number was marginally reduced when compared with mice on control diet. The proliferation index in colonic adenomatous crypts, as reflected by Ki-67 staining, was significantly decreased from 88.14% in control mice to 75.6+/-4% in mice on oenocyanin (P=0.014). Expression of Akt in small intestinal adenomas from Apc(Min) mice on oenocyanin was reduced by 54% (P=0.003), when compared to controls. Oenocyanin anthocyanins and glucuronide metabolites were found in the urine and intestine but not in plasma. CONCLUSIONS: The results suggest that oenocyanin may be a viable and economical alternative to anthocyanin-rich berry extracts for chemopreventive intervention. Akt and pErk might be suitable biomarkers of anthocyanin target organ efficacy.
Assuntos
Adenoma/prevenção & controle , Antocianinas/análise , Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias Intestinais/prevenção & controle , Fitoterapia/métodos , Vitis/química , Adenoma/metabolismo , Adenoma/patologia , Animais , Antocianinas/farmacocinética , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Delphinidin has been reported to inhibit EGFR signalling. To determine whether other receptor tyrosine kinases (RTKs) are also influenced by delphinidin, we examined its ability to inhibit the kinase activity of EGFR, ErbB2, VEGFR-2, VEGFR-3 and IGF1R in a cell-free test system. We found that delphinidin strongly inhibited the protein tyrosine kinase activity of all tested RTKs at low micromolar concentrations. In A431 and PAE cells, ligand-induced phosphorylation of the receptors was also potently suppressed, with a preference for the suppression of the activity of ErbB3 (IC(50) approximately 100 nM) and VEGFR-3 (IC(50) < 50 microM). Thus the inhibition of RTKs by delphinidin is not limited to cell-free assays but is also of relevance in the cellular context. The results indicate that delphinidin acts as a broad-spectrum inhibitor of RTKs. Given the crucial role of the receptors in tumour growth and metastasis, we conclude that delphinidin has the potential to act directly against tumour cells as well as to interfere with key tumour-host interactions, although the suitability of delphinidin as a drug in cancer management may be compromised by its limited stability. Nevertheless, delphinidin may represent a novel lead compound for the development of chemopreventative and chemotherapeutic intervention strategies.
Assuntos
Antocianinas/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dieta , Receptores ErbB/metabolismo , Flavonoides/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Fenóis/farmacologia , Fosforilação , Polifenóis , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Two standardized anthocyanin-rich mixtures were investigated for their ability to inhibit the receptor tyrosine kinases (RTKs) EGFR, ErbB2, ErbB3, VEGFR-2, and VEGFR-3. Both mixtures reduced the kinase activity of recombinant kinase domains of each RTK at concentrations
Assuntos
Antocianinas/análise , Extratos Vegetais/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Vaccinium myrtillus/química , Vitis/química , Animais , Aorta , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais , Feminino , Frutas/química , Humanos , Fosforilação/efeitos dos fármacos , Extratos Vegetais/química , Suínos , Neoplasias VulvaresRESUMO
Anthocyanins, plant pigments in fruits and berries, have been shown to delay cancer development in rodent models of carcinogenesis, especially those of the colorectal tract. Anthocyanins and anthocyanidins, their aglycons, especially cyanidin and delphinidin, have been subjected to extensive mechanistic studies. In cells in vitro, both glycosides and aglycons engage an array of anti-oncogenic mechanisms including anti-proliferation, induction of apoptosis and inhibition of activities of oncogenic transcription factors and protein tyrosine kinases. Anthocyanins and anthocyanidins exist as four isomers, interconversion between which depends on pH, temperature and access to light. Anthocyanidins are much more prone to avid chemical decomposition than the glycosides, and they only survive for minutes in the biophase. These pharmaceutical issues are very important determinants of the suitability of these flavonoids for potential development as cancer chemopreventive drugs, and they have hitherto not received adequate attention. In the light of their robust cancer chemopreventive efficacy in experimental models and their superior stability as compared to that of the aglycons, the anthocyanins seem much more suitable for further drug development than their anthocyanidin counterparts.
Assuntos
Antocianinas/farmacologia , Anticarcinógenos/farmacologia , Neoplasias/prevenção & controle , Animais , Antocianinas/farmacocinética , Anticarcinógenos/farmacocinética , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos como Assunto , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , HumanosRESUMO
In the present study, delphinidin was found to suppress the phosphorylation of the epidermal growth factor receptor (EGFR) within human tumour cells (human colon carcinoma cell line (HT29), human vulva carcinoma cell line (A431)), albeit less effective than the flavonol quercetin. The higher potency of quercetin was also observed downstream on the level of the mitogen-activated protein kinase (MAPK) cascade. In addition, delphinidin, quercetin and (-)-epigallocatechin-3-gallate (EGCG) were found to suppress the phosphorylation of the ErbB2 receptor, with delphinidin exhibiting the strongest inhibitory properties. Their potency to suppress the ErbB2 receptor phosphorylation can be summarised as delphinidin > EGCG > quercetin. The effectiveness of delphinidin against the EGFR and the ErbB2 receptor was comparable, indicating a broader spectrum of activity against receptor tyrosine kinases. At low micromolar concentrations delphinidin showed some preference towards the ErbB2 receptor. In summary, quercetin and delphinidin appear to differ in their activity profile towards the ErbB receptor family members. Whereas quercetin was most effective against the EGFR, delphinidin exhibited some preference towards the ErbB2 receptor.