The aim was to investigate mechanisms contributing to quercetin's previously described effects on cell‐proliferation and ‐differentiation, which contradicted its proposed anticarcinogenic potency. In ...a 10‐day experiment, 40 μM quercetin stabilized by 1 mM ascorbate reduced Caco‐2 differentiation up to 50% (p < 0.001). Caco‐2 RNA from days 5 and 10, hybridized on HG‐U133A2.0 Affymetrix GeneChips®, showed 1743 affected genes on both days (p < 0.01). All 14 Caco‐2 differentiation‐associated genes showed decreased expression (p < 0.01), including intestinal alkaline phosphatase, that was confirmed technically (qRT‐PCR) and functionally (enzyme‐activity). The 1743 genes contributed to 27 pathways (p < 0.05) categorized under six gene ontology (GO) processes, including apoptosis and cell‐cycle. Genes within these GO‐processes showed fold changes that suggest increased cell‐survival and ‐proliferation. Furthermore, quercetin down‐regulated expression of genes involved in tumor‐suppression and phase II metabolism, and up‐regulated oncogenes. Gene expression changes mediated by ascorbate‐stabilized quercetin were concordant with those occurring in human colorectal carcinogenesis (∼︁80–90%), but were opposite to those previously described for Caco‐2 cells exposed to quercetin without ascorbate (∼︁75–90%). In conclusion, gene expression among Caco‐2 cells exposed to ascorbate‐stabilized quercetin showed mechanisms contrary to what is expected for a cancer‐preventive agent. Whether this unexpected in vitro effect is relevant in vivo, remains to be elucidated.
The effect of the flavonoid quercetin and its conjugate rutin was investigated on (biomarkers of) colorectal cancer (CRC). Male F344 rats (n = 42/group) were fed 0, 0.1, 1, or 10 g quercetin/kg diet ...or 40 g rutin/kg diet. Two wk after initial administration of experimental diets, rats were given 2 weekly subcutaneous injections with 15 mg/kg body wt azoxymethane (AOM). At wk 38 post-AOM, quercetin dose dependently (P < 0.05) decreased the tumor incidence, multiplicity, and size, whereas tumor incidences were comparable in control (50%) and rutin (45%) groups. The number of aberrant crypt foci (ACF) in unsectioned colons at wk 8 did not correlate with the tumor incidence at wk 38. Moreover, at wk 8 post-AOM, the number and multiplicity of ACF with or without accumulation of β-catenin were not affected by the 10 g quercetin/kg diet. In contrast, another class of CRC-biomarkers, β-catenin accumulated crypts, contained less β-catenin than in controls (P < 0.05). After enzymatic deconjugation, the plasma concentration of 3′-O-methyl-quercetin and quercetin at wk 8 was inversely correlated with the tumor incidence at wk 38 (r = −0.95, P ≤ 0.05). Rats supplemented with 40 g rutin/kg diet had only 30% of the (3′-O-methyl-) quercetin concentration of 10 g quercetin/kg diet-fed rats (P < 0.001). In conclusion, quercetin, but not rutin, at a high dose reduced colorectal carcinogenesis in AOM-treated rats, which was not reflected by changes in ACF-parameters. The lack of protection by rutin is probably due to its low bioavailability.
Quercetin is a dietary polyphenolic compound with potentially beneficial effects on health. Claims that quercetin has biological effects are based mainly on in vitro studies with quercetin aglycone. ...However, quercetin is rapidly metabolized, and we have little knowledge of its availability to tissues. To assess the long-term tissue distribution of quercetin, 2 groups of rats were given a 0.1 or 1% quercetin diet ~50 or 500 mg/kg body weight (wt) for 11 wk. In addition, a 3-d study was done with pigs fed a diet containing 500 mg quercetin/kg body wt. Tissue concentrations of quercetin and quercetin metabolites were analyzed with an optimized extraction method. Quercetin and quercetin metabolites were widely distributed in rat tissues, with the highest concentrations in lungs (3.98 and 15.3 nmol/g tissue for the 0.1 and 1% quercetin diet, respectively) and the lowest in brain, white fat, and spleen. In the short-term pig study, liver (5.87 nmol/g tissue) and kidney (2.51 nmol/g tissue) contained high concentrations of quercetin and quercetin metabolites, whereas brain, heart, and spleen had low concentrations. These studies have for the first time identified target tissues of quercetin, which may help to understand its mechanisms of action in vivo.
Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific ...methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have
BRAF
p.V600E
mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with
BRAF
p.V600E
mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing
BRAF
p.V600E
with
BRAF
wild type revealed
MEIS1
as the most significant differentially methylated gene (log
2
fold change: 0.89, false discovery rate-adjusted
P
-value 2.8*10
-9
). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (
n
=228) showed a significant association between
BRAF
p.V600E
and
MEIS1
methylation (OR: 13.0, 95% CI: 5.2 - 33.0,
P
<0.0001).
MEIS1
methylation was associated with decreased
MEIS1
gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of
MEIS1
,
MEIS1
D27
, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of
MEIS1
promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight
BRAF
p.V600E
tumor epithelial fractions (50%) showed
MEIS1
promoter methylation, as well as three out of eight
BRAF
p.V600E
stromal fractions (38%). Only one out of six
BRAF
wild type showed
MEIS1
promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion,
BRAF
p.V600E
colon tumors showed significant
MEIS1
promoter methylation, which was associated with decreased
MEIS1
gene expression.
Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific ...methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAFp.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAFp.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAFp.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAFp.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAFp.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAFp.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAFp.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.
Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., ...and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328–13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of α-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human α-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp29-Glu, Asp-Ile-Val-Asp208-Arg, and Asp-Asp-Ser-Asp633-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp633-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of α-adducin and γ-adducin in the MARCKS domain that preceded α-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of α-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of α-adducin at Asp-Asp-Ser-Asp633-Ala.
Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers
and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., ...and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328â13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal
relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential
cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis
of renal proximal tubular epithelial cells, which was associated with the cleavage of α-adducin into a 74-kDa fragment; this
was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged
human α-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp 29 -Glu, Asp-Ile-Val-Asp 208 -Arg, and Asp-Asp-Ser-Asp 633 -Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp 633 -Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of α-adducin and
γ-adducin in the MARCKS domain that preceded α-adducin cleavage and was associated with loss of adducins from adherens junctions;
this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of α-adducin
due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage
of α-adducin at Asp-Asp-Ser-Asp 633 -Ala.
PDF
