Fernando Salvador Moreno

Targeting the epigenome with bioactive food components for cancer prevention.

Epigenetic processes participate in cancer development and likely influence cancer prevention. Global DNA hypomethylation, gene promoter hypermethylation and aberrant histone post-translational modifications are hallmarks of neoplastic cells which have been associated with genomic instability and altered gene expression. Because epigenetic deregulation occurs early in carcinogenesis and is potentially reversible, intervention strategies targeting the epigenome have been proposed for cancer prevention. Bioactive food components (BFCs) with anticancer potential, including folate, polyphenols, selenium, retinoids, fatty acids, isothiocyanates and allyl compounds, influence DNA methylation and histone modification processes. Such activities have been shown to affect the expression of genes involved in cell proliferation, death and differentiation that are frequently altered in cancer. Although the epigenome represents a promising target for cancer prevention with BFCs, few studies have addressed the influence of dietary components on these mechanisms in vivo, particularly on the phenotype of humans, and thus the exact mechanisms whereby diet mediates an effect on cancer prevention remains unclear. Primary factors that should be elucidated include the effective doses and dose timing of BFCs to attain epigenetic effects. Because diet-epigenome interactions are likely to occur in utero, the impact of early-life nutrition on cancer risk programming should be further investigated.

Associations between glutathione peroxidase-1 Pro198Leu polymorphism, selenium status, and DNA damage levels in obese women after consumption of Brazil nuts

OBJECTIVE Alterations in selenium (Se) status may result in suboptimal amounts of selenoproteins, which have been associated with increased oxidative stress levels. The Pro198Leu polymorphism at the glutathione peroxidase-1 (GPx1) gene is supposed to be functional. The response of Se status, GPx activity, and levels of DNA damage to a Se supplementation trial between the genotypes related to that polymorphism was investigated. METHODS A randomized trial was conducted with 37 morbidly obese women. Participants consumed one Brazil nut, which provided approximately 290 μg of Se a day, for 8 wk. Blood Se concentrations, erythrocyte GPx activity, and DNA damage levels were measured at baseline and at 8 wk. The results were compared by genotypes. RESULTS The genotype frequencies were 0.487, 0.378, and 0.135 for Pro/Pro (the wild-type genotype), Pro/Leu, and Leu/Leu, respectively. At baseline, 100% of the subjects were Se deficient, and after the supplementation, there was an improvement in plasma Se (P < 0.001 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), erythrocyte Se (P = 0.00 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), and GPx activity (P = 0.00 for Pro/Pro, P < 0.00001 for Pro/Leu, P < 0.001 for Leu/Leu). In addition, the Pro/Pro group showed a decrease in DNA damage after Brazil nut consumption compared with baseline (P < 0.005), and those levels were higher in Leu/Leu subjects compared with those with the wild-type genotype (P < 0.05). CONCLUSION Consumption of one unit of Brazil nuts daily effectively increases Se status and increases GPx activity in obese women, regardless of GPx1 Pro198Leu polymorphism. However, the evaluated biomarkers showed distinct results in response to the supplementation when the polymorphism was considered.

Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticarcinogenic agent. However, its short half‐life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of “resistant hepatocyte” model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (p < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB‐induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways.

Chemopreventive effects of β-ionone and geraniol during rat hepatocarcinogenesis promotion: distinct actions on cell proliferation, apoptosis, HMGCoA reductase, and RhoA

Chemopreventive activities of the dietary isoprenoids β-ionone (βI) and geraniol (GOH) were evaluated during the promotion phase of hepatocarcinogenesis. Over 5 consecutive weeks, rats received daily 16 mg/100 g body weight (b.w.) of βI (βI group), 25 mg/100 g b.w. of GOH (GOH group), or only corn oil (CO group, controls). Compared to the CO group, the following was observed: only the βI group showed a decrease in the mean number of visible hepatocyte nodules (P<.05); βI and GOH groups had reduced mean number of persistent preneoplastic lesions (pPNLs) (P<.05), but no differences regarding number of remodeling PNL (rPNLs) were observed; only the βI group exhibited smaller rPNL size and percentage of liver sections occupied by pPNLs (P<.05), whereas the GOH group displayed a smaller percentage of liver sections occupied by rPNLs (P<.05); a trend was observed in the βI group, which showed reduced cell proliferation of pPNLs (P<.10), and the GOH group had increased apoptosis in pPNLs and rPNLs (P<.05); only the βI group displayed reduced total plasma cholesterol concentrations (P<.05) and increased hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase mRNA levels (P<.05); only the GOH group had lower hepatic membrane RhoA protein levels (P<.05); both the βI- and GOH-treated groups had higher hepatic concentrations of βI and GOH, respectively (P<.05). Given these data, βI and GOH show promising chemopreventive effects during promotion of hepatocarcinogenesis by acting through distinct mechanism of actions: βI may inhibit cell proliferation and modulate HMGCoA reductase, and GOH can induce apoptosis and inhibit RhoA activation.

Inhibitory Effects of Lutein and Lycopene on Placental Glutathione S-Transferase-Positive Preneoplastic Lesions and DNA Strand Breakage Induced in Wistar Rats by the Resistant Hepatocyte Model of Hepatocarcinogenesis

Inhibitory effects of lutein (LUT) and lycopene (LYC) on hepatic preneoplastic lesions (PNLs) and DNA strand breakage induced in Wistar rats by the resistant hepatocyte (RH) model of hepatocarcinogenesis were investigated. Animals received by gavage during 8 consecutive weeks on alternate days 70 mg/kg body weight of LUT or LYC. Rats treated with only corn oil and submitted to this model were used as controls. At the end of the experiment, treatment of the animals with LUT or LYC resulted in an increase in the respective liver carotenoid concentrations (P < 0.05). Moreover, it tended to reduce the incidence, total number, and multiplicity of hepatocyte nodules compared with the control group, although the differences did not reach statistical significance. Animals treated with LUT or LYC presented also a lower number of hepatic placental glutathione S-transferase-positive (GST-P) PNLs (P < 0.05), which were smaller (P < 0.05) and occupied a smaller area of the liver section (P < 0.05). Finally, hepatic DNA strand breakage evaluated by the comet assay was lower (P < 0.05) in carotenoid-treated animals when compared with the control group. Therefore, the results indicate that LUT and LYC represent promising chemopreventive agents during hepatocarcinogenesis and whose anticarcinogenic actions could be related to a protection against DNA instability.

Inhibitory effects of β-carotene and vitamin A during the progression phase of hepatocarcinogenesis involve inhibition of cell proliferation but not alterations in DNA methylation

The inhibitory effects of β-Carotene and vitamin A administered to rats in the progression phase of the resistant hepatocyte model of hepatocarcinogenesis were investigated. β-Carotene- and vitamin A-treated animals tended to present with a lower incidence of hepatic cancers than controls at sacrifice. Vitamin A, but not β-Carotene, administration also tended to reduce the total number of persistent hepatocyte nodules. Histological examination of sections stained with hematoxylin and eosin confirmed these results. This suggests that both compounds exhibit inhibitory effects during conversion of persistent nodules to cancers, whereas only the retinoid is also capable of inhibiting the evolution of persistent nodules or causing them to regress. Moreover, β-carotene- and vitamin A-treated animals showed lower hepatic bromodeoxyuridine labeling indexes in neoplastic lesions as well as in adjacent normal tissues than controls, suggesting an inhibitory action of these substances on cell proliferation. However, neither β-carotene nor vitamin A administration resulted in substantial alterations in the CCGG sequence methylation pattern of hydroxymethylglutaryl coenzyme A reductase, c-myc, and c-Ha-ras genes, the products of which are related to cell proliferation and carcinogenesis. Therefore, these inhibitory effects of β-carotene and vitamin A on progression of hepatocarcinogenesis do not seem to be related to DNA methylation.

Natural retinoids and β-carotene: from food to their actions on gene expression

Abstract Vitamin A (retinol) is found in foods of animal origin in the form of retinyl ester, or in fruits and vegetables as carotenoids with provitamin A activity, especially β-carotene. Inside the enterocytes, retinol binds to cellular retinol-binding protein, type II (CRBPII), which directs its esterification by the enzyme lecithin:retinol acyltransferase (LRAT). β-Carotene may undergo central or eccentric cleavage with the formation of retinoids, or may be released into the circulation unchanged. The retinyl esters synthesized, as well as β-carotene that has not undergone cleavage, are incorporated into chylomicrons and taken up mainly by hepatocytes. In the liver, retinol may be stored in stellate cells as retinyl esters, be oxidized to retinoic acid, or be liberated toward target cells bound to retinol-binding protein (RBP). All-trans retinoic acid and its 9-cis isomer have binding affinity for nuclear receptors that activate transcription and that bind as dimers to specific nucleotide sequences present in promoters of target genes. Retinoids also modulate gene expression acting at the posttranscriptional level. The existence of nuclear receptors of β-carotene has not been reported thus far. However, it has been demonstrated that β-carotene can also modulate gene expression. Information about the metabolism of retinoids and of β-carotene has also had important repercussions from a clinical viewpoint, such as the administration of all-trans retinoic acid to patients with acute promyelocytic leukemia.

Effects of selenium compounds on proliferation and epigenetic marks of breast cancer cells

Breast cancer is a global public health problem and the most frequent cause of cancer death among women. Mammary carcinogenesis is driven not only by genetic alterations but also by epigenetic disturbances. Because epigenetic marks are potentially reversible they represent promising molecular targets for breast cancer prevention interventions. Selenium is a promising anti-breast cancer trace element that has shown the modulation of DNA methylation and histone post-translational modifications in other malignancies. This study aimed to evaluate the effects of selenium compounds [methylseleninic acid (MSA) and selenite] on cell proliferation and death, expression of the tumor suppressor gene RASSF1A and epigenetic marks in MCF-7 human breast adenocarcinoma cells. Treatment with MSA or selenite markedly inhibited (P<0.05) in a dose-dependent manner the proliferation of MCF-7 cells. MSA induced (P<0.05) G2/M cell arrest while selenite presented the opposite effect. Regarding cell death induction, MSA acted mainly by inducing apoptosis (P<0.05), while selenite only induced necrosis (P<0.05). Furthermore selenite, but not MSA, markedly induced (P<0.05) cytotoxicity and increased (P<0.05) RASSF1A expression. Both selenium compounds inhibited (P<0.05) DNMT1 expression. MSA decreased (P<0.05) H3K9me3 and increased (P<0.05) H4K16ac, while selenite decreased (P<0.05) this latter histone mark. To the best of our knowledge this is the first report showing that selenite and MSA modulate epigenetic marks specifically in breast cancer cells. Our data reinforce the anti-breast cancer potential of selenium that is dependent on its chemical form. Furthermore the data show that epigenetic mechanisms represent relevant molecular targets involved in selenium inhibitory effects in breast cancer cells.

Exposure to lard-based high-fat diet during fetal and lactation periods modifies breast cancer susceptibility in adulthood in rats

The present study investigated whether early life exposure to high levels of animal fat increases breast cancer risk in adulthood in rats. Dams consumed a lard-based high-fat (HF) diet (60% fat-derived energy) or an AIN93G control diet (16% fat-derived energy) during gestation or gestation and lactation. Their 7-week-old female offspring were exposed to 7,12-dimethyl-benzo[a]anthracene to induce mammary tumors. Pregnant dams consuming an HF diet had higher circulating leptin levels than pregnant control dams. However, compared to the control offspring, significantly lower susceptibility to mammary cancer development was observed in the offspring of dams fed an HF diet during pregnancy (lower tumor incidence, multiplicity and weight), or pregnancy and lactation (lower tumor multiplicity only). Mammary epithelial elongation, cell proliferation (Ki67) and expression of NFκB p65 were significantly lower and p21 expression and global H3K9me3 levels were higher in the mammary glands of rats exposed to an HF lard diet in utero. They also tended to have lower Rank/Rankl ratios (P=.09) and serum progesterone levels (P=.07) than control offspring. In the mammary glands of offspring of dams consuming an HF diet during both pregnancy and lactation, the number of terminal end buds, epithelial elongation and the BCL-2/BAX ratio were significantly lower and serum leptin levels were higher than in the controls. Our data confirm that the breast cancer risk of offspring can be programmed by maternal dietary intake. However, contrary to our expectation, exposure to high levels of lard during early life decreased later susceptibility to breast cancer.

β-Carotene and cancer chemoprevention: From epidemiological associations to cellular mechanisms of action

Numerous studies have been conducted in an attempt to clarify the relationship between β-carotene and cancer chemoprevention, assumed to exist on the basis of an inverse association between fruit and vegetable intake, or blood levels of β-carotene, and cancer risk, suggested in observational epidemiologic studies. The chemopreventive action of the carotenoid has been observed in both in vitro and in vivo experimental models of carcinogenesis. Several cellular and molecular mechanisms have been proposed to explain the anticarcinogenic effect of β-carotene, with emphasis on its metabolic conversion to retinoids, its antioxidant action and, more recently, its eventual modulatory action at the gene expression level. On the other hand, in intervention trials, β-carotene was found to be deleterious to smokers. Some cell mechanisms are indicated here to explain this adverse effect of the carotenoid, causing a probable chronic prooxidant action that potentiates the oxidative state present in the lungs of these individuals during an advanced promotional phase of the carcinogenic process. In this respect, more basic research is needed, especially using an animal model of pulmonary carcinogenesis that will permit to investigate in a distinct manner the effect of the carotenoid on the initiation, promotion and progression steps of carcinogenesis. Furthermore, β-carotene may be effective as a chemopreventive agent against cancer if chronically present during or before the early stages of carcinogenesis, in a non-oxidative cellular environment and in physiological amounts, in combination with antioxidant substances, as it is naturally found in a diet rich in fruits and vegetables.