Roberto Martín-Hernández

Expression of MicroRNA-15b and the Glycosyltransferase GCNT3 Correlates with Antitumor Efficacy of Rosemary Diterpenes in Colon and Pancreatic Cancer

Colorectal and pancreatic cancers remain important contributors to cancer mortality burden and, therefore, new therapeutic approaches are urgently needed. Rosemary (Rosmarinus officinalis L.) extracts and its components have been reported as natural potent antiproliferative agents against cancer cells. However, to potentially apply rosemary as a complementary approach for cancer therapy, additional information regarding the most effective composition, its antitumor effect in vivo and its main molecular mediators is still needed. In this work, five carnosic acid-rich supercritical rosemary extracts with different chemical compositions have been assayed for their antitumor activity both in vivo (in nude mice) and in vitro against colon and pancreatic cancer cells. We found that the antitumor effect of carnosic acid together with carnosol was higher than the sum of their effects separately, which supports the use of the rosemary extract as a whole. In addition, gene and microRNA expression analyses have been performed to ascertain its antitumor mechanism, revealing that up-regulation of the metabolic-related gene GCNT3 and down-regulation of its potential epigenetic modulator miR-15b correlate with the antitumor effect of rosemary. Moreover, plasmatic miR-15b down-regulation was detected after in vivo treatment with rosemary. Our results support the use of carnosic acid-rich rosemary extract as a complementary approach in colon and pancreatic cancer and indicate that GCNT3 expression may be involved in its antitumor mechanism and that miR-15b might be used as a non-invasive biomarker to monitor rosemary anticancer effect.

The Ellagic Acid Derivative 4,4′-Di-O-Methylellagic Acid Efficiently Inhibits Colon Cancer Cell Growth through a Mechanism Involving WNT16

Ellagic acid (EA) and some derivatives have been reported to inhibit cancer cell proliferation, induce cell cycle arrest, and modulate some important cellular processes related to cancer. This study aimed to identify possible structure-activity relationships of EA and some in vivo derivatives in their antiproliferative effect on both human colon cancer and normal cells, and to compare this activity with that of other polyphenols. Our results showed that 4,4′-di-O-methylellagic acid (4,4′-DiOMEA) was the most effective compound in the inhibition of colon cancer cell proliferation. 4,4′-DiOMEA was 13-fold more effective than other compounds of the same family. In addition, 4,4′-DiOMEA was very active against colon cancer cells resistant to the chemotherapeutic agent 5-fluoracil, whereas no effect was observed in nonmalignant colon cells. Moreover, no correlation between antiproliferative and antioxidant activities was found, further supporting that structure differences might result in dissimilar molecular targets involved in their differential effects. Finally, microarray analysis revealed that 4,4′-DiOMEA modulated Wnt signaling, which might be involved in the potential antitumor action of this compound. Our results suggest that structural-activity differences between EA and 4,4′-DiOMEA might constitute the basis for a new strategy in anticancer drug discovery based on these chemical modifications.

Modulation of cholesterol-related gene expression by dietary fiber fractions from edible mushrooms

Mushrooms are a source of dietary fiber (DF) with a cholesterol-lowering effect. However, their underlying mechanisms are poorly understood. The effect of DF-enriched fractions from three mushrooms species on cholesterol-related expression was studied in vitro. The Pleurotus ostreatus DF fraction (PDF) was used in mice models to assess its potential palliative or preventive effect against hypercholesterolemia. PDF induced a transcriptional response in Caco-2 cells, suggesting a possible cholesterol-lowering effect. In the palliative setting, PDF reduced hepatic triglyceride likely because Dgat1 was downregulated. However, cholesterol-related biochemical data showed no changes and no relation with the observed transcriptional modulation. In the preventive setting, PDF modulated cholesterol-related genes expression in a manner similar to that of simvastatin and ezetimibe in the liver, although no changes in plasma and liver biochemical data were induced. Therefore, PDF may be useful reducing hepatic triglyceride accumulation. Because it induced a molecular response similar to hypocholesterolemic drugs in liver, further dose-dependent studies should be carried out.

Water-Soluble Compounds from Lentinula edodes Influencing the HMG-CoA Reductase Activity and the Expression of Genes Involved in the Cholesterol Metabolism

A water extract from Lentinula edodes (LWE) showed HMG-CoA reductase inhibitory activity but contained no statins. NMR indicated the presence of water-soluble α- and β-glucans and fucomannogalactans. Fractions containing derivatives of these polysaccharides with molecular weight down to approximately 1 kDa still retained their inhibitory activity. Once digested LWE was applied to Caco2 in transport experiments, no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the Caco2 monolayer was applied to HepG2, some genes were modulated (after 24 h). LWE was also administrated to normo- and hypercholesterolemic mice, and no significant lowering of serum cholesterol levels was observed; but reduction of triglycerides in liver was observed. However, LWE supplementation modulated the transcriptional profile of some genes involved in the cholesterol metabolism similarly to simvastatin, suggesting that it could hold potential as a hypolipidemic/hypocholesterolemic extract, although further dose-dependent studies should be carried out.

Plasma Cholesterol-Lowering Activity of Lard Functionalized with Mushroom Extracts Is Independent of Niemann–Pick C1-like 1 Protein and ABC Sterol Transporter Gene Expression in Hypercholesterolemic Mice

Interest in food matrices supplemented with mushrooms as hypocholesterolemic functional foods is increasing. This study was to (i) investigate the hypocholesterolemic activity of lard functionalized with mushroom extracts (LF) including fungal β-glucans, water-soluble polysaccharides, or ergosterol and (ii) examine the LF influence on transcriptional mechanisms involved in cholesterol metabolism. mRNA levels of 17 cholesterol-related genes were evaluated in jejunum, cecum, and liver of high cholesterol-fed mice. The four tested LFs decreased plasma cholesterol by 22-42%, HDLc by 18-40%, and LDLc by 27-51%, and two of them increased mRNA levels of jejunal Npc1l1 and Abcg5 and hepatic Npc1l1. mRNA levels of other cholesterol-related genes were unchanged. These findings suggest that LF may have potential as a dietary supplement for counteracting diet-induced hypercholesterolemia and could be a source for the development of novel cholesterol-lowering functional foods. However, the cholesterol-lowering effect was unrelated to transcriptional changes, suggesting that post-transcriptional mechanisms could be involved.

A Comprehensive In Silico Analysis of the Functional and Structural Impact of Nonsynonymous SNPs in the ABCA1 Transporter Gene

Disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs), which are important indicators of action sites and effective potential therapeutic approaches. Identification of deleterious nsSNPs is crucial to characterize the genetic basis of diseases, assess individual susceptibility to disease, determinate molecular and therapeutic targets, and predict clinical phenotypes. In this study using PolyPhen2 and MutPred in silico algorithms, we analyzed the genetic variations that can alter the expression and function of the ABCA1 gene that causes the allelic disorders familial hypoalphalipoproteinemia and Tangier disease. Predictions were validated with published results from in vitro, in vivo, and human studies. Out of a total of 233 nsSNPs, 80 (34.33%) were found deleterious by both methods. Among these 80 deleterious nsSNPs found, 29 (12.44%) rare variants resulted highly deleterious with a probability >0.8. We have observed that mostly variants with verified functional effect in experimental studies are correctly predicted as damage variants by MutPred and PolyPhen2 tools. Still, the controversial results of experimental approaches correspond to nsSNPs predicted as neutral by both methods, or contradictory predictions are obtained for them. A total of seventeen nsSNPs were predicted as deleterious by PolyPhen2, which resulted neutral by MutPred. Otherwise, forty two nsSNPs were predicted as deleterious by MutPred, which resulted neutral by PolyPhen2.

Targeting the lipid metabolic axis ACSL/SCD in colorectal cancer progression by therapeutic miRNAs: miR-19b-1 role

An abnormal acyl-CoA synthetase/stearoyl-CoA desaturase (ACSL/SCD) lipid network fuels colon cancer progression, endowing cells with invasive and migratory properties. Therapies against this metabolic network may be useful to improve clinical outcomes. Because micro-RNAs (miRNAs/miRs) are important epigenetic regulators, we investigated novel miRNAs targeting this pro-tumorigenic axis; hence to be used as therapeutic or prognostic miRNAs. Thirty-one putative common miRNAs were predicted to simultaneously target the three enzymes comprising the ACSL/SCD network. Target validation by quantitative RT-PCR, Western blotting, and luciferase assays showed miR-544a, miR-142, and miR-19b-1 as major regulators of the metabolic axis, ACSL/SCD. Importantly, lower miR-19b-1 expression was associated with a decreased survival rate in colorectal cancer (CRC) patients, accordingly with ACSL/SCD involvement in patient relapse. Finally, miR-19b-1 regulated the pro-tumorigenic axis, ACSL/SCD, being able to inhibit invasion in colon cancer cells. Because its expression correlated with an increased survival rate in CRC patients, we propose miR-19b-1 as a potential noninvasive biomarker of disease-free survival and a promising therapeutic miRNA in CRC.

Modulation of miRNA expression in aged rat hippocampus by buttermilk and krill oil

The increasing incidence of age-induced cognitive decline justifies the search for complementary ways of prevention or delay. We studied the effects of concentrates of phospholipids, sphingolipids, and/or 3-n fatty acids on the expression of genes or miRNAs related to synaptic activity and/or neurodegeneration, in the hippocampus of aged Wistar rats following a 3-month supplementation. The combination of two phospholipidic concentrates of krill oil (KOC) and buttermilk (BMFC) origin modulated the hippocampal expression of 119 miRNAs (11 were common to both BMFC and BMFC + KOC groups). miR-191a-5p and miR-29a-3p changed significantly only in the BMFC group, whereas miR-195-3p and miR-148a-5p did so only in the combined-supplemented group. Thirty-eight, 58, and 72 differentially expressed genes (DEG) were found in the groups supplemented with KOC, BMFC and BMFC + KOC, respectively. Interaction analysis unveiled networks of selected miRNAs with their potential target genes. DEG found in the KOC and BMFC groups were mainly involved in neuroactive processes, whereas they were associated with lysosomes and mRNA surveillance pathways in the BMFC + KOC group. We also report a significant reduction in hippocampal ceramide levels with BMFC + KOC. Our results encourage additional in-depth investigations regarding the potential beneficial effects of these compounds.

The transcriptional and mutational landscapes of lipid metabolism-related genes in colon cancer

Metabolic alterations encountered in tumors are well recognized and considered as a hallmark of cancer. In addition to Warburg Effect, epidemiological and experimental studies support the crucial role of lipid metabolism in colorectal cancer (CRC). The overexpression of four lipid metabolism-related genes (ABCA1, ACSL1, AGPAT1 and SCD genes) has been proposed as prognostic marker of stage II CRC (ColoLipidGene signature). In order to explore in depth the transcriptomic and genomic scenarios of ABCA1, ACSL1, AGPAT1 and SCD genes, we performed a transcriptomic meta-analysis in more than one thousand CRC individuals. Additionally we analyzed their genomic coding sequence in 95 patients, to find variants that could orchestrate CRC prognosis. We found that genetic variant rs3071, located on SCD gene, defines a 9.77% of stage II CRC patients with high risk of death. Moreover, individuals with upregulation of ABCA1 and AGPAT1 expression have an increased risk of CRC recurrence, independently of tumor stage. ABCA1 emerges as one of the main contributors to signature’s prognostic effect. Indeed, both high ABCA1 expression and presence of tumoral genetic variants located in ABCA1 coding region, seem to be associated with CRC risk of death. In addition the non-synonymous polymorphism rs2230808, located on ABCA1, is associated with gene expression. Patients carrying at least one copy of minor allele showed higher levels of ABCA1 expression than patients carrying homozygous major allele. This study broaden the prognostic value of ABCA1, ACSL1, AGPAT1 and SCD genes, independently of CRC tumor stage, leading to future precision medicine approaches and “omics”-guided therapies.

3’UTR Polymorphism in ACSL1 Gene Correlates with Expression Levels and Poor Clinical Outcome in Colon Cancer Patients

Strong evidence suggests that lipid metabolism (LM) has an essential role in tumor growth to support special energetic and structural requirements of tumor cells. Recently, overexpression of LM-related genes, apolipoproteins related to metabolic syndrome, and ACSL/SCD network involved in fatty acid activation have been proposed as prognostic markers of colon cancer (CC). Furthermore, activation of this latter lipid network has been recently demonstrated to confer invasive and stem cell properties to tumor cells promoting tumor aggressiveness and patient relapse. With the aim of elucidating whether any genetic variation within these genes could influence basal expression levels and consequent susceptibility to relapse, we genotype, in 284 CC patients, 57 polymorphisms located in the 7 genes of these lipid networks previously associated with worse clinical outcome of CC patients (ABCA1, ACSL1, AGPAT1, APOA2, APOC1, APOC2 and SCD), some of them related to CC aggressiveness. After adjusting with clinical confounding factors and multiple comparisons, an association between genotype and disease-free survival (DFS) was shown for rs8086 in 3’-UTR of ACSL1 gene (HR 3.08; 95% CI 1.69–5.63; adjusted p = 0.046). Furthermore, the risk T/T genotype had significantly higher ACSL1 gene expression levels than patients carrying C/T or C/C genotype (means = 5.34; 3.73; 2.37 respectively; p-value (ANOVA) = 0.019), suggesting a functional role of this variant. Thus, we have identified a “risk genotype” of ACSL1 gene that confers constitutive high levels of the enzyme, which is involved in the activation of fatty acids through conversion to acyl-CoA and has been recently related to increased invasiveness of tumor cells. These results suggest that rs8086 of ACSL1 could be a promising prognostic marker in CC patients, reinforcing the relevance of LM in the progression of CC.