Synergistic Effect of a Flavonoid-Rich Cocoa-Carob Blend and Metformin in Preserving Pancreatic Beta Cells in Zucker Diabetic Fatty Rats.

The loss of functional beta-cell mass in diabetes is directly linked to the development of diabetic complications. Although dietary flavonoids have demonstrated antidiabetic properties, their potential effects on pancreatic beta-cell preservation and their synergistic benefits with antidiabetic drugs remain underexplored. We have developed a potential functional food enriched in flavonoids by combining cocoa powder and carob flour (CCB), which has shown antidiabetic effects. Here, we investigated the ability of the CCB, alone or in combination with metformin, to preserve pancreatic beta cells in an established diabetic context and their potential synergistic effect. Zucker diabetic fatty rats (ZDF) were fed a CCB-rich diet or a control diet, with or without metformin, for 12 weeks. Markers of pancreatic oxidative stress and inflammation, as well as relative beta-cell mass and beta-cell apoptosis, were analyzed. Results demonstrated that CCB feeding counteracted pancreatic oxidative stress by enhancing the antioxidant defense and reducing reactive oxygen species. Moreover, the CCB suppressed islet inflammation by preventing macrophage infiltration into islets and overproduction of pro-inflammatory cytokines, along with the inactivation of nuclear factor kappa B (NFκB). As a result, the CCB supplementation prevented beta-cell apoptosis and the loss of beta cells in ZDF diabetic animals. The observed additive effect when combining the CCB with metformin underscores its potential as an adjuvant therapy to delay the progression of type 2 diabetes.

Maternal Diet Determines Milk Microbiome Composition and Offspring Gut Colonization in Wistar Rats.

Mother's milk contains a unique microbiome that plays a relevant role in offspring health. We hypothesize that maternal malnutrition during lactation might impact the microbial composition of milk and affect adequate offspring gut colonization, increasing the risk for later onset diseases. Then, Wistar rats were fed ad libitum (Control, C) food restriction (Undernourished, U) during gestation and lactation. After birth, offspring feces and milk stomach content were collected at lactating day (L)4, L14 and L18. The V3-V4 region of the bacterial 16S rRNA gene was sequenced to characterize bacterial communities. An analysis of beta diversity revealed significant disparities in microbial composition between groups of diet at L4 and L18 in both milk, and fecal samples. In total, 24 phyla were identified in milk and 18 were identified in feces, with Firmicutes, Proteobacteria, Actinobacteroidota and Bacteroidota collectively representing 96.1% and 97.4% of those identified, respectively. A higher abundance of Pasteurellaceae and Porphyromonas at L4, and of Gemella and Enterococcus at L18 were registered in milk samples from the U group. Lactobacillus was also significantly more abundant in fecal samples of the U group at L4. These microbial changes compromised the number and variety of milk-feces or feces-feces bacterial correlations. Moreover, increased offspring gut permeability and an altered expression of goblet cell markers TFF3 and KLF3 were observed in U pups. Our results suggest that altered microbial communication between mother and offspring through breastfeeding may explain, in part, the detrimental consequences of maternal malnutrition on offspring programming.

Obesity and telomere status in the prognosis of patients with colorectal cancer submitted to curative intention surgical treatment.

The risk of colorectal cancer (CRC) development has been associated with telomere dysfunction and obesity. However, clinical relevance of these parameters in CRC prognosis is not clear. Therefore, the aim of the present study was to evaluate the impact of obesity and telomere status in the prognosis of patients affected by CRC and submitted to curative surgical treatment. According to published data, this is the first work in which obesity and telomere status are jointly considered in relation to CRC prognosis. A prospective study including 162 patients with CRC submitted to curative surgical treatment was performed. Subjects were classified according to their BMI. Telomere status was established through telomere length and telomerase activity evaluation. Statistical analyses were performed using the SPSS software package version 22. Telomere shortening was inversely associated with BMI in patients with CRC. Notably, among patients with CRC, subjects with obesity exhibited less shortening of tumor telomeres than non-obese patients (P=0.047). Patients with shorter telomeres, both in the tumor (median telomere length <6.5 kb) and their non-tumor paired tissues (median telomere length <7.1 kb), had the best clinical evolution, regardless of the Dukes' stage of cancers (P=0.025, for tumor samples; P=0.003, for non-tumor samples). Additionally, subjects with a BMI >31.85 kg/m2 showed the worse clinical outcomes compared with subjects with other BMI values. Interestingly, the impact of BMI showed sex dependence, since only the group of men displayed significant differences in CRC prognosis in relation to obesity status (P=0.037). From the results of the present study, based on a multivariate prediction model to establish prognosis, it was concluded that telomere length is a useful biomarker to predict prognosis in patients with CRC. Regardless of BMI values, the improved clinical evolution was associated with shorter telomeres. The impact of BMI seems to be associated with other factors, such as sex.

Defective liver glycogen autophagy related to hyperinsulinemia in intrauterine growth-restricted newborn wistar rats.

Maternal malnutrition plays a critical role in the developmental programming of later metabolic diseases susceptibility in the offspring, such as obesity and type 2 diabetes. Because the liver is the major organ that produces and supplies blood glucose, we aimed at defining the potential role of liver glycogen autophagy in the programming of glucose metabolism disturbances. To this end, newborns were obtained from pregnant Wistar rats fed ad libitum with a standard diet or 65% food-restricted during the last week of gestation. We found that newborns from undernourished mothers showed markedly high basal insulin levels whereas those of glucagon were decreased. This unbalance led to activation of the mTORC1 pathway and inhibition of hepatic autophagy compromising the adequate handling of glycogen in the very early hours of extrauterine life. Restoration of autophagy with rapamycin but not with glucagon, indicated no defect in autophagy machinery per se, but in signals triggered by glucagon. Taken together, these results support the notion that hyperinsulinemia is an important mechanism by which mobilization of liver glycogen by autophagy is defective in food-restricted animals. This early alteration in the hormonal control of liver glycogen autophagy may influence the risk of developing metabolic diseases later in life.

Antitumor Activity of Quinazolinone Alkaloids Inspired by Marine Natural Products.

Many fungal quinazolinone metabolites, which contain the methyl-indole pyrazino [1,2-b]quinazoline-3,6-dione core, have been found to possess promising antitumor activity. The purpose of this work was to synthesize the enantiomeric pairs of two members of this quinazolinone family, to explore their potential as antitumor and their ability to revert multidrug resistance. The marine natural product fiscalin B (4c), and antienantiomers (4b, 5b, and 5c) were synthesized via a one-pot approach, while the syn enantiomers (4a, 4d, 5a, and 5d) were synthetized by a multi-step procedure. These strategies used anthranilic acid (i), chiral N-protected α-amino acids (ii), and tryptophan methyl esters (iii) to form the core ring of pyrazino[2,1-b]quinazoline-3,6-dione scaffold. Four enantiomeric pairs, with different enantiomeric purities, were obtained with overall yields ranging from 7 to 40%. Compounds 4a⁻d and 5a⁻d were evaluated for their growth inhibitory effect against two tumor cell lines. Differences between enantiomeric pairs were noted and 5a⁻d displayed GI50 values ranging from 31 to 52 µM, which are lower than those of 4a⁻d. Nevertheless, no effect on P-glycoprotein (P-gp) modulation was observed for all compounds. This study disclosed new data for fiscalin B (4c), as well as for its analogues for a future development of novel anticancer drug leads.

Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer.

The role of telomeres and telomerase in colorectal cancer (CRC) is well established as the major driving force in generating chromosomal instability. However, their potential as prognostic markers remains unclear. We investigated the outcome implications of telomeres and telomerase in this tumour type. We considered telomere length (TL), ratio of telomere length in cancer to non-cancer tissue (T/N ratio), telomerase activity and TERT levels; their relation with clinical variables and their role as prognostic markers. We analyzed 132 CRCs and paired non-cancer tissues. Kaplan-Meier curves for disease-free survival were calculated for TL, T/N ratio, telomerase activity and TERT levels. Overall, tumours had shorter telomeres than non-tumour tissues (P < 0.001) and more than 80% of CRCs displayed telomerase activity. Telomere lengths of non-tumour tissues and CRCs were positively correlated (P < 0.001). Considering telomere status and clinical variables, the lowest degree of telomere shortening was shown by tumours located in the rectum (P = 0.021). Regarding prognosis studies, patients with tumours showing a mean TL < 6.35 Kb experienced a significantly better clinical evolution (P < 0.001) and none of them with the highest degree of tumour telomere shortening relapsed during the follow-up period (P = 0.043). The mean TL in CRCs emerged as an independent prognostic factor in the Cox analysis (P = 0.017). Telomerase-positive activity was identified as a marker that confers a trend toward a poor prognosis. In CRC, our results support the use of telomere status as an independent prognostic factor. Telomere status may contribute to explaining the different molecular identities of this tumour type.

Telomere length and telomerase activity in non-small cell lung cancer prognosis: clinical usefulness of a specific telomere status.

BACKGROUND: Considering previous data and the need to incorporate new biomarkers for the prognosis of solid tumours into the clinic, our aim in this work consists of evaluating the potential clinical use of telomeres and telomerase in non-small cell lung cancer (NSCLC). METHODS: Telomere status was established by determination of telomere length using the Terminal Restriction Fragment length method, and telomerase activity by the Telomeric Repeat Amplification Protocol in 142 NSCLCs and their corresponding control samples, obtained from patients submitted to surgery. Group-oriented curves for disease-free survival were calculated according to the Kaplan-Meier method considering telomere length, T/N ratio (telomere length in tumour to control tissue) and telomerase activity. RESULTS: Overall, tumours had significantly shorter telomeres compared with telomeres in control tissues (P = 0.027). More than 80 % of NSCLCs displayed telomerase activity. Regarding prognosis studies, patients whose tumours showed a mean telomere length (MTL) <7.29 Kb or T/N ratio <0.97 showed a significantly poor clinical evolution (P = 0.034 and P = 0.040, respectively). As result of a Cox multivariate analysis including pathologic state and lymph node dissemination, the MTL and T/N ratio emerged as independent significant prognostic factors. CONCLUSIONS: Telomerase activity was identified as a marker of poor prognosis. The novel finding of this study is the independent prognosis role of a specific telomere status in NSCLC patients. According to our results, telomere function may emerge as a useful molecular tool that allow to select groups of NSCLC patients with different clinical evolution, in order to establish personalized therapy protocols.

Prognostic value of telomere function in gastric cancers with and without microsatellite instability.

OBJECTIVE: To identify molecular markers that may be useful in the selection of gastric cancer patients with different prognoses, we investigated telomere function in gastric cancers with and without microsatellite instability (MSI). MATERIALS AND METHODS: We analyzed 83 gastric cancers and its paired-normal tissues to investigate MSI and telomere function. MSI was established using five polymorphic human repeat DNA markers. Telomere function was evaluated by determining telomerase activity, telomere length, and telomere-repeat factors 1 and 2 (TRF1 and TRF2) expression. RESULTS: Patients with high microsatellite instability (MSI-H) gastric cancers showed a significantly better prognosis than those affected by microsatellite stable or low microsatellite instability (MSS/MSI-L) tumors (P = 0.03). The lowest expression levels of TRF1 and TRF2 were associated with MSI-H gastric cancers (P = 0.008 and 0.006, respectively). Moreover, a clear trend toward a worse prognosis was found in the group of patients who had tumors with the shortest telomeres (P = 0.01). Cox multivariate analysis showed that MSI emerged as a protective prognostic factor; MSS/MSI-L tumors conferred a significantly poor prognosis in patients (relative risk = 4.862-fold greater than the MSI-H group) (P = 0.033). Telomere length of gastric tumors less than 2.86 kbp was a factor that led to a poor prognosis (relative risk = 4.420, with respect to tumors showing telomere length ≥ 2.86 kbp) (P = 0.002). CONCLUSION: We propose telomere status as a potential molecular marker with usefulness in the establishment of the prognosis of gastric cancers both for the mutator phenotype and for the suppressor pathway.

B-ring-aryl substituted luotonin A analogues with a new binding mode to the topoisomerase 1-DNA complex show enhanced cytotoxic activity.

Topoisomerase 1 inhibition is an important strategy in targeted cancer chemotherapy. The drugs currently in use acting on this enzyme belong to the family of the camptothecins, and suffer severe limitations because of their low stability, which is associated with the hydrolysis of the δ-lactone moiety in their E ring. Luotonin A is a natural camptothecin analogue that lacks this functional group and therefore shows a much-improved stability, but at the cost of a lower activity. Therefore, the development of luotonin A analogues with an increased potency is important for progress in this area. In the present paper, a small library of luotonin A analogues modified at their A and B rings was generated by cerium(IV) ammonium nitrate-catalyzed Friedländer reactions. All analogues showed an activity similar or higher than the natural luotonin A in terms of topoisomerase 1 inhibition and some compounds had an activity comparable to that of camptothecin. Furthermore, most compounds showed a better activity than luotonin A in cell cytotoxicity assays. In order to rationalize these results, the first docking studies of luotonin-topoisomerase 1-DNA ternary complexes were undertaken. Most compounds bound in a manner similar to luotonin A and to standard topoisomerase poisons such as topotecan but, interestingly, the two most promising analogues, bearing a 3,5-dimethylphenyl substituent at ring B, docked in a different orientation. This binding mode allows the hydrophobic moiety to be shielded from the aqueous environment by being buried between the deoxyribose belonging to the G(+1) guanine and Arg364 in the scissile strand and the surface of the protein and a hydrogen bond between the D-ring carbonyl and the basic amino acid. The discovery of this new binding mode and its associated higher inhibitory potency is a significant advance in the design of new topoisomerase 1 inhibitors.

Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity.

BACKGROUND: Considering previous result in Non-Small Cell Lung Cancer (NSCLC), we investigated in human cancer cells the role of PARP3 in the regulation of telomerase activity. METHODS: We selected A549 (lung adenocarcinoma cell line) and Saos-2 (osteosarcoma cell line), with high and low telomerase activity levels, respectively. The first one was transfected using a plasmid construction containing a PARP3 sequence, whereas the Saos-2 cells were submitted to shRNA transfection to get PARP3 depletion. PARP3 expression on both cell systems was evaluated by real-time quantitative PCR and PARP3 protein levels, by Western-blot. Telomerase activity was determined by TRAP assay. RESULTS: In A549 cells, after PARP3 transient transfection, data obtained indicated that twenty-four hours after transfection, up to 100-fold increased gene expression levels were found in the transfected cells with pcDNA/GW-53/PARP3 in comparison to transfected cells with the empty vector. Moreover, 48 hours post-transfection, telomerase activity decreased around 33%, and around 27%, 96 hours post-transfection. Telomerase activity average ratio was 0.67 ± 0.05, and 0.73 ± 0.06, respectively, with significant differences. In Saos-2 cells, after shRNA-mediated PARP3 silencing, a 2.3-fold increase in telomerase activity was detected in relation to the control. CONCLUSION: Our data indicated that, at least in some cancer cells, repression of PARP3 could be responsible for an increased telomerase activity, this fact contributing to telomere maintenance and, therefore, avoiding genome instability.

Differential expression of senescence and cell death factors in non-small cell lung and colorectal tumors showing telomere attrition.

OBJECTIVE: The main aim of this work is to investigate the expression of factors related to senescence and cell death pathways in non-small cell lung cancers (NSCLCs) and colorectal cancers (CRCs) in relation to telomere status. METHODS: We analyzed 158 tissue samples, 36 NSCLCs, 43 CRCs, and their corresponding control tissues obtained from patients submitted to surgery. Telomere function was evaluated by determining telomerase activity and telomere length. Expression of factors related to senescence, cell death pathways, transformation and tumorigenesis was investigated using arrays. Results were validated by real-time quantitative PCR. RESULTS: Considering tumors with telomere shortening, expression for BNIP3, DAPK1, NDRG1, EGFR, and CDKN2A was significantly higher in NSCLC than in CRC, whereas TP53 was overexpressed in CRC with respect to NSCLC. Moreover, compared to nontumor samples, DAPK1, GADD45A, SHC1, and TP53 were downregulated in the group of NSCLCs with telomere shortening, and no significant differences were found in CRC. CONCLUSIONS: In NSCLC, the failure of pathways which involve factors such as DAPK1, GADD45A, SHC1, and TP53, in response to short telomeres, could promote tumor progression. In CRC, the viability of these pathways in response to short telomeres could contribute to limiting tumorigenesis.

Methylation profiling in non-small cell lung cancer: clinical implications.

The aim of this study was to identify a panel of methylation markers that distinguish non-small cell lung cancers (NSCLCs) from normal lung tissues. We also studied the relation of the methylation profile to clinicopathological factors in NSCLC. We collected a series of 46 NSCLC samples and their corresponding control tissues and analyzed them to determine gene methylation status using the Illumina GoldenGate Methylation bead array, which screens up to 1505 CpG sites from 803 different genes. We found that 120 CpG sites, corresponding to 88 genes were hypermethylated in tumor samples and only 17 CpG sites (16 genes) were hypomethylated when compared with controls. Clustering analysis of these 104 genes discriminates almost perfectly between tumors and normal samples. Global hypermethylation was significantly associated with a worse prognosis in stage IIIA NSCLC patients (P=0.012). Moreover, hypermethylation of the CALCA and MMP-2 genes were statistically associated to a poor clinical evolution of patients, independently of TNM tumor stage (P=0.06, RR=2.64; P=0.04, RR=2.96, respectively). However, hypermethylation of RASSF1 turned out to be a protective variable (P=0.02; RR=0.53). In conclusion, our results could be useful for establishing a gene methylation pattern for the detection and prognosis of NSCLC.

Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer.

BACKGROUND: Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312. RESULTS: The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo. CONCLUSIONS: We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.

Differential colorectal carcinogenesis: Molecular basis and clinical relevance.

Colorectal cancer (CCR) is one of the most frequent cancers in developed countries. It poses a major public health problem and there is renewed interest in understanding the basic principles of the molecular biology of colorectal cancer. It has been established that sporadic CCRs can arise from at least two different carcinogenic pathways. The traditional pathway, also called the suppressor or chromosomal instability pathway, follows the Fearon and Vogelstein model and shows mutation in classical oncogenes and tumour suppressor genes, such as K-ras, adenomatous polyposis coli, deleted in colorectal cancer, or p53. Alterations in the Wnt pathway are also very common in this type of tumour. The second main colorectal carcinogenesis pathway is the mutator pathway. This pathway is present in nearly 15% of all cases of sporadic colorectal cancer. It is characterized by the presence of mutations in the microsatellite sequences caused by a defect in the DNA mismatch repair genes, mostly in hMLH1 or hMSH2. These two pathways have clear molecular differences, which will be reviewed in this article, but they also present distinct histopathological features. More strikingly, their clinical behaviours are completely different, having the "mutator" tumours a better outcome than the "suppressor" tumours.

MMP-7 and SGCE as distinctive molecular factors in sporadic colorectal cancers from the mutator phenotype pathway.

Colorectal cancers (CRCs) from the suppressor and the mutator carcinogenic pathways display distinctive pathological and clinical features that remain not completely understood. In this context, the aim of this work was to study the differential expression of metalloproteinases and adhesion molecules related to cancer invasiveness in both groups of tumours. We analyzed 84 tissue specimens, 42 primary sporadic CRCs obtained from patients who underwent radical surgery, and its corresponding control tissues. According to microsatellite instability, 31 cancers showed low or null microsatellite instability (MSI-L/MSS) and 11 tumours displayed high microsatellite instability (MSI-H). Expression assays were established using the Oligo GEArray(R) human extracellular matrix and adhesion molecules microarray containing 114 genes. Real-time quantitative PCR (RT-qPCR) confirmed expression data from arrays, using TaqMan probes. Results from oligoarray expression analyses indicated that ITGA3, ITGA9, ITGB4, ITGB7 and MMP15 had significantly higher expression levels in MSI-H tumours versus MSS/MSI-L cancers, whereas COL12A1, CSPG2, FN1, MMP-7 and SGCE were down-regulated in tumours with high microsatellite instability when compared to the stable group. After RT-qPCR validation, two of these genes, MMP-7 and SGCE, were confirmed to have statistical differences between the two groups of tumours studied. In both cases, MSI-H tumours displayed significant lower expression levels than MSI-L/MSS tumours. In conclusion, these two distinctive molecular markers could be related to a diminished invasion in colorectal tumours from the mutator pathway, this may contribute to the understanding of the better patient prognosis conferred by this type of tumours.

Expression of Human MDGA1 Increases Cell Motility and Cell-Cell Adhesion and Reduces Adhesion to Extracellular Matrix Proteins in MDCK Cells.

Characterization of the novel human protein MDGA1 (MAM Domain containing Glycosylphosphatidylinositol Anchor-1) has been reported in our laboratory in the past few years. hMDGA1 is a glycoprotein containing 955 aminoacids (137 kDa) attached to the eukaryotic cell membrane by a GPI (Glycosylphosphatidylinositol) anchor and localized specifically into membrane microdomains known as lipid rafts. Moreover, MDGA1 protein contains structural features found in different types of cell adhesion molecules (CAMs) such as the presence of immunoglobulin domains and a MAM domain (Meprin, A5 protein, receptor protein-tyrosine phosphatase µ), suggesting a role of MDGA1 in cell migration and/or adhesion. In order to investigate this aim, stable MDCK cell lines expressing MDGA1 or the truncated proteins IgGPI (lacking the MAM domain) and MAMGPI (lacking Ig domains) were generated. Our results reveal that MDGA1 increases the ability of MDCK cells to migrate, as it contains both Ig and MAM domains which have been implicated in cell motility. In addition, cell adhesion to extracellular matrix proteins, mainly to collagen IV, is reduced by MDGA1 and the IgGPI and MAMGPI truncated proteins. Accordingly, silencing MDGA1 by siRNA revealed a significant increase in adhesion to collagen IV. Furthermore, MDGA1 expression, through the intrinsic properties of the MAM domain, increases cell-cell adhesion independently of the cell monolayer used, suggesting that MDGA1 mediates cell-cell adhesiveness in a heterophilic manner.

Telomere function in colorectal cancer.

Colorectal cancer is the third most common form of cancer and the second leading cause of cancer-related death in the western world. Tumour cells acquire the hallmarks of cancer during the carcinogenic selection process. Cell immortality is one of the principal features acquired during this process which involves the stabilization of telomere length. It is achieved mainly, by telomerase activation. Thus, the discovery of telomeres and telomerase allowed an understanding of the mechanisms by which cells can become immortalized. Different studies have shown that tumour cells have shorter telomeres than nontumour cells and have detected telomerase activity in the majority of tumours. Survival studies have determined that telomere maintenance and telomerase activity are associated with poor prognosis. Taking into account all the results achieved by different groups, quantification and evaluation of telomerase activity and measurement of telomere length may be useful methods for additional biologic and prognostic staging of colorectal carcinoma.