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  • Open access
  • 34 Reads
Role of Gal-3 on cisplatin-induced acute liver injury model

Oxidative stress is a common mechanism in the cytotoxicity of cisplatin, a widely used antineoplastic agent related to hepatotoxicity. In this context, we highlight galectin-3 (Gal-3), a β-galactoside binding protein that regulates the inflammatory response and oxidative stress, and modified citrus pectin (MCP), an inhibitor of Gal-3. Thus, this study evaluates the effect of Gal-3 inhibition with MCP on cisplatin-induced acute liver injury in Wistar rats. Animals were divided into 4 groups (n = 5/group): SHAM – intraperitoneal (i.p.) injection of saline for 3 days; CIS – i.p. injection of cisplatin (10 mg/kg/day) for 3 days; MCP - orogastric gavage with MCP (100 mg/kg/day) for 7 days, followed by saline via i.p.; and MCP+CIS - gavage with MCP for 7 days, followed by cisplastin via i.p. for 3 days. Cisplatin administration caused a significant weight loss in the animals from CIS and MCP+CIS, an effect corroborated by a marked reduction in the glycogen storage in hepatocytes compared to their control groups. Cisplatin also provoked a marked increase in the influx of leukocytes, liver degeneration, ROS production and STAT3 activation in the hepatocytes, plasma levels of cytokines (IL-6, IL-10), and hepatic toxicity biomarkers (ARG1, GSTα, SDH). Cisplatin per se reduced Gal-3 levels, especially in the mitochondria of hepatocytes. On the other hand, the MCP+CIS group also showed increased levels of IL-1β, TNF-α, and GOT1, as well as raised hepatic levels of MDA production and mitochondrial respiratory complex I. In conclusion, inhibition of Gal-3 with MCP did not protect the liver against the deleterious effects of cisplatin, indicating that Gal-3 is important for tissue, cellular and molecular maintenance of the liver.

  • Open access
  • 17 Reads
Interleukin (IL)-11 is involved in the functional liaison between breast tumor cells and the surrounding stroma
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Current advances in molecular profiling methodologies and the accessibility to multi-omics datasets are paving the way toward a better understanding of heterogeneous diseases, including breast cancer (BC). In this regard, we sought to uncover the transcriptional changes triggered by estrogen and insulin in a primary BC cell line (BCAHC-1), which expresses the 46kDa isoform of the estrogen receptor (ER)a and the insulin receptor, as we previously ascertained.

Raw data from RNA sequencing of BCAHC-1 cells were processed by the Bcl2Fastq 2.20 version of the Illumina pipeline, while in silico analyses were performed in R Studio using the TCGA dataset. Real-time PCR, immunoblotting, ELISA and chromatin immunoprecipitation experiments were used to identify the molecular events triggered by estrogen and insulin in BCAHC-1 cells and cancer-associated fibroblasts (CAFs). Furthermore, migration and invasion assays allowed us to ascertain the mechanisms triggering these biological responses upon the aforementioned hormone treatments.

First, we determined that 17b-estradiol (E2) and insulin stimulate a peculiar IL-11 expression and secretion in BCAHC-1 cells. Thereafter, bioinformatics analyses confirmed the up-regulation of IL-11 in ER-positive BCs respect to adjacent normal tissues and its association with worse survival. Next, the involvement of IL-11 in pro-metastatic transduction signaling by pathway enrichment analyses was established. Worthy, we found that the secretion of IL-11 by BCAHC-1 cells prompts an invasive phenotype of CAFs through the up-regulation of genes belonging to the extracellular matrix organization pathway, namely the intercellular adhesion molecule 1 and integrin alpha 5.

Overall, our findings indicate that IL-11 secretion by BC cells may elicit a paracrine action on the surrounding stroma toward invasive properties, suggesting that IL-11 could be considered as a valuable target in comprehensive treatments of ER-positive BC patients.

  • Open access
  • 28 Reads
Mechanistic insights on the anticancer effects of metformin in primary breast cancer cells.
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Metabolic disorders, like obesity, type 2 diabetes (T2D) and metabolic syndrome, have been implicated in breast cancer (BC) progression. In this regard, insulin has been shown to promote mitogenic and metastatic responses in BC through diverse signaling pathways. Moreover, high levels of insulin and elevated expression of its cognate receptor, namely insulin receptor (IR), have been associated with increased BC incidence, resistance to treatments and poor outcome. Metformin (1,1-dimethylbiguanide hydrochloride) is the most commonly prescribed drug for T2D treatment worldwide. Worthy, metformin has been shown to interfere with BC cell growth. In order to provide novel insights through which metformin can elicit anti-cancer responses in BC, we performed bioinformatics analysis as well as TaqMan Gene Expression Assay, flow cytometry, immunofluorescence, immunoblots, 2D and 3D proliferation assays and motility experiments. A naturally immortalized BC cell line (namely BCAHC-1) and important components of the tumor microenvironment, like cancer-associated fibroblasts (CAFs) derived from BC patients, were used as model systems. We found that metformin inhibits the activation of main transduction pathways, the gene expression changes and the proliferative effects induced by insulin in BCAHC-1 cells. Moreover, metformin prevented the insulin-stimulated induction of CXC chemokine receptor 4 (CXCR4), which has been involved in BC metastatic dissemination. Next, metformin suppressed the invasion of CAFs triggered through CXCR4 by insulin stimulated BCAHC-1 cells. Our findings may suggest novel transduction mechanisms involved in the inhibitory effects elicited by metformin in both BC cells and CAFs.

  • Open access
  • 40 Reads
FMO3 misfolding might indirectly block the unfolded protein response via PERK in hepatocytes

Trimethylaminuria (TMAU) is a metabolic syndrome characterized by the accumulation and the excretion of trimethylamine (TMA), synthesized by gut microbiota, which is excreted through sweat, breath, urine and other body fluids, determining an unpleasant rotten fish odor in affected patients. The primary form (TMAU1) is determined by homozygous causative mutations in the FMO3 gene that could impair enzyme function. Frequently, TMAU1 affected patients do not carry causative mutations in homozygous condition. Therefore, we hypothesized that compound heterozygosity and haplotype variants might also cause FMO3 misfolding playing a significant role in FMO3 activity reduction or alteration. FMO3 misfolding might determine hepatocytes ER stress that in this case could be amplified by the TMAO levels reduction. In fact, it is known that TMAO binds the luminal domain of protein kinase R-like endoplasmic reticulum kinase (PERK), activating the unfolded protein response and consequently reducing endoplasmic reticulum stress.

To confirm our hypothesis, we performed a mutational analysis of FMO3 gene in 26 patients by Sanger sequencing. Then, a proteomic in silico analysis, using different platforms and software, was carried out with the final aim of revealing how these variant combinations could influence the enzyme folding, also simulating its dynamic behaviour with the TMA substrate. Results revealed the presence of 17 variants distributed in 26 different haplotypes which might lead to possible impairments of FMO3 activity, probably reducing the interaction time between the enzyme catalytic site and TMA or losing the wild-type binding site.

Since little is still known about the role that the combination of multiple variants could exert on the enzyme activity, our analysis could represent a starting point to unveil new scenarios about the genetic form of TMAU.

  • Open access
  • 113 Reads
The Effect of RAS2 Gene Mutation in Yeast Model

More than 30% of all human malignancies are brought about by mutation in RAS proto-oncogenes (HRAS, KRAS, and NRAS) that are greatly conserved in yeast (RAS1 and RAS2). This makes yeast an efficient single-celled eukaryotic model organism to study their functions. In this current investigation, the null mutation of RAS2 gene was analyzed to find out its deleterious consequences in yeast cells based on their ability to utilize glycerol as its respiratory substrate, mtDNA mutation rate, mtDNA abundance and distribution pattern. Mutant cells grown in YPEG plates demonstrated slight respiratory deficiency than the wild type. Erythromycin-resistant assay was carried out to analyze the spontaneous mitochondrial DNA mutation rate in ras2 mutant and was found greater than the wild type. In addition, the mitochondrial DNA of both strains was also visualized under a fluorescence microscope using DAPI fluorescent stain. It was observed that mtDNA abundance was much lower than that of the wild type. Thus, the present investigation revealed that the deletion of RAS2 gene resulted in mtDNA mutation and depletion.

  • Open access
  • 32 Reads
Metabolic activity of Chlamydomonas reinhardtii cells under diclofenac-induced stress

The non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), are detected in water bodies all over the World. Their presence in the water environment poses a threat to non-target plant organisms, including unicellular green algae. To survive in the contaminated environment, these organisms need to modify their metabolism to be able to cope with NSAIDs-induced stress. Knowledge of algal response to drugs is crucial for environmental protection.

In the present work, we report the response of green alga Chlamydomonas reinhardtii to DCF applied in the concentration 32.7 mg/L, corresponding to toxicological parameter EC10. The algae susceptibility for DCF was estimated based on physiological parameters: population growth, oxidative stress symptoms, and photosynthetic activity. Moreover, the cells’ cultures were analyzed for the appearance of diclofenac transformation products.

We have found that DCF caused a slight decrease in the population growth rate and photosynthetic activity (quantum yield of photosynthesis) of the cells. Further, some symptoms of oxidative stress (singlet oxygen overproduction) were observed. However, in the biomass and culture media wide range metabolites of DCF were discovered. This suggests, that in the presence of relatively low concentration of DCF biochemical activity of the algae was efficient enough to metabolize a part of the drug in the medium. What is important, some of the analyzed transformation products were similar to those formed during the metabolism of DCF by bacteria, while other were characteristic for eucaryotic metabolic pathways.

In conclusion, C. reinhardtii exposed to DCF can keep its metabolic activity on the level sufficient for surviving and for biotransformation of the drug. Our results give rise to the assumption that other algae strains may also have potential to metabolize DCF thus contributing to the remediation of the environment contaminated with pharmaceuticals.

This work was partially supported by National Science Centre Poland [UMO-2019/35/B/NZ9/01567].

  • Open access
  • 13 Reads
Parental genetics communicate with intrauterine environment to reprogram newborn telomeres and immunity

Telomeres, markers for cellular senescence, have been found substantially influenced by parental inheritance. It is well known that genomic stability is preserved by the DNA repair mechanism through telomerase. This study aimed to determine the association between parents-newborn telomere length (TL) and telomerase gene(TERT), highlighting DNA repair combined with TL/TERT polymorphism and immunosenescence of the triad. The mother-father-newborns triad blood samples (n=312) were collected from Ziauddin Hospitals, Pakistan between September 2021-June 2022. The telomere length (T/S ratio) was quantified by qPCR, polymorphism was identified by Sanger sequencing and immunosenescence by flow cytometry. The linear regression was applied for TL and gene association. The newborns had longest TL(2.51+2.87) and strong positive association (R=0.25, p=<0.0001)(transgenerational health effects) with mothers’ TL(1.6+2.00). Maternal demographics; Socioeconomic status, education and occupation, showed significant effects on TL of newborn (p<0.015,0.034,0.04, respectively). The TERT risk genotype CC (rs2736100) was predominant in the triad (0.6, 0.5 0.65, respectively) with a strong positive association with newborn TL (β=2.91, <0.0011). Further analysis highlighted the expression of KLRG 1+ in T-cells with longer TL but less frequent among newborns. The study concludes that TERT, parental TL, antenatal maternal health and immunity has a significantly positive effect on the repair of newborn TL.

  • Open access
  • 23 Reads
Microgravity exposure induces antioxidant barrier deregulation and mitochondrial structure alterations in TCam-2 cells

One of the hallmarks of microgravity-induced alterations in several cell models is an alteration of oxidative balance. Notably, also male germ cells, sensible to oxidative stress, have been shown susceptible to changes of gravitational force. To gain more insights into the mechanisms of male germ cell response to altered gravity, a 3D cell culture model was established from TCam2 cells, a seminoma cell line, and the only available in-vitro model to study mitotically active human male germ cells. TCam2 spheroids were cultured for 24 hours under unitary gravity (UG) or simulated microgravity conditions (SM), which were obtained using the Random Positioning Machine (RPM). Apoptosis and necrosis analyses performed on UG and SM exposed samples, revealed no significant differences of all the cell death markers. Notably, Mitosox assay revealed a significant oxidation of mitochondria, after microgravity exposure, at least at this culture time. In SM treated samples, gene expression levels (evaluated by Real-time PCR) of the main enzymes of the antioxidant barrier, GPX1 and NCF1, are reduced indicating an influence of SM on mitochondria function. Notably, the expression of HMOX, involved in the heme catabolism of mitochondria cytochromes, is increased. SOD, XDH, CYBA, NCF-2, TXN and TXNRD genes were not affected. The ultrastructural analysis by Transmission Electron Microscopy revealed that SM significantly altered TCam2 spheroid mitochondria, which appeared swollen and, in some cases, disrupted. Indeed, mitophagy, or mitochondrial autophagy, appears to be more represented in samples exposed to simulated microgravity. This result seems to be in line with the increase, mediated by the simulated microgravity, of the enzyme HMOX1. All together, these preliminary data demonstrate TCam2 spheroids sensitivity to acute SM exposure, strongly indicating a microgravity-dependent modulation of mitochondria morphology and activity and encourage to perform further investigations on chronical exposure to SM of TCam2 spheroids.

  • Open access
  • 31 Reads
Microgravity exposure alterations of cellular junctions proteins in TCam-2 cells: localization and interaction

One of the most important hazards of space environment is microgravity, which causes alteration in the physiology of different systems, including the reproductive one. It is widely accepted that cytoskeleton is the microgravity-sensitive apparatus of the cells, and that cytoskeletal modifications are responsible of microgravity-triggered cell alterations. We established a 3D free floating culture system from TCam2 cell, a human seminoma cell line, and then exposed the obtained TCam2 spheroids for 24h at unitary gravity (UG) or under simulated microgravity condition (SM), by using the Random Position Machine (RPM). We tested cytoskeletal and junctional features of these samples by western blot and confocal microscopy analysis to elucidate the impact of microgravity on adherent and occluding junctions of TCam2 spheroids. The junctional ultrastructure was studied by Transmission electron microscopy (TEM).
TEM analysis revealed the presence of occluding junctions both in UG or SM samples. Even if western blot revealed no quantitative difference of actin and occludin proteins both in UG and SM exposed samples, fluorescence colocalization analysis showed a significative increase of colocalization area of occludin and actin proteins in the superficial layer of TCam2 spheroids grown in RPM conditions. This result let us speculate that tight junction functionality is different id UG and SM exposed spheroids.
As far as adherent junctions is concerned, TEM analysis revealed adherent junctions both in UG or SM samples. Moreover, we observed by western blot a trend in increase of vimentin expression in SM exposed spheroids. Confocal microscopy analyses confirm this significant increase..
All together these data suggest that simulated microgravity conditions in TCam2 spheroids alters tight junction assembly, while the increase in intermediate filaments structures can in part be associated with an enrichment in adherent junctions. Functional investigation are needed to deeper clarify this hypothesis.

  • Open access
  • 17 Reads
CAVPENET decreases prostate cancer cells proliferation and invasion through modulation of protein phosphatase activity

Prostate cancer (PCa) is a disquieting cause of men’s death worldwide and there is an urgent need to develop new effective therapeutic strategies. Protein phosphatase 1 (PP1) recently emerged as a promising therapeutic target in cancer. In this context, the main goal of this work is to develop peptides to disrupt a key PP1 complex for PCa development, thus impairing PCa progression. The peptide designed to interrupt the interaction between PP1 and caveolin-1 (CAV1) was synthetised using microwave-assisted solid phase synthesis and coupled to penetratin to allow an efficient cell delivery. The efficacy of the synthetised peptide - CAVPENET (and a scrambled homologue - CAVPENET control) was evaluated in vitro, using androgen-dependent (LnCaP) and androgen-independent (PC-3) cell lines. We found that, after 48h incubation, CAVPENET significantly decreases the LnCaP and PC-3 cells viability and invasive ability. A significant decrease in the phosphorylation of AKT at Ser473 was also observed after 48h incubation with CAVPENET. A slightly recover of AKT phosphorylation levels after simultaneously incubation of CAVPENET (10 µM) with tautomycin (10nM) – a highly specific PP1 inhibitor, suggested a role of PP1 in the CAVPENET-induced alterations in AKT phosphorylation. Moreover, incubation with CAVPENET (10µM) + Cantharidin (0.5µM) – a potent and selective PP2A inhibitor, almost completely recover the phosphorylation levels of AKT, suggesting a role for PP2A in the effect of CAVPENET. Altogether, these results highlight the potential of the synthetised peptide to negatively impact the PCa cells proliferation and invasive ability, by interfering with the interaction of CAV1 with PP1 and/or PP2A. Further analyses are now required to confirm the disruption of the interactions and to better elucidate the mechanisms of cells death.

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