Κυριακή, 30 Ιουνίου 2019

Molecular & Cellular Toxicology

Long non-coding RNA RP11-6O2.4 indicates poor prognosis and suppresses cell cycle progression through the p38-MAPK signaling pathway in gastric cancer


Backgrounds: The role of long non-coding RNAs (lncRNA) in gastric cancer (GC) has been highlighted in studies conducted over the past decade. However, the potential clinical value and the mechanisms of action of RP11–6O2.4 in GC have not been thoroughly elucidated to date. The specific aim of the present study was to assess RP11–6O2.4 and to explore its role in human GC.

Methods: Quantitative real-time polymerase chain reaction (qPCR) was performed to analyze the expression levels of RP11-6O2.4 in GC tissues, paired adjacent noncancerous tissues (ANTs) and GC cell lines. In addition, the correlation between RP11–6O2.4 expression and the clinical characteristics and prognosis of patients with GC was statistically analyzed. The effects of RP11-6O2.4 on the GC cell cycle transformation through the p38-MAPK signaling pathway were explored by flow cytometry, qPCR and Western blot analysis after treatment with SB203580, a p38MAPK specific inhibitor, in vitro.

Results: The expression levels of RP11-6O2.4 in GC tissues were significantly lower than the paired ANTs (P<0.05). In addition, RP11–6O2.4 expression was significantly lower in cases with older age, longer maximum tumor diameter, higher ASA grade and deeper invasive depth (P<0.05). RP11–6O2.4 expression was significantly higher in cases with well/middle differentiation than poor/no differentiation; higher in cases without lymph node metastasis than in lymph node metastasis; and higher in cases in stage I/II than in stage III/IV. An in vitro assay showed that RP11–6O2.4 induced G0/G1 phase cell cycle arrest, likely by regulating the p38-MAPK signaling pathway.

Conclusion: The above mentioned data suggested that RP11–6O2.4 was a tumor-suppressor gene in GC. RP11-6O2.4 might play an important role in the cell cycle transformation by regulating the p38-MAPK signaling pathway, thereby representing a specific biomarker and a potential molecular target for the treatment of GC.

Comparison of three human liver cell lines for in vitro drug-induced liver injury assessment: Huh7, HepaRG, and stem cell-derived hepatocytes


Backgrounds: Drug-induced liver injury (DILI) is a major causal factor for failure in clinical trials and withdrawal of drugs from pharmaceutical markets. Therefore, a human cell-based in vitro system has been established to overcome the limitations of preclinical trials. However, previous studies focused on the drug metabolism in each cell line or described to be superior in one single cell line for assessing DILI. In this study, we used Huh7, HepaRG, and Hepatosight-S cells to evaluate the liver toxicity and dysfunction driven by DILI-inducing drugs.

Methods: Cytotoxicity of 17 DILI-inducing drugs was assessed by LDH release assay and the drug-induced liver dysfunction was confirmed by albumin secretion. Furthermore, we analyzed the expression levels of phase I and phase II enzymes and nuclear receptors in each cell lines.

Results: DILI-inducing drugs were differently detected in each cell line because each cell line differs on the expression of drug metabolic enzymes which associated with reactivity on DILI inducing drugs.

Conclusion: To predict the responses of DILI-inducing drugs in human liver cells, using diverse cell lines having different drug metabolic capabilities is more suitable for predicting DILI.

Covalent conjugates of granulin-epithelial precursor-siRNA with arginine-rich peptide for improved stability and intracellular delivery in hepatoma cells


Backgrounds: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. However, little is known about the molecular mechanisms of HCC development, progression, or effective therapeutic approaches. Recently, granulin-epithelin precursor (GEP) has been suggested as a novel factor that can control HCC cell proliferation. Thus, we aimed to develop new cell-permeable and stable genetic agents that can control GEP expression in HepG2 cells.

Methods: Small interfering RNA-GEP (GEP siRNA) was used for the development of therapeutic agents for HCC treatment. GEP siRNA was first modified by adding polyethylene glycol (PEG) at the terminus. Arginine-rich peptide (ARP) was chemically conjugated with PEGylated GEP siRNA. Chemical conjugates of GEP siRNA-PEG-ARP were evaluated for cytotoxicity and bioavailability, including stability against DNase I enzymatic activity and GSH. The conjugate was further evaluated for intracellular distribution and cell proliferation effects.

Results: The optimal conjugation ratio of GEP siRNA to ARP was a 1:10 molar ratio, and stability was confirmed using DNase I and glutathione stability assays. Conjugates showed increased intracellular uptake and distribution in HepG2 cells. Interestingly, HepG2 cells treated with conjugates showed significantly decreased cell proliferation.

Conclusion: These results provide insights into the importance of chemical modification of unstable genetic therapeutics for HCC treatment. Conjugation of PEGylated GEP siRNA with ARP may be a potential HCC therapeutic agent.

Interactive effects between components in binary mixtures of zinc sulfate and iron oxide nanoparticles on Daphnia magna


Backgrounds: The concern in the toxicological impact of nanomaterials on aquatic organisms has grown, due to the high adsorption capacity for (in) organic compounds in the aquatic environment. In order to evaluate the toxicity of mixtures composed with metal ion and metal oxide nanoparticles and the interaction between components in binary mixtures, we tested the mixture toxicity of iron oxide nanoparticles (i.e., PVP-Fe3O4 NPs) and zinc sulfate (ZnSCU) on Daphnia magna.

Methods: The toxicity of binary mixtures with different concentration-combinations were identified by the effective concentration values (ECxmix) based on the concentration-response curves. Concentration addition index (CAI) and effect addition index (EAI) were applied for examining the interaction between components in binary mixtures.

Results: The findings from this study implied the ZnSO4 had a high toxic effect on D. magna more than PVP-Fe3O4 NPs and the synergistic toxicity in binary mixtures were depended on the toxic effects of ZnSO4 and their exposure concentration rather than those of PVP-Fe3O4 NPs. Interestingly, the antagonistic effects between ZnSO4 and PVP-Fe3O4 NPs in binary mixtures showed in the high concentration-combinations suggesting that antagonism in toxicity may be due to the high adsorption capacity of PVP-Fe3O4 NPs for organic compounds.

Conclusion: In this study, synergistic- and antagonistic effects in binary mixtures with various concentrationcombinations will provide important information for elucidating the toxicity mechanism of mixtures composed inorganic compounds and metal oxide nanoparticles (MONPs). However, in order to conduct the risk assessment of environmental nanoparticle, further studies regarding the mixture toxicity of nanomaterials with environmentally relevant concentrations and the interaction between components will be required.

Novel GRN mutations in Koreans with Alzheimer's disease


Backgrounds Alzheimer's disease (AD) and fronto-temporal dementia (FTD) are the two most common neurodegenerative diseases leading to early onset dementia (<65 years). Mutations in the amyloid precursor protein, presenilin, and presenilin 2 genes are involved in some cases of familial early-onset AD (EOAD), while the microtubule-associated protein tau (MAPT) and pro-granulin (GRN) mutations have been mainly identifed in FTD patients. Clinically, FTD was often misdiag-nosed and confused with AD or psychiatric disorders, which could be a challenge in disease diagnosis.

Methods: We performed mutation analysis of GRN in 89 Korean patients with clinically diagnosed EOAD. In silico predictions were also performed for the variants to estimate their role in different disorders.

Results: No pathological mutations in MAPT was identified, but we identified two novel genetic variations in the GRN gene: p.Leu585Phe (c.1767G>T) and c.IVS8+23_+26delTGGG, which occurred independently in two EOAD patients (frequenct of 2/89, 2.2%). Using a combination of clinical and association studies, in silico prediction, and 3-D modeling software, we suggest that both mutations are probably pathogenic and involved in FTD.

Conclusion: Our data suggest that it would be important to re-examine EOAD patients who had been diagnosed when the FTD spectrum was not well described and the causative FTD genes had not yet been identifed. In addition, we propose initially analyzing genes associated with the frst form of suspected dementia and, if the results are negative, studying genes implicated in the other form of dementia.

Roles of integrins in regulating metastatic potentials of cancer cell derived exosomes


Purpose of review: Exosomes are nano-sized extracellular vesicles ranging from 30–150 nm in diameter. Exosomes interact with nearby or distantly localized recipient cells by involving ligand/receptor binding at the surface of recipient cells or fusion with plasma membrane of recipient cells. A number of recent literatures support the role of integrins in mediating metastatic process of cancer cell derived exosomes. While exosomes need to dock on the surface of recipient cells to transmit signals or transfer their contents, the process of docking and uptake of exosomes by recipient cells is poorly understood. This review discusses the recent reports that suggest the potential roles of exosomal integrins in mediating docking and uptake of exosomes, and how these processes are related to metastatic potentials of cancer cell derived exosomes.

Recent findings: Exosomal integrins (α6β4, α6β1, αvβ5, αvβ3, αvβ6) and exosomal integrin ligands (vinculin, fibronectin) have been recently reported to play roles in cancer metastasis. As exosomes are involved in cell-to-cell communication, integrins and their ligands in cancer cell derived exosomes can contribute to progression by selecting target tissues and triggering integrin mediated signaling cascades to form new metastatic niche. Recent studies also suggest that exosomal integrins and their ligands can be targeted for developing exosome based diagnostics and therapies.

Mild NO preconditioning protects H9c2 cells against NO-induced apoptosis through activation of PI3K/Akt and PKA-dependent pathways


Backgrounds: Nitric oxide (NO) plays a key role in cardioprotection. Its role against cardioprotection is dependent on the level of NO. Although it is well known that NO preconditioning has cardioprotective effects, but its mechanism remains unsatisfactory.

Methods: To induce NO preconditioning, H9c2 cells were treated with low NO concentration and subsequently induced apoptosis by high NO. The signalling and anti-apoptotic effects of NO preconditioning were monitored by Western blotting, facs analysis.

Results: Sodium nitroprusside (SNP)-induced cytotoxicity was inhibited by low SNP (0.3 mM) preconditioning. Furthermore, low SNP phosphorylated Akt/FoxO1 in the presence of high SNP (1.5 mM), while phosphorylated Akt/FoxO1 and viability were reversed by PI3K inhibitor. Also, low NO-induced CREB phosphorylation with high NO was inhibited by PKA inhibitor, indicating that NO preconditioning protects NO-induced cytotoxicity in PKA dependently. Apoptotic inhibition with NO preconditioning was accompanied with increased Bcl-2, decreased Bax, and caspase3 activation, which was all reversed by LY294002.

Conclusion: Our results indicate that low NO preconditioning prevent subsequent high NO-induced apoptosis via Akt/PKA/CREB activation in H9c2 cells.

LncRNA MALAT1 protects human umbilical vein endothelial cells against ox-LDL triggered cell death through regulation of MGP


Backgrounds: Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is an lncRNA that has been suggested as a key regulator in the onset of atherosclerosis (AS). This study described the role of MAL-AT1 in oxidized low density lipoprotein (ox-LDL)-induced endothelial cells death.

Methods: Human umbilical vein endothelial cells (HU-VECs) were subjected to ox-LDL, before which the expression of MALAT1 was overexpressed by transfection. CCK-8 assay, flow cytometer detection, and western blot were carried out to evaluate cell viability, apoptosis and autophagy. qRT-PCR and western blot analyses were performed to investigate the regulatory relationship between MALAT1, Matrix Gla protein (MGP) and mTOR signaling to decode the underlying mechanism.

Results: Up-regulation of MALAT1 attenuated ox-LDL-induced HUVECs lose, as evidenced by the promoted cell viability, and the decreased apoptosis rate. This finding was coupled with the down-regulated p53, Bax, active-caspase-3, Beclin-1 and LC3-II, as well as the up-regulated Bcl-2 and p62. Meanwhile, MALAT1 up-regulation promoted the phosphorylation of p70S6K and mTOR, and the expression of MGP. MGP up-regulation exhibited MALAT1-like propoties in preventing ox-LDL-induced cell death and mTOR deactivation. Of contrast, MGP silence affected HUVECs survival and mTOR signaling resulted in contrary impacts.

Conclusion: The present work described that MALAT1 up-regulation prevented ox-LDL-mediated apoptosis and autophagy in HUVECs. The protective effects of MALAT1 might be partially via up-regulating MGP, which led to the activation of mTOR signaling.

Transcriptomics and proteomics analysis of Aβ (1-42)-induced neurotoxicity


Backgrounds: β-Amyloid (Aβ) is a principal constituent of senile plaques in Alzheimer's disease (AD) and induces neuronal cell death. The molecular mechanism of how Aβ evokes neuronal cell death remains complicated, which were investigated in the present study.

Methods: Using the human neuroblastoma cell line SHSY5Y, we investigated the neurotoxic effects of human β-Amyloid 1–42 (Aβ1–42) aggregates on gene expression profile and protein expression profile by using the Agilent GeneChip Human 1A (V2) Oligo MicroArray, Quantitative Real-time PCR, PF-2D and Western blot analysis.

Results: Our results show that Aβ1–42 specifically influences gene and protein expression such as EGR1, eIF5A, PDE8A, ERp57 and ERp5 in pathways associated with apoptotic process, protein translation, cAMP catabolic process and response to endoplasmic reticulum stress.

Conclusion: Although Genes with significant changes in transcriptomic analysis matched very few of the proteins identified in proteomics analysis, our findings will strengthen our knowledge concerning the molecular mechanisms underlying AD.

Prevention and relaxation effects of Liriope platyphylla on bronchial asthma in vitro model by suppressing the activities of MAPK/NF-κB pathway


Backgrounds: Bronchial asthma (BA) is a common type of asthma, defined as a chronic inflammation, hyperplasia, and hypercontraction of the airway. In this study, we investigated whether Liriope platyphylla extract (LPP) can help prevent inflammation, suppress hyperplasia, and support relaxation via regulation of MAPK/NF-κB activity.

Methods: To investigate the inflammation prevention effects, A549 cells, a human airway epithelial cell line, were pretreated with various concentration of LPP and then treated with EGF recombinant (10 ng/mL). Anti-hyperplasia and muscle relaxation experiments were performed with asthma condition human bronchial smooth muscle (hBSM) cells. The effects of LPP on MAPK/NF-κB activity and MAPK/NF-κB-related signaling, such as COX-2 and iNOS expression, was analyzed through western blot analysis. The mRNA expression levels of Th-2-cell-related cytokines, including IL-6 and TNF-α, and contraction-related factors, such as PLCβ, IP3R, and MLCK, were determined by real-time PCR. Results: LPP pretreatment significantly reduced MAPK/NF-κB activity in EGF-induced asthma condition A549 cells. Therefore, there was significant suppression of both COX-2 and iNOS expression. In addition, the mRNA expression of IL-6 and TNF-α was dose depen-dently reduced. Although there was no statistical significance, factors related to contraction showed a tendency to decrease.

Conclusion: Based on our results, we believe that LPP shows good potential for application as a therapeutic agent for asthma through inflammation prevention and bronchial smooth muscle relaxation.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos

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