ResearchPad - mtt-assay https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Decyl caffeic acid inhibits the proliferation of colorectal cancer cells in an autophagy-dependent manner <i>in vitro</i> and <i>in vivo</i>]]> https://www.researchpad.co/article/elastic_article_13874 The treatment of human colorectal cancer (CRC) cells through suppressing the abnormal survival signaling pathways has recently become a significant area of focus. In this study, our results demonstrated that decyl caffeic acid (DC), one of the novel caffeic acid derivatives, remarkedly suppressed the growth of CRC cells both in vitro and in vivo. The inhibitory effects of DC on CRC cells were investigated in an in vitro cell model and in vivo using a xenograft mouse model. CRC cells were treated with DC at various dosages (0, 10, 20 and 40 μM), and cell survival, the apoptotic index and the autophagy level were measured using an MTT assay and flow cytometry analysis, respectively. The signaling cascades in CRC were examined by Western blot assay. The anti-cancer effects of DC on tumor growth were examined by using CRC HCT-116 cells implanted in an animal model. Our results indicated that DC differentially suppressed the growth of CRC HT-29 and HCT-116 cells through an enhancement of cell-cycle arrest at the S phase. DC inhibited the expression of cell-cycle regulators, which include cyclin E and cyclin A proteins. The molecular mechanisms of action were correlated to the blockade of the STAT3 and Akt signaling cascades. Strikingly, a high dosage of DC prompted a self-protection action through inducing cell-dependent autophagy in HCT-116 cells. Suppression of autophagy induced cell death in the treatment of DC in HCT-116 cells. DC seemed to inhibit cell proliferation of CRC differentially, and the therapeutic advantage appeared to be autophagy dependent. Moreover, consumption of DC blocked the tumor growth of colorectal adenocarcinoma in an experimental animal model. In conclusion, our results suggested that DC could act as a therapeutic agent through the significant suppression of tumor growth of human CRC cells.

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<![CDATA[Abrogation of pathogenic attributes in drug resistant <i>Candida auris</i> strains by farnesol]]> https://www.researchpad.co/article/elastic_article_7651 Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and overexpression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. All the isolates were found capable enough to form biofilms on 96-well flat bottom microtiter plate that was further confirmed by MTT reduction assay. In addition, these strains have active drug efflux mechanism which was supported by rhodamine-6-G extracellular efflux and intracellular accumulation assays. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. We further studied the role of farnesol, an endogenous quorum sensing molecule, in modulating development of biofilms and drug efflux in C. auris. The MIC for planktonic cells ranged from 62.5–125 mM, and for sessile cells was 125 mM (4h biofilm) and 500 mM (12h and 24h biofilm). Furthermore, farnesol (125 mM) also suppresses adherence and biofilm formation by C. auris. Farnesol inhibited biofilm formation, blocked efflux pumps and downregulated biofilm- and efflux pump- associated genes. Modulation of C. auris biofilm formation and efflux pump activity by farnesol represent a promising approach for controlling life threatening infections caused by this pathogen.

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<![CDATA[Ectopic Otoconin 90 expression in triple negative breast cancer cell lines is associated with metastasis functions]]> https://www.researchpad.co/article/5c6f1491d5eed0c48467a304

Triple negative breast cancer (TNBC) is an aggressive tumor with propensity to metastasize and poor treatment options. Improving treatment options would be impactful; thus, finding a tumor-specific cell surface protein with metastasis promoting functions that could be knocked out was the goal of this study. The Otoconin 90 gene (OC90), frequently amplified in tumors on chromosome 8q24.22, was identified as a potential therapeutic candidate. Normally OC90 is expressed in the cochlea with no known function in other normal tissues. In silico analysis of The Cancer Genome Atlas (TCGA) multi-tumor RNAseq cohorts revealed that OC90 is expressed in many tumor types at high prevalence and genomic amplification is associated with the elevated mRNA expression. In vitro assays in TNBC cell lines revealed OC90 expression with control over cell viability, apoptosis and invasion. RNA-seq analysis of OC90-siRNA knockdown and OC90-overexpression in BT20, BT549, HCC38 cell lines identified co-expressed transcripts, HMGA2, POLE2 and TRIB3. Altered expression of HMGA2, POLE2 and TRIB3 was predictive of survival among members of the Metabric breast cancer cohort. Thus, OC90 represents a potential therapeutic target whose knockdown could improve the treatment of TNBC.

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<![CDATA[Phorbol ester-induced angiogenesis of endothelial progenitor cells: The role of NADPH oxidase-mediated, redox-related matrix metalloproteinase pathways]]> https://www.researchpad.co/article/5c478cb0d5eed0c484bd3eb3

Endothelial progenitor cells (EPCs) may contribute to ischemia-induced angiogenesis in atherosclerotic diseases. The protein kinase C (PKC) family is involved in the regulation of angiogenesis, however the role of PKCα in EPCs during angiogenesis is unclear. The aim of this study was to evaluate the role of PKCα in EPCs during angiogenesis. Phorbol-12-myristate-13-acetate (PMA), a PKCα activator, significantly increased the activity and expression of matrix metalloproteinases (MMP) -2 and -9 in human (late outgrowth) EPCs in vitro. The MMPs promoted the migratory function and vascular formation of EPCs, which then contributed to neovascularization in a mouse hindlimb-ischemia model. Reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enhanced the expression of MMPs to increase the bioactivity of EPCs during angiogenesis. The mitogen-activated protein kinase (MAPK) signal pathway was associated with the activation of NADPH oxidase. PMA extensively activated the extracellular signal–regulated kinase (Erk) signal pathway to increase the expression of MMP-9. PMA also activated the p38, Erk, and c-Jun N-terminal kinase signal pathways to increase the expression of MMP-2. PMA-stimulated EPCs enhanced neovascularization in a mouse model of hindlimb ischemia via nuclear factor-κB translocation to up-regulation of the expression of MMP-2 and MMP-9. PMA could activate PKCα and promote the angiogenesis capacity of human EPCs via NADPH oxidase-mediated, redox-related, MMP-2 and MMP-9 pathways. The PKCα-activated, NADPH oxidase-mediated, redox-related MMP pathways could contribute to the function of human EPCs for ischemia-induced neovascularization, which may provide novel insights into the potential modification of EPCs for therapeutic angiogenesis.

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<![CDATA[Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation]]> https://www.researchpad.co/article/5c3d00f8d5eed0c48403729d

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch.

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<![CDATA[Optimization of extraction of antioxidant polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm and its cytotoxic activity against murine lymphoid cancer cell line]]> https://www.researchpad.co/article/5c37b784d5eed0c48449027e

The purpose of this study was to optimize the extraction method for polysaccharide from the fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm and to assess the antioxidant and cytotoxic potentials of polysaccharide. In this investigation, polysaccharides from Pleurotus ostreatus (Jacq.) P. Kumm were extricated by utilizing the hot water. One-single factor and response surface methodology was established to optimize the extraction conditions for polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm. Examination of antioxidant activity of Pleurotus ostreatus polysaccharide (POP) was directed by utilizing 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) techniques. Cytotoxicity of POP was evaluated using an MTT assay. The experimental data were fitted to a quadratic equation utilizing multiple regression investigations, and the ideal conditions were as per the following: water/crude material proportion, 26.04 mL/g; an extraction time of 62.08 minutes; and an extraction temperature 70.5°C. Under such conditions, the polysaccharide yield was 5.32 ± 0.12% with the anticipated yield. POP showed good scavenging activity against DPPH radical (p<0.001, EC50 = 1036.38 μg/mL, R2 = 0.8313) and ABTS radicals (p<0.001, EC50 = 824.37 μg/mL, R2 = 0.8223), with a dose (p<0.001)-and-time (p<0.001) dependent cytotoxic potential on Ehrlich ascites carcinoma cell line in vitro. This demonstrated that polysaccharides (POP) had certain cancer prevention agent exercises. In this manner, these examinations give reference to additionally research and reasonable improvement of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide and POP may prove a useful therapeutic agent, due to its robust antioxidant and cytotoxic activity.

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<![CDATA[Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion]]> https://www.researchpad.co/article/5b694661463d7e3867f4ad07

Objectives

To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines.

Materials and methods

An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA

Results

Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches.

Conclusion

The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.

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<![CDATA[Ion channel expression patterns in glioblastoma stem cells with functional and therapeutic implications for malignancy]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbc83

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation, migration, and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme, a highly aggressive brain cancer, suggesting that ion channel expression may be perturbed in this population. However, little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing, we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance, expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally, genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes, gene mutations, survival outcomes, regional tumor expression, and experimental responses to loss-of-function. Together, the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance.

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<![CDATA[MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2]]> https://www.researchpad.co/article/5989dadcab0ee8fa60bba539

Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury.

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<![CDATA[Structurally optimized analogs of the retrograde trafficking inhibitor Retro-2cycl limit Leishmania infections]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be03b6

In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58μM and10.57+/-2.66μM, respectively, which is significantly lower than 40.15μM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.

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<![CDATA[A natural product-like JAK2/STAT3 inhibitor induces apoptosis of malignant melanoma cells]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0209

The JAK2/STAT3 signaling pathway plays a critical role in tumorigenesis, and has been suggested as a potential molecular target for anti-melanoma therapeutics. However, few JAK2 inhibitors were being tested for melanoma therapy. In this study, eight amentoflavone analogues were evaluated for their activity against human malignant melanoma cells. The most potent analogue, compound 1, inhibited the phosphorylation of JAK2 and STAT3 in human melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels. A cellular thermal shift assay was performed to identify that JAK2 is engaged by 1 in cell lysates. Moreover, compound 1 showed higher antiproliferative activity against human melanoma A375 cells compared to a panel of cancer and normal cell lines. Compound 1 also activated caspase-3 and cleaved PARP, which are markers of apoptosis, and suppressed the anti-apoptotic Bcl-2 level. Finally, compound 1 induced apoptosis in 80% of treated melanoma cells. To our knowledge, compound 1 is the first amentoflavone-based JAK2 inhibitor to be investigated for use as an anti-melanoma agent.

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<![CDATA[Cell survival and differentiation with nanocrystalline glass-like carbon using substantia nigra dopaminergic cells derived from transgenic mouse embryos]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc051

Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells. Because the interest in carbon nanomaterials has grown within the last decade in light of a wide variety of applications, the aim of this study was to test and evaluate the suitability and cytocompatibility of a particular nanometer-thin nanocrystalline glass-like carbon film (NGLC) composed of curved graphene flakes joined by an amorphous carbon matrix. This material is a disordered structure with high transparency and electrical conductivity. For this purpose, we used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryos. Cells were cultured either in a powder of increasing concentrations of NGLC microflakes (82±37μm) in the medium or on top of nanometer-thin films bathed in the same culture medium. The metabolism activity of SN4741 cells in presence of NGLC was assessed using methylthiazolyldiphenyl-tetrazolium (MTT) and apoptosis/necrosis flow cytometry assay respectively. Growth and proliferation as well as senescence were demonstrated by western blot (WB) of proliferating cell nuclear antigen (PCNA), monoclonal phosphorylate Histone 3 (serine 10) (PH3) and SMP30 marker. Specific dopaminergic differentiation was confirmed by the WB analysis of tyrosine hydroxylase (TH). Cell maturation and neural capability were characterized using specific markers (SYP: synaptophysin and GIRK2: G-protein-regulated inward-rectifier potassium channel 2 protein) via immunofluorescence and coexistence measurements. The results demonstrated cell positive biocompatibility with different concentrations of NGLC. The cells underwent a process of adaptation of SN4741 cells to NGLC where their metabolism decreases. This process is related to a decrease of PH3 expression and significant increase SMP30 related to senescence processes. After 7 days, the cells increased the expression of TH and PCNA that is related to processes of DNA replication.

On the other hand, cells cultured on top of the film showed axonal-like alignment, edge orientation, and network-like images after 7 days. Neuronal capability was demonstrated to a certain extent through the analysis of significant coexistence between SYP and GIRK2. Furthermore, we found a direct relationship between the thickness of the films and cell maturation. Although these findings share certain similarities to our previous findings with graphene oxide and its derivatives, this particular nanomaterial possesses the advantages of high conductivity and transparency. In conclusion, NGLC could represent a new platform for biomedical applications, such as for use in neural tissue engineering and biocompatible devices.

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<![CDATA[Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines]]> https://www.researchpad.co/article/5989daa6ab0ee8fa60ba7a0b

Introduction

Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43.

Materials and Methods

F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.

Results

E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.

Discussion

These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

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<![CDATA[MicroRNA-223 Increases the Sensitivity of Triple-Negative Breast Cancer Stem Cells to TRAIL-Induced Apoptosis by Targeting HAX-1]]> https://www.researchpad.co/article/5989db3eab0ee8fa60bd5e04

Drug resistance remains a significant challenge in the treatment of triple-negative breast cancer (TNBC). Recent studies have demonstrated that this drug resistance is associated with a group of cells known as cancer stem cells (CSCs), which are believed to determine the sensitivity of tumor cells to cancer treatment. MicroRNAs (miRNAs) are small, non-coding RNAs that play significant roles in normal and cancer cells. MiR-223 reportedly acts as a tumor suppressor in a range of cancers. However, the role of miR-223 in TNBC, especially in triple-negative breast cancer stem cells (TNBCSCs), remains unknown. Here, we found that miR-223 expression was down-regulated in CD44+CD24-/low TNBCSCs compared with non-CSCs. Furthermore, we found that miR-223 overexpression resensitized TNBCSCs to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The HAX-1 gene, which is located in the mitochondria and functions as an anti-apoptotic protein, was found to be directly regulated by miR-223 in MDA-MB-231 cells. We demonstrated that miR-223 overexpression promoted TRAIL-induced apoptosis through the mitochondria/ROS pathway. In conclusion, our results suggest that miR-223 increases the sensitivity of TNBCSCs to TRAIL-induced apoptosis by targeting HAX-1. Our findings have improved our understanding of the role of miR-223 in TNBC and may contribute to TNBC treatment.

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<![CDATA[Study on the Cytotoxic, Genotoxic and Clastogenic Potential of Attalea phalerata Mart. ex Spreng. Oil Pulp In Vitro and In Vivo Experimental Models]]> https://www.researchpad.co/article/5989da58ab0ee8fa60b8f520

Attalea phalerata Mart. ex Spreng. (Arecaceae), popularly known as “bacuri”, is used in Brazilian folk medicine. Its oil is used orally to relieve pulmonary congestion and joint pain. In topical applications, it is applied as an effective hair tonic and anti-dandruff. The in natura pulp and its nuts are used as food because of its nutritional value. Despite its use in folk medicine, there is a lack of data regarding its in vivo/in vitro cytotoxic/genotoxic and clastogenic effects. Therefore, in this study, we evaluated the cytotoxic, genotoxic and clastogenic effects of Attalea phalerata Mart. ex Spreng. oil (APMO) in vitro and in vivo. For the analysis of cytotoxic potential, the Artemia salina and MTT (3-(4,5-dimethizzol-zyl)-2,5-diphenyltetrazolium bromide) assays were performed. Possible cytotoxic, genotoxic and clastogenic effects of APMO intake were determined by performing the comet and micronucleus assays. Male and female Wistar rats were orally treated with doses of 125, 250, 500 or 1000 mg.kg-1 of the APMO daily for 28 consecutive days (four weeks). The results showed that the APMO did not induce cell death in the experiments of Artemia salina and MTT, indicating that it has no cytotoxicity. The APMO did not cause significant damage to the DNA of the rats in the four doses used when compared to the negative control group (saline + Tween® 80). The APMO did not present any significant increase in micronucleated polychromatic erythrocytes (MNPCEs) for the four tested doses. When compared to the positive control group, all groups (comet and micronucleus tests) were statistically different. These data suggest that the administration of Attalea phalerata Mart oil. ex Spreng does not cause cytotoxicity, genotoxicity and clastogenicity in experimental models in vitro and in vivo following oral administration in this study.

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<![CDATA[Effects of Ranolazine on Astrocytes and Neurons in Primary Culture]]> https://www.researchpad.co/article/5989d9f3ab0ee8fa60b6f259

Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.

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<![CDATA[2-Phenylnaphthalene Derivatives Inhibit Lipopolysaccharide-Induced Pro-Inflammatory Mediators by Downregulating of MAPK/NF-κB Pathways in RAW 264.7 Macrophage Cells]]> https://www.researchpad.co/article/5989db19ab0ee8fa60bcdd2f

The anti-inflammatory pharmacological effect of eight 2-phenylnaphthalenes (PNAP-1PNAP-8) on lipopolysaccharide (LPS)-induced RAW 264.7 (a mouse cell line) was investigated. Among them, 6,7-dihydroxy-2-(4′-hydroxyphenyl)naphthalene (PNAP-6) and 2-(4′-aminophenyl)-6,7-dimethoxynaphthalene (PNAP-8) exhibited the best anti-inflammatory activity in this study. PNAP-6 and PNAP-8 not only significantly decreased the expression of inducible nitric oxide synthase and cyclooxygenase-II, but also inhibited the production of nitric oxide, interleukin-6, and tumor necrosis factor-α in LPS stimulated cells. Moreover, PNAP-6 and PNAP-8 inhibited nuclear factor (NF)-κB activation by decreasing the degradation of IκB and nuclear translocation of NF-κB subunit (p65). In addition, PNAP-6 and PNAP-8 also attenuated the phosphorylation of ERK, p38, and JNK. These results suggest that PNAP-6 and PNAP-8 exert anti-inflammatory activities by down regulating NF-κB activation and the mitogen-activated protein kinase signaling pathway in LPS-stimulated Raw 264.7 cells. This is the first study demonstrating that PNAPs can inhibit LPS-induced pro-inflammatory mediators in macrophages cells.

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<![CDATA[Knockdown of Mediator Complex Subunit 19 Suppresses the Growth and Invasion of Prostate Cancer Cells]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbb68

Prostate cancer (PCa) is one of the most common cancers in elderly men. Mediator Complex Subunit 19 (Med19) is overexpressed and plays promotional roles in many cancers. However, the roles of Med19 in PCa are still obscure. In this study, by using immunohistochemical staining, we found higher expression level of Med19 in PCa tissues than in adjacent benign prostate tissues. We then knocked down the Med19 expression in PCa cell lines LNCaP and PC3 by using lentivirus siRNA. Cell proliferation, anchor-independent growth, migration, and invasion were suppressed in Med19 knockdown PCa cells. In nude mice xenograft model, we found that Med19 knockdown PCa cells formed smaller tumors with lower proliferation index than did control cells. In the mechanism study, we found that Med19 could regulate genes involved in cell proliferation, cell cycle, and epithelial-mesenchymal transition, including P27, pAKT, pPI3K, IGF1R, E-Cadherin, N-Cadherin, Vimentin, ZEB2, Snail-1 and Snail-2. Targeting Med19 in PCa cells could inhibit the PCa growth and metastasis, and might be a therapeutic option for PCa in the future.

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<![CDATA[Glucose Uptake Activities of Bis (2, 3-Dibromo-4, 5-Dihydroxybenzyl) Ether, a Novel Marine Natural Product from Red Alga Odonthaliacorymbifera with Protein Tyrosine Phosphatase 1B Inhibition, In Vitro and In Vivo]]> https://www.researchpad.co/article/5989db36ab0ee8fa60bd3339

Background and Aims

Protein tyrosine phosphatase 1B (PTP1B) is a novel therapeutic target for type-2 diabetes, which negatively regulates the insulin signaling transduction. Bis (2, 3-dibromo-4, 5-dihydroxybenzyl) ether (BDDE), a novel bromophenol isolated from the Red Alga, is a novel PTP1B inhibitor. But the anti-diabetic effects are not clear. In the present study, we evaluated the in vitro and in vivo antidiabetic effects of BDDE.

Methods

The insulin-resistant HepG2 cells were used to evaluate the in vitro antidiabetic effects of BDDE. MTT assay was used to determine the safety concentrations in HepG2 cells. Glucose assay kit was used to check glucose uptake after treated with BDDE. Western blotting assay was used to explore the potent mechanisms. The db/db mice were used to evaluate the in vivo antidiabetic effects of BDDE. Body weight, blood glucose, Glycated hemoglobin (HbA1c), lipid profile, and insulin level were checked at the respective time points. Gastrocnemii were dissected and used to analyze the PTP1B and insulin receptor β (IRβ) expression.

Results

BDDE increased the insulin-resisted glucose uptake in HepG2 cells. BDDE also decreased the expression of PTP1B and activated the substrates and downstream signals in insulin signal pathway, such as IRβ, insulin receptor substrate-1/2 (IRS1/2), phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB/Akt). In the db/db mice model, BDDE significantly decreased the blood glucose, HbA1c and triglyceride (TG) levels. BDDE also decreased the expression of PTP1B and activated the phosphorylation of IRβ in gastrocnemii. Moreover, BDDE at high doses downregulated the body weight without affecting food and water intake.

Conclusion

Our results suggest that BDDE as a new PTP1B inhibitor improves glucose metabolism by stimulating the insulin signaling and could be used in the treatment of type-2 diabetes mellitus.

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<![CDATA[Utilization of Molecular Dynamics Simulation Coupled with Experimental Assays to Optimize Biocompatibility of an Electrospun PCL/PVA Scaffold]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcc04

The main focus of this study is to address the possibility of using molecular dynamics (MD) simulation, as a computational framework, coupled with experimental assays, to optimize composite structures of a particular electrospun scaffold. To this aim, first, MD simulations were performed to obtain an initial theoretical insight into the capability of heterogeneous surfaces for protein adsorption. The surfaces were composed of six different blends of PVA (polyvinyl alcohol) and PCL (polycaprolactone) with completely unlike hydrophobicity. Next, MTT assay was performed on the electrospun scaffolds made from the same percentages of polymers as in MD models to gain an understanding of the correlation between protein adsorption on the composite surfaces and their capability for cell proliferation. To perform simulations, two ECM (extracellular matrix) protein fragments, namely, collagen type I and fibronectin, two essential proteins for initial cell attachment and eventual cell proliferation, were considered. To evaluate the strength of protein adsorption, adhesion energy and final conformations of proteins were studied. For MTT analysis, different blends of PCL/PVA electrospun scaffolds were prepared, on which endothelial cells were cultured for one week. Theoretical results indicated that the samples with more than 50% of PCL significantly represented stronger protein adsorption. In agreement with simulation results, experimental analysis also demonstrated that the more hydrophobic the surface became, the better initial cell attachment and cell proliferation could be achieved, which was particularly better observed in samples with more than 70% of PCL.

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