Vimentin Gene Research Articles

Vimentin-Rab7a Pathway Mediates the Migration of MSCs and Lead to Therapeutic Effects on ARDS.

Acute respiratory distress syndrome (ARDS) is difficult to treat and has a high mortality rate. Mesenchymal stem cells (MSCs) have an important therapeutic effect in ARDS. While the mechanism of MSC migration to the lungs remains unclear, the role of MSCs is of great clinical significance. To this end, we constructed vimentin knockout mice, extracted bone MSCs from the mice, and used them for the treatment of LPS-induced ARDS. H&E staining and Masson staining of mouse lung tissue allowed us to assess the degree of damage and fibrosis of mouse lung tissue. By measuring serum TNF-α, TGF-β, and INF-γ, we were able to monitor the release of inflammatory factors. Finally, through immunoprecipitation and gene knockout experiments, we identified upstream molecules that regulate vimentin and elucidated the mechanism that mediates MSC migration. As a result, we found that MSCs from wild-type mice can significantly alleviate ARDS and reduce lung inflammation, while vimentin gene knockout reduced the therapeutic effect of MSCs in ARDS. Cytological experiments showed that vimentin gene knockout can significantly inhibit the migration of MSCs and showed that it changes the proliferation and differentiation status of MSCs. Further experiments found that vimentin's regulation of MSC migration is mainly mediated by Rab7a. Rab7a knockout blocked the migration of MSCs and weakened the therapeutic effect of MSCs in ARDS. In conclusion, we have shown that the Vimentin-Rab7a pathway mediates migration of MSCs and leads to therapeutic effects in ARDS.

Vimentin as a Cap of Invisibility: Proposed Role of Vimentin in Rabbit Hemorrhagic Disease Virus (RHDV) Infection.

Vimentin is an intermediate filament, a cytoskeleton protein expressed mainly in cells of mesenchymal origin. Increasing evidence indicates that vimentin could play a key role in viral infections. Therefore, changes in tissue and extracellular vimentin expression and associated signal trails may determine/protect the fate of cells and the progression of disease caused by viral infection. Rabbit hemorrhagic disease virus (RHDV), genotype GI.1, is an etiological agent that causes a severe and highly lethal disease—RHD (rabbit hemorrhagic disease). This article evaluates the gene and protein expression of vimentin in the tissues (liver, lungs, spleen, and kidneys) and serum of rabbits experimentally infected with two RHDV variants (GI.1a). The VIM mRNA expression levels in the tissues were determined using reverse transcription quantitative real-time PCR (RT-qPCR). In addition, the amount of vimentin protein in the serum was analyzed by an ELISA test. We observed significantly elevated expression levels of VIM mRNA and protein in the liver and kidney tissues of infected rather than healthy rabbits. In addition, VIM mRNA expression was increased in the lung tissues; meanwhile, we observed only protein-enhanced vimentin in the spleen. The obtained results are significant and promising, as they indicate the role of vimentin in RHDV infection and the course of RHD. The role of vimentin in RHDV infection could potentially rely on the one hand, on creating a cap of invisibility against the intracellular viral spread, or, on the other hand, after the damage of cells, vimentin could act as a signal of tissue damage.

Effects of Rhizopus Nigricans Exopolysaccharide on Proliferation, Apoptosis, and Migration of Breast Cancer MCF-7 Cells and Akt Signaling Pathway

Objective. To study the effect of Rhizopus nigricans exopolysaccharide EPS1-1 on the proliferation, apoptosis, and migration of breast cancer MCF-7 cells. Methods. Human breast cancer MCF-7 cells were cultured in vitro and treated with different concentrations of EPS1-1. The effect of EPS1-1 on cell proliferation was tested by the CCK-8 experiment, and the effect of EPS1-1 on cell apoptosis was determined by flow cytometry. And the scratch test was used to detect the impact of EPS1-1 on cell migration. Western blot then was used to measure the expression changes of related proteins in the Akt signaling pathway. Results. Compared with the control group, treatment with EPS1-1 significantly reduced the proliferation, migration, and invasion ability of MCF-7 cells and promoted the apoptosis of MCF-7 cells in a dose-dependent manner. In terms of the underlying mechanism, EPS1-1 can significantly inhibit the phosphorylation of Akt at threonine 308 and serine 473 and cause the expression changes of downstream proliferation-related genes CCND1 and p21, apoptosis-related genes Bcl-2 and Bax, and migration-related genes Vimentin and E-cadherin in terms of their protein levels. Conclusion. EPS1-1 can inhibit the proliferation, migration, and invasion of breast cancer MCF-7 cells and promote the apoptosis of MCF-7 cells by inhibiting the activation of the Akt signaling pathway. Therefore, EPS1-1 can be used as a potential new drug or adjuvant drug for the treatment of breast cancer.

Detection of average methylation level of specific genes by binary-probe hybridization

We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.

Iodothyronine deiodinase 2 (DiO2) regulates trophoblast cell line cycle, invasion and apoptosis; and its downregulation is associated with early recurrent miscarriage

IntroductionTrophoblast development is a crucial event in placentation and pregnancy complications but its underlying mechanisms remain unclear. Thus, we aimed at investigating the role of DiO2 in trophoblast cell line decisions and assessing its placental villous expression in early recurrent miscarriage (ERM) patients. MethodsThe placental villous expression of DiO2 was determined with immunofluorescence. Cell proliferation was measured with the CCK8 kit while cell-cycle and apoptosis were studied with flow-cytometry. Cell migration and invasion were measured with wound-healing and transwell assays, respectively. Gene expression was then assessed with RT-qPCR and western blotting. ResultsDiO2 is expressed in the CTB, PCT, DCT and STB of the placenta. Its overexpression arrested trophoblast cell line proliferation at the G1 phase of the cell-cycle by downregulating cyclin-D1 and PCNA, while promoting apoptosis via increased caspase-3 activity and inhibition of the AKT and ERK1/2 signaling pathways. Also, it augmented trophoblast cell line migration and invasion via the upregulation of N-cadherin, vimentin, fascin-1, twist-1 and other epithelial-mesenchymal transition genes. DiO2 knockdown elicited the opposite effects. Surprisingly, each of these effects of DiO2 manipulation was not mediated by thyroid hormone metabolism. Assessment of the ERM placental villi revealed a downregulation of DiO2, N-cadherin, vimentin, fascin-1 and twist-1. The expression of E-cadherin remained unchanged in these placentae. DiscussionDuring placentation, DiO2 may inhibit trophoblast proliferation while facilitating their differentiation into an invasive phenotype; and that its downregulation may contribute to the shallow trophoblast invasion that precedes ERM. Hence, DiO2 is a potential therapeutic target against ERM.

Vimentin affects colorectal cancer proliferation, invasion, and migration via regulatedby activator protein 1.

Uncontrolled recurrence and metastasis are important reasons for the high mortality rate of malignant tumors. Vimentin is positively correlated with the degree of malignancy of cancer cells. Vimentin is also highly expressed in colorectal cancer (CRC) cells andplays a critical rolein the metastasis and prognosis of CRC. However, the molecular mechanism of vimentin in the progression of CRC is incompletely understood. Therefore, the most active regions (nucleotides: 785-1085 nt) of the vimentin promoter in CRC were identified using luciferase experiments. By transcription factor sequence search and mutation analysis, the activator protein 1 (AP-1) binding site in the region of 785-1085 nt was confirmed. The vimentin promoter activity was enhanced by overexpression of AP-1. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that the binding site was recognized by AP-1. By cell proliferation assay, colony-forming assay, scratch-wound assay, cell migration assay, and cell invasion assay, we demonstrated that the AP-1 overexpression increased CRC cell proliferation, migration, and invasion. However, when vimentin was knocked down by vimentin small hairpin RNA in the CRC cell of AP-1 overexpression, this trend disappeared. Animal experiments and immunohistochemistry showed that AP-1 promoted tumor growth by regulating the vimentin gene. In summary, AP-1 affected metastasis, invasion of CRC cells in vitro, and tumor growth in vivo by activating the vimentin promoter. This study might provide new insights into the molecular mechanisms of the development of CRC and provide potential therapeutic targets for CRC.

Antitumoral effects of Santolina chameacyparissus on Non-Small Cell Lung Cancer Cells

Santolina chameacyparissus (Santo) is an evergreen plant which is traditionally used for its anti-inflammatory effects in various diseases. In this study, we aimed to explore the effects of Santo in non-small cell lung cancer cells. We extracted volatile oil from the plant and evaluated cytotoxicity, apoptosis, and motility effects of the extract on two non-small cell lung cancer (NSCLC) cell lines; one is a patient derived and the other one is a commercially available A549 cells. We also identified its components via GC/MS and investigated possible targets of the major components of the plant using qPCR and docking studies. Cytotoxicity tests showed dose dependent cell killing activity and flow cytometry assays exposed apoptotic effects of Santo. The essential oil also remarkably decreased migration rate of A549 cells, therefore we evaluated the expression levels of epithelial to mesenchymal transition related genes E-cadherin and Vimentin ratio, ZEB1 and SNAIL and another motility related gene Ezrin. Santo did not change the expression of EMT related genes but decreased Ezrin levels. According to the results of the GS/MS analysis, Artemisia ketone and Camphor were identified as major molecules of the extract. Docking analysis also revealed that artemisia ketone, the major component of the Santo extract, potentially showed strong binding to the active site of ezrin protein and both artemisia ketone and camphor had ability to bind DNA. The results of the present study indicate that Santo and its components artemisia ketone and camphor are promising anti-cancer agents, and their potential in targeting DNA and oncogenic proteins in the lung cancer cells seems worth to focus on this plant in cancer related drug discovery science.

Pseudouridylate Synthase 7 Promotes Cell Proliferation and Invasion in Colon Cancer Through Activating PI3K/AKT/mTOR Signaling Pathway.

Colorectal cancer is commonly malignant tumor. Herein, we demonstrate that pseudouridylate synthase 7 (PUS7) is closely related to colon cancer. But the biological role of PUS7 in colon cancer is not known. The present study aims to investigate the effects of PUS7 in colon cancer clinical samples and cells and the related molecular mechanism. A profile data set was downloaded from the Cancer Genome Atlas database, which included data from colon cancer tissue samples and normal tissue samples. The top 200 differentially expressed genes were subsequently investigated by a protein-protein interaction (PPI) network. RT-PCR and western blot assays were used to determine gene expression levels. CCK8 assay, colony formation experiment, transwell and flow cytometry assay were used to determine cell viability, proliferation, invasion, and apoptosis, respectively. PUS7 is a key gene from the most significant module of the PPI network. PUS7 was upregulated in colon cancer tissues and cell lines. Moreover, PUS7 overexpression is significantly related to the poor survival rate for 60 colon cancer's patients. Cell proliferation and invasion was significantly reduced by PUS7 inhibition and promoted by PUS7 overexpression. The protein levels of cleaved caspase-3/9, c-myc, E-cadherin and vimentin genes were significantly regulated in colon cancer cells transfected with PUS7 interference or overexpression. PUS7 overexpression significantly upregulated the phosphorylation levels of PI3K, AKT and mTOR. The results of this study demonstrate thatPUS7 overexpression upregulates cell proliferation, invasion and inhibits cell apoptosis of colon cancer cells via activating PI3K/AKT/mTOR signaling pathway.

The Effect of EZH2 Inhibition through DZNep on Epithelial-Mesenchymal Transition Mechanism.

Although the molecular pathogenesis of hepatocellular carcinoma (HCC) is uncertain, it is known that the epithelial-mesenchymal transition (EMT) mechanism and epigenetic changes have an important role. This study was focused on evaluating the relationship of 3-Deazaneplanocin A (DZNep) with the EMT mechanism, which is a histone methyltransferase inhibitor on HCC and is also known as an enhancer of zeste homolog 2 (EZH2) inhibitor. Cell viability of HepG2 cells (HCC cell line) assessed for DZNep over 72 hours with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, colony-forming assay, apoptosis assay, RNA isolation, cDNA synthesis, and real-time PCR (RT-PCR) were performed to see the effect of DZNep on HepG2 cells. DZNep reduced cell proliferation for 72 hours, also significantly reduced colony formation in addition it increased the total apoptosis. DZNep on EZH2, E-cadherin, N-cadherin, and Vimentin (Vim) gene expressions was given different results by either decreasing or increasing the expressions. In this study, we observed a positive effect of DZNep on apoptosis and TIMP3 expression level and decreased colony formation. However, it gave complicated results with the level of gene expression E-cadherin and TIMP2, increase the level of Vim and MMP2 expression. Therefore, we think that further studies are necessary to clarify the role of DZNep.

The Improvement Effect of Sodium Ferulate on the Formation of Pulmonary Fibrosis in Silicosis Mice Through the Neutrophil Alkaline Phosphatase 3 (NALP3)/Transforming Growth Factor-β1 (TGF-β1)/α-Smooth Muscle Actin (α-SMA) Pathway.

BackgroundPneumoconiosis is a chronic progressive fibrotic interstitial pneumonia for which the pathogenesis and treatment remain unclear. Previous studies showed that sodium ferulate (SF) may have a therapeutic effect, and this study explored the mechanism underlying SF-related improvement.Material/MethodsIn this study, a silicosis mouse model and primary cultured mouse lung fibroblasts were established. Hematoxylin-eosin staining, western blot analysis, quantitative real-time polymerase chain reaction, and Masson staining were used to observe the lung injury, expression of vimentin, and the degree of pulmonary fibrosis. The extracted lung fibroblasts were identified by immunofluorescence. The expression of fibrosis-related genes encoding transforming growth factor-β1 (TGF-β1), neutrophil alkaline phosphatase 3 (NALP3), collagen-1, α-smooth muscle actin (α-SMA), and phosphorylated p38 (p-p38) and p38 proteins were detected by western blot. The effects of SF and the TGF-β pathway agonist SRI-011381 on cell proliferation and the expression of fibrosis-related protein in mouse lung fibroblasts were measured by Cell Counting Kit-8, immunofluorescence, and western blot as needed.ResultsSF reduced the lung lesions in silicosis mice and inhibited the expression of vimentin and fibrosis-related genes, while having no effect on body weight. Vimentin expression was positive in the extracted cells. In vitro experiments showed that SF inhibited the proliferation of lung fibroblasts and the expression of fibrosis-related proteins. In addition, SF partly reversed the opposite regulatory effect of SRI-011381 on lung fibroblasts.ConclusionsSF inhibited lung injury and fibrosis in silicosis mice through the NALP3/TGF-β1/α-SMA pathway.

Inhibition of gastric cancer cell growth, invasion and metastasis by tocotrienolic amide

Purpose: To investigate the effect of tocotrienolic amide on gastric cancer (GC) cell growth and metastasis, and the underlying mechanism of action.Methods: Gastric cancer (GC) cell lines MKN28 and NCI-N87 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin solution at 37 ˚C in a humidified atmosphere of 5 % CO2 and 95 % air. Cell invasion and migration were determined using Transwell and wound healing assays, respectively. Realtime quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used for the determination of changes in the levels of expression of Snail, E-cadherin, vimentin, and microRNA-195-5p (miR-195-5p). In vivo tumor growth inhibition was determined 45 days after establishment of GC xenografts in nude mice.Results: Treatment of MKN28 and NCI-N87 cells with tocotrienolic amide significantly and dosedependently reduced their invasiveness and migratory capacity (p < 0.05). It also significantly and dosedependently downregulated the mRNA and protein expressions of Snail and vimentin, but significantly upregulated E-cadherin expression (p < 0.05). The mRNA expression of miR-195-5p was significantly and dose-dependently upregulated in MKN28 and NCI-N87 cells treated with tocotrienolic amide, but was downregulated after transfection with miR-195-5p inhibitor (p < 0.05). Transfection of MKN28 and NCI-N87 cells with miR-195-5p inhibitor significantly and dose-dependently upregulated mRNA and protein expressions of Snail (p < 0.05). Moreover, treatment of GC mice with TCTA led to significant and dose-dependent reduction in tumor weight and volume (p < 0.05).Conclusion: These results suggest that tocotrienolic amide inhibits the growth and metastasis of GC cells by directly targeting Snail and vimentin genes, and thus can potentially be developed for the management of gastric cancer.
 Keywords: Gastric cancer, Metastasis, Protein expression, Tocotrienolic amide, Tumor volume

Mesenchymal Stem/Stromal-Like Cells from Diploid and Triploid Human Embryonic Stem Cells Display Different Gene Expression Profiles.

Background:hESCs-MSCs open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods:Herein, hESCs-MSCs were characterized by IF technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using RT-PCR technique. FACS was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results:MSCs were derived from diploid and triploid hESC lines using sequential 3D and 2D cultures and characterized with the specific markers. IF showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. The hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Conclusion:Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications.

Tramadol aggravates cardiovascular toxicity in a rat model of alcoholism: Involvement of intermediate microfilament proteins and immune-expressed osteopontin.

Tramadol and alcohol are among commonly abused drugs. Although there are potential dangers reported upon their mixing, there are no previous reports describing this mixture's effects on the cardiovascular system (CVS). The aim was to study the effects of mixed alcohol and tramadol on the CVS of adult male rats. Fifty rats were divided into four groups: control, tramadol-treated group, alcohol-treated, and coadministration groups. Tramadol caused a significant increases in creatine kinase-MB, troponin I, malondialdehyde, protein carbonyl, 8-hydroxy-2'-deoxyguanosine, and a significant decrease in total antioxidant capacitywith histological alterations in sections of the heart and aorta and a significant increase in the area% of collagen fibers while there was a nonsignificant difference in body weight, heart weight, heart weight/body weight ratio, lipid profile, tissue tumor necrosis factor-αand interferon-γ, intermediate microfilament proteins (IFPs) {desmin, vimentin, connexin43} gene expression, mean area% of elastic fibers in aortic tissue and osteopontin expression in cardiac and aortic tissue. Alcohol treatment caused a significant change in all the measured parameters and more damage in histological sections. The changes were highest in the coadministration group. There was a strong positive correlation between the area% of collagen fibers and vimentin gene expression, and the area% of osteopontin expression was positively correlated to connexin43 in cardiac and vascular tissue. Tramadol causes CVS injury mainly through oxidative stresses, while the alcohol effect is multifactorial; mixing both aggravates CVS injury. The study also highlights the role of IFPs and osteopontin-expression in inducing injury.

Vimentin regulation of autophagy activation in lung fibroblasts in response to lipopolysaccharide exposure in vitro.

BackgroundThe activation and assembly of the NLRP3 inflammasome is dependent on the interaction between NLRP3 and the intermediate filament protein vimentin in an acute respiratory distress syndrome (ARDS) model. We investigated the role of vimentin in this process using human fetal lung (HFL-1) fibroblasts with vimentin transfer genes or gene knockdown and lipopolysaccharide (LPS) intervention.MethodsHFL-1 cells [con-vector + LPS, vimentin-pCMV3 (VIM-pCMV3), con-siRNA, and vimentin siRNA (VIM-siRNA)] were treated with LPS. An oxidative stress damage assessment, apoptosis analysis, and quantification of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 by enzyme linked immunosorbent assay (ELISA) were performed. Immunoblotting was used to reveal the autophagy pathway.ResultsWe demonstrated that in response to LPS vimentin expression was lower in the HFL-1 cells with the vimentin gene knocked down. Specifically, an increase in oxidative stress, a decrease in mitochondrial membrane potential, or an increase in calcium ion permeability resulted in an increase in the fibroblast apoptosis rate. In addition, the inflammatory response after vimentin gene knockout was upregulated, as indicated by higher levels of TNF-a, IL-1β, IL-6, and IL-10. Importantly, the mechanism of suppression of vimentin in the lung fibroblasts was caused by a decrease in autophagy, an increase in mitochondrial membrane protein, and a decrease in mitochondrial function, which may contribute to the augmented cellular injury generated during the response to LPS.ConclusionsThis study provides insights into whether vimentin may interfere with the inflammatory cascade by activating the autophagy pathway of mitochondrial lung fibroblasts in the early stage of acute lung injury (ALI).

195 Development of a fetal porcine model for congenital viral infections in the developing CNS

To develop a porcine model for analysis of the effects of congenital viral infections on CNS and spinal (CSF) derived fetal neural precursor (NPC) and neural progenitor cells. Isolation of NPCs from the subventricular zone (SVZ) of the porcine brain or CSF derived from premature (day 104) and post-term (day 135) Cesarean-delivered piglets were plated in 12-well plates. Nonadherent plates were used to generate neurospheres with self-renewal capacity and for long term passages. After a 2-day incubation for neurosphere formation and removal of apoptotic debris, cells were seeded in various conditions including incubation in (1) 10% FBS, (2) Rock Inhibitor (RI)-specific inhibitor of apoptosis, (3) FBS and RI, and (4) Neurobasal media alone (control). After purification and identification of NPCs, cells were then infected with low MOI of clinically isolated first passage ZIKV HN16, then fixed and stained with Flavivirus-specific antibody (MAb 4G2) followed by FITC labeled secondary antibody staining visualization under fluorescence microscopy. Gene expression studies were also performed from RNA from ZIKV infected and mock infected samples to determine effects on genes related to NPC self-renewal and survival. The brain derived cells cultured for 4 days showed three distinct populations including NSCs, NPCs, and a mixed NSC/NPC population. After 21 days of culture, cells shifted from an NSC population toward later within the NPC lineage. Both brain and CSF cells showed relatively equal permissivity to ZIKV viral infection (Fig.1). Moreover, there was no difference in Vimentin gene expression in ZIKV infected cells, while SOX-2 and Nestin gene expression following ZIKV infection was significantly lower (p<0.05; Fig.2). The findings that Nestin expression is downregulated when comparing brain and CSF cells, particularly after ZIKA infection, is of interest since Nestin is postulated to play a role in axonal growth. This study demonstrated the ability to utilize porcine fetal NPCs in a safe and effective manner for the study of NPC development and effects of viral infections

Promoted inhibition of TLR4/miR-155/ NFkB p65 signaling by cannabinoid receptor 2 agonist (AM1241), aborts inflammation and progress of hepatic fibrosis induced by thioacetamide

The endocannabinoid system plays a pivotal role, whether it is promoting or dampening hepatic fibrosis. This study investigated the role of Cannabinoid receptor 2 (CB2) activation by the synthetic analog (AM1241) on revoking the progress of liver fibrosis. Thioacetamide (TAA) was used to induce liver fibrosis in rats for three weeks followed by its concurrent administration with AM1241 at two different doses for another three weeks. Markers for liver function and oxidative stress, hepatic TNF-α, IL-1β and IL-6, qRT-PCR expression of Toll like receptor 4 (TLR4), TGF-β1, α-SMA and microRNA-155 (miR-155) genes, Western blot for protein levels of Vimentin and E-cadherin, immunohistochemical expression of NFκB p65 and histopathology of liver tissue were all investigated. AM1241 administration significantly maintained liver function markers and decreased; malondialdehyde, Vimentin, TLR4, TGF-β1, α-SMA and miR-155 genes expression, NFκB p65 immune-expression and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Additionally, AM1241 significantly increased E-Cadherin level, GSH and SOD content. Histologically, AM1241 limited fibroplasia extension, and broke the itinerary of bridging fibrosis. In conclusion, activation of the CB2 receptors by AM1241 promoted liver regeneration and overrun the progression of liver fibrosis through; inhibition of TLR4/miR-155/NFκB p65 pathway, suppression of pro-inflammatory IL-6, IL-1β and TNF-α, reducing TGF-β1, α-SMA, Vimentin and up-regulating E-Cadherin.

L-carnitine suppresses cisplatin-induced renal injury in rats: impact on cytoskeleton proteins expression.

We designed this work to examine the curative role of L-carnitine (LCAR) in a rat model of cisplatin (CDDP)-induced kidney injury. We induced kidney injury in rats by a single intraperitoneal injection of 5mg/kg of CDDP. Fifteen days post injection, rats were orally supplemented with 354mg/kg of LCAR for another 15days. Kidney tissues were subjected to histo-biochemical analysis along with mRNA gene expression quantification for cytoskeleton proteins encoding genes (vimentin, nestin, and connexin 43) by real-time reverse transcription polymerase chain reaction. LCAR reversed CDDP-induced renal structural and functional impairments. LCAR significantly declined serum urea and creatinine concentrations, restored oxidant/antioxidant balance, reversed inflammation, and antagonized caspase 3-mediated apoptotic cell death in renal tissues. Moreover, LCAR effectively down-regulated cytoskeleton proteins mRNA levels, reflecting amelioration of CDDP-provoked podocyte injury. We concluded that LCAR has a favorable therapeutic utility against CDDP-induced kidney injury.

Inhibition of metastasis and suppression of pERK1/2 and pFAK expression by Solanum xanthocarpum crude extracts in human lung cancer cell line A549 in vitro

Solanum xanthocarpum is an important part of natural remedies for lung in Ayurveda. Lung cancer metastasis is a major challenge in the present time. Expressions and activation of focal adhesion kinase (FAK) and extracellular receptor kinases 1 and 2 (ERK1/2) are key checkpoints in cancer which lead to an invasion, epithelial-mesenchymal transition (EMT) and metastatic progression. In this study, the efficacy of the whole plant and root crude extracts prepared in ethyl acetate and chloroform on highly metastatic lung cancer cell line A549 was evaluated. The effect of crude extracts on cell migration was scrutinized using transwell migration assessment. The phosphorylated forms of proteins, pFAK and pERK1/2 levels, were estimated using ELISA. Zymography technique was utilized to evaluate the activities of MMP-2 and MMP-9 proteins. The EMT markers, vimentin and N-cadherin gene expressions were analyzed using semi-quantitative RT-PCR. The extracts significantly reduced single-cell migration. The expression levels of pFAK and pERK1/2 notably decreased and FAK and ERK1/2 remained unchanged. Moreover, gelatinase activity, vimentin and N-cadherin expression were also significantly reduced in treated cells compared to untreated cells. The crude extracts of S. xanthocarpum impaired the metastatic progression of A549 cells in vitro.

DCLK1 Inhibition Sensitizes Colorectal Cancer Cells to Radiation Treatment.

Colorectal cancer (CRC) is one of the most prevalent diagnosed cancers and a common cause of cancer-related mortality. Despite effective clinical responses, a large proportion of patients undergo resistance to radiation therapy. Therefore, the identification of efficient targeted therapy strategies would be beneficial to overcome cancer radioresistance. Doublecortin-like kinase 1 (DCLK1) is an intestinal and pancreatic stem cell marker that showed overexpression in a variety of cancers. The transfection of DCLK1 siRNA to ‎normal HCT-116 cells was performed, and then cells were irradiated with X-rays. The effects of DCLK1 inhibition on cell survival, apoptosis, cell cycle, DNA damage response (ATM and γH2AX proteins), epithelial-mesenchymal transition (EMT) related genes (vimentin, N‐cadherin, and E-cadherin), cancer stem cells markers (CD44, CD133, ALDH1, and BMI1), and β‐catenin signaling pathway (β‐catenin) were evaluated. DCLK1 siRNA downregulated DCLK1 expression in HCT-116 cells at both mRNA and protein levels (P <0.01). Colony formation assay showed a significantly reduced cell survival in the DCLK1 siRNA transfected group in comparison with the control group following exposure to 4 and 6 Gy doses of irradiation (P <0.01). Moreover, the expression of cancer stem cells markers (P <0.01), EMT related genes (P <0.01), and DNA repair proteins including pATM (P <0.01) and γH2AX (P <0.001) were significantly decreased in the transfected cells in comparison with the nontransfected group after radiation. Finally, the cell apoptosis rate (P <0.01) and the number of cells in the G0/G1 phase in the silencing DCLK1 group was increased (P <0.01). These findings suggest that DCLK1 can be considered a promising therapeutic target for the treatment of radioresistant human CRC.

Risk Factors and Molecular Study of Vimentin Gene (VIM), Associated with Female Breast Cancer in Khartoum, Sudan

<i>Background</i>: The aim of this study was to investigate the possible risk factors and mutations in <I>VIM</I> gene among Sudanese’s breast cancer women in Khartoum State. <i>Methods</i>: This case-control study involved 45 patients with breast cancer and 45controls. It was conducted across three hospitals and two laboratories in Khartoum State. A structural questionnaire was used to obtain data regarding age, family history, menarche, marriage, menopause, pregnancy, nulliparous and parous women, breast-feeding, use of fertility or contraceptive drugs and grade of the disease. DNA from patient and control tissues was extracted using extraction kits. PCR was conducted to amplify <I>VIM</I> gene using specific primers. PCR products were sequenced in order to detect the mutation in <I>VIM</I> gene. Data was analyzed using Pearson’s Chi-square tests to identify risk factors associated with breast cancer. <i>Results</i>: The study showed that the main risk factors associated with breast cancer were family history with first degree relative, menarche, irregularity of menarche, reproductive factors such as pregnancy, breast-feeding and nulliparous. DNA sequencing revealed no mutations in <I>VIM</I> gene associated with breast cancer in Sudanese women in Khartoum State. <i>Conclusion</i>: The association of other risk factors such as menopausal status, age of menopausal, oral contraceptive birth control and fertility hormones needs more illumination and further work. Other genes associated with breast cancer can be investigated.