Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide. It has been reported that histone demethylases are involved in the carcinogenesis of certain types of tumors. Here, we studied the role of one of the histone lysine demethylases, plant homeodomain finger protein 8 (PHF8), in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). Using short hairpin RNA via lentiviral infection, we established stable ESCC cell lines with constitutive downregulation of PHF8 expression. Knockdown of PHF8 in ESCC cells resulted in inhibition of cell proliferation and an increase of apoptosis. Moreover, there were reductions of both anchorage-dependent and -independent colony formation. In vitro migration and invasion assays showed that knockdown of PHF8 led to a reduction in the number of migratory and invasive cells. Furthermore, downregulation of PHF8 attenuated the tumorigenicity of ESCC cells in vivo. Taken together, our study revealed the oncogenic features of PHF8 in ESCC, suggesting that PHF8 may be a potential diagnostic marker and therapeutic target for ESCC.
Mitochondria are a class of dynamic organelles that constantly undergo fission and fusion. Mitochondrial dynamics is governed by a complex molecular machinery and finely tuned by regulatory proteins. During cell injury or stress, the dynamics is shifted to fission, resulting in mitochondrial fragmentation, which contributes to mitochondrial damage and consequent cell injury and death. Emerging evidence has suggested a role of mitochondrial fragmentation in the pathogenesis of renal diseases including acute kidney injury and diabetic nephropathy. A better understanding of the regulation of mitochondrial dynamics and its pathogenic changes may unveil novel therapeutic strategies.
The recent reports of resistance in Plasmodium falciparum to artemisinin derivatives and their partner drugs demand intensive studies toward understanding the molecular mechanisms of resistance. In this study, we examined the in vitro susceptibility of 63 P. falciparum field isolates collected from the China-Myanmar border area to chloroquine (CQ) and piperaquine (PPQ). Parasite isolates remained highly resistant to CQ, with the geometric mean 50% inhibitory concentration (IC50) of 252.7 nM and a range of 51.9 to 1,052.0 nM. In comparison, these parasites had a geometric mean IC50 of 28.4 nM for PPQ, with a fairly wide range of 5.3 to 132.0 nM, suggesting that certain parasite isolates displayed relatively high levels of resistance to PPQ. Interestingly, within the 4 years of study, the parasites exhibited a continuous decline in susceptibilities to both CQ and PPQ, and there was a significant correlation between responses to CQ and PPQ (Pearson correlation coefficient = 0.79, P < 0.0001). Consistent with the CQ-resistant phenotype, all parasites carried the pfcrt K76T mutation, and most parasites had the CVIET type that is prevalent in Southeast Asia. In contrast, pfmdr1 mutations were relatively rare, and no gene amplification was detected. Only the pfmdr1 N1042D mutation was associated with resistance to CQ. For the pfmrp1 gene, four substitutions reached relatively high prevalence of >22%, and the I876V mutation was associated with reduced sensitivity to CQ. However, we could not establish a link between PPQ responses and the polymorphisms in the three genes associated with quinoline drug resistance.
Currently, cartilage repair remains a major challenge for researchers and physicians due to its limited healing capacity. Cartilage regeneration requires suitable cells; these must be easily obtained and expanded, able to produce hyaline matrix with proper mechanical properties, and demonstrate sustained integration with native tissue. At present, there is a wide variety of possible cell sources for cartilage regeneration; this review explores the diversity of sources for cartilage forming cells and the distinctive characteristics, advantages, limitations, and potential applications of each cell source. We place emphasis on cell sources used for in vitro and clinical studies.
cell sources; cartilage regeneration; stem cells; chondrocytes
Acute myeloid leukemia (AML) is the most common malignant myeloid disorder of progenitor cells in myeloid hematopoiesis and exemplifies a genetically heterogeneous disease. The patients with AML also show a heterogeneous response to therapy. Although all-trans retinoic acid (ATRA) has been successfully introduced to treat acute promyelocytic leukemia (APL), it is rather ineffective in non-APL AML. In our present study, 1200 off-patent marketed drugs and natural compounds that have been approved by the Food and Drug Administration (FDA) were screened for anti-leukemia activity using the retrovirus transduction/transformation assay (RTTA). Furazolidone (FZD) was shown to inhibit bone marrow transformation mediated by several leukemia fusion proteins, including AML1-ETO. Furazolidone has been used in the treatment of certain bacterial and protozoan infections in human and animals for more than sixty years. We investigated the anti-leukemic activity of FZD in a series of AML cells. FZD displayed potent antiproliferative properties at submicromolar concentrations and induced apoptosis in AML cell lines. Importantly, FZD treatment of certain AML cells induced myeloid cell differentiation by morphology and flow cytometry for CD11b expression. Furthermore, FZD treatment resulted in increased stability of tumor suppressor p53 protein in AML cells. Our in vitro results suggest furazolidone as a novel therapeutic strategy in AML patients.
A susceptibility locus for tuberculosis, a re-emerging infectious disease throughout the world, was previously discovered to exist on chromosome 11p15. IFITM3 gene encoding for interferon inducible transmembrane protein 3, is located at 11p15. It acts as an effector molecule for interferon-gamma, which is essential for anti-tuberculosis immune response. In order to investigate the association between susceptibility to TB and genetic polymorphisms of the IFITM3 core promoter, a case-control study including 368 TB patients and 794 healthy controls was performed in Han Chinese children in northern China. The rs3888188 polymorphism showed significant association with susceptibility to TB. The rs3888188 G allele, acting recessively, was more frequent in TB patients (95% confidence interval: 1.08–1.56, Bonferroni P-value: 0.039). We further assessed the effect of rs3888188 polymorphism on IFITM3 transcription in vitro. As based on luciferase promoter assays, the promoter activity of haplotypes with rs3888188 G allele was lower than that of haplotypes with rs3888188 T allele. Moreover, peripheral-blood mononuclear cells carrying rs3888188 GG genotype showed a reduced IFITM3 mRNA level compared to cells carrying TT or GT genotype. In conclusion, rs3888188, a functional promoter polymorphism of IFITM3, was identified to influence the risk for pediatric TB in Han Chinese population.
Diabetic nephropathy is a well-known complication of diabetes and is a leading cause of chronic renal failure in the Western world. It is characterized by the accumulation of extracellular matrix in the glomerular and tubulointerstitial compartments and by the thickening and hyalinization of intrarenal vasculature. The various cellular events and signaling pathways activated during diabetic nephropathy may be similar in different cell types. Such cellular events include excessive channeling of glucose intermediaries into various metabolic pathways with generation of advanced glycation products, activation of protein kinase C, increased expression of transforming growth factor β and GTP-binding proteins, and generation of reactive oxygen species. In addition to these metabolic and biochemical derangements, changes in the intraglomerular hemodynamics, modulated in part by local activation of the renin-angiotensin system, compound the hyperglycemia-induced injury. Events involving various intersecting pathways occur in most cell types of the kidney.
hyperglycemia; advanced glycation products; protein kinase C; GTP-binding proteins; reactive oxygen species; hypertension; tubulointerstitial fibrosis
One of the major problems facing clinical nephrology currently throughout the world is an exponential increase in patients with end-stage renal disease (ESRD), which is largely related to a high incidence of diabetic nephropathy. The latter is characterized by a multitude of metabolic and signaling events following excessive channeling of glucose, which leads to an increased synthesis of extracellular matrix (ECM) glycoproteins resulting in glomerulosclerosis, interstitial fibrosis and ultimately ESRD. With the incidence of nephropathy at pandemic levels and a high rate of ESRD, physicians around the world must treat a disproportionately large number of diabetic patients with up-to-date innovative measures. In this regard, identification of genes that are crucially involved in the progression of diabetic nephropathy would enhance the discovery of new biomarkers and could also promote the development of novel therapeutic strategies. Over the last decade, we focused on the recent methodologies of high-throughput and genome-wide screening for identification of relevant genes in various animal models, which included the following: (1) single nucleotide polymorphism-based genome-wide screening; (2) the transcriptome approach, such as differential display reverse transcription polymerase chain reaction (DDRT-PCR), representational difference analysis of cDNA (cDNA-RDA)/suppressive subtractive hybridization, SAGE (serial analysis of gene expression) and DNA Microarray; and (3) the proteomic approach and 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectroscopic analysis. Several genes, such as Tim44 (translocase of inner mitochondrial membrane-44), RSOR/MIOX (renal specific oxidoreductase/myo-inositol oxygenase), UbA52, Rap1b (Ras-related GTPase), gremlin, osteopontin, hydroxysteroid dehydrogenase-3β isotype 4 and those of the Wnt signaling pathway, were identified as differentially expressed genes in kidneys of diabetic rodents. Functional analysis of these genes and the subsequent translational research in the clinical settings would be very valuable in the prevention and treatment of diabetic nephropathy. Future trends for identification of the biomarkers and therapeutic target genes should also include genome scale DNA/histone-methylation profiling, metabolomic approaches (e.g. metabolic phenotyping by 1H spectroscopy) and lectin microarray for glycan profiling along with the development of robust data-mining strategies.
Epithelial–mesenchymal transition (EMT) is thought to contribute to the progression of renal tubulointerstitial fibrosis. Norcantharidin (NCTD) is a promising agent for inhibiting renal interstitial fibrosis. However, the molecular mechanisms of NCTD are unclear. In this study, a unilateral ureteral obstruction (UUO) rat model was established and treated with intraperitoneal NCTD (0.1 mg/kg/day). The UUO rats treated with NCTD showed a reduction in obstruction-induced upregulation of α-SMA and downregulation of E-cadherin in the rat kidney (P<0.05). Human renal proximal tubule cell lines (HK-2) stimulated with TGF-β1 were treated with different concentrations of NCTD. HK-2 cells stimulated by TGF-β1 in vitro led to downregulation of E-cadherin and increased de novo expression of α-SMA; co-treatment with NCTD attenuated all of these changes (P<0.05). NCTD reduced TGF-β1-induced expression and phosphorylation of Smad2/3 and downregulated the expression of Snail1 (P<0.05). These results suggest that NCTD antagonizes tubular EMT by inhibiting the Smad pathway. NCTD may play a critical role in preserving the normal epithelial phenotype and modulating tubular EMT.
High-glucose-based peritoneal dialysis solution (PDS) is considered to be one of the primary causes for the increase of ionic permeability in peritoneum as detected by transmesothelial electrical resistance (TER) measurements and claudin-1 expression. However, the mechanism is not clear. The aim of this study is to test the hypothesis that high-glucose PDS induces hyperpermeability in human peritoneal mesothelial cell (HPMC) monolayer by mitochondrial respiratory chain complex III pathway.
HPMCs were cultured in a 1 : 1 mix of Dulbecco’s modified Eagle’s medium (DMEM) and PDS containing 1.5% and 4.25% glucose for 24 h. A 1 : 1 mixture of 160 mg/L glutathione and 4.25% glucose PDS was also added as an antioxidant group. TER measurement and immunostaining and western blot analysis of claudin-1 expression were examined for detection of permeability damage in HPMCs. MitoSOX™ Red staining and respiratory chain complexes’ activities were determined for detection of mitochondrial reactive oxygen species (ROS) production and mitochondrial complexes’ activities.
TER decreased in a time-and concentration-dependent manner after culture with high-glucose PDS for 24 h. Claudin-1 was also downregulated. Complex III activity was inhibited accompanied by increasing mitochondrial ROS generation. These changes were partially prevented by glutathione.
These findings demonstrate that mitochondrial respiratory complex III pathway has crucial importance in maintaining permeability of HPMCs, which might reveal a valuable target for novel therapies to fight hyperpermeability of peritoneum during the prolonged PD treatment.
peritoneal dialysis; human peritoneal mesothelial cell; respiratory chain complex; mitochondria; permeability
Widely used rod rotation and translation techniques for idiopathic scoliosis (IS) are effective in correcting spinal coronal deformity. Bilateral apical vertebral derotation technique by vertebral column manipulation (VCM) and vertebral coplanar alignment (VCA) technique are two strategies for three-dimensional (3D) correction for IS. The purpose of this study is to compare the post-surgical results and technical features of the bilateral apical vertebral derotation technique by VCM against the VCA technique in patients with Lenke type 1 IS.
Forty-eight patients with Lenke type 1 IS were enrolled in the present prospective clinical assay. They were divided into groups A (bilateral apical vertebral derotation technique by VCM, n=24) and B (VCA technique, n=24). Radiographic parameters measured before and after surgery included the Cobb angle, thoracic kyphosis, and apical vertebral rotation. Scoliosis Research Society (SRS)-22 scores were evaluated during the final follow-up. The differences in the demographics, surgical details, and radiographic measurements between the two groups were determined using a T test. The Mann–Whitney U test was used to evaluate the differences in the SRS-22 scores. A value of P<0.05 was considered statistically significant.
In the coronal plane, a significant difference was found in the correction rate of the major curve (group A: 84.8%, group B: 78.4%; P=0.045) and in the Cincinnati Correction Index between two groups (group A: 2.21, group B: 1.98; P=0.047). In the sagittal plane, no difference was found in the postoperative thoracic kyphosis between the two groups (P=0.328). In the transverse plane, no difference was found between the two groups in the correction rates of the rotation angle sagittal (P=0.298), rib hump (P=0.934), apical vertebral body-to-rib ratio (P=0.988), or apical rib spread difference (P=0.184). Patients underwent follow up for an average of 21.9 and 22.2 months in groups A and B, respectively. Results obtained at the final follow-up indicated no significant loss of correction. No differences were found in the SRS-22 scores between the two groups. No aortic or neurological complications were observed.
The 3D deformity of the spine was effectively corrected using the bilateral apical vertebral derotation technique by VCM and the VCA technique, and encouraging post-surgical results were obtained for patients with Lenke type 1 IS. The two techniques were effective in allowing 3D correctional force that was applied in different ways.
Idiopathic scoliosis; Three-dimensional correction; Bilateral apical vertebral derotation; Vertebral column manipulation; Vertebral coplanar alignment
To compare the efficacy and safety of TDR to that of the fusion for the treatment of lumbar degenerative disc disease (LDDD).
Randomized controlled trials comparing TDR with any other intervention for LDDD were acquired by a comprehensive search in PubMedCentral, MEDLINE, EMBASE, BIOSIS, ClinicalTrials.gov, and the FDA trials register. Methodologic quality was assessed and relevant data were retrieved, and appropriate meta-analysis was performed. Two review authors independently selected studies, extracted data, and assessed risk of bias. Results and upper bounds of confidence intervals were compared with predefined clinically relevant differences.
Six relevant randomized controlled trials (RCTs) involving 1,603 patients were identified and reported two year follow-up results. Patients in TDR group compared with lumbar fusion group demonstrated significant improvements in ODI, VAS scores and complication rates at the two year follow-up. Meanwhile, except for operating time in anterior group, intra-operative blood loss, operating time in posterior group, and reoperation rate were without clinical significance between the two groups. In addition, the range of motion (ROM) was maintained within normal ranges after TDR.
The results showed the TDR has significant safety and efficacy comparable to lumbar fusion at two year follow-up. Although superiority compared to fusion could not be proved, by comparing clinical symptoms relieved, motion preserved, and the low reoperation rate during long-term follow-up on TDR, TDR was considered safe and effective. Therefore, the authors suggest adopting TDR on a large scale; with failure of TDR, interbody fusion would be performed.
Metastatic melanoma of the spine usually occurs as vertebral metastatic melanoma or intramedullary spinal cord metastatic melanoma. The present study reports a case of easily misdiagnosed delayed metastatic intraspinal extradural melanoma of the lumbar spine. A 67-year-old female patient presented with lower back pain accompanied by progressive intermittent claudication. Magnetic resonance imaging (MRI) suggested compression of the lumbar spinal cord caused by an extradural mass. The mass showed T2-hypointensity, T1-hypointensity and slight enhancement following a gadolinium-contrast injection. The patient had been diagnosed with a vulvar melanoma 13 years previously and had also undergone a resection of this tumor. A current diagnosis of a lumbar stenosis resulting from hypertrophy of the ligamentum flavum was suspected. However during corrective surgery, a dark gray solid mass was observed. An L3 laminectomy and removal of the tumor was performed. The tumor was confirmed to be a malignant melanoma by histopathological investigation. The patient was treated with radiotherapy and immunotherapy. At the final 13-month follow-up, the patient showed no signs of recurrence. It may be concluded that an early diagnosis of metastatic melanoma was prevented by delayed metastasis, the location of the mass and its unusual appearance in MRI scans. In such cases, early surgical removal and an appropriate comprehensive treatment are critical for patient survival. These observations suggest that caution should be used in the diagnosis of similar cases.
metastatic melanoma; lumbar spine; extradural; delayed metastasis; lumbar stenosis; misdiagnosed
The Caenorhabditis elegans SH3 domain interactome was mapped and compared with the yeast SH3 interactome. Orthologous SH3 domain-mediated interactions are highly rewired, but the general function of the SH3 domain network is conserved between the two species
C. elegans Src homology 3 (SH3) domain interactome was mapped using stringent yeast two-hybrid, resulting in a total of 1070 interactions among 79 out of 84 worm SH3 domains and 475 proteins.SH3 domain binding specificities were profiled for 36 worm SH3 domains using peptide phage display.The yeast and worm SH3 domain interactomes are significantly enriched in endocytosis proteins, but the specific interactions mediated by orthologous SH3 domains are highly rewired.Using the worm SH3 interactome, we identified new endocytosis proteins in worm and human.
Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.
network evolution; phage display; protein interaction conservation; SH3 domains; yeast two-hybrid
The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes.
The cell membrane is a busy place, with cell-surface proteins continually added and removed according to the needs of the cell. Each protein extends a polypeptide tail into the cell cytoplasm. When a protein is to be removed from the cell surface, its tail recruits a protein complex known as the AP2 adaptor to the membrane. AP2 then recruits a coat protein called clathrin, which forms a spherical scaffold around the adaptor, the target protein and the surrounding membrane, enclosing them inside a vesicle that breaks off from the membrane and enters the cell.
Endocytosis is particularly common in neurons, which use it as a means of recycling proteins at synapses—the contact points between nerve cells. However, it is unclear whether synaptic-vesicle recycling also involves clathrin and AP2. To address this question, Gu et al. examined mutant nematode worms (C. elegans) in which the composition of AP2 had been altered.
AP2 has four subunits, called α, β2, μ2 and σ2, and Gu et al. produced worms that lack either the α- or μ2-subunit, or both. Few worms that lacked both subunits survived. Surprisingly, however, worms that lacked just one subunit were viable, despite previous evidence that AP2 requires all four subunits to be functional. Nevertheless, these single mutants produced 30% fewer synaptic vesicles compared to wild-type worms. To examine the consequences of both subunits being absent, Gu et al. rescued the double mutants by selectively expressing AP2 in their skin. These animals—which still lack AP2 in their nervous systems—produced 70% fewer synaptic vesicles than their wild-type counterparts.
The results show that AP2 does not need all four of its subunits and that it can exist as two semi-independent hemicomplexes. Moreover, Gu et al. show that C. elegans uses at least two endocytotic mechanisms (AP2-dependent and independent) to recycle vesicles and so maintain synaptic function.
apa-2; apm-2; synaptic vesicle endocytosis; AP2; C. elegans
One advantage of the nematode Caenorhabditis elegans as a model organism is its suitability for in vivo optical microscopy. Imaging C. elegans often requires animals to be immobilized to avoid movement-related artifacts. Immobilization has been performed by application of anesthetics or by introducing physical constraints using glue or specialized microfluidic devices. Here we present a method for immobilizing C. elegans using polystyrene nanoparticles and agarose pads. Our technique is technically simple, does not expose the worm to toxic substances, and allows recovery of animals. We evaluate the method and show that the polystyrene beads increase friction between the worm and agarose pad. We use our method to quantify calcium transients and long-term regrowth in single neurons following axotomy by a femtosecond laser.
Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products.
We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice.
Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.
Saccharomyces cerevisiae; Vaccine; Myostatin; Chromosomal integration
Plasmablastic lymphoma (PBL) is a rare aggressive B-cell lymphoproliferative disorder, which has been characterized by the World Health Organization as a new entity. Although PBL is most commonly seen in the oral cavity of human immunodeficiency virus (HIV)-positive patients, it can also be seen in extra-oral sites in immunocompromised patients who are HIV-negative. Here we present a rare case of PBL of the small intestine in a 55-year-old HIV-negative male. Histopathological examination of the excisional lesion showed a large cell lymphoma with plasmacytic differentiation diffusely infiltrating the small intestine and involving the surrounding organs. The neoplastic cells were diffusely positive for CD79a, CD138 and CD10 and partly positive for CD38 and epithelial membrane antigen. Approximately 80% of the tumor cells were positive for Ki-67. A monoclonal rearrangement of the kappa light chain gene was demonstrated. The patient died approximately 1.5 mo after diagnosis in spite of receiving two courses of the CHOP chemotherapy regimen. In a review of the literature, this is the first case report of PBL with initial presentation in the small intestine without HIV and Epstein-Barr virus infection, and a history of hepatitis B virus infection and radiotherapy probably led to the iatrogenic immunocompromised state.
Plasmablastic lymphoma; Small intestine; Human immunodeficiency virus; Differential diagnosis
Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies.
Targeted Therapy; Therapy Resistance; Cancer Stem Cells; Raf; Akt; PI3K; mTOR; AMPK; Metformin
Background. microRNA (miRNA, miR) are thought to interact with multiple mRNAs which are involved in the EMT process. But the role of miRNAs in peritoneal fibrosis has remained unknown. Objective. To determine if miRNA589 regulates the EMT induced by TGFβ1 in human peritoneal mesothelial cell line (HMrSV5 cells). Methods. 1. Level of miR589 was detected in both human peritoneal mesothelial cells (HPMCs) isolated from continuous ambulatory peritoneal dialysis (CAPD) patients' effluent and HMrSV5 cells treated with or without TGFβ1. 2. HMrSV5 cells were divided into three groups: control group, TGFβ1 group, and pre-miR-589+TGFβ1 group. The level of miRNA589 was determined by realtime PCR. The expressions of ZO-1, vimentin, and E-cadherin in HPMCs were detected, respectively. Results. Decreased level of miRNA589 was obtained in either HPMCs of long-term CAPD patients or HMrSV5 cells treated with TGFβ1. In vitro, TGFβ1 led to upregulation of vimentin and downregulation of ZO-1 as well as E-cadherin in HMrSV5 cells, which suggested EMT, was induced. The changes were accompanied with notably decreased level of miRNA589 in HMrSV5 cells treated with TGFβ1. Overexpression of miRNA589 by transfection with pre-miRNA589 partially reversed these EMT changes. Conclusion. miRNA589 mediates TGFβ1 induced EMT in human peritoneal mesothelial cells.
Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells.
Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1.
Conclusion and Implications
This investigation demonstrates that PI3-K/Akt activation is an important signaling in resveratrol-mediated activation of AMPK phosphorylation and SIRT1 expression, and inhibition of phosphorylation of CREB and MAPKs activation, proinflammatory mediators and cytokines production in response to LPS in RAW 264.7 cells.
To discover and develop novel natural compounds with therapeutic selectivity or that can preferentially kill cancer cells without significant toxicity to normal cells is an important area in cancer chemotherapy. Kushen, the dried roots of Sophora flavescens Aiton, has a long history of use in traditional Chinese medicine to treat inflammatory diseases and cancer. Kushen alkaloids (KS-As) and kushen flavonoids (KS-Fs) are well-characterized components in kushen. KS-As containing oxymatrine, matrine, and total alkaloids have been developed in China as anticancer drugs. More potent antitumor activities were identified in KS-Fs than in KS-As in vitro and in vivo. KS-Fs may be developed as novel antitumor agents.
Inflammation plays a major role in the destruction of cartilage in osteoarthritis (OA), with the interaction of multiple mediators, immune cells, fibroblasts and chondrocytes. Current 2D studies in vitro with cell lines, as well as animal models, are limited in terms of providing insight into pathogenic mechanisms related to the human system. Hence, an in vitro human 3D cartilage tissue system was established to study the impact of inflammatory mediators on chondrocytes and matrices as an initial approach to emulating early stages of OA. An in vitro 3D humancartilage tissue system was established by culturing primary chondrocytes in silk protein porous scaffolds up to 21 days in static culture, with and without cytokine (IL-1β and TNF-α) exposure or with the use of macrophage conditioned medium (MCM). To assess chondrocyte responses, transcript levels, histology and immunohistochemistry were used to assess changes in cell viability and in cartilage matrix composition, including collagen type II and aggrecan. Chondrocyte hypertrophy and apoptosis were assessed via collagen type X and caspase-3. RT-PCR revealed that the cytokines and the MCM regulated matrix-related gene expression of chondrocytes, but with different outcomes. For anabolic-encoding genes, MCM suppressed collagen type II and up-regulated aggrecan. In contrast, the cytokines suppressed aggrecan formation and had no effect on collagen type II. For catabolic-encoded genes, both cytokines and MCM upregulated MMP1, MMP3, MMP13 and ADAMTS4, with cytokines preferentially upregulating MMP13 and MCM upregulating ADMTS4. MCM down-regulated ADAMTS5. In addition, MCM stimulation led to hypertrophy and apoptosis of chondrocytes, outcomes not found with the cytokine treatment group. A decrease in aggrecan content with cytokines and MCM stimulation was found, while MCM resulted in greater reduction than the cytokine treatment. The results demonstrated that OA-like features, such as changes in matrix synthesis gene expression, increase of collagense gene expression and loss of aggrecan, were initiated within this 3D chrondrocyte human tissue system upon stimulation of the cultures with cytokines and MCM. MCM was a better inducer of immune-related features of OA, because besides the features found with cytokine stimulation, the MCM treatment also initiated collagen X expression and deposition and apoptosis of chondrocytes, important features of human OA. The results obtained with this new in vitro tissue model provide an initial step towards the development of an early stage OA system to allow for more systematic study and insight into the origins and outcomes with this disease.
Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.
When cargo is internalized from the cell surface by endocytosis, it enters a series of intracellular organelles called endosomes. Endosomes sort cargo, such that some cargos are sent to the lysosome for degradation, while others are recycled to the plasma membrane. Small GTPase proteins (Rabs) are well-known master regulators of endosome function. As cargo moves through the endosomal system, it must pass from the domain controlled by one Rab-GTPase to the domain controlled by another. Little is known about how transitions along the recycling pathway are controlled, or if Rab transitions are necessary for cargo recycling. Here we identified a group of proteins that act on recycling endosomes to deactivate the early acting GTPase RAB-5. Disruption of any of these proteins interferes with recycling. Our work shows that RAB-5 deactivation is important for cargo recycling, and it provides some of the first mechanistic insight into how changes in Rabs can be controlled during endocytic recycling. Importantly, several proteins that we found contribute to this recycling function have roles in other cellular processes, such as cell migration and the removal of cell corpses. Therefore our work also suggests that endocytic recycling could contribute to these processes in previously unsuspected ways.