Dioscin induces osteosarcoma cell apoptosis by upregulating ROS-mediated P38 MAPK signaling.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Many patients with osteosarcoma readily develop resistance to chemotherapy and have an extremely dismal prognosis. Dioscin, a saponin, is known to exhibit potent anticancer activities and induce cellular death of a variety of cancer types. However, the inhibitory effect of dioscin on osteosarcoma cells and its underlying mechanisms have not been fully elucidated. We investigated the responses of human U2-OS and MG63 osteosarcoma cells to dioscin with regard to proliferation, apoptosis, migration, and invasion, and studied the effect of dioscin on MAPK-related proteins by western blot analysis assays. Dioscin inhibited osteosarcoma cell proliferation, migration, and invasion. Moreover, it induced osteosarcoma cell apoptosis via reactive oxygen species (ROS)-dependent apoptotic signaling. N-acetylcysteine, a reactive oxygen species inhibitor, suppressed dioscin-induced apoptosis, indicating that ROS play an essential role in dioscin-induced apoptosis. Western blot analysis assays showed that p38 MAPK was upregulated after dioscin treatment, and that dioscin induced apoptosis by upregulating ROS-mediated p38 MAPK signaling. Our study suggests that dioscin possesses antitumor activities against human osteosarcoma cells, inhibits osteosarcoma cell proliferation, migration and invasion, and induces osteosarcoma cell apoptosis through upregulating ROS-mediated p38 MAPK signaling. This study may provide a new therapeutic strategy and potential clinical applications for the treatment of osteosarcoma.

Anatomical characteristics and potential gene mutation sites of a familial recurrent patellar dislocation.

BACKGROUND: Recurrent patellar dislocation is the result of anatomical alignment and imbalance of restraint of bone and soft tissue. We investigate the anatomical characteristics of the knee joint in a family of patients with recurrent patella dislocation, and to screen the possible pathogenic genes in this family by whole exome sequencing in 4 patients and 4 healthy subjects, so as to provide theoretical basis for the pathogenesis of this disease. METHODS: The data related to patella dislocation were measured by imaging data. The peripheral blood DNA of related family members was extracted for the whole exome sequencing, and then the sequencing results were compared with the human database. By filtering out synonymous variants and high-frequency variants in population databases, and then integrating single nucleotide non-synonymous variants of family members, disease-causing genes were found. RESULTS: All patients in this family have different degrees of abnormal knee anatomy, which is closely related to patella dislocation. The sequencing results of patients and normal persons in this patella dislocation family were compared and analyzed, and the data were filtered through multiple biological databases. Find HOXB9 (NM_024017.4:c.404A>G:p.Glu135Gly),COL1A1(NM_000088.3:c.3766G>A:p.Ala1256Thr),GNPAT(NM_014236.3:c1556A>G:p.Asp519Gly),NANS(NM_018946.3:c.204G>C:p.Glu68Asp),SLC26A2(NM_000112.3:c.2065A>T:p.Thr689Ser) are nonsynonymous variants (MISSENSE). Through Sanger sequencing, the identified mutations in HOXB9 and SLC26A2 genes were only present in samples from patients with recurrent patellar dislocation. CONCLUSIONS: The patients with recurrent patellar dislocation had markedly abnormal knee anatomy in this family. HOXB9 gene and SLC26A2 gene were found to be the possible pathogenic genes or related genes for patella dislocation.

Ganoderma lucidum Exerts an Anticancer Effect on Human Osteosarcoma Cells via Suppressing the Wnt/ß-Catenin Signaling Pathway.

Background: Current treatment of osteosarcoma is limited in part by side effects and low tolerability, problems generally avoided with traditional Chinese medicine. Ganoderma lucidum, a traditional Chinese medicine with antitumor effects, offers a potential alternative, but little is known about its molecular mechanisms in osteosarcoma cells. Objective: To investigate the effect of G lucidum on osteosarcoma cells and its mechanism. Methods: Osteosarcoma MG63 and U2-OS cells were treated with G lucidum, followed by assays for cell proliferation (Cell Counting Kit-8), colony formation, and apoptosis (Alexa Fluor 647-Annexin V/propidium iodide, flow cytometry). Migration and invasion of cells were assessed by wound healing and Transwell invasion assays, and the effect of G lucidum on Wnt/ß-catenin signal transduction was studied by real-time quantitative polymerase chain reaction, western blot, and dual-luciferase assay. Results:G lucidum inhibited the proliferation, migration, and invasion, and induced apoptosis of human osteosarcoma MG63 and U2-OS cells. Dual-luciferase assay showed that G lucidum suppressed the transcriptional activity of T-cell factor/lymphocyte enhancer factor in the Wnt/ß-catenin signaling pathway. Moreover, G lucidum blocked Wnt/ß-catenin signaling by inhibiting the Wnt co-receptor LRP5 and Wnt-related target genes, such as ß-catenin, cyclin D1, C-Myc, MMP-2, and MMP-9. At the same time, when Wnt/ß-catenin was inhibited, the expression of E-cadherin was upregulated. Conclusions: Our results suggest that G lucidum broadly suppresses osteosarcoma cell growth by inhibiting Wnt/ß-catenin signaling.

Cisplatin induces chemoresistance through the PTGS2-mediated anti-apoptosis in gastric cancer.

It has been proposed that the aberrant expressions of the classical apoptosis-related genes and the subsequent decrease of apoptosis contribute to the development of cisplatin resistance in gastric cancer. However, little is known about the correlation and the molecular regulation mechanisms of cisplatin and the apoptosis-related gene expressions. Herein, we first identified the expressions of the anti-apoptotic BCL2 and the prostaglandin-endoperoxide synthase-2 (PTGS2) genes, which were abundant in the gastric carcinoma and associated with poor patient survival, were closely related with the resistance against cisplatin. Further investigations revealed that PTGS2 served as an essential mediator involved in the developing process of the resistance against cisplatin via mediating the inhibition effects of cisplatin on BCL2 expression. Mechanistically, cisplatin induced PTGS2 expression through ROS/NF-κB pathway. In addition, PTGS2 mediated cisplatin-induced BCL2 expression and subsequent resistance to apoptosis via PGE2/EP4/MAPKs (ERK1/2, P38) axis. Analysis of the clinical specimens demonstrated that PTGS2 and BCL2 were positively correlated in human gastric cancer. Moreover, in the xenograft models, inhibition of PTGS2 by celecoxib significantly augmented the cytotoxic efficacy of cisplatin in the resistant gastric cancer via suppression of PTGS2 and BCL2 expressions regulated by ERK1/2 and P38 signal axis, suggesting PTGS2 might be employed as an adjunctive therapeutic target for reversal of the chemoresistance in a subset of cisplatin resistant gastric cancer.

Stenosing tenosynovitis : Evaluation of percutaneous release with a specially designed needle vs. open surgery.

PURPOSE: The purpose of this study was to evaluate the effectiveness of conventional open surgery and percutaneous release with a specially designed needle for treating stenosing tenosynovitis in terms of cure, relapse and complication rates. METHODS: In this study 89 fingers from 76 patients were randomly assigned and allocated to one of the treatment groups. A total of 37 patients were treated with open surgery in group 1 and 39 patients with percutaneous release using a specially designed needle in group 2. A patient-based 4-inch visual analogue scale (VAS), Quinnell grading (QG), disability of arm shoulder and hand (DASH) score and finger total joint range of motion (FTROM) score were evaluated before treatment and after 7, 30 and 180 days. When finger QG scores were equal or greater than 2 points at follow-up at 180 days this was defined as recurrence.. RESULTS: There were no significant differences between the two groups (P > 0.05) in terms of VAS, DASH and QG scores and the degree of FTROM. At 7 days all the data were significantly different (p < 0.05) compared with preoperative data, 30 days was significantly different (p < 0.05) compared with 7 days while at 180 days no significant differences could be found (p > 0.05) compared with 30 days. The recurrence rate in group 1 was 4.65% and 6.55% in group 2. CONCLUSION: The percutaneous release and open surgery methods displayed similar effectiveness regarding the cure and recurrence of trigger finger disorder. The use of a specially designed needle for release is a safe and reliable method.

Reliability, Validity, and Responsiveness of the Chinese Version of the Knee Injury and Osteoarthritis Outcome Score (KOOS) in Patients with Anterior Cruciate Ligament Reconstruction in Mainland China.

OBJECTIVE: The aim of this study was to perform a cross-cultural adaption of the KOOS into Chinese and to evaluate its psychometric properties in patients with anterior cruciate ligament reconstruction (ACL reconstruction) in mainland China. DESIGN: A cross-sectional study. SETTING: Patients completed the Chinese version of the KOOS and the SF-36 questionnaire three times. We evaluated the reliability, checked the validity, and assessed the responsiveness. PARTICIPANTS: A total of 42 patients who had undergone ACL reconstruction. MAIN OUTCOME MEASURES: The results of the questionnaire survey. RESULTS: The Chinese version of the KOOS was well accepted, with ideal test-retest reliability and internal consistency. The test-retest reliability was significant, with high ICC values ranging from 0.888 to 0.941. Additionally, we found that the internal consistency was adequate, with Cronbach's alpha coefficient ranging from 0.740 to 0.975. All a priori hypotheses were supported by a high correlation between the KOOS and SF-36. Furthermore, responsiveness was demonstrated since the ES and SRM between subscales following ACL reconstruction was found in the expected pattern. CONCLUSIONS: The Chinese version of the KOOS showed psychometric properties demonstrating acceptable reliability and validity similar to the original version. We conclude that the Chinese version is a reliable and valid instrument for research and clinical assessments of ACL reconstruction patients in mainland China.

Ligustrazine Exerts Cardioprotection in Animal Models of Myocardial Ischemia/Reperfusion Injury: Preclinical Evidence and Possible Mechanisms.

Ligustrazine (Lig) is one of the main effective components of Ligusticum Chuanxiong Hort, which possesses a variety of biological activities in the cardiovascular system. Here, we conducted a preclinical systematic review to investigate the efficacy of Lig for animal models of myocardial ischemia/reperfusion injury and its possible mechanisms. Twenty-five studies involving 556 animals were identified by searching 6 databases from inception to August 2017. The methodological quality was assessed by using Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist. All the data were analyzed using Rev-Man 5.3 software. As a result, the score of study quality ranged from 2 to 6 points. Meta-analyses showed Lig can significantly decrease the myocardial infarct size, cardiac enzymes and troponin compared with control (P < 0.01). The possible mechanisms of Lig for myocardial infarction are antioxidant, anti-inflammatory, anti-apoptosis activities and improving coronary blood flow and myocardial metabolism. In conclusion, the findings indicated that Lig exerts cardio protection through multiple signaling pathways in myocardial ischemia/reperfusion injury.

A Preclinical Systematic Review and Meta-Analysis of Astragaloside IV for Myocardial Ischemia/Reperfusion Injury.

Astragaloside IV (AS-IV), the major pharmacological extract from Astragalus membranaceus Bunge, possesses a variety of biological activities in the cardiovascular systems. Here, we aimed to evaluate preclinical evidence and possible mechanism of AS-IV for animal models of myocardial ischemia/reperfusion (I/R) injury. Studies of AS-IV in animal models with myocardial I/R injury were identified from 6 databases from inception to May, 2018. The methodological quality was assessed by using CAMARADES 10-item checklist. All the data were analyzed using Rev-Man 5.3 software. As a result, 22 studies with 484 animals were identified. The quality score of studies ranged from 3 to 6 points. Meta-analyses showed AS-IV can significantly decrease the myocardial infarct size and left ventricular ejection fraction, and increase shortening fraction compared with control group (P < 0.01). Significant decreasing of cardiac enzymes and cardiac troponin and increasing of decline degree in ST-segment were reported in one study each (P < 0.05). Additionally, the possible mechanisms of AS-IV for myocardial I/R injury are promoting angiogenesis, improving the circulation, antioxidant, anti-inflammatory and anti-apoptosis. Thus, AS-IV is a potential cardioprotective candidate for further clinical trials of myocardial infarction.

Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways.

BACKGROUND/AIMS: Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro. METHODS: BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting. RESULTS: Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin. CONCLUSION: These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.

Chinese Herbal Medicine for Vascular Dementia: A Systematic Review and Meta-Analysis of High-Quality Randomized Controlled Trials.

BACKGROUND: Vascular dementia (VaD) is the second common form of dementia and Chinese herbal medicine (CHM) has been used for aging-related disorders for thousands of years. However, there is still a lack of scientific evidence using CHM for VaD. OBJECTIVE: To conduct a systematic review to assess the current evidence available for the effectiveness and safety of CHM for VaD. METHODS: Six databases were searched for high-quality randomized-controlled clinical trials that met the requirements of at least 4 of the 7 domains of the Cochrane risk of bias tool from their inception to February 2017. RevMan 5.3 was applied for data analysis. RESULTS: Forty studies with 42 comparisons and 3,572 individuals were included. The studies investigated the CHM versus placebo (n = 4), CHM versus western conventional treatment (WCT) (n = 36), and CHM plus WCT versus WCT (n = 2). Meta-analysis showed that CHM for VaD could improve Mini-Mental State Examination (MMSE), the Activities of Daily Living, Hasegawa's dementia scale, and clinical effective rate but had statistically similar effect based on Blessed Behavior Scale (BBS) outcome when compared with WCTs. When compared with placebo, CHMs were more beneficial in improving MMSE but showed no significant difference in BBS scores. CHM as adjuvant therapy exerted an additive anti-VaD benefit on MMSE scores. The participants of CHM group had fewer adverse events than that of the placebo group or WCT group. CONCLUSION: The findings of the present study support, at least to an extent, that CHM can be recommended for routine use for treatment of VaD.

RACK1 Silencing Induces Cell Apoptosis and Inhibits Cell Proliferation in Hepatocellular Carcinoma MHCC97-H Cells.

This study aimed to explore the effects of RACK1 gene silencing on the apoptosis and proliferation of hepatocellular carcinoma (HCC) MHCC97-H cells. After transfecting MHCC97-H cells with siRNA, RACK1 gene silencing model was established. The cells were divided into blank group, siRNA group and empty plasmid group, respectively. The mRNA and protein expressions of RACK1, cyclin D1 and BAX were determined by qRT-PCR and Western blotting. CCK-8 assay, flow cytometry and FITC-Annexin V/PI staining were used to determine cell viability, cell cycle and cell apoptosis, respectively. The results of qRT-PCR and Western blotting suggested that when compared with the blank group and the empty plasmid group, the mRNA and protein expressions of RACK1 and Cyclin D1 decreased significantly while the mRNA and protein BAX expressions increased substantially in the siRNA group (all P < 0.05). The results of CCK-8 assay revealed that the siRNA group exhibited significantly lower cell viability when compared with the blank group and the empty plasmid group (both P < 0.05); and the cell viability in the siRNA group decreased gradually with the increase of time. The results of flow cytometry and FITC-Annexin V/PI staining indicated that when compared with the blank group and the empty plasmid group, the proportion of cells in S phase was markedly lower and the apoptosis rate was significantly higher in the siRNA group (both P < 0.05). Our study suggests that inhibition of RACK1 could suppress cell proliferation and induce apoptosis in HCC MHCC97-H cells.

All-trans-retinoic acid activates SDF-1/CXCR4/ROCK2 signaling pathway to inhibit chondrogenesis.

Recent studies have indicated that ATRA inhibits chondrogenesis and can lead to congenital clubfoot (CCF). The molecular mechanism of ATRA-induced chondrogenesis is not clear. As RhoA/ROCK and SDF-1/CXCR4 signaling play important molecular roles for a variety of cellular processes, we hypothesized that RhoA/ROCK2 and SDF-1/CXCR4 signaling are involved in ATRA-induced chondrogenesis in rat embryo hind limb bud mesenchymal cells (rEHBMCs). We found that ATRA dose-dependently inhibits proliferation and expression of chondrogenic transcription factors (SOX9 and COL2A1) in rEHBMCs. In contrast, ATRA increases the expression of ROCK2, SDF-1 and CXCR4. Pharmacological inhibition of ROCK signaling and SDF-1/CXCR4 signaling by Y27632 and AMD3100, respectively, resulted in elevated expression of SOX9 and COL2A1. In addition, we found that disturbing SDF-1/CXCR4 signaling by AMD3100 decreases ATRA-induced ROCK2 expression. In vivo studies we also confirm that SOX9 expression of early-stage cartilage progenitors in the proliferative zone and COL2A1 expression in prehypertrophic chondrocytes are decreased in ATRA-treated rat embryo hind limb. Together, these results show that ATRA activates SDF-1/CXCR4/ROCK2 signaling to inhibit chondrogenesis to lead to CCF by suppressing differentiation through down-regulation of SOX9 and COL2A1 expression in rat embryo hind limb bud mesenchymal cells.

Antitumor activity of resveratrol against human osteosarcoma cells: a key role of Cx43 and Wnt/ß-catenin signaling pathway.

Osteosarcoma is a high-grade bone sarcoma with strong invasive ability. However, treatment with traditional chemotherapeutic drugs is limited by low tolerability and side effects. Resveratrol has been reported previously to have selective antitumor effect on various tumor cells while little is known about its effects and underlying mechanism in osteosarcoma biology. In this study, we found that resveratrol inhibits proliferation and glycolysis, induces apoptosis and reduces the invasiveness of U2-OS cells in vitro. After treatment with resveratrol, the expression of related Wnt/ß-catenin signaling pathway target genes, such as ß-catenin, c-myc, cyclin D1, MMP-2 and MMP-9, was downregulated and an increased E-cadherin level was observed as well. Additionally, the dual luciferase assay results also indicated that resveratrol suppressed the activity of Wnt/ß-catenin signaling pathway. Interestingly, we noticed that the expression of connexin 43 (Cx43) increased with the prolongation of resveratrol treatment time. To further investigate the relationship between Cx43 and the Wnt/ß-catenin signaling pathway in osteosarcoma, we used lentiviral-mediated shRNA to knockdown the expression of Cx43. Knockdown of Cx43 activated the Wnt/ß-catenin signaling pathway, promoted proliferation and invasion, and inhibited apoptosis of U2-OS cells. Taken together, our results demonstrate that the antitumor activity of resveratrol against U2-OS cells in vitro occurs through up-regulating Cx43 and E-cadherin, and suppressing the Wnt/ß-catenin signaling pathway. Moreover, Cx43 expression is negatively related to the activity of the Wnt/ß-catenin pathway in U2-OS cells.

Human Basic Fibroblast Growth Factor Inhibits Tau Phosphorylation via the PI3K/Akt-GSK3ß Signaling Pathway in a 6-Hydroxydopamine-Induced Model of Parkinson's Disease.

BACKGROUND: Basic fibroblast growth factor (bFGF) has been increasingly investigated due to its neuroprotection in neurodegenerative disorders. Because there are still no cures for any of these disorders, it is crucial to identify new therapeutic targets and screen potential drugs. The increased phosphorylation of tau at Ser396 leads to intracellular tau accumulation, which forms neurofibrillary tangles in Parkinson's disease (PD). In this study, neuroprotection by bFGF was observed, and the mechanisms related to its regulation of phosphorylated tau were investigated. METHODS: bFGF-loaded liposome carriers were intranasally administered to rats. The neuroprotective effects of bFGF were assessed in a PD model induced by 6-hydroxydopamine (6-OHDA) in vivo and in vitro. The phosphorylation of tau was measured, and the PI3K/Akt-GSK3ß signaling pathway was investigated. RESULTS: Our study demonstrated that liposomes markedly assisted in the delivery of bFGF to the striatum and substantia nigra of rats and enhanced the neuroprotective effects of bFGF on dopaminergic neurons. bFGF treatment significantly ameliorated the behavioral deficits induced by 6-OHDA, rescued the loss of tyrosine hydroxylase-positive neurons and increased the number of Nissl bodies. bFGF reduced the phosphorylation of tau and GSK3ß and increased the phosphorylation of PI3K/Akt. CONCLUSION: Liposomes markedly assisted in the delivery of bFGF to the brain and enhanced the neuroprotective effects of bFGF by inhibiting the phosphorylation of tau. bFGF down-regulated the phosphorylation of tau by increasing the phosphorylation of GSK3ß via the PI3K/Akt signaling pathway. These findings provide a new vision of bFGF as a potential therapy for PD.

Assessment of the embryotoxicity of four Chinese herbal extracts using the embryonic stem cell test.

Rhizoma Atractylodes macrocephala, Radix Isatidis, Coptis chinensis and Flos Genkwa are common herbal remedies used by pregnant woman in China. In this study, their potential embryotoxicity was assessed using the embryonic stem cell test (EST) and a prediction model. The potential embryotoxicity of the herbs was based on three endpoints: the concentrations of the compounds that inhibited the proliferation of 50% of embryonic stem cells (ESCs) (IC50ES), the concentrations that inhibited 50% of 3T3 cells (IC503T3), and the concentrations that inhibited the differentiation of 50% of ESCs (ID50ES). The results revealed that Rhizoma Atractylodes macrocephala and Radix Isatidis are non-embryotoxic compounds. Coptis chinensis extracts appeared to demonstrated weak embryotoxicity, and Flos Genkwa exhibited strong embryotoxicity. These results may be useful in guiding the clinical use of these herbs and in expanding the application of the EST to the field of traditional Chinese medicine.

Detecting the developmental toxicity of bFGF in the embryonic stem cell test using differential gene expression of differentiation-related genes.

Basic fibroblast growth factor (bFGF) is a mitogenic cytokine that can stimulate mesoderm-and neuroectoderm-originated cell proliferation. This study was performed to investigate the effects of bFGF on cell differentiation and the expression of specific markers at different embryonic developmental stages. We firstly evaluated the embryotoxic potential of bFGF in vitro using a modified EST protocol. Sequentially, we further investigated how bFGF impact the different tissue-special genes and proteins expressions during the differentiation of murine ES cells in vitro and attempt to reveal the effects of bFGF on differentiation processes. This analysis was focused on key tissue- and stage-specific genes involved in ectodermal, mesodermal, and endodermal differentiation, including ectodermal-specific gene Nestin, Oligo2 and Syn, mesodermal-specific gene MHC and MyoD, and endodermal-specific gene GATA6, TTR and ALB, as well as undifferentiated gene Sox-2 and Oct-4. The results demonstrate that bFGF could promote expression of ectodermal-specific genes and protein, but suppress the expressions of endoderm-specific and some mesoderm-specific gene and protein. A conclusion can be drawn that bFGF exhibits weak embryotoxicity and mainly promotes ES cell differentiation towards the ectodermal lineages but suppress differentiation into endoderm lineages. These opposing effects of bFGF on the embryonic development of the three germ layers may be related to its weak embryotoxic potential. More specifically, inhibition of expression of the endodermal-specific markers transthyretin (TTR), and albumin (ALB) by bFGF may be of more value in detecting the embryotoxic potential of bFGF.

Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity.

Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.

[Pharmacokinetics of a fusion protein for human acidic fibroblast growth factor and transcriptional activator protein in rat and its penetration across blood-brain barrier].

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.

The effect of transforming growth factor-beta1 on nasopharyngeal carcinoma cells: insensitive to cell growth but functional to TGF-beta/Smad pathway.

OBJECTIVES: This study explored the response of nasopharyngeal carcinoma cells to TGF-beta1-induced growth suppression and investigated the roles of the TGF-beta/Smad signaling pathway in nasopharyngeal carcinoma cells. METHODS: The cells of nasopharyngeal carcinoma cell line CNE2 were treated with TGF-beta1. The growth responses of CNE2 cells were analyzed by MTT assay. The mRNA expression and protein subcellular localization of the TGF-beta/Smad signaling components in the CNE2 were determined by real time RT-PCR and immunocytochemical analysis. RESULTS: We found that the growth of CNE2 cells was not suppressed by TGF-beta1. The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level. TGF-beta type II receptor and Smad7 had no change compared to the normal nasopharyngeal epithelial cells. In addition, Smad2 was phosphorylated to pSmad2, and the activated pSmad2 translocated into the nucleus from the cytoplasm, while the inhibitory Smad-Smad7 translocated from the nucleus to the cytoplasm after TGF-beta1 stimulation. CONCLUSION: The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional. Further work is needed to address a more detailed spectrum of the TGF-beta/Smad signaling pathway in CNE2 cells.

Rapamycin inhibits cholangiocyte regeneration by blocking interleukin-6-induced activation of signal transducer and activator of transcription 3 after liver transplantation.

Cholangiocyte proliferation is necessary for biliary recovery from cold ischemia and reperfusion injury (CIRI), but there are few studies on its intracellular mechanism. In this process, the role of rapamycin, a new immunosuppressant used in liver transplantation, is still unknown. In order to determine whether rapamycin can depress cholangiocyte regeneration by inhibiting signal transducer and activator of transcription 3 (STAT3) activation, rapamycin (0.05 mg/kg) was administered to rats for 3 days before orthotopic liver transplantation. The results indicated that cholangiocytes responded to extended cold preservation (12 hours) with severe bile duct injures, marked activation of the interleukin-6 (IL-6)/STAT3 signal pathway, and increased expression of cyclin D1 until 7 days after transplantation, and this was followed by compensatory cholangiocyte regeneration. However, rapamycin treatment inhibited STAT3 activation and resulted in decreased cholangiocyte proliferation and delayed biliary recovery after liver transplantation. On the other hand, rapamycin showed no effect on the expression of IL-6. We conclude that the IL-6/STAT3 signal pathway is involved in initiating cholangiocytes to regenerate and repair CIRI. Rapamycin represses cholangiocyte regeneration by inhibiting STAT3 activation, which might have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation. Insights gained from this study will be helpful in designing therapy using rapamycin in clinical patients after liver transplantation.