Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM:To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity. METHODS:In this study,we designed a secretory protein by adding a secretory signal peptide(SP) to the N terminus of Transactivating Transcription(TAT)-apoptin(SP-TAT-apoptin),to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model. RESULTS:In human liver carcinoma HepG2 cells,SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48 h,however,it translocated into the nuclear compartment and caused massive apoptotic cell death,as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not,however,cause any cell death in non-malignant human umbilical vein endothelial cells(HUVECs). Most importantly,the conditioned medium from Chinese hamster ovary(CHO) cells transfected with SP-TAT-apoptin also induced significant cell deathin HepG2 cells,but not in HUVECs. CONCLUSION:The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells,and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

Epithelial-mesenchymal transition mediated tumourigenesis in the gastrointestinal tract

Epithelial-mesenchymal transition (EMT) is a highly conserved process that has been well characterised in embryogenesis. Studies have shown that the aberrant activation of EMT in adult epithelia can promote tumour metastasis by repressing cell adhesion molecules,including epithelial (E)-cadherin. Reduced intracellular adhesion may allow tumour cells to disseminate and spread throughout the body. A number of transcription proteins of the Snail superfamily have been implicated in EMT. These proteins have been shown to be over-expressed in advanced gastrointestinal (GI) tumours including oesophageal adenocarcinomas,colorectal carcinomas,gastric and pancreatic cancers,with a concomitant reduction in the expression of E-cadherin. Regulators of EMT may provide novel clinical targets to detect GI cancers early,so that cancers previously associated with a poor prognosis such as pancreatic cancer can be diagnosed before they become inoperable. Furthermore,pharmacological therapies designed to inhibit these proteins will aim to prevent local and distant tumour invasion.

Endoplasmic reticulum stress transducer old astrocyte specifically induced substance contributes to astrogliosis after spinal cord injury

Old astrocyte specifically induced substance （OASIS） is an endoplasmic reticulum （ER） stress transducer specifically expressed in astrocytes and osteoblasts. OASIS regulates the differentiation of neural precursor cells into astrocytes in the central nervous system. This study aimed to elucidate the involvement of ER stress responses stimulated via OASIS in astrogliosis following spinal cord injury. In a mouse model of spinal cord contusion injury, OASIS mRNA and protein expression were evaluated at days 7 and 14. A significant increase in OASIS mRNA on day 7 and an increase in protein on days 7 and 14 was observed in injured spinal cords. Immunostaining on day 7 revealed co-localization of OASIS and astrocytes in the periphery of the injury site. Furthermore, anti-OASIS small interfering RNA （siRNA） was injected at the injury sites on day 5 to elucidate the function of OASIS. Treatment with anti-OASIS siRNA caused a significant decrease in OASIS mRNA on day 7 and protein on days 7 and 14, and was associated with the inhibition of astrogliosis and hindlimb motor function recovery. Results of our study show that OASIS expression synchronizes with astrogliosis and is functionally associated with astrogliosis after spinal cord injury.

Edaravone protects against oxygen-glucose-serum deprivation/restoration-induced apoptosis in spinal cord astrocytes by inhibiting integrated stress response

We previously found that oxygen-glucose-serum deprivation/restoration（OGSD/R） induces apoptosis of spinal cord astrocytes, possibly via caspase-12 and the integrated stress response, which involves protein kinase R-like endoplasmic reticulum kinase（PERK）, eukaryotic initiation factor 2-alpha（eIF2α） and activating transcription factor 4（ATF4）. We hypothesized that edaravone, a low molecular weight, lipophilic free radical scavenger, would reduce OGSD/R-induced apoptosis of spinal cord astrocytes. To test this, we established primary cultures of rat astrocytes, and exposed them to 8 hours/6 hours of OGSD/R with or without edaravone（0.1, 1, 10, 100 μM） treatment. We found that 100 μM of edaravone significantly suppressed astrocyte apoptosis and inhibited the release of reactive oxygen species. It also inhibited the activation of caspase-12 and caspase-3, and reduced the expression of homologous CCAAT/enhancer binding protein, phosphorylated（p）-PERK, p-eIF2α, and ATF4. These results point to a new use of an established drug in the prevention of OGSD/R-mediated spinal cord astrocyte apoptosis via the integrated stress response.

Association between homeobox protein transcript antisense intergenic ribonucleic acid genetic polymorphisms and cholangiocarcinoma

BACKGROUND Cholangiocarcinoma(CCA)represents a rare but highly aggressive malignancy that is often challenging to diagnose,especially in early stages.The role of existing tumor biomarkers for CCA diagnosis,remains controversial due to their low sensitivity and specificity.Increasing evidence has implicated long non-coding ribonucleic acid polymorphisms with cancer susceptibility in a variety of tumor types.The association between long non-coding ribonucleic acid homeobox protein transcript antisense intergenic ribonucleic acid(HOTAIR)polymorphisms and CCA risk has not been reported yet.AIM To investigate the influence of HOTAIR variants on the risk of CCA development.METHODS We conducted a case-control study in which three HOTAIR single nucleotide polymorphisms(rs920778,rs4759314 and rs7958904)were genotyped in a Greek cohort.Our study population included 122 CCA patients(80 males and 42 females)and 165 healthy controls.The polymorphisms under investigation were examined in peripheral blood samples.RESULTS HOTAIR rs4759314 AG and GG genotypes were associated with a significantly increased CCA risk[P=0.004,odds ratio:3.13;95%confidence interval:1.65-5.91 and P=0.005,odds ratio:12.31;95%confidence interval:1.48-101.87,respectively].However,no significant associations of HOTAIR rs920778,and rs7958904 were detected.Similarly,we found no significant associations between rs4759314 AA genotype and CCA susceptibility.CONCLUSION HOTAIR rs4759314 AG and GG genotypes may be implicated with CCA development and may serve as a potential diagnostic biomarker.

Structural components of the nuclear body in nuclei of Allium cepa cells

Nuclear bodies have long been noted in interphase nuclei of plant cells, but their structural component, origin and function are still unclear by now. The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 microm in diameter. They possibly were formed in nucleolus and subsequently released, and entered into nucleoplasm. Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins. Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF). Based on these data, we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities, instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm, and may also be involved in splicing of pre-mRNAs.

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole(Cynoglossus semilaevis)

BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp,which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR（qRT-PCR）. The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages（egg, larva, juvenile and fingerling stages）.The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch（dph）; then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to ＋109. The predicted transcription factor binding sites（E-box binding factors, zinc finger transcription factor, etc.） in this region might participate in the transcriptional regulation of the bmp2 gene.

Sumoylation of SUVR2 contributes to its role in transcriptional gene silencing

The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remodeling proteins CHR19,CHR27, and CHR28(CHR19/27/28), which are also involved in transcriptional gene silencing. Here our study demonstrated that SUVR2 is almost fully mono-sumoylated in vivo. We successfully identified the exact SUVR2 sumoylation site by combining in vitro mass spectrometric analysis and in vivo immunoblotting confirmation. The luminescence imaging assay and quantitative RT-PCR results demonstrated that SUVR2 sumoylation is involved in transcriptional gene silencing. Furthermore, we found that SUVR2 sumoylation is required for the interaction of SUVR2 with CHR19/27/28, which is consistent with the fact that SUMO proteins are necessary for transcriptional gene silencing. These results suggest that SUVR2 sumoylation contributes to transcriptional gene silencing by facilitating the interaction of SUVR2 with the chromatin-remodeling proteins CHR19/27/28.

Expression of COX-2 and transcription factor CCAAT enhancer binding proteinβin refractory sinusitis with nasal polyps and its significance

Objective To study the expression and significance of COX-2 and C/EBP-βin refractory sinusitis with nasal polyps,and to explore the relationship between them and the recurrence of sinusitis with nasal polyps.Methods The protein expression of COX-2 and C/EBP-βin 20 cases of refractory sinusitis with nasal polyps,20 cases of sinusitis with nasal polyps and 20 cases of normal nasal mucosa were detected by western blot,and the relationship between the two was compared.Results The expression levels of COX-2 and C/EBP-βin refractory sinusitis with nasal polyps were significantly different from those in refractory sinusitis with nasal polyps(P<0.05);The expression levels of COX-2 and C/EBP-βin sinusitis tissues with nasal polyps were significantly different from those in normal nasal mucosa tissues(P<0.05);The expression levels of COX-2 and C/EBP-βin each group were significantly correlated(P<0.05).Conclusions The high expression of COX-2 and C/EBP-βmay be closely related to postoperative recurrence of sinusitis patients with nasal polyps.Both may be used as objective indicators to judge the postoperative follow-up and recurrence tendency of patients with sinusitis with nasal polyps..

Glucocorticoid receptor and heat shock protein 90 in peripheral blood mononuclear cells from asthmatics

Objective To investigate the expression of glucocerticoid receptor (GR) and heat shock protein 90(HSP90) mRNA in peripheral blood mononuclear cells (PBMCs) from steroid-sensitive (SS), steroiddependent (SD) and steroid-resistant (SR) asthmatics patients, and to evaluate the role of GR and HSP90in the pathogenesis of SR.Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expressions of GR and HSP90 mRNA in PBMC stimulated with IL-2 and/or IL-4 from 10 normal volunteers,10 SS, 5 SD and 6 SR patients.Results The expressions of GR and HSP90 mRNA were the highest in PBMC from SR patients (0.730±0.171 and 1.122±0.165, respectively) compared with the normals (P＜0.05). The second was from SS patients (0.359±0.350 and 0.885±0.250, respectively). The lowest was from the SD patients (0.017±0.008 and 0.078 ± 0.039, respectively). The ratio of HSP90/GR in SR was significantly lower than that in the others, and it suggested that the expression of HSP90 mRNA was not sufficient in this group of patients. When PBMC from SS, SD and SR was incubated with IL-2 or IL-4 alone, no changes in GR and HSP90 mRNA expression were observed. While incubated with combination of IL-2 and IL-4, a significantly higher expression of GR mRNA was observed in all asthmatics, and a significantly higher expression of HSP90 mRNA was observed only in SS and SD patients.Conclusion The lowering of HSP90/GR ratio may be one of the causes of SR. IL-2 and IL-4 may play roles in the imbalance of HSP90/GR.

Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development

Background Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of fotic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase （DHFR） gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 （HAND2） and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations. Methods Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression. Results DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos （P〈0.05）. Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos. Conclusions DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

Ginseng-Derived Panaxadiol Saponins Promote Hematopoiesis Recovery in Cyclophosphamide-Induced Myelosuppressive Mice:Potential Novel Treatment of Chemotherapy-Induced Cytopenias

Objective：To investigate the potential efficacy of panaxadiol saponins component（PDS-C）,a biologically active fraction isolated from total ginsenosides,to reverse chemotherapy-induced myelosuppression and pancytopenia caused by cyclophamide（CTX）.Methods：Mice with myelosuppression induced by CTX were treated with PDS-C at a low-（20 mg/kg）,moderate-（40 mg/kg）,or high-dose（80 mg/kg） for 7 consecutive days.The level of peripheral white blood cell（WBC）,neutrophil（NEU） and platelet（PLT） were measured,the histopathology and colony formation were observed,the protein kinase and transcription factors in hematopoietic cells were determined by immunohistochemical staining and Western blot.Results：In response to PDS-C therapy,the peripheral WBC,NEU and PLT counts of CTX-induced myelosuppressed mice were significantly increased in a dose-dependent manner.Similarly,bone marrow histopathology examination showed reversal of CTX-induced myelosuppression with increase in overall bone marrow cellularity and the number of hematopoietic cells（P〈0.01）.PDS-C also promoted proliferation of granulocytic and megakaryocyte progenitor cells in CTX-treated mice,as evidenced by significantly increase in colony formation units-granulocytes/monocytes and-megakaryocytes（P〈0.01）.The enhancement of hematopoiesis by PDS-C appears to be mediated by an intracellular signaling pathway,this was evidenced by the up-regulation of phosphorylated mitogen-activated protein kinase（p-MEK） and extracellular signal-regulated kinases（p-ERK）,and receptor tyrosine kinase（C-kit） and globin transcription factor 1（GATA-1） in hematopoietic cells of CTX-treated mice（P〈0.05）.Conclusions：PDS-C possesses hematopoietic growth factor-like activities that promote proliferation and also possibly differentiation of hematopoietic progenitor cells in myelosuppressed mice,probably mediated by a mechanism involving MEK and ERK protein kinases,and C-kit and GATA-1 transcription factors.PDS-C may potentially be a novel treatment of myelosuppression and pancytopenia caused by chemotherapy.

Reverse effect of Semaphorin-3F on rituximab resistance in diffuse large B-cell lymphoma via the Hippo pathway

Background:Exploring the underlying mechanism of rituximab resistance is critical to improve the outcomes of patients with diffuse large B-cell lymphoma(DLBCL).Here,we tried to identify the effects of the axon guidance factor semaphorin-3F(SEMA3F)on rituximab resistance as well as its therapeutic value in DLBCL.Methods:The effects of SEMA3F on the treatment response to rituximab were investigated by gain-or loss-of-function experiments.The role of the Hippo pathway in SEMA3F-mediated activity was explored.A xenograft mouse model generated by SEMA3F knockdown in cells was used to evaluate rituximab sensitivity and combined therapeutic effects.The prognostic value of SEMA3F and TAZ(WW domain-containing transcription regulator protein 1)was examined in the Gene Expression Omnibus(GEO)database and human DLBCL specimens.Results:We found that loss of SEMA3F was related to a poor prognosis in patients who received rituximab-based immunochemotherapy instead of chemotherapy regimen.Knockdown of SEMA3F significantly repressed the expression of CD20 and reduced the proapoptotic activity and complement-dependent cytotoxicity(CDC)activity induced by rituximab.We further demonstrated that the Hippo pathway was involved in the SEMA3F-mediated regulation of CD20.Knockdown of SEMA3F expression induced the nuclear accumulation of TAZ and inhibited CD20 transcriptional levels via direct binding of the transcription factor TEAD2 and the CD20 promoter.Moreover,in patients with DLBCL,SEMA3F expression was negatively correlated with TAZ,and patients with SEMA3F^(low)TAZ^(high)had a limited benefit from a rituximab-based strategy.Specifically,treatment of DLBCL cells with rituximab and a YAP/TAZ inhibitor showed promising therapeutic effects in vitro and in vivo.Conclusion:Our study thus defined a previously unknown mechanism of SEMA3F-mediated rituximab resistance through TAZ activation in DLBCL and identified potential therapeutic targets in patients.