Overexpression of vascular endothelial growth factor enhances the neuroprotective effects of bone marrow mesenchymal stem cell transplantation in ischemic stroke.

Although bone marrow mesenchymal stem cells (BMSCs) might have therapeutic potency in ischemic stroke, the benefits are limited. The current study investigated the effects of BMSCs engineered to overexpress vascular endothelial growth factor (VEGF) on behavioral defects in a rat model of transient cerebral ischemia, which was induced by middle cerebral artery occlusion. VEGF-BMSCs or control grafts were injected into the left striatum of the infarcted hemisphere 24 hours after stroke. We found that compared with the stroke-only group and the vehicle- and BMSCs-control groups, the VEGF-BMSCs treated animals displayed the largest benefits, as evidenced by attenuated behavioral defects and smaller infarct volume 7 days after stroke. Additionally, VEGF-BMSCs greatly inhibited destruction of the blood-brain barrier, increased the regeneration of blood vessels in the region of ischemic penumbra, and reducedneuronal degeneration surrounding the infarct core. Further mechanistic studies showed that among all transplant groups, VEGF-BMSCs transplantation induced the highest level of brain-derived neurotrophic factor. These results suggest that BMSCs transplantation with vascular endothelial growth factor has the potential to treat ischemic stroke with better results than are currently available.

A Practical Method for Determination of Nine Nucleosides in Tricholoma matsutake by UPLC/MS and Quantitative Analysis of Multicomponents Using Single Marker Method.

Nucleosides can be used as quality evaluation indicators of Tricholoma matsutake. In this work, a new ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS) strategy for quantitative analysis of multiple components using a single marker (QAMS) was proposed to determine nine nucleosides (adenosine, cytidine, guanosine, inosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyuridine) in T. matsutake. Guanosine was set as the internal reference substance, whose content in T. matsutake was determined using the conventional external standard method. Relative correction factors (RCFs) between guanosine and eight other nucleosides were measured. The concentrations of the eight components were calculated with the obtained RCFs by QAMS. An ultrasonic extraction method is used for sample preparation. This method was validated to be sensitive, precise, and accurate with the LODs of 0.31-1.9 ng, the overall intraday and interday variations less than 4.08%, and the overall recovery over 89.0%. The correlation coefficients (r 2) of the calibration curves were higher than 0.9918. The values of vector angle analysis were above 0.99845, which indicates no significant differences between the conventional external standard method and the present QAMS method. As far as we know, this is also the first report of UPLC/MS analysis of nucleoside compounds by QAMS, providing an efficient and feasible quality assessment method for other natural products containing nucleosides.

MicroRNA-224 is upregulated in HepG2 cells and involved in cellular migration and invasion.

BACKGROUND AND AIM: MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics. METHODS: We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 (miR-224) can influence the biological behaviors of HepG2 cells. RESULTS: MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK4 and MMP9, which were involved in the invasion of tumor, was found. CONCLUSIONS: Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.

Rapid induction of PC3/BTG2 gene by hepatopoietin or partial hepatectomy and its mRNA expression in hepatocellular carcinoma.

BACKGROUND: The anti-proliferative gene, PC3 (pheoch-romocytoma cell 3)/BTG2 (B-cell translocation gene 2), is one of the early growth response genes and belongs to the BTG/Tob protein family. This study aimed to assess the effects of recombinant human hepatopoietin (HPO) and partial hepatectomy on rapidly induced expression of immediate-early genes and to investigate the expression of PC3/BTG2 mRNA in hepatocellular carcinoma (HCC) at different stages of progression. METHODS: After a rat model of partial hepatectomy was established, we investigated gene expression within 1 hour after 2/3 partial hepatectomy by representational difference analysis and in a primary cultured hepatocyte system. The expression levels of PC3/BTG2 from liver tissues of the rat model were assessed by RT-PCR and Northern blotting. Meanwhile, the expression of BTG2 mRNA in a tissue microarray of HCC was determined by in situ hybridization. RESULTS: The PC3/BTG2 gene was rapidly induced after 2/3 partial hepatectomy and its expression peaked within 1-2 hours after operation. HPO rapidly induced the expression of the genes c-fos, LRF-1, and PC3 in primary cultured rat hepatocytes, which might be one of the molecular mechanisms by which HPO stimulates hepatocyte proliferation. Positive BTG2 mRNA expression was detected in 71.19% (42/59) of the HCC samples and in 75% (3/4) of the normal liver tissue samples obtained from the region around the HCC tissues. PC3/BTG2 mRNA was located mainly in the cytoplasm of HCC cells and its expression was related to the degree of differentiation. CONCLUSIONS: Recombinant human HPO and partial hepatectomy rapidly induce the expression of the PC3/BTG2 gene. PC3/BTG2 mRNA is highly expressed in HCC cells and its expression is related to the degree of cell differentiation. The abnormal expression of PC3/BTG2 is closely related to the genesis and development of HCC, so PC3/BTG2 may play an important role in these processes.

[The expression of B-cell translocation gene 2 in diethylnitrosamine-induced primary hepatocellular carcinoma rat model.].

OBJECTIVE: To investigate the expression and role of B-cell translocation gene 2(BTG2) in the carcinogenesis of hepatocellular carcinoma (HCC). METHODS: Modified Diethylnitrosamine (DEN)-induced primary hepatocellular carcinoma rat model was established. The expression of BTG2, p53 and cyclinD1 was detected by RT-PCR, western blot and immunohistochemistry. RESULTS: The BTG2 protein was predominantly localized in the nucleus, with faint cytoplasmic staining in normal liver cells; however, it is mainly a cytoplasmic protein in HCC cells. BTG2 was over-expressed during the early stage after DEN treatment, the expression level peaked at 5 weeks and then it gradually decreased to the normal level after 16 weeks. The expression of cyclin D1 and cyclin E was increased gradually after DEN treatment, and peaked at 16 weeks and 5 weeks respectively. A significant increase in p53 was not observed until 5 weeks after DEN treatment, and it gradually decreased after 16 weeks. CONCLUSIONS: Decreased expression of BTG2 may be an important step in carcinogenesis of HCC. BTG2 may positively regulate p53 expression and negatively regulate cyclin D1 expression in the carcinogenesis of HCC.

INPP4B promotes colorectal cancer cell proliferation by activating mTORC1 signaling and cap-dependent translation.

BACKGROUND AND OBJECTIVE: Inositol polyphosphate 4-phosphatase type II (INPP4B) is over-expressed in CRC tissues, and emerges as an oncogene. However, the mechanism by which INPP4B regulates CRC cell proliferation remains largely unclear. In this study, we aimed to investigate the regulatory mechanisms of INPP4B in CRC. MATERIALS AND METHODS: The expression levels of mRNA were detected by qRT-PCR. The expression levels of protein were determined by Western blot. Cell Counting Kit-8 (CCK-8) assays and BrdU incorporation assays were performed to evaluate cell proliferation abilities. Bicistronic luciferase assays and the m7GTP pull down assay were performed to measure the cap-dependent translation in cells. RESULTS: INPP4B promotes CRC cell proliferation by increasing mTORC1 activity. Furthermore, it was shown that the activation of mTORC1 signaling by INPP4B led to increased cap-dependent translation, which is essential for INPP4B-mediated CRC cell proliferation. Finally, it was demonstrated that increased AKT and serum and glucocorticoid-inducible kinase 1 activity contributed to the activation of cap-dependent translation induced by INPP4B. CONCLUSION: Collectively, the present study reveals INPP4B promotes colorectal cancer cell proliferation by activating mTORC1 signaling and cap-dependent translation.

[The expression of Tec and the level of its phosphorylation in primary hepatic carcinomas].

OBJECTIVES: To detect the expressions of Tec tyrosine kinase in hepatocellular carcinoma and the levels of phosphorylation of tyrosine kinase in liver cancer tissues, paracancerous tissues and normal liver tissues and to find the significance of their differences. METHODS: 200 specimens of tissues, including liver cancer tissues, surrounding liver tissues not more than 1.5 cm from the cancers, and normal liver tissues were investigated for Tec protein expression and Tec phosphorylation by tissue microarrays and immunohistochemistry (SP method). RESULTS: The positive immunohistochemical stainings of Tec in cancerous tissues and non-cancerous tissues showed no obvious differences, nevertheless, the immunostaining levels in liver cancer tissues were much higher than in non-cancerous tissues and they correlated with the grading of tumors (P < 0.05). The phosphorylation of Tec was significantly expressed in liver cancer tissues (73%) in comparison with other tissues (42%, 10% both P < 0.05), but it did not correlate with any clinicopathological characteristics. CONCLUSION: Overexpression of Tec is associated with the tumorigenesis and development of liver cancer; inhibiting Tec or degrading Tec phosphorylation directly might affect the progression of liver cancer.

Roles of PD-1, Tim-3 and CTLA-4 in immunoregulation in regulatory T cells among patients with sepsis.

OBJECTIVE: This study aims to elucidate the roles of PD-1, Tim-3 and CTLA-4 in sepsis. METHODS: Sepsis patients (n = 182) were selected as sepsis group and divided into three subgroups: mild sepsis group, severe sepsis group and septic shock group; 185 healthy volunteers were enrolled as control group. Flow cytometry and blood routine examination were performed for T lymphocytes and surface co-stimulatory molecules expressions. Pearson correlation test was applied for the correlation of co-stimulatory molecules expressions on T lymphocytes with critical illness in sepsis. Logistic regression analysis was conducted for risk factors in sepsis. RESULTS: Heart rate and WBC in subgroups were higher than control group (P < 0.05). The differences in APACHE II, SAP II and SOFA score among subgroups were statistically significant (P < 0.05). Compared with control group, lymphocyte ratio and percentage of CD4(+) T cells reduced in subgroups (P < 0.05). The differences in expression levels of CD4(+)PD-1(+), CD8(+)PD-1(+), and CD8(+)CTLA-4(+) showed statistical significances (P < 0.05). Apparently, expression levels of CD4(+)TIM-3(+), CD8(+)TIM-3(+), CD4(+)PD-1(+), CD8(+)PD-1(+), and CD4(+)CTLA-4(+) were positively correlated with APACHE II score (all P < 0.05). Logistic regression analysis showed that heart rate and expression level of CD4(+)PD-1(+) might be risk factors while the percentage of CD4(+) T cells might be a protective factor for sepsis (P < 0.05). CONCLUSION: PD-1 aggravates immune responses consistent with promotion of T cell exhaustion in sepsis. Expression level of CD4(+)PD-1(+) and heart rate are potential risk factors while percentage of CD4(+) T cells is a possible protective factor for sepsis.

[Effect of lovastatin on the expression of IkappaBalpha and cell-cycle regulating protein in MCF-7 cells].

OBJECTIVE: To study the effect of lovastatin on the expression of IkappaBalpha and cell-cycle regulating proteins in MCF-7 cells. METHODS: MCF-7 cells were treated with 4, 8 and 16 micro mol/L lovastatin for 48 - 72 h. The distribution of cell cycles was assayed by flow cytometry (FCM). The protein expression of IkappaBalpha, CDK4, p16, pRb in cytoplasm and IkappaBalpha in the nucleus were detected by Western blot. RESULTS: Lovastatin could arrest cellcycle in the G(0)/G(1) phase in a dose- and time-dependent manner, obviously lowering the expression of IkappaBalpha, CDK4 and pRb protein level in the cytoplasm and increasing IkappaBalpha in the nucleus, but not on p16 protein level. CONCLUSION: Lovastatin can induce the arrest of cell cycle in G(0)/G(1) phase by affecting the expression of IkappaBalpha and cell-cycle regulating protein in MCF-7 cells.

Nm23-H1 protein binds to APE1 at AP sites and stimulates AP endonuclease activity following ionizing radiation of the human lung cancer A549 cells.

Non-metastatic protein-23 homolog-1 (Nm23-H1) is a multifunctional protein with DNase and histidine protein kinase activities. Human apurinic endonuclease-1 (APE1) is the AP endonuclease DNA base excision repair (BER) enzyme involved in several important cellular functions. Since the relationship between Nm23-H1 and APE1 proteins is unclear, we evaluated their interaction at different time points after irradiating human lung cancer A549 cells with X-rays. We found that Nm23-H1 and APE1 overexpression was induced by irradiation in a dose- and time-dependent manner. Subcellular distribution pattern of both proteins was reversed after irradiation. After irradiation, APE1 that initially showed nuclear localization was gradually increased in the cytoplasm, whereas Nm23-H1 that mainly showed cytoplasmic localization was gradually increased in the nuclei of A549 cells. Nm23-H1 and APE1 interaction was demonstrated by His-pull-down and co-immunoprecipitation assays. The presence of Nm23-H1/APE1 complex in X-ray-irradiated A549 cells was also detected by DNA affinity precipitation analysis of a DNA fragment containing an AP site. Although the AP endonuclease activity of Nm23-H1 was too weak to be detected, the AP endonuclease activity of APE1 was increased with the enhanced Nm23-H1 expression. In conclusion, our data point to a mechanism by which Nm23-H1 protects cells against oxidative stress through the engagement of DNA BER enzyme APE1.

Clinical study of recombinant adenovirus-p53 combined with fractionated stereotactic radiotherapy for hepatocellular carcinoma.

OBJECTIVE: The purpose of our study was to evaluate the feasibility and treatment outcomes of recombinant adenovirus-p53 (rAd-p53, trademarked as Gendicine) combined with fractionated stereotactic radiotherapy (fSRT) in treatment of primary hepatocellular carcinoma (HCC). METHODS: We randomly enrolled 40 patients with HCC treated by fSRT alone (fSRT group) or rAd-p53 combined with fSRT (combined group). Tumor size was 2-5.2 cm (average 3.2 cm). We prescribed 50 Gy in 10 fractions at the 50%-80% isodose line of the planning target volume for 2 weeks in two groups. The combined group was treated with two intratumoral injections of rAd-p53 on day 1 and 8 while fSRT started on day 3. Tumor response was assessed after treatment using modified WHO criteria. The follow-up period was 11-44 months (median 35 months). RESULTS: The overall response rate of fSRT group was 70%, with 4 patients showing complete response (20%), 10 partial response (50%) and 6 stable disease (30%). Correspondingly the overall response rate of combined group was 85%, with 7 patients showing complete response (35%), 10 partial response (50%) and 3 stable disease (15%). The 1-year survival rates of fSRT group and combined group were 70.0% and 90.0%, respectively. The 1-year disease-free survival rates of fSRT group and combined group were 65% and 85%, respectively. These treatments were well tolerated, because grade 3 or 4 toxicity was not observed. CONCLUSION: These results suggest that rAd-p53 combined with fSRT is a relatively safe and effective method for treating primary hepatocellular carcinoma compared with only fSRT. Thus, rAd-p53 combined with fractionated SRT may be preferred as a choice of local treatment for primary HCC when the patients are inoperable or when the patients refuse operation.

[The expression of platelet-derived growth factor-A and its receptor in acute radiation-induced skin ulcer in rats].

OBJECTIVE: To investigate the dynamic expression of platelet-derived growth factor-A (PDGF-A) and its receptor alpha (PDGFR-alpha) in different acute radiation-induced skin ulcers, and to explore the underlying mechanism involved in retarded healing of the ulcer. METHODS: The model of acute radiation-induced skin ulcers in rats was replicated with 50 Gy 60Co gamma rays to the skin (radiation group, R, n = 55), rats with full - thickness skin excision wounds as control group (T, n = 55), and 5 normal rats to serve as normal control (NC) group. The expression of PDGF-A and PDGFR-alpha protein and PDGF-A mRNA was respectively assessed by means of histochemistry and in situ RT-PCR. RESULTS: No PDGF-A expression was identified in the rat skin in NC group. The expression of PDGF-A and PDGFR were reduced in R group during inflammatory responsive and granulation formation periods (14 - 28 days after radiation, the IA value of PDGF-A varied from 14.0 +/- 1.2 to 20.3 +/- 1.2 compared with that in T group in which the IA value of PDGF-A at the same period (3 - 9 days after injury) varied from 20.0 +/- 1.6 to 28.3 +/- 1.0, and reduced gradually during scar formation period (55 days after radiation). CONCLUSION: The reduction of PDGF-A and PDGFR-expression may be partially involved in the mechanism of retarded healing of acute radiation-induced skin ulcers.