ATM Signaling Pathway Is Implicated in the SMYD3-mediated Proliferation and Migration of Gastric Cancer Cells

PURPOSE: We previously found that the histone methyltransferase suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3 (SMYD3) is a potential independent predictive factor or prognostic factor for overall survival in gastric cancer patients, but its roles seem to differ from those in other cancers. Therefore, in this study, the detailed functions of SMYD3 in cell proliferation and migration in gastric cancer were examined. MATERIALS AND METHODS: SMYD3 was overexpressed or suppressed by transfection with an expression plasmid or siRNA, and a wound healing migration assay and Transwell assay were performed to detect the migration and invasion ability of gastric cancer cells. Additionally, an MTT assay and clonogenic assay were performed to evaluate cell proliferation, and a cell cycle analysis was performed by propidium iodide staining. Furthermore, the expression of genes implicated in the ataxia telangiectasia mutated (ATM) pathway and proteins involved in cell cycle regulation were detected by polymerase chain reaction and western blot analyses. RESULTS: Compared with control cells, gastric cancer cells transfected with si-SMYD3 showed lower migration and invasion abilities (P<0.05), and the absence of SMYD3 halted cells in G2/M phase and activated the ATM pathway. Furthermore, the opposite patterns were observed when SMYD3 was elevated in normal gastric cells. CONCLUSIONS: To the best of our knowledge, this study provides the first evidence that the absence of SMYD3 could inhibit the migration, invasion, and proliferation of gastric cancer cells and halt cells in G2/M phase via the ATM-CHK2/p53-Cdc25C pathway. These findings indicated that SMYD3 plays crucial roles in the proliferation, migration, and invasion of gastric cancer cells and may be a useful therapeutic target in human gastric carcinomas.

Morphological changes of bone in the progress of rat chronic fluorosis / 中国地方病学杂志

ObjectiveTo observe the morphological changes of bone in the progress of chronic fluorosis.MethodsWistar rats were randomly divided into three groups,30 rats in each group:normal control group,experimental group Ⅰ and experimental group Ⅱ according to body weight.Rats in normal control group drank distilled water freely.Experimental group Ⅰ and group Ⅱ drunk distilled water with sodium fluoride preparation of fluorine containing ion 100,150 mg/L solution for six months,respectively.Bone mineral density was detected by X-ray,bone morphological changes were observed under light microscope and bone histomorphometric parameters were calculated using image analysis software.ResultsThe bone mineral density values were different statistically between the three groups after feeding for 2 and 4 months(F =19.79,3.28,all P ＜ 0.05).However no significant difference was found after feeding for 6 months(F =1.80,P ＞ 0.05).The bone mineral density of experimental group Ⅰ (0.20 ± 0.03,0.21 ± 0.03) was significantly higher than that of the normal control group(0.17 ± 0.03,0.20 ± 0.04) after feeding for 2 and 4 months.The bone mineral density of experimental group Ⅱ (0.21 ± 0.02) was lower than that of normal control group(0.22 ± 0.03) after feeding for 6 months.The bone lamella in experimental group Ⅰ was arranged disorderly,the number of osteocytes increased with their nucleus atrophy and the osteoblasts were more than that of control grouo which arranged in layers observed under light microscooy.In exoerimental group Ⅱ,the bone lamella was bent deformation,the number of osteocytes had decreased with their nucleus shrinking or even disappeared and the number of osteoclasts had increased significantly observed under light microscopy.In experimental group Ⅰ,the mean trabecular density [(0.33 ± 0.03)％] increased and the mean trabecular separation,thickness [( 163.57 ± 1.99),(59.26 ± 7.18 ) μm] decreased compared with that of normal control group [(0.31 ± 0.02)％,(186.60 ± 2.90)μm,(86.42 ± 1.48)μm,all P ＜ 0.05].In experimental group Ⅱ,the mean trabecular density[(0.26 ± 0.02)％] decreased,the mean trabecular thickness[(71.42 ± 10.77)μm] reduced compared with that of normal control group[(0.31 ± 0.02)％,(86.42 ± 1.48)μm].ConclusionsExcess fluoride can damage bone tissue.Low doses of fluoride can stimulate osteoblast activity and enhance osteogenesis.The activity of osteoblasts is great than that of osteoclasts.High doses of fluoride can stimulate both osteoblasts and osteoclasts activity,but mainly the activity of osteoclasts,and bone resorption increases.

Astrocyte activation and Bcl-2 protein expression induced by lipopolysaccharide in mouse brain / 中南大学学报(医学版)

OBJECTIVE@#To investigate the changes of anti-apoptotic protein Bcl-2 expression in neurons and activation of brain astroglial cells, and the relationship between astrocytes and neurons in mice after a single intracerebroventricular (ICV) stereotaxic injection of lipopolysaccharide (LPS).@*METHODS@#C57BL/6J mice of different ages were divided into a control group and an experiment group. Immunohistochemistry to Bcl-2 and that to GFAP were conducted to observe the expression of Bcl-2 protein in neurons and GFAP in astrocytes in the brain at different time-points after the LPS injection. The glial cell type expressing Bcl-2 was characterized with immunofluorescence double labeling.@*RESULTS@#GFAP-immunoreactive cells in the control mice were observed mainly within hippocampal formation, piriform, entorhinal cortex, septum, striatum, amygdaloid nucleus, subcortical white matter, as well as in the main fiber tracts. At 24 h after the LPS treatment there was no obvious difference in GFAP immunoreactivity compared with the controls. Astrocytes were markedly activated in periventricular brain regions such as hippocampus, the hypothalamic parenchyma surrounding the third ventricle, with larger cell body and hypertrophic processes 2 days after the endotoxin treatment. After the LPS injection, Bcl-2 positive cells were distributed widely in the brain, such as in the cortex (primary and secondary motor cortex, somatosensory cortex), hypothalamic parenchyma surrounding the third ventricle, diagonal band, hippocampus, septum and the red nucleus of the midbrain. At these sites, Bcl-2 induction increased significantly 2 days after the ICV LPS injection, with some subregional differences, peaking on 4th day. No immunofluorescent double labeling cells for GFAP and Bcl-2 were observed in the brain of the mice after the LPS administration, but merging GFAP positive astrocytes and Bcl-2 positive neurons were seen. Double staining for Bcl-2 and GFAP also showed that the projections of activated astrocytes were found in the sheath of Bcl-2 positive neurons 4 days after the ICV LPS administration.@*CONCLUSION@#LPS can activate astroglial cells and upregulate of Bcl-2 expression in the neurons in the mouse brain, which may participate in the administration of central nervous system to central-immunity stimulated regulation and the protective response to the inflammatory stimulus. The projections of activated astrocytes are found in the sheath of Bcl-2 positive neurons, indicating that there is close relationship between astrocytes and neurons.

Effect of precursor of brain-derived neurotrophic factor on hippocampal neuron survival and its mechanism / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of precursor of brain-derived neurotrophic factor (proBDNF) on cultured hippocampal neuron and its intracellular mechanism.@*METHODS@#The hippocampal neurons were dissociated from E18 rats and cultured in neurobasal medium, and then the cells were treated with proBDNF, preBDNF(propeptide of proBDNF) and proBDNF antiserum,respectively. Thirty minutes, 1 hour or 48 hours later, the cells were stained with Nissl solution, and the immunocytochemistry methods of ELK-p (Ets domain protein), ErK2(extracellular signal regulated kinase) and c-fos were performed.@*RESULTS@#The expressions of ELK-p, ErK and c-fos were significantly upregulated in the cultured hippocampal neurons after they were treated with proBDNF protein,and the immuno-positive staining was also obvious in some nuclei. While the endogenous proBDNF was neutralized by proBDNF antiserum treatment, the expressions of ELK-p, ErK and c-fos were downregulated and many cells showed swelling and vasoculation. The immunoreactivity in preBDNF treated cells was similar to that in normal cultured cells.@*CONCLUSION@#proBDNF plays an important role in sustaining the hippocampal neuron survival through upregulating the ELK and ErK pathways.

Expression of eNOS in the endothelial cells during the arteriogenesis of pig hind-limbs / 中南大学学报(医学版)

OBJECTIVE@#To detect the expression and activity of eNOS during the arteriogenesis of hind-limbs of pigs.@*METHODS@#The right femoral artery was ligated, and the left femoral artery shamly operated under routine surgical procedures. Animals were sacrificed after two weeks. The expression and activity of eNOS in collateral vessels were studied by confocal immunofluorescence with antibodies against eNOS and phosphorylated eNOS (P-eNOS) respectively.@*RESULTS@#In normal small arteries, the expression of eNOS was very low, and the staining was very weak. In growing collateral vessels, the expression of eNOS was significantly up-regulated, showing very strong positive staining. The expression of P-eNOS was also high. Dural immunostaining showed that eNOS and P-eNOS were colocalized in the endothelial cells.@*CONCLUSION@#eNOS is up-regulated and activated during arteriogenesis, suggesting that eNOS can exert the possible role in mediating the proliferation of endothelial cells and the inflammation, and contribute to the collateral vessel growth.

Effect of polygonum multiflorum thunb on BDNF expression in rat hippocampus induced by amyloid beta-protein (Abeta) 1-40 / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of polygonum multiflorum thunb (PMT) on the expression of brain-drived neurtrophic factor (BDNF) in hippocampus CA1 in rats induced by Abeta1-40.@*METHODS@#The rat model was established by injecting Abeta1-40 into the right dorsal hippocampus in rats, and their behavior was observed by Y-maze. The morphological changes of rat hippocampus were examined by the Nissl staining. The expression of BDNF in hippocampus CA1 neurons was also examined by immunohistochemistry and Western blot.@*RESULTS@#After the administration of Abeta1-40, the times of rats learning how to elude electric stimulation were significantly increased, and the expression of BDNF in hippocampus CA1 were obviously reduced. The aggregation of Abeta1-40 in hippocampus and the morphological changes of CA1 hippocampus were found. After the treatment with PMT for 30 days, the times learning how to elude electric stimulation were obviously reduced, while the expression of BDNF in hippocampus CA1 was also increased.@*CONCLUSION@#PMT can reverse the down-regulation of BDNF expression in hippocampus CA1 induced by Abeta1-40.

Apoptosis of breast cancer cell line MCF-7 cells induced by gemcitabine and radiation / 中南大学学报(医学版)

OBJECTIVE@#To investigate the apoptosis of human breast cell line MCF-7 cells induced by gemcitabine and radiation.@*METHODS@#The MTT method was applied to study the growth inhibition of MCF-7 treated with gemcitabine, radiation, gemcitabine and radiation. The apoptosis index (AI) was analyzed by flow cytometry. The morphology of the MCF-7 cells apoptosis was observed by transmission electron microscopy.@*RESULTS@#When MCF-7 cells were treated with gemcitabine at different concentrations for 24 h, the cell growth inhibition rate was increased in a concentration-dependent manner. The apoptotic indexes (AI) of MCF-7 of four groups by flow cytometry revealed. The AI of (R+D) group was significantly different from those of the radiation group and the gemcitabine group (P<0.05). Condensed chromation, nuclear fragmentation and apoptotic body of MCF-7 cells were found by transmission electron microscope.@*CONCLUSION@#The apoptosis of human breast cancer cell line, MCF-7 cells, could be induced by gemcitabine. Gemcitabine can significantly enhance the radiation-induced apoptosis of MCF-7 cells.

Expression of brain-derived neurotrophic factor in hippocampus of ovariectomized mouse / 中南大学学报(医学版)

OBJECTIVE@#To explore the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus after ovariectomy in mice.@*METHODS@#BDNF levels were detected by immunohistochemistry combined image analysis in hippocampal CA regions and dentate gyrus of ovariectomized mice.@*RESULTS@#The expression of BDNF in hippocampus of mice decreased significantly after the ovariectomy after 4 days. The recovery BDNF expression started 14 days after the ovariectomy and after 28 days, the expression of BDNF in hippocampus recovered to the normal level.@*CONCLUSION@#The decrease of estrogen in ovariectomized mice can weaken the expression of BDNF in hippocampus during the early stage.

Pathway for macrophage invasion into the tunica media and the intima in vein graft / 中南大学学报(医学版)

OBJECTIVE@#To test whether the macrophages in vein grafts may invade into the vascular wall from the adventitia apart from the lumen.@*METHODS@#Expression of CD68 (a marker of macrophage) and CD31 (a marker of endothelial cells) in 30 vein grafts were detected by confocal immunofluroscence. Images were processed with Silicon Graphics Octane.@*RESULTS@#In normal veins, there were a few CD68 positive cells in the adventitia, but few CD68 positive cells were found in the media and the intima. In the diseased vein, CD68 positive cells were significantly increased and present in all layers of the vascular wall, the adventitia, media and intima, extending from the adventitia to the media. There was a close relationship between the penetration of CD68 positive cells into the media from the adventitia and the angiogenesis in the media.@*CONCLUSION@#The adventitia may be an important pathway for macrophage invasion.