Relationship between apoptosis,proliferation and expression,mutation of related genes in breast cancer

AIM:To study the relationship between apoptosis, proliferation and expression,mutation of related genes in breast cancer.METHODS:Methods of TUNEL, immunohistochemical S-P and PCR-SSCP were used respectively to study apoptotic index (AI), mitotic index(MI), expression of Bcl-2,p53,c-erbB-2,PCNA,Ki67,TopoⅡ and mutation of p53 in 54 cases of breast cancer.RESULTS:AI and MI were 9.40±3.78 and 5.96±2.36, respectively. There was a significant direct correlation between them(r=0.46.P＜0.01).High expression of Bcl-2,PCNA,Ki67,TopoⅡ coincided with high AI,MI(P＜0.01). High expression of p53,c-erbB-2 and mutation of p53 coincided with high MI(P＜0.01). Type of p53 mutation coincided with AI(P＜0.05).CONCLUSION:Disturbance of gene control between apoptosis and proliferation is related with expression,mutation of related genes in breast cancer.     

Effects of survivin inhibitor YM155 on mitochondrial apoptotic pathway of retinoblastoma Y79 cells

AIM: To investigate the effects of survivin inhibitor YM155 {4,9-dihydro-1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide} on the apoptosis, mitochondrial membrane potential (Δψ_m) and cytochrome C (Cyt C) of retinoblastoma Y79 cells, and to analyze the mitochondrial mechanisms of apoptosis.METHODS: Y79 cells were cultured in vitro and treated with YM155 at concentrations of 0, 0.5, 1, 2, 4 and 8 nmol/L. The cells in control group were treated without YM155. The proliferation of Y79 cells were measured by CCK-8 assay and bromodeoxyuridine (BrdU) labeling assay. Y79 cells were randomly divided into 4 groups:control group (with equal volume of RPMI-1640 nutrient medium), positive control group (10 nmol/L topotecan), low-dose (1 nmol/L) YM155 group and high-dose (2 nmol/L) YM155 group. The effects of YM155 on the apoptosis, the changes of Δψ_m, the mitochondrial distribution and the protein level of Cyt C in the Y79 cells were evaluated by flow cytometry with Annexin V-FITC/PI staining, JC-1 staining, immunofluorescence analysis and Western blot, respectively. RESULTS: Compared with control group, YM155 significantly inhibited the proliferation of Y79 cells and induced apoptosis (P<0.05). YM155 significantly reduced Δψ_m of the Y79 cells, promoted Cyt C which released from mitochondria to the cytosol and reduced the protein level of Cyt C in the mitochondria (P<0.05). CONCLUSION: YM155 inhibits Y79 cell proliferation and induces apoptosis, and the possible mechanisms may be involved in the mitochondrium-mediated apoptotic pathway.     

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons

AIM: To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was identified by PCR, and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass, reactive oxygen species (ROS) and ATP production, cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining, flow cytometry, ATP metabolic assay kit and TUNEL. RESULTS: Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons (P<0.05), enhanced the ability of ATP synthesis (P<0.01), inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION: PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis, inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.     

Arnt2 nucleocytoplasmic translocation in the process of CGNs apoptosis induced by low potassium

AIM：To analyze the changes of Arnt2 subcellular localization in the process of cerebellar granule neurons (CGNs) apoptosis induced by low concentration of potassium.METHODS：Western blotting was used to detect the variation of Arnt2 proteins in nuclei extracts of CGNs in the process of apoptosis induced by low concentration of potassium.Laser scanning confocal microscopy (LSM) was used to detect the changes of Arnt2 subcellular localization.RESULTS：In nuclei extracts of CGNs,when treated with low concentration of potassium,Arnt2 protein was up-regulated obviously at 30 min and peaked at 1 h,returned to the level of normal control at 6 h.LSM analysis result showed that Arnt2,which located in normal CGNs nuclei,was translocated into cytoplasm after induced by low concentration of potassium gradually.At the late stage of apoptosis,Arnt2 proteins located in cytoplasm and overlapped with HSP60 completely,which was regarded as a protein of mitochondria localization.CONCLUSIONS：Arnt2 protein translocates from nuclei into cytoplasm (probably into mitochondria) during CGNs apoptosis evoked by low concentration of potassium;suggesting that Arnt2 is involved in apoptosis probably by transmitting apoptotic or survival signals evoked by potassium directly.     

灵芝中肿瘤细胞凋亡诱导剂的研究

灵芝水提物（LZ）对8种肿瘤细胞增殖的抑制作用具有剂量依赖关系.通过透析和离子交换凝胶过滤层析方法从LZ中分离出活性组分LZ-DN-2-2和LZ-DW-2-a-3.LZ、LZ-DN-2-2 和LZ-DW-2-a-3可剂量依赖地诱导SW620细胞凋亡,使细胞增殖周期停滞于G0期.     

Effect of AMD3100 on dengue virus type 2-induced apoptosis in Eahy926 cells

AIM: To investigate the role of AMD3100 (an inhibitor of CXCR4) in dengue virus type 2 (DV2)-induced apoptosis in human umbilical vein endothelial cell line Eahy926. METHODS: The expression of factor Ⅷ in Eahy926 cells was examined by immunohistochemistry staining. The cells were divided into untreated group, DV2 infection group and DV2+AMD3100 group. Flow cytometric analysis was used to detect the expression of CXCR4 in Eahy926 cells 24 h, 36 h, 48 h and 60 h after DV2 infection. In addition, the percentage of apoptotic cells was also analyzed by flow cytometry. Immunofluorescence was performed to detect the phosphatidylserine (PS) on the surface of Eahy926 cells.RESULTS: Eahy926 cells were factor Ⅷ-positive. Compared with untreated group, the expression of CXCR4 increased in DV2 infection group, most markedly 48 h after infection (66.13%±10.30%, P<0.05). The percentage of apoptotic Eahy926 cells after DV2 infection was the highest at 36 h (29.85%±15.78%, P<0.05). The percentage of DV2-induced apoptotic cells in DV2+AMD3100 group was higher than that in DV2 infection group. The green fluorescence-labeled cells in DV2 infection group and DV2+AMD3100 group were more than those in untreated group. CONCLUSION: DV2 infection induces apoptosis and increases the expression of CXCR4 in Eahy926 cells. AMD3100, the inhibitor of CXCR4, may be a promoter of apoptosis in Eahy926 cells after DV2 infection.     

Factors involved in regulation of polymorphonuclear neutrophils apoptosisin vitro

AIM:This study aimed at elucidated the possibility that prevent tissue from secondary injury by regulating polymorphonuclear neutrophil (PMN) apoptosisin vitro. METHODS:Neutrophils, isolated from peripheral blood, were incubated with sodium arsenite (Ars), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), burned serum and traumatic serum, respectively. Apoptosis rate, expression of CD11b, respiratory burst and concentration of Ca²⁺ were then measured. RESULTS:The elevation of PMN apoptosis rate was Ars concentration dependent, but activated PMN became insensitive to Ars. IL-6 delayed PMN apoptosis (compared with control at 24 h,P＜0.05), inhibited CD11b expression. Burned and traumatic serum had more significant effects on PMN compared with IL-6. CONCLUSION:PMN was observed for the first time to resist spontaneous apoptosis when activated by LPS/TNF, and became insensitive to apoptosis-inducing substance Ars. IL-6, burned serum and traumatic serum could delay PMN apoptosis and recover PMN functions partly.     

Effect of intraoperative dexmedetomidine intervention on perioperative neurocognitive disorders in aged mice

AIM To investigate the effect of dexmedetomidine (DEX) on perioperative neurocognitive disorders (PND) in aged mice and its mechanism. METHODS The PND animal model was established by hepatic left lateral lobectomy in C57BL/6J aged mice, and the effects of intraoperative DEX intervention on postoperative cognitive behavior in aged mice were evaluated by Morris water maze and Y maze experiments. The effects of intraoperative DEX intervention on the changes of neurons in hippocampal CA1 region of aged mice were observed by Nissl staining and TUNEL staining. The effect of intraoperative DEX intervention on long-term potentiation in hippocampal CA1 region was recorded by patch-clamp technique. The effects of intraoperative DEX intervention on hippocampal astrocyte activation in aged mice were detected by immunofluorescence and immunohistochemistry. The effects of intraoperative DEX intervention on the protein levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and cleaved caspase-3 in hippocampal CA1 region of aged mice were determined by Western blot. RESULTS Intraoperative DEX intervention attenuated postoperative cognitive dysfunction in aged mice (P<0.01). Intraoperative DEX intervention significantly inhibited surgery-induced hippocampal neuron loss (P<0.01), reversed surgery-induced decrease in field excitatory postsynaptic potential amplitude, and reduced surgery-induced increases in the protein levels of TNF-α, IL-6 and cleaved caspase-3 in hippocampal CA1 region of aged mice (P<0.01). CONCLUSION Dexmedetomidine protects hippocampal neurons and improves postoperative cognitive function in aged mice by inhibiting hippocampal astrocyte activation and reducing neuronal inflammation and neuronal apoptosis.     

Crosstalk between autophagy and apoptosis induced by Rip2 and its mechanisms in human pancreatic cancer cells

AIM To investigate the crosstalk between autophagy and apoptosis caused by receptor-interacting protein 2 (Rip2) and its underling mechanisms in human pancreatic cancer cells. METHODS Plasmids (pEGFP-C2 and pEGFP-Rip2) were transfected into human pancreatic cancer Panc-1 cells by jetPRIME method. The Panc-1 cells transfected with pEGFP-Rip2 were treated with 3-methyladenine (3-MA), an autophagy inhibitor. The apoptotic rate was analyzed by flow cytometry. The levels of apoptosis-associated proteins were measured by Western blot. The activity of caspase-8, -9 and -3 was examined by colorimetric method. Moreover, the Panc-1 cells transfected with pEGFP-Rip2 were treated with Z-VAD-FMK, a broad inhibitor of caspases. Subsequently, the levels of autophagy- and PI3K/Akt/mTOR signaling pathway-related proteins were assessed by Western blot. The autophagosomes were observed under transmission electron microscope. RESULTS (1) The apoptotic rate in pEGFP-Rip2 group markedly increased as compared with control group and pEGFP-C2 group, while the apoptotic rate in pEGFP-Rip2+3-MA group was further elevated compared with pEGFP-Rip2 group (P<0.05). Meanwhile, the protein levels of Fas, Bax and cytoplasmic cytochrome c (Cyt-c) were significantly increased, and the protein expression of Bcl-2 was markedly reduced in pEGFP-Rip2+3-MA group as compared with pEGFP-Rip2 group (P<0.05). The activity of caspase-8, -9 and -3 in pEGFP-Rip2+3-MA group was higher than that in pEGFP-Rip2 group. (2) The protein expression of beclin-1 and LC3-Ⅱ was significantly increased and more accumulated autophagosomes were observed under transmission electron microscope in pEGFP-Rip2+Z-VAD-FMK group as compared with pEGFP-Rip2 group. Furthermore, the protein levels of p-mTOR and p-Akt in pEGFP-Rip2+Z-VAD-FMK group were markedly reduced compared with pEGFP-Rip2 group, while no significant difference of mTOR and Akt protein expression was found between the 2 groups. CONCLUSION Inhibition of autophagy promotes apoptosis induced by Rip2 in the pancreatic cancer cells. Its mechanism may be associated with the further activation of the intrinsic and extrinsic apoptotic pathways. Suppression of apoptosis accelerates autophagy induced by Rip2 in the pancreatic cancer cells, and the mechanism may be related to the further down-regulation of PI3K/Akt/mTOR signaling pathways. There is a mutual antagonistic effect between autophagy and apoptosis caused by Rip2 in pancreatic cancer cells.     

Effect of nicotine in vitro on expressions of apoptosis-related genes in human lung adenocarcinoma cells by cDNA microarray

AIM: To investigate effect of nicotine on growth of human lung adenocarnoma cells and expressions of apoptosis-related gene. METHODS: Lung adenocarcinoma cell line, SPC-A-1, was cultured in the presence of various concentrations (1-1 000 μg/L) of nicotine for 48 hours. MTT was applied to evaluate effect of nicotine in vitro on growth of SPC-A-1 cell line. After SPC-A-1 cells were treated with 100 μg/L for 48 hours, cDNA expression profile microarray was used to detect the expressions of 451 apoptosis-related genes in SPC-A-1 cell line. RESULTS: Significant proliferation in SPC-A-1 cells treated with nicotine (1-10 μg/L) was observed, but this effect decreased with increase in concentration of nicotine in culture. Growth inhibition rate of 1, 10, 100, 1 000 μg/L of nicotine was 27%, -40%, -40% and -93%. Microarray detection showed that significantly different expressions appeared in 80 of 451 apoptosis-related genes. 29 apoptosis-promoted genes and 26 apoptosis-inhibited genes were up-regulated significantly (CY3/CY5>2.0), and 25 genes were significantly down-regulated (CY3/CY5<0.5). CONCLUSION: Nicotine may promote growth of human lung adenocarcinoma cell through regulating many apoptosis-related gene expressions.