推荐一个优秀的“凋亡”网站

凋亡是机体维持细胞增生与死亡平衡的一个重要机制，在维持内环境稳定与组织发育的基本生命过程中发挥着重要作用。这一平衡的任何紊乱将导致多种严重的病理过程，包括癌症。因而凋亡是近年细胞生物学、分子生物学等生物医学多个领域的研究热点，有关文献急剧增加，相关知识动态更新甚快，有时几乎令人目不暇接。工作中接触到不少凋亡相……

Downregulation of MUC1 Inhibits Proliferation and Promotes Apoptosis by Inactivating NF-κB Signaling Pathway in Human Nasopharyngeal Carcinoma

Objective To investigate the effect of mucin 1(MUC1)on the proliferation and apoptosis of nasopharyngeal carcinoma(NPC)and its regulatory mechanism.Methods The 60 NPC and paired para-cancer normal tissues were collected from October 2020 to July 2021 in Quanzhou First Hospital.The expression of MUC1 was measured by real-time quantitative PCR(qPCR)in the patients with PNC.The 5-8F and HNE1 cells were transfected with siRNA control(si-control)or siRNA targeting MUC1(si-MUC1).Cell proliferation was analyzed by cell counting kit-8 and colony formation assay,and apoptosis was analyzed by flow cytometry analysis in the 5-8F and HNE1 cells.The qPCR and ELISA were executed to analyze the levels of TNF-αand IL-6.Western blot was performed to measure the expression of MUC1,NFкB and apoptosis-related proteins(Bax and Bcl-2).Results The expression of MUC1 was up-regulated in the NPC tissues,and NPC patients with the high MUC1 expression were inclined to EBV infection,growth and metastasis of NPC.Loss of MUC1 restrained malignant features,including the proliferation and apoptosis,downregulated the expression of p-IкB、p-P65 and Bcl-2 and upregulated the expression of Bax in the NPC cells.Conclusion Downregulation of MUC1 restrained biological characteristics of malignancy,including cell proliferation and apoptosis,by inactivating NF-κB signaling pathway in NPC.

Prognositic value of anoikis and tumor immune microenvironment-related gene in the treatment of osteosarcoma

Objective:Osteosarcoma is a highly aggressive primary malignant bone tumor commonly seen in children and adolescents,with a poor prognosis.Anchorage-dependent cell death(anoikis)has been proven to be indispensable in tumor metastasis,regulating the migration and adhesion of tumor cells at the primary site.However,as a type of programmed cell death,anoikis is rarely studied in osteosarcoma,especially in the tumor immune microenvironment.This study aims to clarify prognostic value of anoikis and tumor immune microenvironment-related gene in the treatment of osteosarcoma.Methods:Anoikis-related genes(ANRGs)were obtained from GeneCards.Clinical information and ANRGs expression profiles of osteosarcoma patients were sourced from the therapeutically applicable research to generate effective therapies and Gene Expression Omnibus(GEO)databases.ANRGs highly associated with tumor immune microenvironment were identified by the estimate package and the weighted gene coexpression network analysis(WGCNA)algorithm.Machine learning algorithms were performed to construct long-term survival predictive strategy,each sample was divided into high-risk and low-risk subgroups,which was further verified in the GEO cohort.Finally,based on single-cell RNA-seq from the GEO database,analysis was done on the function of signature genes in the osteosarcoma tumor microenvironment.Results:A total of 51 hub ANRGs closely associated with the tumor microenvironment were identified,from which 3 genes(MERTK,BNIP3,S100A8)were selected to construct the prognostic model.Significant differences in immune cell activation and immune-related signaling pathways were observed between the high-risk and low-risk groups based on tumor microenvironment analysis(all P<0.05).Additionally,characteristic genes within the osteosarcoma microenvironment were identified in regulation of intercellular crosstalk through the GAS6-MERTK signaling pathway.Conclusion:The prognostic model based on ANRGs and tumor microenvironment demonstrate good predictive power and provide more personalized treatment options for patients with osteosarcoma.

TPOL triggers apoptosis with mitochondrial injury through activating a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signaling

AIM:To explore the influence of ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate(TPOL)on cell apoptosis and its potential mechanism.METHODS:HEK293T cells sensitive to TPOL were treated with different concentrations of TPOL with or without exposure to light radiation,before treatment with various inhibitors,N-acetyl-Lcysteine(NAC),pifithrin-αand Z-DVED-FMK.Cell viability was measured by CCK-8 assay.Annexin V/propidium iodide staining was used to count the number of apoptotic cells.DCFH-DA staining was used to detect reactive oxygen species(ROS)levels,and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry.The expression of apoptosis-related proteins and cell cycle-regulated molecules was measured by Western blot.RESULTS:TPOL enhanced the apoptosis of HEK293T cells in a dose-dependent manner(P<0.05),with a decrease in Bcl-2 and increases in Bax and cytochrome C(Cyto C),followed by up-regulation of activated caspase-9 and caspase-3,and the cleavage of PARP(P<0.05).The TPOL-enhanced cleavage of caspase-3 and PARP was rescued by Z-DVED-FMK(P<0.01).TPOL also led to a rapid increase in ROS,a reduction in mitochondrial membrane potential,and the release of Cyto C(P<0.01),all of which could be reversed by the ROS scavenger NAC.Moreover,the TPOL-caused alterations in p21,p27,Rb,and CDK2 were also recovered by the p53 inhibitor pifithrin-α(P<0.05).The TPOL-induced changes in Bax,Bcl-2,cleaved caspase-9,activated caspase-3,and cleaved PARP were subsequently rescued by pretreatment with pifithrin-α(P<0.05).CONCLUSION:TPOL can induce cellular apoptosis with ROS-mediated mitochondrial membrane damage through the activation of a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signal axis.

Zuogui Jiangtang Jieyu Formula ameliorating hippocampal neuronal apoptosis in diabetic rats with depression by inhibiting JNK signaling pathway

Objective To investigate the effect of Zuogui Jiangtang Jieyu Formula(左归降糖解郁方,ZJJF)on hippocampal neuron apoptosis in diabetic rats with depression and to ascertain whether its mechanism involves the regulation of JNK signaling pathway.Methods(i)A total of 72 specific pathogen-free(SPF)grade male Sprague Dawley(SD)rats were randomly divided into six groups,with 12 rats in each group:control,model,metformin(Met,0.18 g/kg)+fluoxetine(Flu,1.8 mg/kg),and the high-,medium-,and low-ZJJF dosages(ZJJF-H,20.52 g/kg;ZJJF-M,10.26 g/kg;ZJJF-L,5.13 g/kg)groups.All groups except control group were injected once via the tail vein with streptozotocin(STZ,38 mg/kg)combined with 28 d of chronic unpredictable mild stress(CUMS)to establish diabetic rat models with depression.During the CUMS modeling period,treatments were administered via gavage,with control and model groups receiving an equivalent volume of distilled water for 28 d.The efficacy of ZJJF in reducing blood sugar and alleviating depression was evaluated by measuring fasting blood glucose,insulin,and glycated hemoglobin levels,along with behavioral assessments,including the open field test(OFT),forced swim test(FST),and sucrose preference test(SPT).Hippocampal tissue damage and neuronal apoptosis were evaluated using hematoxylin-eosin(HE)staining and terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling(TUNEL)staining.Apoptosis-related proteins Bax,Bcl-2,caspase-3,and the expression levels of JNK/Elk-1/c-fos signaling pathway were detected using Western blot and real-time quantitative polymerase chain reaction(RT-qPCR).(ii)To further elucidate the role of JNK signaling pathway in hippocampal neuronal apoptosis and the pharmacological effects of ZJJF,an additional 50 SPF grade male SD rats were randomly divided into five groups,with 10 rats in each group:control,model,SP600125(SP6,a JNK antagonist,10 mg/kg),ZJJF(20.52 g/kg),and ZJJF(20.52 g/kg)+Anisomycin(Aniso,a JNK agonist,15 mg/kg)groups.Except for control group,all groups were established as diabetic rat models with depression,and treatments were administered via gavage for ZJJF and intraperitoneal injection for SP6 and Aniso for 28 d during the CUMS modeling period.Behavioral changes in rats were evaluated through the OFT,FST,and SPT,and hippocampal neuron damage and apoptosis were observed using HE staining,Nissl staining,TUNEL staining,and transmission electron microscopy(TEM).Changes in apoptosis-related proteins and JNK signaling pathway in the hippocampal tissues of rats were also analyzed.

The Chinese Herbal Formula Weichang’an Reduces the Proliferation of Human Gastric Cancer Cells via Mitochondrial Apoptosis

Objective The aim of the study was to investigate the effects of Chinese herbal formula Weichang’an(WCA)on the proliferation and mitochondria-mediated apoptosis of human gastric cancer cells.Methods Cell Counting Kit-8(CCK8)was used to evaluate the antiproliferative activity ofWCA on MKN45 cells;Giemsa staining was used to investigate cell colony formation;flow cytometry was used to analyze cell cycle,apoptosis rate,and caspases activation;and Hoechst staining was used to analyze the morphology of cell nuclei.The mitochondrial membrane potential was analyzed by both flow cytometric measurements and fluorescence microscopy.Western blot was used to analyze the protein levels of pro-caspase-3,Bcl-2,Bax,and Bcl-X.MKN45 cells were subcutaneously injected into the right forelimb of 16 nude mice to establish the tumor xenograft model,and a total of 12 nude mouse tumor xenograft models were successfully created.The nude mice were divided into the control group and the WCA-treated group.The two groups received normal saline and WCA treatment at 35.49 g/kg by a single daily oral gavage for 21 days.The body weight and tumor size of the nude mice were measured twice a week.The ultrastructural changes of subcutaneous tumors were observed by a transmission electron microscope.Results WCA can suppress cell proliferation and colony formation.It can also induce changes in the mitochondrial membrane potential and increase the activities of caspases-3,caspases-8,and caspases-9 in MKN45 cells.WCA induced S-phase arrest and apoptosis in MKN45 cells,and decreased the expression levels of antiapoptotic proteins Bcl-2 and Bcl-X in MKN45 cells,while increasing the expression levels of the proapoptotic proteins Bax and pro-caspase-3 compared with the control group.WCA inhibited the growth of a xenografted MKN45 tumor in nude mice,and the protein levels of Bax and pro-caspase-3 were significantly increased,while Bcl-X and Bcl-2 were reduced compared with the control group.The differences are statistically significant(p<0.05).Conclusion WCA could suppress the proliferation of gastric cancer cells via mitochondrial apoptosis.

Induction of apoptosis and G2/M cell cycle arrest by oridonin in human gastric cancer BGC-823 cells

Aim To investigate in vitro apoptosis-induction effects of oridonin on gastric tumor cells BGC-823 and its effects on cell cycle, mitochondrial membrane potential and intracellular Ca^2＋ to shed light on the mode of its anticancer action. Methods The MTT method was used to investigate the inhibitory effect of oridonin on BGC-823 cells. The apoptosis-induction effect was evaluated by confocal laser microscopy and flow cytometry. The change of mitochondrial membrane potential and the increase of intracellular Ca^2＋ were assessed by fluorescence probe rhodamine123 and Fluo 3-AM, respectively, with flow cytometry. The expression of apoptosis and cell cycle related proteins was studied using western blotting. Results Oridonin inhibited BGC-823 cells growth with IC50 of 22.21 p, mol.L^-1. It induced apoptosis in a dose-dependent manner. In addition, it decreased mitochondria membrane potential, increased intracellular Ca^2＋, and activated pro-caspase 3. BGC-823 cells were arrested in G2/M cell cycle phase with lower expression of cyclin A protein. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. Conclusion Oridonin inhibits the proliferation of BGC-823 cells through G2/M cell cycle arrest and apoptosis induction, which is mediated by influx of Ca^2＋, up-regulation of p53, activation of caspase-3, and down-regulation of cyclin A.

Pinacidil reduces neuronal apoptosis following cerebral ischemia-reperfusion in rats through both mitochondrial and death-receptor signal pathways

Objective To investigate effect of pinacidil, an ATP sensitive potassium channel （KATP） opener, on the neuronal apoptosis and its signaling transduction mechanism following focal cerebral ischemia-reperfusion in rats. Methods One hundred male Wistar rats were randomly divided into four groups： A, sham-operated group; B, ischemia-reperfusion group; C, KATe opener treatment group; and D, KATe opener and blocker treatment group. The middle cerebral artery occlusion （MCAO） model was established by using the intraluminal suture occlusion method, neuronal apoptosis was determined by TUNEL staining, and expressions of caspase-8, caspase-9 and caspase-3 mRNA were detected by in situ hybridization. Results （1） The numbers of apoptotic neurons at 12 h, 24 h, 48 h, and 72 h were significantly less in group C than in groups B and D （P 〈 0.01 or P 〈 0.05）; and there was no difference between groups B and D at all time points （P 〉 0.05）. （2） The expressions of caspase-3 mRNA and caspase-8 mRNA at all times and the expressions of caspase-9 mRNA at 12 h, 24 h, 48 h, 72 h were significantly lower in group C than in groups B and D （P 〈 0.01 or P 〈 0.05）; and there were no differences between groups B and D at all time points （P 〉 0.05）. Conclusions KATP opener can significantly decrease the neuronal apoptosis and the expressions of caspase-3, caspase-8 and caspase-9 mRNAs following cerebral ischemiareperfusion. The neuronal apoptosis may be decreased by the inhibition of both mitochondrial and death-receptor signal pathways.

Correlation of free radical level and apoptosis after intracerebral hemorrhage in rats

Objective To investigate the correlation of perihematomal free radical level and neuronal apoptosis following the intracerebral hemorrhage （ICH）. Methods Animals were randomly divided into 4 groups： sham operation group, model group, 1 mg/kg edaravone group, and 3 mg/kg edaravone group. Each group was then divided into seven subgroups, in which the rats were correspondingly killed at 6 h, 12 h, 24 h, 48 h, 72 h, 7 d or 14 d （n = 1 in each subgroup of the sham group, and n = 6 in each subgroup of the other 3 groups）. By Horseley-Clarke technique, autoblood （80 μL） were administered into the left caudate putamen of SD rats in a double administration-withdrawal way. Rats in the sham group were needled in but not administered with autoblood. The ICH model was then evaluated by Bederson＇s scale. Around the hematoma, the levels of malonaldehyde （MDA） and hydroxyl radical were tested by spectrophotometer, and the process of apoptosis was tested by terminal deoxynucleofidyltransferase-mediated dUTP-biotin nick end labeling （TUNEL） method. Results （1） ICH significantly increased the levels of MDA and hydroxyl radicals. Significant differences in MDA and hydroxyl radical contents were observed among the four groups. （2） In the sham group, a small number of TUNEL-positive cells were found. In the other three groups, the TUNEL-positive cells were observed at 6 h, increased significantly at 24 h, and reached peak level at 3 d, then fell profoundly at 7 d, but remained detectable at 14 d. （3） The positive correlation existed between apoptosis and free radical level （r = 0.2003）, and existed between apoptosis and MDA content （r = 0.6563） in the brain. Conclusion Post-hemorrhagic apoptosis was related to the production of free radicals, indicating that the elevated free radicals following the ICH could induce neuron and glial cell apoptosis.

Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation of Goose Granulosa Cells

Inhibin a is one of the candidate genes that control the ovulation in poultry. To study the genetic effects of inhibin a on apoptosis and proliferation of goose granulosa cells cultured in vitro, two RNA interference （RNAi） expression vectors, psiRNA-INHal and psiRNA-INHα2, were constructed to knock down inhibin α gene expression. After 48 h of transfection, the efficiency of these two RNAi expression vectors was examined by fluorescence microscopy. Meanwhile, inhibin protein expression levels, apoptosis indexes （AI） and proliferation indexes （PI） of granulosa cells were analyzed by flow cytometry. In addition, the supernatants were collected to assay the concentrations of estrogen （E2） and progesterone （P） by radioimmunoassay. The results showed that the expression level of inhibin a in the RNAi group were decreased 30%--40% than those in the control groups （P 〈0.05） and the apoptosis indexes and proliferation indexes in the RNAi groups were significantly higher than those in the control groups （P 〈0.05）. However, the E2 concentrations in the RNAi groups were lower than those in the control groups （P 〈0.05）. These results indicate that inhibin a has antagonistic effect on granulosa cell apoptosis.

Apoptosis of Human Hepatoma Cells Induced by Gynostemma Pentaphyllum Makino

Objective： To identify the proapoptotic effects of Gynostemma pentaphyllum Makino （GpM） on human hepatoma cells. Methods： The effects of GpM on the cell apoptosis of human hepatoma cell line Huh-7 was assessed by flow cytomety. The expression of Bcl-2, Bcl-XL, Bax and Bad molecules in hepatoma cells treated with GpM was detected by Western blot. Results： After treatment with 20 mg/mL GpM for 24 h, 56% of Huh-7 cells were undergoing apoptosis, while cell death was only observed in 12% of humaa fibroblast cells treated with GpM. Western blot demonstrated that, Bcl-2 was markedly decreased in Huh-7 cells treated with GpM. Whereas, Bax was significantly up-regulated in Huh-7 cells treated with GpM. Conclusion： Treatment of human hepatoma cells with GpM induced apoptosis through the down-regulation of Bcl-2, and up-regulation of Bax.

Apoptosis Rate and Objective Diagnosis of Drug Resistance of Ovarian Cancer Cell Lines

Objective： To investigate whether the change of drug resistance degree could be evaluated by apoptotic rate in ovarian cancer cell lines. Methods： Human epithelia ovarian cancer cell line A2780 and its platinum （DDP） resistance cell line AD4 were used. They were divided into 4 groups respectively （A2780-DDP group, A2780-DDP＋VRM group, AD4-DDP group and AD4-DDP＋VRM group）. 3-（4, 5- dimethylthiazol-2-yl）-2, 5-diphenyl-2H-tetrazolium bromide （MTT） was used to measure the multiple of drug resistance. The expression of drug-resistance genes （mdrl, TopoⅡα and GSTπ） was detected by using reverse transcription polymerase chain reaction （RT-PCR）. Semi-quantity assay was proceed by rate the of multidrug resistance genes to G3 PDH gene. Apoptosis was measured by DNA gel electrophoresis and flow cytometry respectively. The advantages and disadvantage of evaluating drug-resistance with these three methods were analyzed. Results： The 50% inhibition concentration （IC50） of A2780 and AD4 was 19.2 μg/mL and 66 μg/mL respectively, and the resistance fold of the AD4 was 3.4. Some drug-resistance genes could be detected by RT-PCR in A2780 and AD4 cell lines. The expression of mdrl was only （0.09±0.03）×10^-2 ： 1 and （0.10±0.02） × 10^-2：1 respectively （rate to G3 PDH gene） with the difference being not significant between them. The expression of TopoⅡα in the A2780 cells was （2.60±0.12）×10^-2：1 and （0.11±0.03）× 10^-2：1 in the AD4 cells respectively with the difference between them being significant. On the contrary, the expression of GSTπ in A2780 cells was lower than in AD4 cells, and the ratio was （0.11±0.03）×10^-2：1 and （3.13±0.14）×10^-2：1 respectively with tile difference being significant between them. There was no significant difference among the genes expression after the drugs were given for 6 h, 12 h and 24 h. couldn＇t reflect the change of drug-resistance timely. DNA gel electrophoresis used to detect apoptosis was only a qualitative analysis. Different drug resistance degrees may be detected by flow cytometry as early as few hours after drugs were given, which realized the earlier and quantities detection of drug resistance. Conclusion： Detection of apoptosis with flow cytometry may not be affected by the variety of drug-resistance genes, suggested this was a general, quantitative and objective method to reflect drug resistance.

Study on Induction of Microcystis aeruginosa Cell Apoptosis by Signal Molecule N-acetyl-homoserine Lactones(AHLs)

[Objective]The relationship between signal molecule N-acety-homoserine lactones（AHLs） and Microcystis aeruginosa cell apoptosis was studied.[Method]With M.aeruginosa as the test materials treated by 5 μmol/L N-acety-homoserine lactones（AHLs）,the morphology of cell apoptosis was observed through staining with DAPI.[Result]Microcystis aeruginosa cell apoptosis was induced by signal molecule N-acetyhomoserine lactones（AHLs） with the concentration of 1 μmol/L to inhibit the growth and proliferation of Microcystis aeruginosa.[Conclusion] The results provided the important scientific basis and new management ideas for the treatment of water bloom of Microcystis aeruginosa.

Expression of PPAR-γ in Lung Cancer and the Role of PPAR-γ in Apoptosis of Lung Cancer

Objective: To investigate the relationship between peroxisomeproliferators-activated receptor-gamma (PPAR-γ) and clinical pathology of lung cancer, and to studythe inhibitory effect of PPAR-γ activators on lung cancer growth and the mechanism of inducingapoptosis of lung cancer. Methods: Expression of PPAR-γ in 15 cases of non-cancerous lung tissuesand 64 cases of lung cancer tissues was detected by using immunohistochemistry. The average A valueswere measured by using image analysis. The expression of PPAR-γ in lung cancer cells was detectedby using RT-PCR. After being treated with PPAR-γ activators, apoptosis was detected by using flowcytometry, and meanwhile, the change in caspase-3 activity was detected by caspase-3 kits. Results:Expression levels of PPAR-γ in cancerous tissues were higher than those in non-cancerous lungtissues. In four types of lung cancer, the sequence of PPAR-γ expression from high to low levelswas small-cell lung cancer, squamous carcinom, large-cell lung cancer, adenocarcinoma in turns. Theexpression of PPAR-γ was correlated with differentiation and postoperative TNM staging of lungcancer tissues, but not with lymph node metastasis. PPAR-γ expressed in two lung cancer cell lines,which could induce apoptosis of lung cancer cells after being treated with PPAR-γ activators, andcaspase-3 activity in cells treated with PPAR-γ activators was significantly increased. Conclusion:PPAR-γ is correlated with clinical pathology and apoptosis of lung cancer closely, and activatedPPAR-γ can increase caspase-3 activity to induce cells apoptosis. PPAR-γ will be able to become anew target for therapy of lung cancer in the future.

Cell Apoptosis Induced by Oligosaccharide in Suspension Cultures of Taxus chinensis

Taxol production of Taxus chinensis (Pilger) Rehd. var. mairei (Lemee et Lévl.) Cheng et L. K. Fu induced by oligosaccharide from Fusarium oxysporum f. vasinfectum (Atkinson) Snyder et Hansen was studied in suspension cultures, and it was found that oligosaccharide triggered cell apoptosis. Under transmission electron microscope the following morphological changes were observed: cell shrinkage, condensation of cytoplasm, nuclear fragmentation, and the increase of high electron density bodies in vacuole in great quantity. In oligosaccharide_treated cells, agarose gel electrophoresis revealed that DNA was digested into oligonucleosomal fragments that were times of 200 bp appearing as DNA ladders. Control cells were in normal physiological state, they were intact, abundant in organelle and with integral nucleus DNA, and the rate of taxol biosynthesis in these cells was very low. After the oligosaccharide to the culture system, the defense system of cells was elicited and the secondary metabolism was strengthened, i.e. phenolics were accumulated in the medium, the activity of polyphenol oxidase (PPO) was increased quickly and secondary wall of cells was thickened. The activity of L _phenylalanine ammonia lyase (PAL), the critical enzyme of the phenylpropanoid pathway, was increased promptly 1 h after elicitation. The rate of taxol production was improved sharply and the maximal taxol concentration at 72 h was six times that of control. Appearance of cell apoptosis was accompanied with the highest concentration of taxol in suspension cultures.

Regulation of Bcl-2 Family in TIP30-Induced Apoptosis in Hepatoblastoma Cells

Objective: To investigate the role of TIP30 in the apoptotic signal pathwayin HepG2, and Hep3B and Hu-7 hepatoblastoma cell lines. Methods: In order to confirm whether TIP30conducted Bcl-2 family was involved in apoptosis signal pathway, MTT assay, in situ 3' end labellingof DNA assay and Western blot were carried out to detect the diverse apoptotic function of TIP30and the regulation of Bcl-2 family. Results: TIP30 induced apoptosis as evidenced by morphologicalchanges in hepatoblastoma cells, which was accompanied by up-regulating Bax and Bad proteins andstimulating them from cytoplasm to mitochondria, and down-regulating Bcl-xl, while it had no effecton the level of Bak protein. Conclusion: TIP30 induced apoptosis partly by modulating the proteinlevels of members of Bcl-2 family in hepatoblastoma cells. Elucidating the mechanism by which TIP30induces cell death might establish it as an anticancer factor.

Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity

Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium （MPP^＋） induced pheochromocytoma （PC12） cell death and the potential mechanisms. Methods pcDNA3.1（＋）-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC 12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-（4,5- dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results （1） The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1（＋）-14-3-3 plasmids transfected into PC 12 cells. （2） MPP^＋ caused a decrease of cell viability in a dose-dependent manner. At 100μmol/L MPP^＋, cell viability reduced approximately 50%. （3） The caspase activity increased along with the MPP^＋ concentrations rising and reached its maximum value （0.34 μmol/mg protein） at 100 μmol/L MPP＊. However caspase activity decreased significantly when the MPP^＋ concentration exceeded 100 μmol/L. （4） Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC 12 cells treated with 100μmol/L MPP^＋ from 26.5% to 8.6%. （5） Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC 12 cells with 100 μmol/L MPP^＋. Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP^＋-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson＇s disease.

Liriodendrin protects SH-SY5Y cells from dopamine-induced cytotoxicity

Aim To investigate the effect of liriodendrin, an extract from Fraxinus sielboldiana blume belonging to the Oleaceae family, on dopamine-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Methods Cell viability was processed when treated with 50 μmol·L^-1 of dopamine for 24 h by MTT assay. Early apoptosis, late apoptosis/necrosis were analyzed by flow cytometry using Annexin V-FITC and propidium iodide （PI） double-staining, respectively. Generation of reactive oxygen species （ROS） was assessed by DCFH-DA, an oxidation-sensitive fluorescent probe. To evaluate mitochondrion membrane potential （Δψm） using flow cytometry with the fluorescent dye Rhodamine 123. The transcriptional level of P53 was studied using RT- PCR. Results The dopamine-induced loss of cell viability was significantly attenuated by liriodendrin treatment at the concentration of 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 mol·L^-1. The protective effects of liriodendrin （10^-7, 10^-6 and 10^-5 mol·L^-1） on dopamine-induced cytotoxicity may be ascribed to its anti-oxidative properties by reducing ROS level and anti-apoptotic effect via protection of Δψm. In addition, the effect of liriodendrin may involve the P53 pathway in apoptosis. Conclusion Liriodendrin may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases such as Parkinson＇s disease （PD）

Neuroprotective Effects of Bushen Decoction Against Glutamate-Induced Neurotoxicity in PC12 Cells

Aim The enhanced effect of Bushen (Kidney-tonifying) decoction (BS) oncultured PC12 cell proliferation and its antagonistic action on neurotoxicity induced by glutamatewere investigated by serum pharmacological method of the Chinese material medica (CMM) in vitro.Methods The effect of BS on cultured PC12 cell activity and its antagonistic action on neurotoxicityinduced by glutamate was observed by MTT method. Flow cytometry and fluorescence microscopetechniques were employed to observe the antagonistic effect of BS on early period apoptosis of PC12cells induced by glutamate. Results The serum with BS was able to enhance activity of PC12 cells andexert antagonistic effect on glutamate-induced neurotoxicity. Meanwhile, these beneficial effectsproduced by BS were found to be the strongest in 20% concentration of in serum BS. Moreover, it caninhibit apoptosis of PC12 cells induced by glutamate , which occurs in the early period. ConclusionBS may exert a potential neuroprotective effect.

Effects of Promoter Region 5’CpG Island Demethylation on the Biological Behavior of Human Colorectal Cancer RKO Cells in Vitro

OBJECTIVE To explore the relationship between the methylation status of the promoter 5＇CpG island region and the biological behavior of human colorectal cancer RKO cells in vitro. METHODS RKO cells were treated with a selective DNA methyltransferase inhibitor-5-aza-2＇-deoxycytidine （5-aza-CdR） for 72 h. Methylationspecific PCR （MSP）, T-A cloning and DNA sequence analysis were used to determinate the 5＇CpG island methylation status of the p16/CDKN2 tumor suppressor gene. Cell growth, morphological changes and apoptosis were analyzed by the MTT assay, flow cytometry, fluorescence staining and electron microscopy. RESULTS The 5＇CpG island of the p16/CDKN2 tumor suppressor gene in RKO cells was a typically hypermethylated. The DNA methyltransferase inhibitor （5-Aza-CdR） effectively reversed the hypermethylation status of the promoter region. With demethylation, RKO cell growth was suppressed, the cells doubling times were prolonged （P〈0.01） and apoptosis was induced, which showed a relationship. CONCLUSION A selective DNA methyltransferase （DNMT） inhibitor can inhibit proliferation by demethylation in 5＇CpG islands, and may be a potential new therapy target for colorectal cancer.