Induction of apoptosis by TPA and VP—16 is through translocation of TR3

AIM：To investigate the role of TR3 in induction of apoptosis in gastric cancer cells.METHODS:Human gastric cancer cell line,MGC80-3,was used .Expression of TR3 mRNA and its protein was detected by Northern blot and Western blot Localization of TR3 protein was showed by imunofluorescence analysis under laser-scanning confocal microscope.Apoptotic morthology was observed by DAPL fluorescence staining,and apoptotic index was couted manong 10000 cells randomly Stable transfection assay was carried out by Lipofectamine.RESULTS:Treatment of MGC80-3 cells with TPA and VP-16 resulted in apoptosis,accompanied by the repression of Bcl-2 protein in atime-dependent manner,At the same time,TPA and VP-16 also up-regulated expression level of TR3 mRNA in MGC80-3 cells that expressed TR3 mRNA When antisense-TR3 expression vector was transfected into the cells,expression of TR3 protein was repressed.In this case,TPA and VP-16 did not induce apoptosis.In addition,TPA and VP-16-induced apotosis involved in translocation of TR3,IN MGC80-3 cells,TR3 localized concentrative in nucleus,after treatment of cells with TPA and Vp-16,TR3 translocated from nuclesu to cytosol obviously,However,When this nuclear translocaion was blocked by LMB apoptosis was not occurred in MGC80-3 cells even in the presence of TPA and VP-16.CONCLUSION:Induction of apoptosis by TPA and VP-16 is through induction of TR3 expression and translocation of TR3 from nucleus to cytosol,Which may be a novel signal pathway for TR3,and represent the new biological function of TR3 to exert its effect on apoptosis in gastric cancer cells.     

Roles of PLC-γ2 and PKCα in TPA-induced apoptosis of gastric cancer cells

AIM: To investigate the roles of PLCγ2 and PKCα in TPA-induced apoptosis of gastric cancer cells. METHODS: Human gastric cancer cell line MGCS0-3 was used. Protein expression levels of PLCγ2 and PKCα were detected by Western blot. Protein localization of PLCγ2 and PKCα was shown by immunofluoscence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among I 000 cells randomly. RESULTS: Treatment of gastric cancer cells MGCS0-3 with TPA not only up-regulated expression of PLC-γ2 protein, but also induced PLC-γ2 translocation from the cytoplasm to the nucleus. However, this process was not directly associated with apoptosis induction. Further investigation showed that PKCa translocation from the cytoplasm to the nucleus was correlated with initiation of apoptosis. To explore the inevitable linkage between PLC-γ2 and PKCα during apoptosis induction, PLC inhibitor U73122 was used to block PLC-γ2 translocation,in which neither stimulating PKCα translocation nor inducing apoptosis occurred in MGC80-3 cells. However, when U73122-treated cells were exposed to TPA, not only PLC-γ2, but also PKCα was redistributed. On the other hand, when cells were treated with PKC inhibitor alone, PLC-γ2 protein was still located in the cytoplasm. However, redistribution of PLC-γ2 protein occurred in the presence of TPA, no matter whether PKC inhibitor existed or not. CONCLUSION: PLC-γ2 translocation is critical in transmittingTPA signal to its downstream molecule PKCα. As an effector, PKCα directly promotes apoptosis of MGC80-3 cells. Therefore, protein translocation of PLCγ2 and PKCα is critical event in the process of apoptosis induction.     

Roles of PLC-γ2 and PKCα in TPA-induced apoptosis of gastric cancer cells

AIM: To investigate the roles of PLCγ2 and PKCα in TPA-induced apoptosis of gastric cancer cells.METHODS: Human gastric cancer cell line MGC80-3 was used. Protein expression levels of PLCγ2 and PKCα were detected by Western blot. Protein localization of PLCγ2 and PKCα was shown by immunofluoscence analysis under laserscanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among 1 000 cells randomly.RESULTS: Treatment of gastric cancer cells MGC80-3 with TPA not only up-regulated expression of PLC-γ2 protein,but also induced PLC-γ2 translocation from the cytoplasm to the nucleus. However, this process was not directly associated with apoptosis induction. Further investigation showed that PKCa translocation from the cytoplasm to the nucleus was correlated with initiation of apoptosis. To explore the inevitable linkage between PLC-γ2 and PKCα during apoptosis induction,PLC inhibitor U73122 was used to block PLC-γ2 translocation,in which neither stimulating PKCα translocation nor inducing apoptosis occurred in MGC80-3 cells. However, when U73122treated cells were exposed to TPA, not only PLC-γ2, but also PKCα was redistributed. On the other hand, when cells were treated with PKC inhibitor alone, PLC-γ2 protein was still located in the cytoplasm. However, redistribution of PLC-γ2 protein occurred in the presence of TPA, no matter whether PKC inhibitor existed or not.CONCLUSION: PLC-γ2 translocation is critical in transmitting TPA signal to its downstream molecule PKCα. As an effector,PKCα directly promotes apoptosis of MGC80-3 cells.Therefore, protein translocation of PLCγ2 and PKCα is critical event in the process of apoptosis induction.     

Selective tropism of liver stem cells to hepatocellular carcinoma in vivo

AIM： To investigate the selective tropism of liver stem cells to hepatocellular carcinoma （HCC） in an animal model and its feasibility as a vector to deliver therapeutic genes for targeted therapy of HCC.
METHODS： WB-F344, a kind of rat liver stem cell, was infected with recombinant virus to establish a cell line with stable, high-level expressing enhanced green fluorescent protein （EGFP）. An animal model of HCC in Wistar rats was established by implanting HCC cells （CBRH7919） combined with an immunosuppressive drug. EGFP labeled liver stern cells were injected into caudal veins of the animals and distribution was observed at different time points after injection. SDF-1 and c-kit expression in non-tumor liver and tumor tissue were analysed by immunohistochemistry for the relationshiop between the expression and migration of liver stem cells. Furthermore, hepatic stern cells were injected via the portal vein, hepatic artery, caudal vein, or directly into the pericancerous liver tissue, respectively, and effects on migration, localization, and proliferation of the hepatic stern cells within the tumor tissue were observed and analyzed.
RESULTS： Recombinant adenovirus could deliver the EGFP gene to hepatic stem cells. A new stem cell line, named WB-EGFP, was established that stably expressed EGFP. WB-EGFP cells still showed selective tropism towards HCC and EGFP expression was stable in vivo. According to immunohistochemistry results, SDF-1 may not be related to the mechanisms of tropism of hepatic stem cells. Different application sites affected the distribution of liver stem cells. Injection via the portal vein was superior with regard to selective migration, localization, and proliferation of the hepatic stem cells within the tumor tissue.
CONCLUSION： Liver stem cells have the biological behavior of selective migration to HCC in vivo and they could localize and proliferate within HCC tissue stably expressing the target gene. Liver stem cells are a potential tool for a targeted gene therapy o     

Expression of exogenous rat collagenase in vitro and in a rat model of liver fibrosis

AIM：The present study was conducted to test the hypothesis that the introduction of the collagenase gene into tissue culture cells and into a rat model of liver fibrosis would result in the expression of enzymatically active product.METHODS:FLAG-tagged full-length rat collagenase cDNA was PCRamplified and cooned into a mammalian expression vector.NIH3T3 cells were then transiently transfected with this construct.Expression of exogenous collagenase mRNA was assessed by RT-PCR,and the exogenous collagenase detected by Western blotting using anti-FLAGmonoclonal antibody.Enzymatic activity was detected by gelatin zymography,To determine the effects of exogenous collagenase production in vovo,the construct was bound to glycosyl-poly-L-lysine and then transduced into rats that had developed liver fibrosis as a result of CCl4 plus ethanol treatment.The hepatic expression of the construct and its effect on the formation of liver fibrosis were demonstated usingRT-PCR and immunohistochemistry.RESULTS:It was found that exogenously expressed rat collagenase mRNA could be detected in NIH3T3 cells following transfection.Enzymatically active collagenase could also be detected in the culture medium.The recombinant plasmid was also expressed in rat liver after in vivo gene transfer.Expression of exogenous rat collagenase correlated with decreased deposition of collagen types IandⅢin the livers of rats with experimentally induced liver fibrosis.CONCLUSION:The expression of active exogenous rat collagenase could be achievad in vitro and in vivo,It was suggested that in vivo expression of active exogenous collagenase may have therapeutic effects on the formation of liver fibrosis.     

Abnormal endogenous pain modulation and somatic and visceral hypersensitivity in female patients with irritable bowel syndrome

AIM： To investigate the role of endogenous pain modulatory mechanisms in the central sensitization implicated by the visceral hypersensitivity demonstrated in patients with irritable bowel syndrome （IBS）. Dysfunction of modulatory mechanisms would be expected to also result in changes of somatic sensory function.
METHODS： Endogenous pain modulatory mechanisms were assessed using heterotopic stimulation and somatic and visceral sensory testing in IBS. Pain intensities （visual analogue scale, VAS 0-100） during suprathreshold rectal distension with a barostat, cold pressor stimulation of the foot and during both stimuli simultaneously （heterotopic stimulation） were recorded in 40 female patients with IBS and 20 female healthy controls.
RESULTS： Rectal hypersensitivity （defined by 95% Cl of controls） was seen in 21 （53%）, somatic hypersensitivity in 22 （55%） and both rectal and somatic hypersensitivity in 14 of these IBS patients. Heterotopic stimulation decreased rectal pain intensity by 6 （-11 to -1） in controls, but increased rectal pain by 2 （-3 to ＋6） in all IBS patients （P 〈 0.05） and by 8 （-2 to ＋19） in IBS patients with somatic and visceral hypersensitivity （P 〈 0.02）.
CONCLUSION： A majority of IBS patients had abnormal endogenous pain modulation and somatic hypersensitivity as evidence of central sensitization.     

Degradation of retinoid X receptor α by TPA through proteasome pathway in gastric cancer cells

AIM: To investigate and determine the mechanism and signal pathway of tetradecanoylphorbol-1, 3-acetate (TPA) in degradation of RXRα.METHODS: Gastric cancer cell line, BGC-823 was used in the experiments. The expression level of R XRα protein was detected by Western blot. Nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation.Localization and translocation of RXRα were observed under laser-scanning confocal microscope through labeling specific anti-RXRα antibody and corresponding immunofiuorescent antibody as secondary antibody. Different inhibitors were used as required.RESULTS: In BGC-823 cells, RXRα was expressed in the nucleus. When cells were treated with TPA, expression of RXRα was repressed in a time-dependent and TPAconcentration-dependent manner. Meanwhile, translocation of RXR from the nucleus to the cytoplasm occurred, also in a time-dependent manner. When cells were pre-incubated with proteasome inhibitor MG132 for 3 hrs, followed by TPA for another 12 hrs, TPA-induced RXRα degradation was inhibited. Further observation of RXRα translocation in the presence of MG132 showed that MG-132 could block TPAinduced RXRα redistribution. Conversely, when RXRαtranslocation was inhibited by LMB, an inhibitor for blocking protein export from the nucleus, TPA could not repress expression of RXRα.CONCLUSION: TPA could induce the degradation of RXRα protein in BGC-823 cells, and this degradation is time-and TPA-concentration-dependent. Furthermore, the degradation of RXRα by TPA is via a proteasome pathway and associated with RXRα translocation from the nucleus to the cytoplasm.     

Phosphorylated vasodilator-stimulated phosphoprotein is localized on mitotic spindles of the gastric cancer cell line SGC-7901

INTRODUCTION The spindle is a vital apparatus of mitotic cells. The mechanism of its formation has attracted great research attention. In centrosome-containing cells, centrosomes are thought to be instrumental for organization of the spindle poles and det…     

线粒体损伤在幽门螺杆菌诱导胃癌细胞凋亡中的作用

目的研究线粒体途径在幽门螺杆菌(Hp)诱导胃癌细胞凋亡过程中的作用。方法采用Westernblotting检测细胞内线粒体DNA细胞色素氧化酶(COX)Ⅰ蛋白表达的含量;流式细胞术测定细胞凋亡和线粒体膜电位变化。结果Hp培养滤液呈剂量和时间依赖性地直接诱导SGC-7901细胞凋亡,随着其浓度的增加,细胞的凋亡率呈上升的趋势。Hp培养滤液处理SGC-7901细胞4h后,线粒体膜电位开始降低,8h后下降更明显,12h已基本降至最低,24h后无明显变化。Hp培养滤液作用胃癌细胞后,线粒体DNACOXⅠ蛋白表达明显低于对照组(632·8±40·6vs895·1±44·2,P<0·05)。结论线粒体途径可能在Hp诱导SGC-7901胃癌细胞凋亡过程中起重要作用。     

钙周期素结合蛋白的细胞周期依赖性转位现象

目的研究钙周期素结合蛋白（CacyBP／SIP）在胃癌细胞SGC7901中亚细胞定位与细胞周期的关系。方法利用双胸腺嘧啶脱氧核苷（胸苷）阻滞法将胃癌细胞SGC790l进行细胞周期同步化,流式细胞术检测细胞周期同步化效果。免疫荧光染色法观察CacyBP／SIP在不同时期的亚细胞定位。蛋白质印迹法检测CacyBP／SIP总蛋白及核蛋白在不同时期的表达水平。结果采用双胸苷阻滞法使SGC7901细胞阻滞于G,／s期,撤药后培养4h,细胞进入S期。大多数细胞在8～12h时进入G2／M期,16h又重新进入G1期。免疫荧光染色发现,在G1和S期时,CacyBP／SIP主要分布在细胞浆中,而在以G2期为主的细胞中,CacyBP／SIP聚集在核周或进入胞核,说明CacyBP／SIP存在细胞周期依赖性的核转位。在细胞的各时相中CacyBP／SIP总蛋白无明显变化,但在G2期时CacyBP／SIP的核蛋白表达水平增高。结论CacyBP／SIP存在细胞周期依赖的转位现象,可能参与了G2／M期的调控作用。     

Akt和核因子κB信号通路在胃癌细胞化学治疗耐药中的作用

目的 探讨Akt、核因子(NF)-κB信号通路在胃癌细胞化学治疗耐药中的作用,以及Akt、NF-κB信号通路的相互作用关系.方法 采用化学治疗药物阿霉素、足叶乙甙及两药联合应用Akt抑制剂(Wortmannin)或NF-κB抑制剂(MG-132)分别作用于胃癌细胞(SGC-7901).用四甲基偶氮唑盐比色法(MTT法)检测SGC-7901细胞增长率;TUNEL法和膜联蛋白V/碘化丙啶(PI)双标法检测肿瘤细胞凋亡;免疫细胞化学法检测NF-κB/P65蛋白表达;电泳迁移率实验(EMSA)法检测NF-κB-DNA结合活性的变化;Western印迹法检测磷酸化Akt或磷酸化NF-κB抑制因子(IκB)α蛋白的表达.结果 ①阿霉素和足叶乙甙均能明显抑制SGC-7901细胞的生长,并呈时间、剂量依赖性;分别联合应用Wortmannin或MG-132后能进一步抑制其生长.②化学治疗药物剂量依赖性地诱导SGC-7901细胞凋亡和NF-κB或Akt活化,联合应用MG-132或Wortmannin均能增强化学治疗药物的诱导凋亡作用和抑制NF-κB或Akt活化作用.③Wortmannin可明显抑制NF-κB的活化,而MG-132对Akt的活化无明显影响.结论 化学治疗药物诱导SGC-7901细胞凋亡的同时诱导Akt、NF-κB活化,抑制Akt、NF-κB的活化可增加化学治疗药物的疗效.     

Synergistic effect of oxymatrine and angiogenesis inhibitor NM-3 on modulating apoptosis in human gastric cancer cells

AIM: To investigate the synergistic effect of oxymatrine(OM) and angiogenesis inhibitor NM-3 on modulatingapoptosis in human gastric cancer cell lines SGC-7901,MKN-45, MKN-74. METHODS: Human gastric cancer lines SGC-7901,MKN-45, MKN-74 were treated with OM in the absenceand presence of NM-3. The inhibitory rates weredetected by MTT assay. Synergistic effect of OM andNM-3 on the growth of survivin, bcl-2, bax and p53 inSGC-7901 cells were examined by semiquantitative RT-PCR and Western blotting, and their growth inhibitoryeffects were also observed on SGC-7901 tumor xenograftin nude mice.RESULTS: OM combined with NM-3 exhibited asynergistic inhibitory effect on the growth of SGC-7901,MKN-45 and MKN-74 cells in a time-dependent manner.Twenty-four hours after treatment with OM, NM-3 aloneand their combination, mRNA expression of survivin andbcl-2 in SGC-7901 cells decreased, p53 mRNA expressionincreased. OM (4 g/L) combined with NM-3 significantlyincreased the expression of p53 mRNA and decreasedthe expression of survivin and bcl-2 compared witheither agent alone (193% ± 34% vs 129% ± 12%;44% ± 18% vs 92% ± 18%; 36 ± 17% vs 93% ± 23%,P < 0.05). Western blotting showed that the synergisticeffect of OM and NM-3 on protein translation of survivin,bcl-2 and p 53 was in accordance with their mRNAs.Furthermore, OM/NM-3 combination obviously exhibitedantitumor growth effect in xenografted human gastriccancer cells SGC-7901 compared with either agent alone.CONCLUSION: OM combined with NM-3 has synergisticinhibitory effects on human gastric cancer cells in vitro and can suppress the growth of xenografted human gastric cancer cells SGC-7901 in vivo.     

Periostin过表达诱导人胃癌细胞化疗耐药性的机制研究

背景：前期研究发现periostin过表达可增强人胃癌细胞株SGC-7901对顺铂和5-氟尿嘧啶（5-Fu）诱导的细胞凋亡的抵抗能力。目的：探讨periostin过表达诱导人胃癌细胞化疗耐药性的可能机制。方法：Periostin稳转组和空载体稳转组SGC-7901细胞分别以5μmol／L顺铂或10μmol／L5-Fu处理24h或不予化疗药物处理,其中periostin稳转组在化疗药物处理前可接受或不接受Akt特异性抑制剂MK-2206（1μmol／L）预处理30min。以蛋白质印迹法检测各组细胞的总Akt、磷酸化Akt（p-Akt）、p53蛋白表达,流式细胞术检测细胞凋亡。结果：periostin稳转组SGC．7901细胞Akt磷酸化水平明显高于空载体稳转组,两组间总Akt无明显差异。在经顺铂或5-Fu处理的各组SGC．7901细胞中,periostin稳转组p53蛋白表达和细胞凋亡率明显低于空载体稳转组,而MK-2206预处理可在一定程度上逆转periostin对p53表达的抑制作用及其诱导的凋亡保护效应。结论：通过激活Akt通路抑制p53表达可能是periostin过表达诱导人胃癌细胞化疗耐药性的机制之一,有望作为胃癌治疗的潜在靶点。     

RhoA and RhoC -siRNA inhibit the proliferation and invasiveness activity of human gastric carcinoma by Rho/PI3K/Akt pathway

AIM： To evaluate the effects of adenovirus-mediated gene transfer of RhoA siRNA and RhoC siRNA on proliferation and invasion of SGC7901 cells by Rho/ PI3K/Akt pathway.
METHODS： Plasmid of RhoA siRNA and RhoC siRNA were constructed and transfected into SGC7901 cells. siRNA and LY294002 （PI3K inhibitor） were designed as the control group. The mRNA and protein expressions of RhoA and RhoC were respectively detected with RT-PCR and western blotting. In order to find out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB/C nude mice with RhoA and RhoC-siRNA, and tumor control rate （%） in nude mice was calculated.
RESULTS： RhoA and RhoC siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 Cells.The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines are on the decline after treatment of Ad-RhoA and RhoC-siRNA （12.64 ±3.27 vs 87.38±17.38, P 〈 0.05）. The values of 490 nm wavelength light absorption were different in the five groups. The number of alive cells in the group of RhoA and RhoC-siRNA was lower than others in the 6^th d （0.71 ± 0.01 vs 3.82± 0.11 P 〈 0.05）. The apoptosis rate of transfected RhoA and RhoC-siRNA group with FACS were 19.07% ± 1.78 and there were significant differences between treated and control groups （19.07 ± 1.78% vs 1.23± 0.11%, P 〈 0.01）. The tumor transplantation experiment in BALB/C nude mice showed intratumoral injection of RhoA or RhoC siRNA can inhibit tumor growth.
CONCLUSION： RhoA and RhoC siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a PI3K/Akt pathway. These results provide a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.     

Dynamic changes in the localization of thermally unfolded nuclear proteins associated with chaperone-dependent protection

Molecular chaperones are involved in the protection of cells against protein damage through their ability to hold, disaggregate, and refold damaged proteins or their ability to facilitate degradation of damaged proteins. Little is known about how these processes are spatially coordinated in cells. Using a heat-sensitive nuclear model protein luciferase fused to the traceable, heat-stable enhanced green fluorescent protein (N-luc-EGFP), we now show that heat inactivation and insolubilization of luciferase were associated with accumulation of N-luc-EGFP at multiple foci throughout the nucleus. Coexpression of Hsp70, one of the major mammalian chaperones, reduced the formation of these small foci during heat shock. Instead, the heat-unfolded N-luc-EGFP accumulated in large, insoluble foci. Immunofluorescence analysis revealed that these foci colocalized with the nucleoli. Time-lapse analysis demonstrated that protein translocation to the nucleolus, in contrast to the accumulation at small foci, was fully reversible upon return to the normal growth temperature. This reversibility was associated with an increase in the level of active and soluble luciferase. Expression of a carboxyl-terminal deletion mutant of Hsp70(1-543), which lacked chaperone activity, had no effect on the localization of N-luc-EGFP, which suggests that the Hsp70 chaperone activity is required for the translocation events. Our data show that Hsp70 not only is involved in holding and refolding of heat-unfolded nuclear proteins but also drives them to the nucleolus during stress. This might prevent random aggregation of thermolabile proteins within the nucleus, thereby allowing their refolding at the permissive conditions and preventing indirect damage to other nuclear components.