MiR-204-3p overexpression inhibits gastric carcinoma cell proliferation by inhibiting the MAPK pathway and RIP1/MLK1 necroptosis pathway to promote apoptosis

BACKGROUND Gastric carcinoma(GC)is the third most frequent cause of cancer-related death,highlighting the pressing need for novel clinical treatment options.In this regard,microRNAs(miRNAs)have emerged as a promising therapeutic strategy.Studies have shown that miRNAs can regulate related signaling pathways,acting as tumor suppressors or tumor promoters.AIM To explore the effect of miR-204-3p on GC cells.METHODS We measured the expression levels of miR-204-3p in GC cells using quantitative real-time polymerase chain reaction,followed by the delivery of miR-204-3p overexpression and miR-204-3p knockdown vectors into GC cells.CCK-8 was used to detect the effect of miR-204-3p on the proliferation of GC cells,and the colony formation ability of GC cells was detected by the clonal formation assay.The effects of miR-204-3p on GC cell cycle and apoptosis were detected by flow cytometry.The BABL/c nude mouse subcutaneous tumor model using MKN-45 cells was constructed to verify the effect of miR-204-3p on the tumorigenicity of GC cells.Furthermore,the study investigated the effects of miR-204-3p on various proteins related to the MAPK signaling pathway,necroptosis signaling pathway and apoptosis signaling pathway on GC cells using Western blot techniques.RESULTS Firstly,we found that the expression of miR-204-3p in GC was low.When treated with the lentivirus overexpression vector,miR-204-3p expression significantly increased,but the lentivirus knockout vector had no significant effect on miR-204-3p.In vitro experiments confirmed that miR-204-3p overexpression inhibited GC cell viability,promoted cell apoptosis,blocked the cell cycle,and inhibited colony formation ability.In vivo animal experiments confirmed that miR-204-3p overexpression inhibited subcutaneous tumorigenesis ability in BABL/c nude mice.Simultaneously,our results verified that miR-204-3p overexpression can inhibit GC cell proliferation by inhibiting protein expression levels of KRAS and p-ERK1/2 in the MAPK pathway,as well as inhibiting protein expression levels of p-RIP1 and p-MLK1 in the necroptosis pathway to promote the BCL-2/BAX/Caspase-3 apoptosis pathway.CONCLUSION MiR-204-3p overexpression inhibited GC cell proliferation by inhibiting the MAPK pathway and necroptosis pathway to promote apoptosis of GC cells.Thus,miR-204-3p may represent a new potential therapeutic target for GC.

Oridonin induces apoptosis in gastric cancer through Apaf-1,cytochrome c and caspase-3 signaling pathway

AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.

Effect of morphine preconditioning on neuronal apoptosis following cerebral ischemia/reperfusion injury

Apoptosis, a form of neuronal damage, takes place following cerebral ischemia/reperfusion injury and caspase-3 plays an important role in apoptosis. Studies have shown that morphine preconditioning influences neuronal apoptosis and related protein expression following cerebral ischemia/reperfusion injury. In the present study, neuronal degeneration was attenuated, and the number of apoptotic cells and caspase-3 expression decreased following morphine preconditioning in a rat model of cerebral ischemia/reperfusion injury. Moreover, pathological changes were attenuated with increasing morphine doses, as well as the number of apoptotic cells and caspase-3 expression. Results from the present study revealed that morphine preconditioning reduced ischemic brain injury and improved cerebral ischemic tolerance in a dose-dependent manner. The anti-apoptotic mechanism of morphine is closely related to Caspase-3.

Niuhuang(Bovis Calculus)-Shexiang(Moschus)combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explore its anti-HCC mechanism.Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro.SMMC-7721 was divided into the BC-Moschus group and the control group,and different doses(rude drug dosage 0.625,1.25,2.5,and 5 mg/m L)of BC-Moschus extract were used for the intervention.The proliferation ability of HCC cells was detected using the Cell Counting Kit-8(CCK-8)assay,and the migration ability was detected by a wound healing assay.A subcutaneous xenograft model was prepared using nude mice with human HCC.Specific pathogen-free-grade BALB/c nude mice(5-week-old)were randomly divided into the following groups(n=6 per group):control(0.9%physiological saline 0.2 m L/d),BC-Moschus[BC 45.5 mg/(kg·d)+Moschus 13 mg/(kg·d)],and cisplatin(DDP,intraperitoneal injection5 mg/kg per week)groups.All groups were administered for 14 d.The volume and mass of the subcutaneous xenografts in nude mice were observed.The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway,apoptosis-associated factor p70 S6 Kinase(S6K),Bax,Bcl-2,caspase-3,and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR(RT-qPCR)and Western blot.Terminal Deoxynucleotidy Transferase-Mediated d UTP NickEnd Labeling(TUNEL)was used for quantitative analysis of apoptotic cells.Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone,and the inhibition effect was dose-and time-dependent(P<0.01).The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration(P<0.01).In the subcutaneous xenograft model of nude mice with human HCC,we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group(P<0.01).The levels of the PI3K/AKT/m TOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased(P<0.01).The expression level of the anti-apoptotic gene Bcl-2 was downregulated(P<0.05),and the expression of the pro-apoptotic gene Baxand apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated(P<0.01).The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC(P<0.01).Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice.The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway,regulation of apoptosis-related protein caspase-3,caspase-9,Bcl-2,and Bax expression,and promotion of apoptosis.

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line

Attrora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma （ESCC）. It has been demonstrated that cells overexpressing Attrora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Attrora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly （ADPribose） polymerase （PARP） in Attrora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A＇s effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.

Effect of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid in combination with carboplatin on gastric carcinoma growth in vivo

AIM： To investigate the effects of 2-（8-hydroxy-6- methoxy-l-oxo-lH-2-benzopyran-3-yl） propionic acid （NM-3） alone and in combination with carboplatin on tumor growth and apoptosis in mouse models of human gastric cancer constructed by subcutaneous implantation of histologically intact tumor tissue. METHODS： Human gastric cancer SGC-7901 tissues were implanted into the dorsal subcutis of nude mice. One week after tumors reached to a volume of 50-100 mm3 for around 1 wk, these mice were randomly divided into 8 groups （n = 10）. NM-3 was injected peritoneally at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg every other day for 5 wk, combined with carboplatin （5 mg/kg） every third day for 4 wk. As controls of combined treatment, another 4 groups of mice were injected with either NM-3 at 10 mg/kg, 20 mg/kg or 40 mg/kg, or with carboplatin alone （5 mg/kg）. The control mice received normal saline. Tumor weight, tumor growth inhibition （TGI）, and intratumoral microvessel density （MVD） were evaluated. Apoptosis of human gastric cancer was detected by TUNEL method and flow o/tometry analysis, respectively. RESULTS： The mean tumor volume （692.40 ± 58.43 mm3, 548.30 ± 66.02 mm3, 382.13 ± 43.52 mm3） after treatment with carboplatin combined NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg was lower than that after treatment with either NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg or with carboplatin alone. Compared with the normal saline group, NM-3 administered at 10 mg/kg, 20 mg/kg or 40 mg/kg significantly reduced the tumor weight in these groups （P 〈 0.05）. Carboplatin used alone at 5 mg/kg showed minimal effects. But NM-3 in combination with carboplatin had greater effects of tumor weight than either NM-3 or carboplatin alone. NM-3 alone at the dose 10 mg/kg or in combination with carboplatin had no obvious effects on body changes. Two mice died of diarrhea in each of the two groups treated with 40 mg/kg NM-3 or with 40 mg/kg NM-3 in combination with carboplatin. A significant increase in apoptosis was observed in the NM-3 treated groups, and the effect was more significant in the groups treated with carboplatin in combination with NM-3 at 10 mg/kg, 20 mg/kg and 40 mg/kg, than in the control group. The induction of apoptosis was positively associated with the dose of NM-3. NM-3 significantly reduced the neomicrovascular formation of gastric cancer. The MVD was lower in the groups treated with NM-3 or with NM-3 in combination with carboplatin than in the group treated with carboplatin or in the normal saline group （P 〈 0.05）. CONCLUSION： The results suggest that the inhibitory effect of NM-3 on gastric cancer growth is mediated through decreased angiogenesis and the increased induction of apoptosis. Furthermore, NM-3 alone at the dose of 10 mg/kg or in combination with carboplatin has no obvious effects on body changes, indicating that NM-3 in combination with carboplatin may be effective in the treatment of gastric cancer. The toxicity of NM-3 needs further studies.

Effects of ATRA, Acitretin and Tazarotene on Growth and Apoptosis of Tca8113 Cells

Summary：To investigate the effects of ATRA, acitretin and tazarotene on the growth and apoptosis of human tongue squamous cell carcinoma cell line Tca8113. The effect of retinoids on growth of Tca8113 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis with double staining with Annexin V-FITC and PI, and active caspase-3 analysis with the staining of FITC-conjugated monoclonal rabbit anli-active caspase-3 antibody were made by flow cytometer. Streptavidin-biotin complex （SABC） immunocytochemical assays were employed for the detections of Bax/Bcl-2 proteins expressions. Our results showed that the retinoids inhibited growth of Tca8113 cells in a dose-and time-dependent manner with maximal inhibition 24 h after treatment of 10 5 mol/L. 10^-5 mol/L retinoids altered cell cycle distribution of Tca8113 cells, revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. 10^-5 mol/L retinoids significantly induced apoptosis of Tca8113 cells （all P〈0.05）, elevated the cells population with detectable active caspase-3 （P〈 0.05 for all）, increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly （all P〈0.05）. Acitretin played a most prominent role among the retinoids. It is concluded that the inhibition of cell cycle progress of Tca8113 cells by ATRA, acitretin and tazarotene is one of the possible mechanisms for proliferation arrest of TcaS113 cells elicited by the retinoids. The retinoids mediate apoptosis in TcaS113 cells that may be caspase-dependent through mitochondria pathway. High concentration retinoids inhibit growth of Tca8113 cells in vitro by interfering with proliferation and inducing apoptosis of cells. Acitretin may be an alternative medicine for the prevention and treatment of tongue squamous cell carcinoma.

Sodium rosmarinate can inhibit hepatocellular carcinoma in vitro and in vivo through increasing caspase-3 and decreasing sirtl

Aim To investigate the antitumor activities and mechanisms of sodium rosmarinate against hepatocellu- lar carcinoma. Methods Antitumor activities of sodium rosmarinate were measured on HepG2, HNE, K562, HCT116, and SW480 cells by MTT assay in vitro at 5 mg· L^-1, 25 mg · L^-1 , and 50 mg · L^-1. Nude mice and H22 cells were employed to establish a liver tumor-bearing murine model. Sodium rosmarinate were intraperitoneal- ly administered at 25 mg. kg^-1 and 50 mg · kg^-1 for 10 days. Body weights, organ indexes, white blood cells, lymphocytes, and neutrophils of peripheral blood were measured. Murine tumors were histologically stained. TNF- ot and IFN-γ in serum were detected by ELISA. Expressions of Caspase-3, Bcl-2, Bax, Sift1, NF-KB p65, NF-KB pS0, and p-IKB-ot were measured by realtime-PCR and Western blot. Results Sodium rosmarinate showed obvi- ous inhibition on proliferation of HepG-2 cells at 5 mg · L^-1 But no inhibition effects were observed on HNE K562, HCT116, and SW480 cells. Body weights and organ indexes of nude mice with H22 cells were not changed by sodium rosmarinate. But sodium rosmarinate showed significant inhibition rate at 25 mg · kg^-1（45.67% ） and 50 mg · kg ^-l （ 52. 82% ）. Necrosis was aggregated by sodium rosmarinate as shown by H＆E staining. TNF-α and IFN-γ in murine serum were significantly increased by sodium rosmarinate at both doses （P 〈 0.05）. Expressions of caspase-3 were significantly increased （P 〈 0.01 ）. Sodium rosmarinate were shown to increase expressions of Bax, decrease expressions of Bcl-2, and significantly decrease Bacl-2/Bax ratio to induce apoptosis. Expressions ofSirtl, NF-KB p65, NF-KB p50, and p-IKB-αwere all decreased dose-dependently. Discussion Sodium rosmari- hate were demonstrated to inhibit hepatocellular carcinoma in vivo and in vitro through up-regulating TNF-α, IFN- γ , Bcl-2/Bax ratio, and down-regulating Sirtl and NF-KB related genes dose dependently, which may be devel- oped a promising drug for treatment of hepatocellular carcinoma.

NF-kB抑制剂PDTC对人胃癌SGC-7901细胞凋亡及Livin和Caspase-3表达的影响

目的:观察核因子-kB(NF-kB)抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)对人胃癌SGC-7901细胞生长及凋亡的影响,研究其对凋亡抑制蛋白Livin和Caspase-3表达水平的调控,探讨PDTC对人胃癌SGC-7901细胞凋亡影响的机制。方法:不同浓度的PDTC作用于SGC-7901细胞不同时间后,MTT法测定其对SGC-7901细胞增殖的抑制率;流式细胞仪观察SGC-7901细胞的凋亡情况;real time PCR法和Western blot法检测SGC-7901细胞Livin和Caspase-3 mRNA和蛋白的表达情况。结果:PDTC作用不同时间后对SGC-7901细胞生长有抑制作用,并诱导SGC-7901细胞凋亡,呈剂量-时间依赖性;PDTC可降低Livin mRNA及蛋白的表达,增加Caspase-3 mRNA及蛋白的表达,且Livin的表达量与PDTC的作用呈剂量,时间负相关。结论:PDTC可诱导人胃癌细胞株SGC-7901凋亡,其机制可能与PDTC抑制NF-kB信号转导通路,下调Livin表达继而上调Caspase-3表达有关。

survivin和caspase-3在胃癌中的表达以及与凋亡的关系

目的 :评价胃癌组织中survivin和caspase 3的表达情况以及与细胞凋亡的关系。 方法 :采用免疫组化的方法 ,对 6 0例无术前放、化疗史的胃癌患者的手术标本进行survivin和caspase 3检测 ,并用原位末端标记法标记凋亡细胞。 结果 :survivin主要在癌组织中表达 ,caspase 3在正常粘膜的表达高于癌组织 ,survivin的表达强度与组织学分级及临床病理分期有关 (P <0 .0 1 )。caspase 3的表达强度随组织分化程度增高而增强 ;而在不同临床病理分期间无显著差异 (P >0 .75 )。胃癌组织中survivin与caspase 3的表达无直线相关关系 ,survivin阳性组凋亡指数明显低于阴性组 (P <0 .0 0 5 ) ,survivin阴性、caspase 3阳性组平均凋亡指数最高为 1 .5 3%。 结论 :survivin的表达与胃癌分化程度及病理分期密切相关 ,可作为提示预后的重要指标。survivin作为凋亡抑制蛋白显著抑制癌组织的细胞凋亡 ,但不能完全抑制caspase

吗啡对人胃癌MGC-803细胞caspase-3表达的影响

目的观察吗啡对人胃癌MGC-803细胞凋亡及caspase-3表达的影响。方法胃癌MGC-803细胞分为两组:对照组和吗啡组。对照组不加任何药物,吗啡组将吗啡加入细胞培养液中使吗啡终浓度为0.1μmol/L。孵育24h后应用流式细胞仪检测细胞凋亡情况,并应用半定量逆转录-聚合酶链反应(RT-PCR)和Western blot技术检测胃癌MGC-803细胞中caspase-3基因mRNA和蛋白的表达情况。结果吗啡组胃癌细胞凋亡率为(17.20±2.95)%,明显高于对照组的(11.40±2.86)%(P<0.05)。RT-PCR和Western blot结果显示,吗啡组胃癌MGC-803细胞中caspase-3mRNA和蛋白表达增加。结论 0.1μmol/L吗啡通过增加caspase-3的表达而促进胃癌细胞的凋亡。

核转录因子κB抑制剂PDTC对胃癌细胞株增殖和凋亡相关蛋白caspase-3表达影响的实验研究

目的:探讨核转录因子NF κB活性特异性抑制剂PDTC(吡咯烷二硫代氨基甲酸盐)对胃癌细胞增殖和凋亡相关蛋白caspase 3表达的影响。方法:利用TransAMTM NF κBP65活性测定试剂盒,检测PDTC作用胃癌细胞株AGS、SGC 7901 12h、24h后,NF κB亚基P65活性的变化。采用噻唑蓝 (MTT)比色法观察PDTC作用 24h、48h、72h,对细胞增殖的影响。用Hoechst33258荧光染色观察凋亡细胞核形态变化,利用免疫印迹法 (Western blot法)检测细胞中活化型caspase 3蛋白表达的变化。结果:经PDTC处理的实验组胃癌细胞NF κB的活性受到抑制(P<0. 01),与对照组相比,细胞的增殖活性明显受到抑制(P<0. 05)。经PDTC处理后的实验组细胞呈现典型的凋亡核固缩表现,胞核呈致密浓染的颗粒状或块状荧光,胞质检测到活性caspase 3蛋白的表达。结论:核因子NF κB参与胃癌细胞的增殖与凋亡调控,PDTC可通过抑制NF κB活性上调caspase 3表达,从而诱导细胞的凋亡。

Caspase-3、Bcl-2和Bak在胃癌中的表达及意义

目的 研究Caspase-3、Bcl-2、Bak等凋亡相关基因蛋白在胃癌中的表达，与胃癌临床病理特征和预后的相关性。方法 将100例无术前放化疗史的胃癌手术标本和30例对照组胃粘膜制成组织芯片，取样针直径2．0mm。用免疫组化方法分别检测Caspase-3、Bcl-2、Bak的表达；用原位末端标记法进行细胞凋亡指数检测。对随访14个月～13年的47例作生存分析。结果 获得2个组织芯片蜡块，分别含114和116个位点。Caspase-3在正常粘膜的表达（90％）显著高于胃癌组织（56％）（P〈0．01），其表达与胃癌组织学分化、血管神经侵犯相关（P〈0．05），与淋巴结转移和国际抗癌联盟（UICC）临床分期及预后无关。Bcl-2和Bak在胃癌中的表达分别为42％和47％，表达均与组织学分化相关（P〈0．05），与淋巴结转移、血管神经侵犯、UICC分期及预后无关。结论 Caspase-3、Bcl-2和Bak均参与细胞凋亡调控和胃癌生物学进程，但其表达与胃癌预后无关。UICC分期仍然是胃癌的独立预后指标。

Caspase-3、Bim和c-Cbl在胃癌中的表达及其意义

目的:探讨Caspase-3、Bim和c-Cbl等凋亡基因蛋白在胃癌中的表达及其与胃癌发生、发展及生物学行为的关系。方法:应用免疫组织化学SP法,检测124例胃癌组织中Caspase-3、Bim和c-Cbl蛋白的表达。结果:Caspase-3、Bim和c-Cbl在胃癌组织中的阳性表达率分别为45.2%、37.9%和71.0%;Caspase-3的阳性表达随组织分化程度增高而增强(P<0.05);Bim的阳性表达随TNM临床分期和浸润深度而降低(P<0.05);c-Cbl阳性表达随TNM临床分期和浸润深度而增高(P<0.05);Caspase-3和Bim的表达呈正相关(r_s=0.403,P<0.05),而Caspase-3、Bim和c-Cbl的表达均呈负相关(r_s=-0.185,r_s=-0.442,P<0.05)。结论:Caspase-3和Bim在胃癌中呈低表达,c-Cbl在胃癌中呈高表达。Caspase-3、Bim和c-Cbl表达与胃癌某些临床病理特征密切相关,Bim和c-Cbl表达与胃癌预后有关,似可作为胃癌预后的指标,Caspase3,Bim和c-Cbl可能参与了细胞凋亡调控和胃癌生物学进程。

Caspase-3 mRNA在胃癌中的表达及其与细胞凋亡的关系

目的:通过观察Caspase-3mRNA在胃癌组织、非典型增生组织及正常胃粘膜中的表达,探讨Caspase-3及其所介导的细胞凋亡在胃癌发病中的作用。方法:应用原位杂交法和TUNEL染色法检测75例胃癌组织、68例癌旁非典型增生组织和10例正常胃粘膜组织中Caspase-3mRNA的表达和上述组织的凋亡指数。结果:Caspase-3mRNA在胃癌组织和非典型增生组织中的阳性率(38.67％、20.59％)显著低于正常胃粘膜(60％);TUNEL染色证实,胃癌及非典型增生组织的凋亡指数显著低于正常胃粘膜(P<0.05),Caspase-3mRNA阳性组的凋亡指数显著高于阴性组(P<0.05)。结论:由Caspase-3及其所介导的细胞凋亡与胃癌的发生和演进有关。

Caspase-3、Bcl-2和Bak在胃癌中的表达及意义

目的研究Caspase--3．Bcl--2、Bak等凋亡相关基因蛋白在胃癌中的表达与胃癌临床病理特征和预后的相关性。方法将100例无术前放化疗史的胃癌手术标本和30例对照组胃粘膜制成组织芯片，取样针直径2．0mm。用免疲组化方法分别检测Caspasc-3、Rot--2．Rak的表达，甩原位末端标记法进行细胞凋亡指数检测。对随访14个月至13年的47倒作生存分析。结果获得2个组织芯片蜡块，分别含114和116个位点。Caspase-3在正常粘膜的表达（90％）显著高于胃癌组织（56％）（P〈0．01），其表达与胃癌组织学分化、血管神经侵犯相关（P〈0．05），与淋巴结转移和国际抗癌联盟（LICC）临床分期及预后无关。Bcl--2和Bak在胃癌中的表达分别为42％和47％，表达均与组织学分化相关（P〈0．05），与淋巴结转移、．血管神经侵犯、UICC分期及预后无关。结论Caspasc-3、Bcl-2和Bak均参与细胞凋亡调控和胃癌生物学进程，但其表达与胃癌预后无关。UICC分期仍然是胃癌的独立预后指标。

Fas、FasL及Caspase-3在维甲酸诱导胃癌细胞凋亡中的作用

目的:探讨维甲酸诱导胃癌细胞BGC-803凋亡的作用及其与Fas、FasL、Caspase-3表达的关系.方法:以0.001、0.01、0.1、1、10、20μmol/L的维甲酸作用BGC-803细胞72h后,MTT法检测维甲酸对BGC-803细胞的生长抑制作用;流式细胞术分析维甲酸对胃癌BGC-803细胞凋亡的诱导作用;Hoechst33342/PI双荧光染色观察细胞凋亡;RT-PCR法检测Fas、FasL、Caspase-3基因的mRNA表达变化.结果:0.1、1、10、20μmol/L的维甲酸作用BGC-803细胞72h后,较对照组(未加药)能显著抑制细胞增殖(32.61%、44.42%、48.14%、51.15%vs0.657%,均P<0.01);20μmol/L维甲酸作用BGC-803细胞12、24和48h后,G2/M期细胞比例显著增加,出现凋亡特征性的亚G1峰;细胞出现染色质凝集、核膜破裂等凋亡特征;作用48h后,Fas、FasL、Caspase-3mRNA表达水平均较对照组显著上调.结论:Fas、FasL、Caspase-3参与了维甲酸诱导胃癌细胞凋亡的调控过程.,

Harmine induces apoptosis in HepG2 cells via mitochondrial signaling pathway

BACKGROUND:Harmine has antitumor and antinociceptive effects,and inhibits human DNA topoisomerase.However no detailed data are available on the mechanisms of action of harmine in hepatocellular carcinoma.This study aimed to investigate the effects of harmine on proliferation and apoptosis and the underlying mechanisms in the human hepatocellular carcinoma cell line HepG2.METHODS:The proliferation of HepG2 cells was determined by the cell counting kit-8 (CCK-8) assay and the clone formation test.The morphology of HepG2 cells was examined using fluorescence microscopy after Hoechst 33258 staining Annexin V/propidium iodide (PI) was used to analyze apoptosis and PI to analyze the cell cycle.Western blotting was used to assess expression of the apoptosis-regulated genes Bcl-2,Bax,Bcl-xl,Mcl-1,caspase-3,and caspase-9 Mitochondrial transmembrane potential (Ψ m) was determined using JC-1.RESULTS:Harmine inhibited the proliferation of HepG2 cells in a dose-dependent manner.Hoechst 33258 staining revealed nuclear fragmentation and chromosomal condensation,cell shrinkage,and attachment loss in HepG2 cells treated with harmine.The percentage of the sub/G1 fraction was increased in a concentration-dependent manner,indicating apoptotic cell death.PI staining showed that harmine changed the cell cycle distribution,by decreasing the proportion of cells inG0/G1 and increasing the proportion in S and G2/M.Harmine induced apoptosis in a concentration-dependent manner,with rates of 20.0%,32.7% and 64.9%,respectively.JC-1 revealed a decrease in Ψ m.Apoptosis of HepG2 cells was associated with caspase-3 and caspase-9 activation,down-regulation of Bcl-2,Mcl-1,and Bcl-xl,and no change in Bax.CONCLUSIONS:Harmine had an anti-proliferative effect in HepG2 cells by inducing apoptosis.Mitochondrial signal pathways were involved in the apoptosis.The cancer-specific selectivity shown in this study suggested that harmine is a promising novel drug for human hepatocellular carcinoma.

姜黄素诱导人胃腺癌MGC80-3细胞凋亡研究

以 5、10和 2 0 μmol/ L 姜黄素处理 MGC80 - 372 h,细胞存活率随药物浓度及处理时间依赖性下降 ,并呈现典型的凋亡细胞形态 ;以 0、10、2 0及 30 μmol/ L姜黄素处理细胞 2 4 h后 ,2 0 μmol/ L以上的处理组 DNA琼脂糖电泳显示均有典型的凋亡梯形条带 ,同样条件处理后分别对断裂及未断裂 DNA作定量测定 ,计算细胞凋亡率分别为 7.3%、39.2 %、50 .5%和 6 4 .3% ,表明姜黄素具有诱导MGC 80 - 3细胞凋亡的显著作用 .流式细胞术检测显示经 10及 30μmol/ L姜黄素处理的 MGC 80 - 3细胞周期主要阻滞于 G2 / M期 ,提示细胞的 G2 / M期阻滞与姜黄素诱导 MGC80 - 3细胞凋亡有关 .

三氧化二砷诱导胃癌细胞AGS的凋亡及对STAT3、VEGF表达的影响

目的:探讨三氧化二砷(arsenic trioxide,As2O3)诱导胃癌细胞AGS的凋亡及对信号转导子与转录激活子基因(signal transducers and activators of transcription 3,STAT3)和血管内皮生长因子基因(vascular endothelial growth factor,VEGF)表达的影响。方法:分别用1、5、10μmol/LAs2O3处理AGS细胞,在培养24、48、72h后,以MTT法检测细胞的生长增殖,以流式细胞术和TUNEL法检测细胞凋亡,以ELISA、免疫组化和实时荧光定量PCR检测凋亡过程中相关基因STAT3、VEGF表达的变化。结果:(1)AGS细胞经As2O3作用后,细胞增殖受到明显抑制,且呈剂量和作用时间依赖关系;(2)FCM术检测在细胞周期G1期前出现亚二倍体凋亡峰;细胞周期分析显示G2/M期阻滞;(3)TUNEL标记发现DNA链的断裂;(4)在AGS细胞凋亡过程中,STAT3和VEGF表达下调,其中10μmol/L浓度的As2O3作用最强。结论:As2O3抑制胃癌细胞AGS增殖并诱导凋亡,其机制可能与细胞周期阻滞和下调STAT3和VEGF基因表达有关。