羟基喜树碱诱导胃癌细胞凋亡的作用机制初步研究

目的：研究羟基喜树碱（HCPT）诱导胃癌细胞的凋亡作用及对凋亡相关基因p53,c-myc,bcl-2,bcl-xl和bcl-xs表达的影响,探讨其诱导胃癌细胞凋亡的作用机制。方法：应用TUNEL染色、流式仪、免疫组化和RT－PCR技术等研究HCPT对胃细胞SGC－7901和MKN－45的诱导凋亡作用和对凋亡相关有达的影响。结果：HCPT作用于细胞后,可看到较为典型的细胞凋亡的形态学变化;细胞核固缩,染色质凝集,呈新月型紧核膜周边,核碎裂,染色质片段化,凋亡小体形成等。流式细胞仪DNA直方图上出现典型的亚二倍体的“凋亡峰”。流式细胞仪计数显示,10μg/ml的HCPT诱导胃癌细胞SGC－7901和MKN－45的凋亡率为21．88％和12．34％。TUNEL染色法显示,细胞凋亡指数在1．865－9．54％之间。免疫组化和RT－PCR结果显示：HCPT能够明显下调SGC－7901细胞的P53和bcl-2基因的蛋白和mRNA表达,对SGC－7901细胞的c-myc,bcl-xl和bcl-xs基因的蛋白表达无影响。HCPT作用后MKN－45细胞的p53蛋白和mRNA的表达增加,对MKN－45细胞的bcl-2,c-myc,bcl-xl和bcl-xs基因的表达无影响。结论：HCPT能够诱导胃癌细胞凋亡,可能是通过调控胃癌细胞的P53和bcl-2的表达而诱导胃癌细胞凋亡。     

Trichosanthin‐induced apoptosis in gastric cancer is related to the downexpression of bcl‐2

OBJECTIVE : To study: (i) the induction of apoptosis in gastric cancer cells by trichosanthin; and (ii) the relationship between apoptosis and the expression of bcl‐2. METHODS : During in vitro experiments, morphological studies and the terminal deoxynucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labeling (TUNEL) method were used to detect apoptosis in gastric adenocarcinoma cell line SGC‐7901 before and after trichosanthin treatment. An immunohistochemical staining method and northern blot hybridization were used to detect the expression of the apoptosis‐related gene bcl‐2 before and after trichosanthin treatment. RESULTS : When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL, 36 h), they presented some typical apoptotic morphological changes that were observed by fluorescent staining. These morphological changes included nuclear condensation and nucleosomal fragments forming a lunate body under the nuclear membrane. When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL) for 36, 42 or 48 h, TUNEL staining revealed a significant increase in the apoptotic index (AI), from 3.78 ± 1.11%, 3.98 ± 1.12% and 3.85 ± 1.08%, to 11.30 ± 2.33%, 10.22 ± 2.00% and 11.18 ± 1.85% (P < 0.01), respectively. When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL, 32 h), immunohistochemical staining revealed a decreased expression of the bcl‐2 protein product: the staining density decreased from ++/+++ to –/+ (P < 0.01). When SGC‐7901 cells were treated with trichosanthin (0.1 μg/mL, 24 h), northern blot hybridization showed a decreased expression of bcl‐2 RNA: hybridization decreased from 35.19 ± 2.34 to 22.27 ± 3.90 (P < 0.01). CONCLUSIONS : Trichosanthin is able to induce apoptosis in gastric cancer. The apoptosis may be mediated by the downexpression of the apoptosis‐related gene bcl‐2.     

RNAi‐mediated silencing of ezrin gene reverses malignant behavior of human gastric cancer cell line SGC‐7901

OBJECTIVE: To investigate the effects of ezrin targeting gene of RNA interference (RNAi) on human gastric cancer cell line SGC‐7901 in vitro. METHODS: The highly metastatic human gastric cancer cell line SGC‐7901 transfected with a small interfering (siRNA) lentivirus vector was selected for this research study. Expressions of ezrin mRNA and ezrin protein in the SGC‐7901 cells were detected using RT‐PCR and Western blot. Cell apoptosis was observed using flow cytometry. Transwell invasion and the cell adhesion test were used to verify the effect of RNAi on ezrin expression in the human gastric cancer cell line SGC‐7901 in vitro. RESULTS: Ezrin gene targeting via a RNAi‐mediated lentivirus vector had obvious inhibitory effects on ezrin expression in the human gastric cancer cell line SGC‐7901. The results of the RT‐PCR show the obvious inhibition of ezrin mRNA expression in Eai and Ebi groups (0.22 ± 0.01 vs 0.95 ± 0.04, P < 0.05; 0.31 ± 0.01 vs. 0.95 ± 0.04, P < 0.05). Western blot analysis revealed a 72.35 ± 3.74% reduction of the ezrin protein level after interference with the ezrin targeting gene. Moreover, the inhibition of ezrin expression clearly inhibited SGC‐7901 cell migration and invasion, and improved cell adhesion as well as increased sensitivity to camptothecin‐induced apoptosis. CONCLUSION: Ezrin gene targeting by RNAi can inhibit the metastatic growth and migration of SGC‐7901 human gastric cancer cells.     

Investigation of the sensitivities of distinct gastric cancer cells to parvovirus H‐1 induced cytotoxicity

OBJECTIVE: To investigate the sensitivities of distinct gastric cancer cells to parvovirus H‐1 induced cytotoxicity and the possible mechanism(s). METHODS: There were six distinct differentiated gastric cancer cell lines: HGC27 (undifferentiated), BGC823 (undifferentiated), MKN45 (poorly differentiated), AGS (poorly differentiated), SGC7901 (moderately differentiated) and MKN28 (well differentiated). The cell cycle distributions were measured by flow cytometry and the differential sensitivities of the six distinct gastric cancer cells after H‐1 virus infection were detected by MTT assay. RT‐PCR was used to detect viral NS1 gene expression in all six gastric cancer cell lines. RESULTS: The S phase ratios of HGC27, BGC823, MKN45, AGS, SGC7901 and MKN28 were 24.72%, 30.15%, 27.10%, 29.03%, 31.82% and 33.73%, respectively. HGC27 cells were sensitive to H‐1 virus induced cytotoxicity, followed by SGC7901 cells. MKN45 and AGS cells were moderately sensitive and MKN28 cells were insensitive. However, BGC823 cells were resistant to H‐1 virus induced cytotoxicity. The expressions of viral NS1 were higher in HGC27, BGC823, MKN45 and SGC7901 cells, and lower in AGS and MKN28 cells. CONCLUSIONS: The sensitivities of the distinct gastric cancer cells to H‐1 virus induced cytotoxicity were markedly different. In general, the poorly differentiated cells showed an enhanced sensitivity to H‐1 virus attack compared with well‐differentiated ones. The enhanced sensitivity of poorly versus well‐differentiated gastric cancer cells to H‐1 virus is related in part to the enhanced capacity of the former for NS1 protein production and accumulation. The undifferentiated BGC823 cells were resistant to H‐1 virus triggered cytotoxicity. It may further verify that not all tumor cells are sensitive to H‐1 virus lytic effects.     

Induction of apoptosis by arsenic trioxide and hydroxy camptothecin in gastriccancer cells in vitro

AIM To study the effects of arsenic trioxide and HCPT on different degrees of differentiated gastric cancer cells (SGC-7901, MKN-45, MKN-28)with respect to both cytotoxicity and induction of apoptosis in vitro. ~ODS The cytotoxicity of As2O3 and HCPT on gastric cancer cells was determined by MTTassay. Morphologic changes of apoptosis of gastric cancer cells were observed by light microscopy and transmission electron microscopy. Apoptosis and cell cycle changes of gastric cancer cells induced by HCPT and As2O3 were investigated by TUNEL method and flow cytometry. RESULTS As2O3 and HCPT had remarkable cytotoxic effects on different degrees of differentiated gastric cancer cells. The IC50 of As2O3 on well differentiated gastric cancer cell MKN-28, moderately differentiated gastric cancer cell SGC-7901, and poorly differentiated gastric cancer cell MKN-28 were 8. 91 μmol/L, 10. 57 μmol/L, and 11.65 μmol/L, respectively. The IC50 of HCPT on MKN-28, SGC-7901, and MKN-45 were 9. 35 rg/L, 10. 21 rg/L, and 12. 63 mg/L respectively after 48 h treatment. After 12 h of exposure to both drugs, gastric cancer cells exhibited morphologic features of apoptosis, including cell shrinkage, nuclear condensation,and formation of apoptotic bodies. A typical subdiploid peak before G0/G1 phase was observed by flow cytometry. The apoptotic rates of SGC7901, MKN-45, and MKN-28 were 13. 84%, 22.52%, and 9. 68%, respectively after 48 h exposure to 10 μmol/L As2O3. The apoptotic rates of SGC-7901, MKN-45, and MKN-28 were 21.88%, 12.35%, and 30. 26%, respectively after 48 h exposure to 10 mg/L HCPT. The apoptotic indice were 7% - 15% as assessed by TUNEL method. The effect of As2O3 on SGC-7901 showed remarkable cell cycle specificity, which induced cell death in G1 phase, and blocked G2/M phase. HCPT also showed a remarkable cell cycle specificity, by inducing cell death and apoptosis in G1 phase and arrest of proliferation at S phase. CONCLUSION AS2O3 and HCPT exhibit significant cytotoxicity on gastric cancer cells by induction of apoptosis. As2O3 and HCPT might have a promising prospect in the treatment of gastric cancer, which needs to be further studied.     

HCTB006联合5-FU对胃癌细胞系MKN28、7901的作用及其机制

目的:探讨肿瘤坏死因子相关诱导配体受体(DR5)的单克隆抗体HCTB006联合5-FU对人胃癌细胞系7901、MKN28的作用以及机制.方法:用ATPlite法检测HBCT006单药组、5-FU单药组及两药物合用对胃癌细胞存活率的影响,研究两者之间的关系;采用流式细胞技术检测胃癌细胞系7901以及MKN28表面DR5的表达水平;Westernblot检测上述3组用药后胃癌细胞内XIAP,caspase3的变化.结果:胃癌细胞系7901、MKN28对HCTB006不敏感;5-FU对二者的增殖抑制作用具有时间以及浓度依赖效应;联合用药组具有很好的协同抑制胃癌细胞系增殖的效果,且具有浓度依赖效应,与给药次序无关.流式细胞技术检测胃癌细胞系7901,MKN28表面死亡受体DR5的表达依次为:93.8%以及87.7%.免疫迹印结果表明,联合用药组可以引发胃癌细胞内凋亡抑制蛋白XIAP的降解,激活最终凋亡执行蛋白caspase3,引起细胞死亡.结论:HTB006与5-FU联合应用具有协同杀伤胃癌细胞的作用.胃癌细胞7901、MKN28对于HCTB006的敏感程度与细胞表面DR5的表达量不相关;联合用药作用机制可能与细胞内抑制凋亡蛋白XIAP降解有关.     

Effects of non‐cytotoxic drugs on the growth of multidrug‐resistance human gastric carcinoma cell line

OBJECTIVE: To investigate the effects of the non‐cytotoxic drug (cyclooxygenase‐2 (COX‐2) inhibitor and octreotide) on growth of the multidrug‐resistant human gastric carcinoma cell line SGC‐7901/ADR. METHODS: The effects of non‐cytotoxic drug on the growth of SGC‐7901 and SGC‐7901/ADR cells were evaluated by ³H‐thymidine incorporation assay. The apoptosis of cells was measured by the TdT‐mediated dUTP nick end‐labeling assay (TUNEL) and flow cytometric assay. Western blotting was used to analysis the expression of cyclooxygenase (COX‐2) protein in SGC‐7901 cells and SGC‐7901/ADR cells and P‐glycoprotein (P‐gp) from SGC‐7901/ADR cells with variable treatments. Activator protein‐1 binding activity was examined by electrophoretic mobility shift assay. RESULTS: ³H‐thymidine incorporation into SGC‐7901/ADR cells treated with celecoxib was significantly lower than that of control group (471.3 ± 79.7 cpm vs 917.5 ± 130.8 cpm, P < 0.05). When combined with octreotide, celecoxib presented lower ³H‐thymidine incorporations than its used alone and decreased to 53.3% of that amount original. Either celecoxib or the combination group markedly induced apoptosis in SGC‐7901/ADR cells. COX‐2 protein in the SGC‐7901/ADR cells was higher than in that of the SGC‐7901 cells (1.543 ± 0.052 vs 0.564 ± 0.021, P < 0.05). The inhibition of P‐gp could be achieved with celecoxib alone and combination with octreotide (0.486 ± 0.012, 0.252 ± 0.014 vs 0.941 ± 0.033, P < 0.05). Moreover, AP‐1 binding activity could be suppressed by non‐cytotoxic drug and showed a synergistic effect. CONCLUSION: The combination of non‐cytotoxic drug significantly improved the inhibitive effects on the growth of multidrug‐resistant human gastric cancer cells.     

19‐peptide,a fragment of tumstatin,inhibits the growth of poorly differentiated gastric carcinoma cells in vitro and in vivo

Background and Aim: This study investigated whether 19‐peptide, a fragment of tumstatin, inhibited the growth of gastric tumor cells in vitro and in vivo. Methods: 19‐peptide was expressed in bacteria and purified with Sephadex G‐15. SGC7901 gastric carcinoma cells and human umbilical‐vein endothelial cells (HUVECs) were exposed to 19‐peptide in vitro, and their viability was evaluated by biochemical and histopathological analysis. In vivo, pieces of solid tumor derived from SGC7901 cells were inoculated into the gastric serosa of 36 nude mice, with a biological glue to hold them in place. Twenty‐eight days after injection of 19‐peptide, the mice were killed. The tumors were measured and examined by western blotting, histopathology, and terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling assay. Results: 19‐peptide induced apoptosis of many SGC7901 cells but few HUVECs in vitro. In vivo, after the application of 19‐peptide, significant tumor cell apoptosis was observed in the center of the tumors, tumor volume was reduced significantly (P < 0.001), and the invasion and migration of cancer cells was reduced. PTEN was increased in the treatment group and phospho‐Akt (pAkt) was decreased in the control group. Conclusions: These results suggest that 19‐peptide inhibits the growth and metastases of poorly differentiated gastric carcinoma cells, primarily by inducing apoptosis. The apoptotic mechanism could be related to anoikis and the PTEN/Akt pathway.     

Anti‐cancer activity of anti‐p185HER‐2 ricin A chain immunotoxin on gastric cancer cells

Background and Aim: Overexpression of the human epidermal growth factor receptor 2 (HER‐2) protein has been detected in gastric cancer and has been associated with an unfavorable prognosis. We investigated the anti‐cancer effects of anti‐p185^HER‐2 ricin A chain (RTA) immunotoxin, alone or in combination with 5‐flurouracil on SGC7901‐HER‐2+ cells. Methods: SGC7901‐HER‐2+ cells were obtained by transfecting SGC7901 cells with HER‐2‐pcDNA3.1. Anti‐p185^HER‐2‐RTA was prepared by chemical conjugation of anti‐HER‐2 monoclonal antibody (mAb) and RTA. The SGC7901‐HER‐2+ cells were incubated with RTA, anti‐p185^HER‐2‐RTA, and/or 5‐flurouracil. The effects of drugs on cells were evaluated by MTT assay and Annexin V‐fluorescein isothiocyanate and propidium iodide double staining flow cytometry. The expression of caspase‐3, caspase‐9, cyclooxygenase‐2, and nuclear factor‐κB/p65 were assayed by western blot. SGC7901‐HER‐2+ cells were transplanted into BALB/c nude mice to produce solid tumors in an attempt to study the immunotoxin activity in vivo. Results: In vitro, anti‐p185^HER‐2‐RTA inhibited cell growth and induced apoptosis in SGC7901‐HER‐2+ cells. Anti‐p185^HER‐2‐RTA enhanced caspase‐3 and caspase‐9 activity, while downregulating the expression of cyclooxygenase‐2 and nuclear factor‐κB/p65. Its combination with 5‐flurouracil further inhibited the growth of SGC7901‐HER‐2+ cells. In vivo, our data showed that anti‐p185^HER‐2‐RTA significantly inhibited the growth of SGC7901‐HER‐2+ cells‐transplanted tumors. Conclusions: Anti‐p185^HER‐2‐RTA inhibits the growth of SGC7901‐HER‐2+ cells. The effect may be related to the activation of caspase‐3 and caspase‐9 and inhibition of cyclooxygenase‐2 and nuclear factor‐κB/p65. Anti‐p185^HER‐2‐RTA plus 5‐FU enhance anti‐cancer activity, suggesting useful clues for further study for the treatment of HER‐2 positive gastric cancers.     

癌性锚蛋白重复序列在胃癌细胞凋亡中的作用

目的 探讨癌性锚蛋白重复序列(Gankyrin)在人胃癌细胞的表达,以及在尼美舒利诱导细胞凋亡过程中的改变.方法 培养不同分化程度的人胃癌细胞系[MKN28(高分化)、AGS(低分化)、MKN45(低分化)和SGC7901(中分化)],以尼美舒利处理细胞,应用四甲基偶氮唑盐试验和流式细胞术检测细胞活力及细胞凋亡,实时PCR和Western印迹法检测Gankyrin基因和蛋白表达.结果 在4种不同分化程度的人胃癌细胞系中,均存在不同水平的Gankyrin基因和蛋白表达.尼美舒利以时间-剂量依赖方式抑制AGS、FYGC7901细胞增殖.与对照组相比,尼美舒利400 μmol/L处理48 h可诱导AGS、SGC7901细胞显著凋亡(细胞凋亡率分别为0.57%±0.19%比23.30%±2.50%和0.88%±0.17%比16.80%±1.55%,P均<0.01).在AGS细胞凋亡过程中,Gankyrin基因和蛋白表达水平下降,以尼美舒利作用后24 h(0.0035±0.0014)和36 h(0.0980±0.0160)改变最为显著(对照组为0.4690±0.1190,P值均<0.01).结论 在人胃癌细胞中存在Gankyrin基因和蛋白表达.Gankyrin可能参与尼美舒利诱导的AGS胃癌细胞凋亡.     

&beta;-elemene enhances the radiosensitivity of gastric cancer cells by inhibiting Pak1 activation

AIM: To explore the potential of β-elemene as a radiosensitizer for gastric cancer cells and the underlying mechanisms. METHODS: SGC7901, MKN45, MKN28, N87, and AGS human gastric cancer cell lines were used to screen for radioresistant gastric cancer cell lines. A 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was used to determine the effects of β-elemene and IPA-3 on cell viability in MKN45 and SGC7901 gastric cancer cell lines. A clonogenic survival assay and annexin V-FITC/PI apoptosis detection assay were used to evaluate cellular radiosensitivity and radiation-induced cell death, respectively. A proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), was employed to screen the proteins regulated by β-elemene pretreatment prior to ionizing radiation (IR) in SGC7901 gastric cancer cell line. IPA-3 was used as a specific small molecule inhibitor of p21-activated protein kinase 1 (Pak1) to target Pak1 signaling. Protein levels of PAK1IP1 (p21-activated protein kinase-interacting protein 1), total Pak1 (t-Pak1), phospho-Pak1 (T423), phospho-ERK1/2 (Thr202/Tyr204), and cleaved caspase-3 (17 kDa) were assessed by western blotting. RESULTS: MKN45 and SGC7901 gastric cancer cell lines were relatively more resistant to IR. β-elemene pretreatment decreased clonogenic survival following IR in MKN45 and SGC7901 gastric cancer cell lines. Additionally, β-elemene pretreatment prior to IR increased radiation-induced cell death compared with IR alone in MKN45 (10.4% ± 0.9% vs 34.8% ± 2.8%, P < 0.05) and SGC7901 (11.6% ± 0.9% vs 46.7% ± 5.2%, P < 0.05) human gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Through iTRAQ analysis and western blot validation, we found that β-elemene upregulated PAK1IP1 and downregulated phospho-Pak1 (T423) and phospho-ERK1/2 in SGC7901 gastric cancer cells. IR increased the level of phospho-Pak1 (T423). Pretreatment with β-elemene decreased radiation-induced Pak1 and ERK1/2 phosphorylation. Inhibition of Pak1 using IPA-3 decreased clonogenic survival following IR. In addition, IPA-3 increased radiation-induced cell death in MKN45 (13.4% ± 0.3% vs 26.6% ± 1.0%, P < 0.05) and SGC7901 (16.0% ± 0.6% vs 37.3% ± 1.7%, P < 0.05) gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Western blotting showed that IPA-3 decreased radiation-induced Pak1 and ERK1/2 phosphorylation. CONCLUSION: This is the first demonstration that β-elemene enhances radiosensitivity of gastric cancer cells, and that the mechanism involves inhibition of Pak1 signaling.     

胃癌细胞对细小病毒H-1敏感性差异的实验研究

目的 探讨不同胃癌细胞株对细小病毒细胞毒作用的敏感性差异及可能的机制。方法共选用HGC27(未分化)、BGC823(未分化)、MKN45(低分化)、AGS(低分化)、SGC7901(中分化)和MKN28(高分化)等6株不同分化状态的胃癌细胞株，用流式细胞仪分析其各自的细胞周期，H-1病毒感染后采用MTT方法检测不同胃癌细胞株对其细胞毒作用的敏感性差异，用RT-PCR来检测H-1病毒中的非结构蛋白基因(NS-1)在6株不同胃癌细胞中的表达。结果 HGC27、BGC823、MKN45、AGS、SGC7901和MKN28等不同分化状态细胞株中，S期细胞的比率分别为24．72％，30．15％，27．10％，29．03％，31．82％和33．73％。其中HGC27细胞对H-1病毒的细胞毒作用敏感；SGC7901细胞其次；MKN45、AGS细胞对H-1病毒的细胞毒作用中等敏感；MKN28细胞对H-1病毒的细胞毒作用不敏感；而BGC823则对H-1病毒的细胞毒作用抵抗。病毒NS-1的mRNA在HGC27、BGC823、MKN45和SGC7901等细胞中的表达水平较高，而在AGS和MKN28中的表达水平却较低。结论 H-1病毒的细胞毒作用在不同的胃癌细胞株中的差异显著。总体上，与高分化细胞株MKN28细胞相比，分化差的细胞对细小病毒H-1的细胞毒作用敏感性增加。其机制至少部分与分化差细胞中病毒NS-1蛋白的产生和积聚能力增高相关。未分化的BGC823细胞对H-1病毒的细胞毒作用抵抗，进一步证实并非所有的肿瘤细胞都对细小病毒的溶胞性作用敏感。     

Transduction of Fas gene or Bcl-2 antisense RNA sensitizes cultured drug resistant gastric cancer cells to chemotherapeutic drugs

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Effect of parthenolide on proliferation and apoptosis in gastric cancer cell line SGC7901

OBJECTIVE: To investigate the effect of parthenolide (PAR) on proliferation and apoptosis in gastric cancer cell line SGC7901. METHODS: Human gastric cancer cell line SGC7901 cells were incubated with various concentration of PAR. After various periods of incubation, the proliferation of SGC7901 cells was assessed by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide methyl thiazolyl tetrazolium (MTT) assay. Apoptosis was measured by the annexin V‐fluorescein isothiocyanate fluoresceine isothiocyanate (FITC)/propidium iodide (PI) double labeled staining method and the morphology of the cell was observed under a fluorescent microscope. Mitochondrial potential was measured by flow cytometry after Rhodamine 123 staining. The expressions of cytochrome C and the Bcl‐2 family of proteins, including Bcl‐2, Bax, Bid and tBid were measured by Western blot. Caspase 3 and 8 activities were measured by enzyme‐linked immunosorbent assay. RESULTS: Treatment with PAR induced apoptosis as confirmed by annexin V‐FITC/PI assay. PAR‐induced apoptosis was associated with intracellular events including the decline of mitochondrial potential, increased release of cytochrome C from the mitochondria, decreased expression of Bcl‐2, increased expression of Bax, Bid and tBid and activation of caspase 3 and 8. CONCLUSION: These results suggest that possibly via activation of the mitochondrial pathway, PAR causes mitochondrial damage leading to the release of cytochrome C and by regulating the expression of the Bcl‐2 family of proteins and activating caspases which leads to results in apoptotic cell death in SGC7901 cells. Our results might be helpful in formulating new therapeutic approaches using Chinese herbal medicine.     

Suppression of P-gp induced multiple drug resistance in a drug resistant gastric cancer cell line by overexpression of Fas

AIM:To observe the drug sensitizing effect and related mechanisms of fas gene transduction on human drug-resistant gastric cancer cell SGC7901/VCR (resistant to Vincristine).METHODS:The cell cycle alteration was observed by FACS. The sensitivity of gastric cancer cells to apoptosis was determined by in vitro apoptosis assay. The drug sensitization of cells to several anti-tumor drugs was observed by MTT assay. Immunochemical method was used to show expression of P-gp and Topo II in gastric cancer cells.RESULTS:Comparing to SGC7901 and pBK-SGC7901/VCR, fas-SGC7901/VCR showed decreasing G2 cells and increasing S cells, the G2 phase fraction of pBK-SGC7901/VCR was about 3.0 times that of fas -SGC7901/VCR but S phase fraction of fas -SGC7901/VCR was about 1.9 times that of pBK-SGC7901/VCR, indicating S phase arrest of fas-SGC7901/VCR. FACS also suggested apoptosis of fas-SGC7901/VCR.fas-SGC7901/VCR was more sensitive to apoptosis inducing agent VM-26 than pBK-SGC7901/VCR. MTT assay showed increased sensitization of fas-SGC7901/VCR to DDP, MMC and 5-FU, but same sensitization to VCR according to pBK-SGC7901/VCR. SGC7901, PBK-SGC7901/VCR and fas -SGC7901/VCR had positively stained Topo II equally. P-gp staining in pBK-SGC7901/VCR was stronger than in SGC7901, but there was little staining of P-gp in fas-SGC7901/VCR.CONCLUSION:fas gene transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree, possibly because of higher sensitization to apoptosis and decreased expression of P-gp.     

Growth inhibition and apoptosis of gastric cancer cell lines by Anemarrhena asphodeloides Bunge

In this study, we aimed to determine the growth inhibition and the induction of apoptotic cell death brought about by the herb Anemarrhena asphodeloides Bunge in gastric cancer cell lines, and to clarify the mechanism of this apoptosis. Water-soluble ingredients of A. asphodeloides, and the gastric cancer cell lines, MKN45 and KATO-III, were used in vitro. Growth inhibition, induction of cell death, morphological features, the presence of DNA ladders, increases in caspase-3-like activity, the effects of a caspase-3 inhibitor on apoptotic cell death, and the release of cytochrome c by A. asphodeloides were analyzed. A. asphodeloides inhibited the growth and decreased the viability of the gastric cancer cell lines. The viability of normal skin fibroblasts in the presence of low concentrations of A. asphodeloides was higher than that of gastric cancer cells. Apoptotic bodies and DNA ladders were observed to be induced in MKN45 and KATO-III by A. asphodeloides. The caspase 3 inhibitor, Ac-DEVD-CHO, inhibited the apoptotic cell death of gastric cancer cells induced by A. asphodeloides. The caspase 3-like activity in MKN45 and KATO-III cells increased after the addition of A. asphodeloides. Cytochrome c was released from mitochondria into the cytosol 8 h after the addition of A. asphodeloides, and reached a peak at 16 h. The peak of cytochrome c release was earlier than that of caspase 3-like activity. We concluded that A. asphodeloides inhibited the growth of the gastric cancer cell lines MKN45 and KATO-III and induced apoptosis. The apoptosis of MKN45 and KATO-III cells induced by A. asphodeloides was associated with the release of cytochrome c from the mitochondria, followed by an increase in caspase 3-like activity. Received: December 10, 1999 / Accepted: September 1, 2000     

氧化砷诱发胃癌细胞株凋亡的初步研究

目的在三氧化二砷（Ａｓ２Ｏ３）治疗ＡＰＬ的基础上，进一步探讨Ａｓ２Ｏ３能否诱发胃癌细胞株凋亡。方法采用荧光标记法，经流式细胞仪和荧光显微镜观察Ａｓ２Ｏ３对ＭＫＮ４５和ＳＧＣ７９０１胃癌细胞株的凋亡诱发率和形态学改变。结果发现Ａｓ２Ｏ３诱发胃癌细胞凋亡率高于５－Ｆｕ的作用；形态学见凋亡细胞呈荧光标记阳性，细胞内出现斑块状荧光，体积缩小。结论Ａｓ２Ｏ３可诱发胃癌细胞凋亡，有必要进一步探索其对胃癌治疗的价值。     

NDRG-1异常甲基化及对胃癌细胞恶性生物学行为影响的研究

目的:探讨N-myc下游调节基因1(NDRG-1)甲基化对胃癌细胞恶行生物学行为的影响.方法:用免疫组织化学法检测NDRG-1在胃癌组织及癌旁组织中的表达;qRT-PCR与Western blot检测胃癌细胞SGC7901及胃正常黏膜上皮细胞RGM-1中NDRG-1的表达,MSP法检测胃癌组织及癌旁组织、胃癌细胞及正常...     

转染Zbtb7a基因对人胃癌SGC7901细胞增殖、凋亡的影响及其机制

目的 观察转染Zbtb7a基因对人胃癌细胞系SGC7901增殖及凋亡的影响,并探讨其机制.方法 将pcDNA3.1-Zbtb7a和pSilencer 3.1-H1-mk用Lipofectamine2000转染SGC7901细胞,采用RT-PCR和Western Blot法检测MK mRNA及蛋白表达,CCK-8试剂盒和平板克隆法检测细胞增殖,流式细胞仪Annexin V-PI染色检测细胞凋亡.结果 转染Zbtb7a后,SGC7901细胞中MK表达水平明显升高,细胞增殖能力明显增强（P＜0.05）,并且0.5ng/mL TRAIL诱导的细胞凋亡受到抑制（P＜0.05）.Zbtb7a高表达后干扰MK的表达,细胞增殖及克隆能力则明显降低（P＜0.05）,TRAIL诱导的细胞凋亡数也明显增加（P＜0.05）.结论 Zbtb7a可以通过上调MK的表达,促进SGC-7901细胞增殖以及抑制TRAI诱导的细胞凋亡.