cytochrome-c-t and Stomach-Neoplasms

To investigate the antitumour efficacy of luteolin on gastric cancer (GC) and study the mechanism underpinning the action.. Effects of luteolin on cell growth inhibition, apoptosis, and cell cycle arrest in MKN45 cells were investigated using the cell counting kit-8 assay. Changes in the mitochondrial membrane potential after luteolin treatment were assessed using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanineiodi-de (JC-1) staining. To investigate whether apoptotic effect by luteolin is related to the phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene (PI3K/Akt) pathway, cells were additionally treated with LY294002, a PI3K/Akt pathway inhibitor. Moreover, the expressions of apoptosis-related proteins, namely B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein (Bax), Akt, p-Akt, caspase-3, and cytochrome C, were detected after luteolin treatment.. The study revealed that in MKN45 cells, luteolin could inhibit the cell proliferation in a time- and dose-dependent manner; block the cell cycle in the S-phase; induce apoptosis; reduce the mitochondrial membrane potential; increase the expression of Bax, caspase-3, and cytochrome C; and decrease the expression of Bcl-2 and p-Akt. Luteolin might be involved in the PI3K/Akt signalling pathway, indicating that this pathway could be a therapeutic target for GC treatment.. Luteolin could inhibit the proliferation of GC cells and block the cell cycle in the S-phase. The mechanism of inducing apoptosis in these cells was related to the PI3K/Akt signalling pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Luteolin; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms

Rosmarinic acid (RA) is a well-known phenolic acid widely present in over 160 species of herbal plants and known to exhibit anti-tumor effects on breast, prostate, and colon cancers in vitro. However, its effect and mechanism in gastric cancer and liver cancer are unclear. Moreover, there is no RA report yet in the chemical constituents of Rubi Fructus (RF). In this study, RA was isolated from RF for the first time, and the effect and mechanism of RA on gastric and liver cancers were evaluated using SGC-7901 and HepG2 cells models. The cells were treated with different concentrations of RA (50, 75, and 100 μg/mL) for 48 h, and the effect of RA on cell proliferation was evaluated by the CCK-8 assay. The effect of RA on cell morphology and mobility was observed by inverted fluorescence microscopy, cell apoptosis and cell cycle were determined by flow cytometry, and the expression of apoptosis-related proteins cytochrome C, cleaved caspase-3, Bax, and Bcl-2 was detected by western blotting. The results revealed that, with an increase in the RA concentration, the cell viability, mobility, and Bcl-2 expression decreased, while the apoptosis rate, Bax, cytochrome C, and cleaved caspase-3 expression increased, and SGC-7901 and HepG2 cells could be induced to arrest their cell cycle in the G0/G1 and S phases, respectively. These results together indicate that RA can induce apoptosis of SGC-7901 and HepG2 cells through the mitochondrial pathway. Thus, this study supplements the material basis of the anti-tumor activity of RF and provides an insight into the potential mechanism of RA-inducing apoptosis of gastric cancer SGC-7901 cells and liver cancer HepG2 cells, thereby facilitating further developmental studies on and the utilization of the anti-tumor activity of RF.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cytochromes c; Hep G2 Cells; Humans; Liver Neoplasms; Male; Proto-Oncogene Proteins c-bcl-2; Rosmarinic Acid; Stomach Neoplasms

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease-activating factor 1 (APAF1)-binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA-binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3-mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase-9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC.

Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Biomarkers; Caspase 9; Cytochromes c; Drug Resistance, Multiple; Humans; Methyltransferases; RNA, Long Noncoding; RNA, Small Interfering; Stomach Neoplasms

α-Hederin, a monodesmosidic triterpenoid saponin, exhibited promising antitumor potential against a variety of human cancer cell lines. However, few related studies about effects of α-hederin on gastric cancer are available. Herein, our results showed that α-hederin significantly inhibited the proliferation of gastric cancer cells and arrested the cell cycle in G1 phase in vitro (p < .05). Further research of the potential mechanism reflected that α-hederin could induce intracellular glutathione decrement, adenosine triphosphate level, and mitochondrial membrane potential variation via inducing reactive oxygen species accumulation during the apoptosis of gastric cancer cells. Moreover, the detection of mitochondrial and cytosol proteins with apoptosis-inducing factor, apoptosis protease activating factor-1, and cytochrome C showed an increase in the cytosol, followed by a decrease of Bcl-2 levels and increases of caspase-3, caspase-8, caspase-9, and Bax, which revealed that α-hederin induced apoptosis via triggering activation of the mitochondrial pathway. Furthermore, the above changes were amplified when pretreated with buthionine sulfoximine, whereas attenuated in the group pretreated with NAC than α-hederin alone (p < .05). In addition, α-hederin significantly inhibited the growth of xenografted gastric tumors with favorable safety. In conclusion, α-hederin could inhibit the proliferation and induce apoptosis of gastric cancer accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Glutathione; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oleanolic Acid; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Saponins; Stomach Neoplasms

BACKGROUND Piperine has been reported to inhibit proliferation and induce apoptosis in various cancer cells. This study aimed to explore the efficacy and underlying mechanism of piperine in human gastric cancer. MATERIAL AND METHODS MTT assay was performed to examine the effect of piperine (concentrations of 0-300 μM) on the proliferation of human gastric cancer SNU-16 cells and normal human gastric epithelial GES-1 cells. Flow cytometry and Western blot were used to determine cell apoptosis and the expression level of protein (Cyto C, cleaved PARP, cleaved caspase-3, Bax, Bcl-2, Bad, Bcl-xl, PI3K, pPI3K, Akt, and pAkt), respectively. To further investigate the anti-tumor mechanism of piperine in SNU-16 cells, we used a small-molecule Akt activator SC79 in this study. The in vivo mechanism of piperine against gastric cancer was evaluated using a xenograft tumor model. RESULTS The results showed that piperine inhibited proliferation and induced apoptosis of SNU-16 cells. Piperine upregulated the protein expression of Bax, Bad, Cyto C, cleaved PARP, and cleaved caspase-3, but downregulated the protein expression of Bcl-2, Bcl-xl, pPI3k, and pAkt. However, SC79 reversed the function of piperine on the apoptosis-related proteins. An in vivo study revealed that, compared with the control group, the tumor volume of mice treated with piperine was significantly reduced. Piperine enhanced cleaved caspase-3 expression but decreased Ki-67 expression in a dose-dependent manner. Moreover, the nontoxicity effect of piperine was confirmed by H&E staining analysis in kidney and heart tissues of mice. CONCLUSIONS Our findings suggest that piperine inhibits proliferation and induces apoptosis of human gastric cancer cells through inhibition of the PI3K/Akt signaling pathway.

Topics: Alkaloids; Animals; Antineoplastic Agents; Apoptosis; bcl-Associated Death Protein; bcl-X Protein; Benzodioxoles; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Piperidines; Poly(ADP-ribose) Polymerases; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stomach Neoplasms; Tumor Burden; Xenograft Model Antitumor Assays

Benzyl isothiocyanate (BITC) is known to inhibit the metastasis of gastric cancer cells but further studies are needed to confirm its chemotherapeutic potential against gastric cancer. In this study, we observed cell shrinkage and morphological changes in one of the gastric adenocarcinoma cell lines, the AGS cells, after BITC treatment. We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay, a cell viability assay, and found that BITC decreased AGS cell viability. Reactive oxygen species (ROS) analyses using 2',7'-dichlorofluorescin diacetate (DCFDA) revealed that BITC-induced cell death involved intracellular ROS production, which resulted in mitochondrial dysfunction. Additionally, cell viability was partially restored when BITC-treated AGS cells were preincubated with glutathione (GSH). Western blotting indicated that BITC regulated the expressions of the mitochondria-mediated apoptosis signaling molecules, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cytochrome c (Cyt c). In addition, BITC increased death receptor DR5 expression, and activated the cysteine-aspartic proteases (caspases) cascade. Overall, our results showed that BITC triggers apoptosis in AGS cells via the apoptotic pathways involved in ROS-promoted mitochondrial dysfunction and death receptor activation.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Biological Products; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Isothiocyanates; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Stomach Neoplasms

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colchicine; Cytochromes c; Dose-Response Relationship, Drug; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stomach Neoplasms; Xenograft Model Antitumor Assays

Ionizing radiation (IR) is the main modality for locoregional control of unresectable gastric cancer (GC). [6]-Gingerol is an active major phenolic compound isolated from ginger (Zingiber officinale Roscoe), and it has been demonstrated to possess antitumor activity in previous studies. In the present study, we aimed to evaluate the potential activity of [6]-gingerol as a radiosensitizer and to further explore the underlying mechanism. A CCK-8 assay revealed that [6]-gingerol inhibited the cell viability of HGC-27 cells in a dose-dependent manner (P<0.05). Colony formation assay indicated that pretreatment of [6]-gingerol prior to IR decreased the clonogenic survival of HGC-27 cells. Notably, the combination of [6]-gingerol with IR enhanced IR-induced cell cycle arrest at the G2/M phase compared with IR alone (41.3% in IR alone vs. 53.5% in [6]-gingerol+IR; P=0.006), and increased IR-induced apoptosis compared with IR alone (9.6% in IR alone group vs. 15.1% in [6]-gingerol+IR; P=0.07). DAPI staining detected the apoptotic nuclear morphological changes in the cells treated with [6]-gingerol and/or IR. Furthermore, western blotting and qRT-PCR revealed that [6]-gingerol pretreatment following IR downregulated the protein expression of cyclin B1, cyclin A2, CDC2 and cyclin D1, upregulated the mRNA expression of p27, and induced active caspase-9, active caspase-3 and cytochrome c. In conclusion, the present study demonstrated that [6]-gingerol enhanced radiosensitivity of GC cells, and that the mechanisms involved at least G2/M phase arrest and apoptosis induction.

Topics: Caspases; Catechols; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chemoradiotherapy; Cytochromes c; Dose-Response Relationship, Drug; Fatty Alcohols; Gene Expression Regulation, Neoplastic; Humans; Radiation-Sensitizing Agents; Stomach Neoplasms

Ginger (Zingiber officinale Roscoe), a monocotyledonous herb, is widely used as an herbal medicine owing to the phytoconstituents it possesses. In the current study, the quantity of [6]-gingerol, the major phenolic ketone, in the fresh ginger and dried ginger rhizome was found to be 6.11 µg/mg and 0.407 µg/mg. Furthermore, [6]-gingerol was assessed for its antiapoptotic effects in human gastric adenocarcinoma (AGS) cells evidenced by acridine orange/ethidium bromide staining technique and Annexin-V assay. An increase in reactive oxygen species (ROS) generation led to a decrease in mitochondrial membrane potential (MMP) and subsequent induction of apoptosis. Results disclose that perturbations in MMP are associated with deregulation of Bax/Bcl-2 ratio at protein level, which leads to upregulation of cytochrome-c triggering the caspase cascade. These enduringly suggest that [6]-gingerol can be effectively used for targeting the mitochondrial energy metabolism to manage gastric cancer cells.

Topics: Acridine Orange; Adenocarcinoma; Annexin A5; Apoptosis; bcl-2-Associated X Protein; Caspases; Catechols; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, High Pressure Liquid; Cytochromes c; Ethidium; Fatty Alcohols; Humans; Membrane Potential, Mitochondrial; Plant Extracts; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Stomach Neoplasms; Up-Regulation; Zingiber officinale

4-[4''-(2'', 2'', 6'', 6''-tetramethyl-l''-piperidinyloxy) amino]-4'-demethyl-epipodophyllotoxin (GP7) is a new semi-synthesized nitroxyl spin-labeled derivative of podophyllotoxin with anti-leukemic and anti-osteosarcoma effects. The purpose of the present study is to investigate the anti-gastric cancer (GC) effects of GP7 and the possible involvement of caspase pathway in GP7-induced apoptotic DNA fragmentation in human GC cells.. Effects of GP7 on the proliferation of human GC cell lines MKN28, AGS, BGC-823 and HGC-27 in different degrees of differentiation and normal human gastric epithelial cell line GES-1 were studied by MTT assay and compared with the effects of etoposide. Effects of GP7 on cell viability and heat shock protein 90 expression of BGC-823 and HGC-27 cells were analyzed by trypan blue exclusion test and western blotting, respectively. Effects of GP7 on apoptotic DNA fragmentation and caspase pathway of BGC-823 and HGC-27 cells were detected by agarose gel electrophoresis, colorimetric assay and western blotting. Caspase-3 inhibitor was used to manipulate the activity of caspase-3.. GP7 inhibited concentration- and time-dependently the proliferation of human GC cells, and the inhibitory effect of GP7 on the proliferation of BGC-823 or HGC-27 cells was 1.15- or 1.21-fold higher than that of etoposide. GP7 downregulated heat shock protein 90, improved the anti-GC effects of adriamycin, cisplatin, 5-fluorouracil and their combinations, induced apoptotic DNA fragmentation, activations of caspase-9 and -3 but not -8, cytochrome-c release and BID cleavage in BGC-823 and HGC-27 cells. Caspase-3 inhibitor abrogated GP7-induced BID cleavage, decreased cytochrome-c release, caspase-9 and -3 activities and apoptotic DNA fragmentation but increased cell viability in BGC-823 and HGC-27 cells.. Our findings indicate that GP7 is a promising anti-GC derivative of podophyllotoxin, and GP7-induced apoptosis in human GC cells may be mediated by mitochondrial pathway with caspase-3-dependent BID cleavage.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Etoposide; Fluorouracil; Humans; Mitochondria; Podophyllotoxin; Spin Labels; Stomach Neoplasms

7-O-Geranylquercetin (GQ) is a novel O-alkylated derivate of quercetin. In this study, we evaluated its apoptosis induction effects in human gastric cancer cell lines SGC-7901 and MGC-803 and explored the potential molecular mechanisms. The results demonstrated that GQ lowered viability of SGC-7901 and MGC-803 cells in a dose- and time-dependent manner without apparent cytotoxicity to human gastric epithelial cell line GES-1. GQ could induce apoptosis in SGC-7901 and MGC-803cells, and arrest the gastric cancer cells at G2/M phase. Mechanism study showed that GQ triggered generation of reactive oxygen species (ROS), then activated p38 and JNK signaling pathways, subsequently led to mitochondrial impairment by regulating the expression of Bcl-2, Bcl-xl and Bax, and finally promoted the release of cytochrome c and the activation of caspases to induce apoptosis. In addition, Z-VAD-FMK (caspase inhibitor) could reverse GQ-induced apoptosis. SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) could rescue GQ-induced cell death and attenuate mitochondrial signal pathway activation. Furthermore, NAC (ROS inhibitor) could rescue GQ-induced cell death, reduce ROS generation, decrease the phosphorylation of p38 and JNK, and then attenuate the activation of mitochondrial signal pathway. Taken together, GQ induces caspase-dependent apoptosis in gastric cancer cells through activating ROS-MAPK mediated mitochondrial signal pathway. This study highlights the potential use of GQ as a gastric cancer therapeutic agent.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cytochromes c; G2 Phase; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Quercetin; Reactive Oxygen Species; Signal Transduction; Stomach Neoplasms

Oridonin is one of the most important antitumor active ingredients of Rabdosia rubescens. Recently published studies from our laboratory have demonstrated that oridonin was able to arrest human gastric cancer SGC-7901 cells at G2/M phase. However, little is known about inducing apoptosis in gastric cancer. The aim of this study was to investigate the effect of oridonin on antineoplastic capability of SGC-7901 cells and the detailed molecular mechanism of oridonin-mediated intrinsic pathway of apoptosis. Cell proliferation was assessed by MTT assay while apoptosis induced by oridonin was determined by Hoechst 33342 staining assay and Annexin V/PI double staining assay. Early apoptotic rate was stained by Annexin V/PI and detected by flow cytometry. Apoptosis pathway was analyzed by western blot analysis of Bcl-2, Bax, cytochrome c and caspase-3 expression. The results showed that oridonin was able to inhibit the SGC-7901 cell proliferation, the 50% growth inhibition (IC50) was 22.74 µM. Oridonin could induce cell apoptosis of SGC-7901 cells and the early apoptotic rates induced by 0, 20, 40, 80 µmol/l oridonin were 1.53±0.67, 3.33±0.29, 84.80±0.82 and 96.43±0.51%, respectively. Western blot analysis revealed that oridonin downregulated Bcl-2 protein (the anti-apoptotic factor) and upregulated Bax protein (pro-apoptotic factor), eventually leading to a reduction in the ratio of Bcl-2/Bax proteins. Furthermore, oridonin induced the release of cytochrome c from the mitochondria to the cytosol and the activation of caspase-3. Taken together, the current study suggested that oridonin induced apoptosis in SGC-7901 cells via the mitochondrial signal pathway, which may represent one of the major mechanisms of oridonin-mediated apoptosis in SGC-7901 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Diterpenes, Kaurane; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stomach Neoplasms

Gastric cancer is one of the leading causes for cancer death. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. It has been reported that zerumbone has the anti-cancer effects in various malignant cells. However, the effect and the mechanism of zerumbone on melanoma cells is still largely unknown. In the study, we determined the actions of zerumbone on the human gastric cancer cell line SGC-7901.We also observed the mechanism by which zerumbone induced gastric cancer cell apoptosis. Our data indicated that zerumbone significantly inhibited the growth of human gastric cancer cells in a dose-dependent manner and apoptosis was the main cause of decreased cell viability in zerumbone -treated cells. The treatment with zerumbone downregulated Cyp A and Bcl-2 levels, upregulated Bax levels, and caused Cytochrome c (Cyt-C) to release, activating Caspase-3. In summary, our study suggests that zerumbone mightinduced human gastric cancer cells apoptosis through down-regulating Cyp A and mitochondria-mediated pathways.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cyclophilin A; Cytochromes c; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Sesquiterpenes; Signal Transduction; Stomach Neoplasms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses multidrug resistance (MDR) and induces apoptosis in MDR gastric carcinoma cells. In our previous study, cisplatin proved to be a sensitizing agent for TRAIL. To study the synergistic effects of cisplatin and TRAIL, we investigated the mechanism by which TRAIL reverses multidrug resistance, the role of c-myc in modulating the death receptors DR4 and DR5 and the relationship between cisplatin and cytochrome c (cyt c) release in SGC7901/VCR and SGC7901/DDP cells. We found that after treatment with TRAIL, the DNA-PKcs/Akt/GSK-3β pathway, which is positively correlated with the levels of MDR1 and MRP1, was significantly inhibited and that this tendency can be abolished by Z-DEVD-FMK (a specific caspase 3 inhibitor). We also found that suppression of c-myc by siRNA reduced the expression of DR4 and DR5 and that transfection with a pAVV-c-myc expression vector increased the expression of DR4 and DR5. Moreover, cisplatin increased the expression of c-myc in the presence of TRAIL, and there is a clear increase in cyt c release from mitochondria with the increasing concentrations of cisplatin. Meanwhile, the intrinsic death receptor pathway of caspase 9, as well as the common intrinsic and extrinsic downstream target, caspase 3, was potently activated by the release of cyt c. Together, we conclude that in TRAIL-treated MDR gastric carcinoma cells, cisplatin induces the death receptors DR4 and DR5 through the up-regulation of c-myc and strengthens the activation of caspases via promoting the release of cyt c. These effects would then be responsible for the TRAIL sensitization effect of cisplatin.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Cell Proliferation; Cisplatin; Cytochromes c; Humans; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; Receptors, TNF-Related Apoptosis-Inducing Ligand; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Stomach Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

Bufalin is an active compound in the traditional Chinese medicine Chan Su, which has been shown to induce apoptosis in a range of cancer cell types. However, certain gastric cancer cells are known to be resistant to bufalin. Intracellular secreted protein acidic and rich in cysteine (SPARC) regulates proliferation and apoptosis. This study aimed to evaluate the role of SPARC in bufalin-induced apoptosis in SGC7901 and MGC803 gastric cancer cells. SGC7901 cells with high SPARC expression were more resistant to bufalin than MGC803 cells with low SPARC expression. This resistance was significantly reversed by small interfering (si)RNA-mediated knockdown of SPARC. Furthermore, it was shown that SPARC negatively regulated bufalin-induced intrinsic apoptosis by protecting mitochondrial integrity, decreasing the release of cytoplasmic cytochrome c and increasing the ratio of Bcl-2/Bax. In addition, SPARC overcame bufalin-induced G2/M phase arrest by increasing levels of Cyclin B1 and Cyclin A protein expression. SPARC also activated cellular survival signals, including Src and Akt, but not extracellular signal-regulated kinase. This study demonstrated that SPARC antagonizes bufalin-induced apoptosis via inhibition of the intrinsic apoptosis pathway, inhibition of cell cycle arrest and activation of certain pathways involved in proliferation. This provides novel evidence for SPARC as a potential target by which to sensitize gastric cancer cells to bufalin.

Topics: Apoptosis; bcl-2-Associated X Protein; Bufanolides; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin B1; Cytochromes c; G2 Phase Cell Cycle Checkpoints; Humans; M Phase Cell Cycle Checkpoints; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria; Osteonectin; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Signal Transduction; src-Family Kinases; Stomach Neoplasms

Hydrogen sulfide (H2S) can be synthesized in mammalian cells by cystathionine γ-lyase (CSE) and/or cystathionine β-synthase (CBS). Both CSE and CBS are expressed in rat gastric tissues but their role in human gastric neoplasia has been unclear. The aims of the present study were to detect CSE and CBS proteins in human gastric cancer and determine the effect of exogenous NaHS on the proliferation of gastric cancer cells. We found that both CSE and CBS proteins were expressed in human gastric cancer cells and upregulated in human gastric carcinoma mucosa compared with those in noncancerous gastric samples. NaHS induced apoptosis of gastric cancer cells by regulating apoptosis related proteins. Also, NaHS inhibited cancer cell migration and invasion. An antigastric cancer role of H2S is thus indicated.

Topics: Aged; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cystathionine beta-Synthase; Cystathionine gamma-Lyase; Cytochromes c; Female; Gastric Mucosa; Humans; Hydrogen Sulfide; Male; Microscopy, Fluorescence; Middle Aged; Real-Time Polymerase Chain Reaction; Stomach Neoplasms; Up-Regulation

The tumor-suppressor gene cyclin-dependent kinase inhibitor 1B (P27) is downregulated in gastric cancer cells mainly through proteolytic degradation mediated by the SKP-Cullin1-F-Box (SCF) complex. But the correlation between its downregulation and gastric cancer prognosis still remains indefinite. MLN4924, an anti-tumor agent, which suppresses the SCF complex by inhibiting Cullin1 neddylation, emerges as a promising tool to elucidate its functions in gastric cancer cells. In this study, MLN4924 induced significant growth inhibition of gastric cancer cells in a dose-dependent manner, along with the simultaneous accumulation of P27 and cell cycle abnormalities such as G2/M arrest. Importantly, we found that P27 silencing in MLN4924-treated cells resulted in an enhancement of growth inhibition both in vitro and in vivo. Mechanism analysis revealed the antagonism effects of antioxidants to this excess apoptosis, suggesting reactive oxygen species (ROS) overproduction especially in the mitochondria was the principal cause of the augmentation. Moreover, the robust ROS attacked the mitochondria to initiate collapse of the mitochondrial membrane permeability and the exportation of apoptosis-inducing factor (AIF), IAP-binding mitochondrial protein (SMAC/DIABLO) and cytochrome c. Finally, we also found that P27 knockdown affected the expression profile of several critical BH3 family members to amplify the mitochondrial dysfunction and apoptosis. In summary, our findings unveiled a protective role of P27 by maintaining mitochondrial membrane permeability in MLN4924-treated gastric cancer cells, and therefore highlighted the potential combination of MLN4924 with P27 inhibition to improve its therapeutic efficacy.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cullin Proteins; Cyclin-Dependent Kinase Inhibitor p27; Cyclopentanes; Cytochromes c; Embryo, Nonmammalian; F-Box Proteins; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Pyrimidines; Reactive Oxygen Species; RNA, Small Interfering; S-Phase Kinase-Associated Proteins; Signal Transduction; Stomach Neoplasms; Xenograft Model Antitumor Assays; Zebrafish

In this study, the anti-proliferative effect of physcion, an anthraquinone derivative isolated and characterized from both terrestrial and marine sources, against human gastric cancer SGC-7901 cells was investigated and the underlying mechanisms were explored. Physcion reduced SGC-7901 cell viability in a dose- and time-dependent manner, as demonstrated by MTT assay. It triggered the mitochondrial/caspase apoptotic pathway indicated by loss of mitochondrial membrane potential and cytochrome c release. Moreover, physcion induced a sustained activation of the phosphorylation of AMPK, and compound C (an inhibitor of AMPK) significantly reversed physcion-induced apoptosis in SGC-7901 cells. In addition, physcion provoked the generation of reactive oxygen species (ROS) in SGC-7901 cells, while the antioxidant N-acetyl cysteine almost completely blocked physcion-induced AMPK activation and apoptosis. Taken together, these findings suggest that physcion induces apoptosis through a ROS/AMPK-dependent mitochondrial pathway.

Topics: Acetylcysteine; AMP-Activated Protein Kinases; Apoptosis; Blotting, Western; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Emodin; Humans; Membrane Potential, Mitochondrial; Phosphorylation; Reactive Oxygen Species; Signal Transduction; Stomach Neoplasms

This study was to investigate the effect of Sargassum fusiforme polysaccharides (SFPS-B2) on the proliferation and apoptosis of human gastric cancer cell line SGC-7901. Cells were treated with different concentrations of SFPS-B2. MTT and flow cytometry (FCM) assays were performed to evaluate the effect of SFPS-B2 on the cell growth and apoptosis. Inverted fluorescent microscope was used to observe cell morphology. Laser scanning confocal microscope (LSCM) was used to analyze intracellular calcium ion concentration, mitochondrion permeability transition pore (MPTP) and mitochondrial membrane potential (MMP). Spectrophotometer was applied to quantify the activity of Caspase-9 and Caspase-3. FCM was used to determine the expressions of Bcl-2, Bax and cytochrome C. It was shown that SFPS-B2 inhibited the growth of SGC-7901. After the treatment for 72 h, the cell apoptosis morphology was obvious, which showed that cell protuberance and apoptotic body appeared, and the cytoplasm was concentrated; the apoptotic peak appeared and the apoptotic rate increased in a dose-dependent manner. After the treatment for 24 h, SFPS-B2 activated intracellular MPTP and decreased MMP. It also increased the activity of Caspase-9 and Caspase-3, down-regulated the expression of Bcl-2 and up-regulated the expression of Bax, induced the release of Cyt-C. SFPS-B2 induced SGC-7901 apoptosis through a mitochondrial-mediated pathway, suggesting it may be an agent for cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Cytoplasm; Drug Screening Assays, Antitumor; Humans; Membrane Potential, Mitochondrial; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Polysaccharides; Proto-Oncogene Proteins c-bcl-2; Sargassum; Stomach Neoplasms; Time Factors

Glycyrrhetinic acid (GA), the main chemical constituents of licorice, has shown remarkable anticancer activity. However, the side effects limit its widespread use. 11-DOGA is produced through reduction of GA 11-carbonyl to 11-hydroxyl to reduce its side effects, although its anticancer activities are largely unknown. Here, we report that the functional mechanisms of GA and 11-DOGA in gastric cancers, as well as the comparison between these two drugs' pharmacological potential. Firstly, we found that GA and 11-DOGA significantly inhibits the viabilities of gastric cancer cells in dose- and time-dependent manners. Both GA and 11-DOGA induce gastric cancer cells apoptosis and cell cycle arrest in G2 phase by upregulation of p21 and downregulation of cdc2 and cyclin B1. Further studies show that GA and 11-DOGA-induced apoptosis in gastric cancer cells is associated with BID translocation from nucleus to mitochondria. Moreover, GA and 11-DOGA could effectively inhibit tumor formation of gastric cancer cells in nude mice. Comparing with 11-DOGA, GA presents higher toxicity toward gastric cancer cells both in vivo and in vitro. Thus, the elucidation of the functional mechanisms of GA and 11-DOGA-induced attenuation of gastric cancer growth suggests a possible therapeutic role of GA and its derivatives.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; G2 Phase; Glycyrrhetinic Acid; Humans; Mice, Nude; Protein Transport; Stomach Neoplasms; Xenograft Model Antitumor Assays

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the principal pungent component in hot peppers. The role of capsaicin in carcinogenesis is quite controversial. Although some investigators suspect that capsaicin is a carcinogen, co-carcinogen, or tumor promoter, others have reported that it has chemopreventive and chemotherapeutic effects. The present study aimed to evaluate the cytotoxicity and chemosensitizing activities of capsaicin alone and on 5-flourouracil (5-FU)-treated gastric cancer cells. In this study, the gastric cancer cell line HGC-27 was used and capsaicin used as a chemosensitizer and 5-flourouracil (5-FU) was used as chemotherapeutic. Cytotoxicity and chemosensitizing activities were analyzed with MTT assay; supernatant levels of LDH and glucose were detected as biochemical markers of cell viability; cytochrome c and AIF were evaluated with western blot; and additionally, wound-healing assays were employed. Results suggested that capsaicin had significant anticancer abilities; such capsaicin were capable of causing multifold decreases in the half maximal inhibitory concentration IC50 value of 5-FU. The continuing controversy surrounding consumption or topical application of capsaicin clearly suggests that more well-controlled epidemiologic studies are needed to evaluate the safety and efficacy of capsaicin use. In summary, the present study demonstrated that capsaicin has the potential to be used for treating gastric carcinoma with 5-FU in vitro.

Topics: Antineoplastic Combined Chemotherapy Protocols; Capsaicin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Fluorouracil; Humans; Stomach Neoplasms

Curcumin, a polyphenol compound derived from the rhizome of the plant Curcuma longa L. has been verified as an anticancer compound against several types of cancer. However, understanding of the molecular mechanisms by which it induces apoptosis is limited. In this study, the anticancer efficacy of curcumin was investigated in human gastric adenocarcinoma SGC-7901 cells. The results demonstrated that curcumin induced morphological changes and decreased cell viability. Apoptosis triggered by curcumin was visualized using Annexin V-FITC/7- AAD staining. Curcumin-induced apoptosis of SGC-7901 cells was associated with the dissipation of mitochondrial membrane potential (MMP) and the release of cytochrome c into the cytosol. Furthermore, the down-regulation of Bcl-2 and up-regulation of Bax that led to the cleavage of caspase-3 and increased cleaved PARP was observed in SGC-7901 cells treated with curcumin. Therefore, curcumin-induced apoptosis of SGC-7901 cells might be mediated through the mitochondria pathway, which gives the rationale for in vivo studies on the utilization of curcumin as a potential cancer therapeutic compound.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Curcuma; Curcumin; Cytochromes c; Down-Regulation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Permeability; Plant Preparations; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stomach Neoplasms; Up-Regulation

Raltitrexed is a specific inhibitor of thymidylate synthase (TS), which has been considered as a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In the present study, the apoptosis mechanisms of raltitrexed in SGC7901 human gastric cancer cells were investigated. The cytotoxic activity of raltitrexed on SGC7901 cells was determined by cell counting kit-8 (CCK-8) assay. The CCK‑8 assay indicated that raltitrexed inhibits SGC7901 cell growth in a dose- and time-dependent manner. The morphological changes were observed by fluorescent microscopy, and characteristic morphological changes, including nuclear shrinkage and apoptotic bodies, were observed following Hoechst 33258 staining. The effects on apoptosis, cell cycle, mitochondrial transmembrane potential and reactive oxygen species (ROS) were measured by flow cytometry. The analysis revealed that raltitrexed exerted a growth inhibitory effect by inducing time-dependent apoptosis and cell-cycle arrest at the G0/G1 phase. In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Raltitrexed‑induced caspase‑3‑dependent apoptosis was identified using a caspase-3 activity assay and pretreatment with the caspase-3 inhibitor, Ac‑DEVD‑CHO (sequence, Ac-Asp-Glu-Val-Asp-CHO). The activity of caspase-3 was analyzed with a spectrometer. The protein expression levels of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and TS were examined by western blot and the mRNA expression level of TS was detected by quantitative polymerase chain reaction. The analysis revealed that the protein levels of Bax, cytochrome c and cleaved caspase‑3 were significantly increased by raltitrexed, while Bcl-2 expression levels were reduced. Furthermore, raltitrexed increased the expression of the TS protein and mRNA in a time‑dependent manner. These results indicate that raltitrexed induces the apoptosis of SGC7901 cells through the caspase‑3‑dependent mitochondrial signaling pathway and upregulates the expression of the TS protein and mRNA.

Topics: Antimetabolites, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cytochromes c; G1 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Stomach Neoplasms; Thiophenes; Thymidylate Synthase

A pair of inverted repeated sequences of the gene survivin was designed for stable double-stranded RNA establishment. After stable transfection, the biological behaviors of gastric cancer cells were observed. The interference rates of survivin-targeting siRNA (siRNA-survivin) in BGC823, MKN45, SGC7901, and cisplatin-resistant SGC7901 groups were 55.363 ± 3.974, 71.433 ± 3.774, 69.433 ± 7.336, and 76.767 ± 3.541%, respectively, compared with those in the control group. After siRNA-survivin interference, survivin protein expression noticeably decreased, apoptotic rates markedly increased, and cell proliferation was inhibited to varying degrees. Mitochondrial cytochrome C protein expression decreased and the levels of cytoplasmic cytochrome C and caspase-3 increased, which showed significant differences compared with values before transfection. pRNA-shSU eukaryotic expression vectors were constructed. After plasmid transfection, green fluorescent protein expression increased and survivin protein expression noticeably increased in BGC823 and SGC7901. siRNA-survivin promotes GC cell apoptosis and inhibits cell proliferation by downregulating survivin mRNA and protein expression. The underlying mechanisms are correlated with a decrease in mitochondrial cytochrome C and cytoplasmic cytochrome C and caspase-3.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cytochromes c; Drug Resistance, Neoplasm; Humans; Inhibitor of Apoptosis Proteins; Microscopy, Fluorescence; Mitochondrial Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Stomach Neoplasms; Survivin; Transfection

Capparis spinosa L., a Uygur medicine, had been shown to have anti-tumor activity in our early experiments with an N-butanol extract (CSBE) as its active fraction. However, the mechanisms responsible for its effects are not clearly understood. Here, we report that treatment of SGC-7901 cells with CSBE resulted in dose-dependent reduction of cell viability and induction of apoptosis.. To observe the inhibitory and killing effects of CSBE on SGC-7901, the SRB method was adopted, apoptosis being observed by electron microscopy. To clarify the mechanisms of apoptosis, Western blot and enzyme-labeled methods were used to examine the release of cytochrome c (Cyt c) and the activation of the caspase cascade.. By electron microscopy, apoptotic morphologic changes were detectable after CSBE administration. In this study, it was also demonstrated that CSBE induced apoptosis in SGC-7901 cells by inhibiting mPTP open, mitochondrial cytochrome c release, caspase-9 and caspase-3 activation.. The findings indicated that CSBE induces apoptosis through mitochondrial pathway.

Topics: 1-Butanol; Apoptosis; Blotting, Western; Capparis; Caspases; Cytochromes c; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Plant Extracts; Stomach Neoplasms; Tumor Cells, Cultured

Ursolic acid, extracted from the traditional Chinese medicine bearberry, can induce apoptosis of gastric cancer cells. However, its pro-apoptotic mechanism still needs further investigation. More and more evidence demonstrates that mitochondrial translocation of cofilin-1 appears necessary for the regulation of apoptosis. Here, we report that ursolic acid (UA) potently induces the apoptosis of gastric cancer SGC-7901 cells. Further mechanistic studies revealed that the ROCK1/PTEN signaling pathway plays a critical role in UA-mediated mitochondrial translocation of cofilin-1 and apoptosis. These findings imply that induction of apoptosis by ursolic acid stems primarily from the activation of ROCK1 and PTEN, resulting in the translocation of cofilin-1 from cytoplasm to mitochondria, release of cytochrome c, activation of caspase-3 and caspase-9, and finally inducing apoptosis of gastric cancer SGC-7901 cells.

Topics: Amides; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cofilin 1; Cytochromes c; Enzyme Activation; Humans; Medicine, Chinese Traditional; Mitochondria; Protein Transport; PTEN Phosphohydrolase; Pyridines; rho-Associated Kinases; Stomach Neoplasms; Triterpenes; Ursolic Acid

This investigation aimed to determine effects of celecoxib on the cell cycle kinetics of the gastric cancer cell line MGC803 and the mechanisms involved by assessing expression of cytochrome C and caspase-9 at the protein level.. Cell proliferation of MGC803 was determined by MTT assay after treatment with celecoxib. Apoptosis was assessed using fluorescence staining and cell cycle kinetics by flow cytometry. Western blotting was used to detect the expression of caspase-9 protein and of cytochrome C protein in cell cytosol and mitochondria.. Celecoxib was able to restrain proliferation and induce apoptosis in a dose- and time- dependent manner, inducing G0/G1 cell cycle arrest, release of cytochrome C into the cytosol, and cleavage of pro-caspase-9 into its active form.. Celecoxib can induce apoptosis in MGC803 cells through a mechanism involving cell cycle arrest, mitochondrial cytochrome C release and caspase activation.

Topics: Apoptosis; Blotting, Western; Caspase 9; Celecoxib; Cell Cycle; Cell Proliferation; Cyclooxygenase 2 Inhibitors; Cytochromes c; Flow Cytometry; Humans; Kinetics; Pyrazoles; Stomach Neoplasms; Sulfonamides; Tumor Cells, Cultured

Alpinetin is a natural flavonoid widely distributed in Zingiberaceae. Previous studies have demonstrated that alpinetin markedly inhibits tumour growth. However, the molecular mechanisms underlying the antitumour effects of alpinetin are unclear. Bcl‑2‑associated X protein (Bax) translocation is known to activate the mitochondrial‑dependent endogenous apoptosis pathway. The aim of the current study was to investigate the roles of Bax and the mitochondrial pathway during alpinetin‑induced gastric cancer cell apoptosis and the effects of alpinetin on the cell cycle. Human gastric cancer cells were treated with various doses of alpinetin and an MTT assay was performed to measure cell viability, flow cytometry to measure the apoptotic rate, changes in the cell cycle and mitochondrial membrane potential and western blot analysis to detect the expression levels of relevant proteins. Results demonstrate that alpinetin induces apoptosis in human gastric cancer cells in a dose‑ and time‑dependent manner. During the early stages of apoptosis, alpinetin may alter mitochondrial membrane potential leading to release of cytochrome c from mitochondria, activation of caspase family members and ultimately apoptosis of human gastric cancer cells. Results of the present study indicate that alpinetin‑induced human gastric cancer cell apoptosis is associated with the mitochondrial pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cytochromes c; Flavanones; G2 Phase Cell Cycle Checkpoints; Humans; M Phase Cell Cycle Checkpoints; Membrane Potential, Mitochondrial; Mitochondria; Stomach Neoplasms; Zingiberaceae

The aim of this study was to analyze the immunoexpression of (Smac) DIABLO, AIF, cytochrome c, Ki-67 and cleaved caspase-3 in gastric cancer. A tissue microarray (TMA) paraffin block was constructed using gastric adenocarcinoma tissue and adjacent normal adjacent mucosa from 87 patients who had not previously undergone radiotherapy or chemotherapy. Immunohistochemistry was used to evaluate the protein levels. Samples were positive for (Smac) DIABLO in 37 (45.6%) and 37 (46.8%), for AIF in 31 (36.9%) and 36 (45.6%), for cytochrome c in 60 (68.9%) and 44 (54.4%), for Ki-67 in 63 (72.4%) and 52 (61.9%) and for cleaved caspase-3 in 21 (24.1%) and 3 (3.4%) cases of tumor and adjacent normal tissues, respectively. Our results suggest that increased expression of Ki-67 and cleaved caspase-3 could contribute to carcinogenesis. The expression of these proteins indicates an attempt of cells to maintain tissue homeostasis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Biomarkers, Tumor; Cytochromes c; Female; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Male; Middle Aged; Mitochondrial Proteins; Stomach Neoplasms; Tissue Array Analysis

Gastric cancer is one of the most common cancers in the world. The "economically developed countries" life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate.. A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy.. Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.

Topics: Animals; Apoptosis; Caspases; Cattle; Cell Cycle Checkpoints; Cell Line, Tumor; Cultured Milk Products; Cytochromes c; DNA Fragmentation; Enzyme Activation; Humans; Probiotics; Propionibacterium; Reactive Oxygen Species; Stomach Neoplasms

Prohibitin (PHB), an evolutionarily-conserved protein, has been found to be over-expressed in gastric cancer and be closely related with tumor malignancy. In this study, to investigate the relationship between PHB expression and cell apoptosis in the BGC823 gastric cancer cell line, low and high expression PHB in BGC823 cells was accomplished using RNA interference technology and gene transfer techniques. Cell proliferation, cell cycling, apoptosis, Bax, Bcl-2 and Cyt.c protein expression and the activation of Caspase-3,9 were assessed after 48 h. Over-expression of PHB gene in BGC823 cells resulted in slow cell growth, cell arrest in G2 phase, and an increased apoptosis ratio while the opposite was found for PHB under-expressing cells. In PHB over-expressing cells, the expression of Bax gene was increased, the expression of Bcl-2 was decreased, the activation level of Caspase-3, 9 was increased, but the activation level of Caspase-8 demonstrated no change. These results indicate that PHB induced apoptosis through the mitochondrial pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Cycle; Cell Proliferation; Cytochromes c; Flow Cytometry; Humans; Mitochondria; Prohibitins; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Stomach Neoplasms; Tumor Cells, Cultured

To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.. The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4, 5-dimethylthiazol-2-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 fragmentation (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.. 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 that the 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 dose-dependent 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.. 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.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dactinomycin; Diterpenes, Kaurane; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Isodon; Medicine, Chinese Traditional; Microscopy, Fluorescence; Phycoerythrin; Plant Extracts; Signal Transduction; Stomach Neoplasms; Time Factors

This study was designed to investigate the effect of juglone on the apoptosis of human gastric cancer SGC-7901 cells. The cytotoxic activity of juglone on SGC-7901 cells was tested by the sulforhodamine B (SRB) assay. The morphological changes in the cells were observed by transmission electron microscopy (TEM). The apoptotic rate, the level of reactive oxygen species (ROS), mitochondrial transmembrane potential and the expression of cytochrome c protein were detected by flow cytometry (FCM). The expression of Bcl-2 and Bax proteins were examined by Western blot. Caspase 3 activity was determined with a microplate reader. Our results were as follows: the GI(50) values for SGC-7901 cells were 36.51 ± 1.05 μmol/L (24h) and 25.37 ± 1.19 μmol/L (48 h). After 24h of exposure to juglone (5, 10, 15 and 20 μmol/L), the cells presented the typical morphological changes of apoptosis, and the rate of apoptosis was found to increase in a dose-dependent manner. After cells were treated with juglone at the same dose for 24h, the level of ROS was significantly higher, the expression of Bcl-2 was significantly down-regulated and the expression of Bax was significantly up-regulated compared to the control. The mitochondrial transmembrane potential was significantly lower, and the expression of the cytochrome c protein was significantly higher relative to the control. Caspase 3 was activated in a concentration-dependent manner. In conclusion, juglone can induce apoptosis in SGC-7901 cells through a mitochondrial pathway that seems to be mediated by the generation of ROS and a reduction in the Bcl-2/Bax ratio.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Stomach Neoplasms

Noscapine plays an important role in the regulation of cell growth and death. It has been reported to potentiate the anti-tumor effect by inducing apoptosis in various malignant cells. However, the mechanism of inducing apoptosis in gastric cancer cells by this agent remains to be clarified.. In the study, we investigated the signaling pathways by which noscapine induces apoptosis in gastric cancer cell lines. Apoptosis of four human gastric cancer cell lines was induced by treatment with noscapine.. Our results indicate that noscapine induced a dose-dependent apoptosis of these cells. The treatment with noscapine upregulated Bax and Cytochrome c (Cyt-c) protein, downregulated Bcl-2 protein. Caspase-3 and caspase-9 were activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, in xenograft tumor mouse model, noscapine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay.. These data of the study suggest that noscapine induces apoptosis in gastric cancer cells via mitochondrial pathways.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Down-Regulation; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Noscapine; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms; Up-Regulation; Xenograft Model Antitumor Assays

To investigate the inhibition effect of Ganoderma applanatum extract (GAEAE) on human gastric cancer cell lines and apoptosis mechanism, Alamar Blue assay was used to assess the inhibition and apoptosis-inducing effect of the GAEAE on proliferation of SGC-7901 cells, the DNA ladders of the apoptosis cells was done, the mRNA expression of p53, Bax, Bcl-2 and JNK were analysed by reverse transcription-polymerase chain reaction. The levels of the Cyt C and the p53, Bax/Bcl-2, JNK proteins and the caspase-3 activity in the SGC-7901 cells were measured with ELISA kits. Our data showed that the GAEAE markedly inhibited the proliferation of SGC-7901 cells in a dose-dependent manner. The expression of Bcl-2 protein was down-regulated and Bax, c-jun, p53 protein expressions were up-regulated by the GAEAE treatment in SGC-7901 cells. The activity of caspase-3 was markedly increased and the Cty C was markedly released into cytoplasm from the mitochondria after GAEAE action. In conclusion, our results indicated that the GAEAE could enhance the sensitivity of SGC-7901 cells to the c-jun, p53, Bax and Bcl-2 induced apoptosis and provided a promising approach to anti-human gastric cancer therapy with Ganoderma applanatum.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Ganoderma; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 4; Mitochondria; Oxazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-jun; Reishi; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Tumor Suppressor Protein p53; Xanthenes

This study was designed to investigate the effect of pseudo-G-Rh2, a novel metabolite of ginsenoside Rh2, on the apoptosis of SGC-7901 human gastric cancer cells. Pseudo-G-Rh2 demonstrated antitumor activity and significantly inhibited the proliferation of SGC-7901 cells in a concentration-dependent manner. After treatment with pseudo-G-Rh2, SGC-7901 cells showed typical apoptotic morphological features, such as chromatin condensation and DNA fragmentation. Pseudo-G-Rh2 could induce mitochondrial membrane potential loss, which led to the release of cytochrome c (Cyt c), Smac/Diablo and apoptosis-inducing factor (AIF) to the cell cytoplasm. Furthermore, pseudo-G-Rh2 exposure not only decreased the expression of the Bcl-2 protein but also increased the expression of the Bax protein and the activities of caspase-9 and caspase-3 in SGC-7901 cells. These results demonstrated that pseudo-G-Rh2 inhibited the proliferation of SGC-7901 cells by initiating apoptosis. Pseudo-G-Rh2-induced apoptosis was associated with a drop in the mitochondrial transmembrane potential, down-regulation of Bcl-2, up-regulation of Bax and activation of caspase-9 and caspase-3.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Ginsenosides; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Stomach Neoplasms

A number of pathogenic bacteria target mitochondria to modulate the host's apoptotic machinery. Studies here revealed that infection with the human gastric pathogen Helicobacter pylori disrupts the morphological dynamics of mitochondria as a mechanism to induce host cell death. The vacuolating cytotoxin A (VacA) is both essential and sufficient for inducing mitochondrial network fragmentation through the mitochondrial recruitment and activation of dynamin-related protein 1 (Drp1), which is a critical regulator of mitochondrial fission within cells. Inhibition of Drp1-induced mitochondrial fission within VacA-intoxicated cells inhibited the activation of the proapoptotic Bcl-2-associated X (Bax) protein, permeabilization of the mitochondrial outer membrane, and cell death. Our data reveal a heretofore unrecognized strategy by which a pathogenic microbe engages the host's apoptotic machinery.

Topics: Animals; Apoptosis; Bacterial Proteins; bcl-2-Associated X Protein; Cell Line; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Dynamins; Fibroblasts; Flow Cytometry; Green Fluorescent Proteins; GTP Phosphohydrolases; HeLa Cells; Helicobacter Infections; Helicobacter pylori; Host-Pathogen Interactions; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Microscopy, Fluorescence; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Mutation; Stomach Neoplasms

The underlying mechanisms of alpha-tocopheryl succinate (alpha-TOS)-mediated apoptosis are not understood in detail, although the redox-silent vitamin E analog is a potent apoptogen and anti-cancer agent. Our previous studies showed the important role of Fas signaling in apoptosis induced by the mitocan. The objective of the present study was to investigate whether apoptosis triggered by alpha-TOS in gastric carcinomas cells involves both mitochondria- and death receptor-dependent pathways. alpha-TOS induced apoptosis and mitochondrial permeability transition in a concentration- and time-dependent manner. As a consequence, cytochrome c and the apoptosis-inducing factor were released and caspases were activated. Bax was translocated from the cytosol to mitochondria and Bid was cleaved into its truncated form, tBid. Knocking down Bid by RNAi and Fas antisense oligodeoxynucleotides resulted in a decreased release and cleavage. The results imply that Bid may serve as a critical integrating factor of the death receptor and mitochondrial pathway in alpha-TOS-mediated apoptosis.

Topics: alpha-Tocopherol; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; fas Receptor; Humans; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oligonucleotides, Antisense; Protein Transport; Receptors, Death Domain; RNA Interference; Signal Transduction; Stomach Neoplasms; Time Factors

Interferon regulatory factor-1 (IRF-1) is a transcription factor that acts as a tumor suppressor and causes apoptosis in cancer cells. We evaluated IRF-1-induced apoptosis in gastric cancer cell lines. We established stable clones in AGS cells that have a tetracycline-inducible IRF-1 expression system. We used these clones and recombinant adenovirus expressing IRF-1 to explore the mechanism of IRF-1-induced apoptosis in gastric cancer. Expression of IRF-1 causes apoptosis in gastric cancer cell lines as shown by phosphatidylserine exposure and cleavage of caspase-8, caspase-3, and Bid with the mitochondrial release of cytochrome c. However, inhibition of caspase-8 and Bid did not inhibit apoptosis and did not decrease cleaved caspase-9 or mitochondrial release of cytochrome c. We then show that IRF-1 upregulates PUMA (p53 upregulated modulator of apoptosis), which is known to activate apoptosis by the intrinsic pathway; this can be p53-independent. IRF-1 binds to distinct sites in the promoter of PUMA and activates PUMA transcription. Moreover, molecular markers of mitochondrial apoptosis are eliminated in PUMA knockout and knockdown cells and phosphatidylserine exposure is decreased dramatically. Finally, we show that IFN-gamma induces IRF-1-mediated upregulation of PUMA in cancer cells. We conclude that IRF-1 can induce apoptosis by the intrinsic pathway independent of the extrinsic pathway by upregulation of PUMA.

Topics: Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Caspases; Cell Line, Tumor; Cytochromes c; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Interferon Regulatory Factor-1; Mitochondrial Proteins; Phosphatidylserines; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins; Stomach Neoplasms; Transcriptional Activation; Up-Regulation

Combined chemotherapy of 5-fluorouracil (5FU) and mitomycin c (MMC) is clinically used for gastric cancer, but the precise conditions and molecular mechanism of these agents when used together remain unclear. We examined the administration sequence of combining 5FU with MMC to maximize toxicity against a human gastric cancer cell line, and then investigated the possible molecular mechanisms underlying the observed toxic effects.. Human gastric cancer MKN45 cells were treated with a combination of 5FU and MMC, and the changes in cell viability and apoptosis-related proteins were determined by a tetrazolium dye-based cytotoxicity assay and Western blot analysis, respectively. The intracellular levels of reactive oxygen species (ROS) were monitored using a fluorescent probe or by a cytochrome c reduction assay.. Pretreatment for 24 h with 5FU augmented the toxic effect of MMC in MKN45 cells. The synergic effect was mediated mainly via ROS formation and the p53-dependent apoptotic pathway, leading to mitochondrial dysfunction and caspase activation. In vitro experiments using extracts of the treated cells showed superoxide anion generation in a redox cycle of MMC, involving alterations in superoxide dismutase.. Pretreatment with 5FU enhanced the MMC-induced toxicity against gastric cancer cells via alterations in antioxidant enzymes with resulting ROS generation. This observation will need confirmation in the clinical setting.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Synergism; Fluorouracil; Humans; Mitochondria; Mitomycin; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Stomach Neoplasms; Superoxide Dismutase; Superoxides; Tumor Suppressor Protein p53

It is known that the vacuolating cytotoxin (VacA) could induce apoptosis. However, the mechanism remained to be elucidated. The aim of this study is to investigate the role of Bcl family of proteins (Bcl-2 and Bax) and the mitochondrial voltage-dependent anion channel (VDAC) in VacA-induced apoptosis of AGS cells.. Plasmid pGBKT7-VacA p58 was constructed and transfected into the AGS cells. RT-PCR and Western blotting were used to determine the expressions of cytochrome c, caspase-3, Bax, Bcl-2 and VDAC1 mRNA and proteins.. VacA p58 can induce cytochrome c release and activate caspase-3 in AGS cells. It up-regulated the expressions of Bax and VDAC1 mRNA and proteins, and decreased the expression of Bcl-2 in AGS cells.. VacA p58 induces apoptosis in AGS cells. This apoptotic process is associated with the up-regulation of Bax/VDAC1 and downregulation of Bcl-2. These findings suggest that the release of cytochrome c by VacA p58 is mainly through VDAC-dependent and Bcl-2 family-dependent pathways.

Topics: Apoptosis; Bacterial Proteins; Caspase 3; Cell Line, Tumor; Cytochromes c; Down-Regulation; Genotype; Humans; Neoplasms, Glandular and Epithelial; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms; Up-Regulation; Voltage-Dependent Anion Channel 1

The abnormal accumulation and activation of the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON), has been implicated in tumorigenesis and metastasis in epithelial tumors including gastric cancer. This study examined whether the sequence-specific small interfering RNA (siRNA) suppression of the RON expression could induce apoptotic cell death, and investigated the involved molecular mechanisms. Sequence-specific siRNA effectively suppressed the RON expression at both the mRNA and protein levels. Silencing of the RON expression significantly inhibited gastric cancer cell proliferation and induced apoptosis in a time-dependent manner. The induction of apoptosis was confirmed by the ladder-patterned DNA fragmentation, the presence of cleaved and condensed nuclear chromatin and the increased number of annexin V-positive cells. RON-targeted siRNA effectively inhibited the constitutive nuclear factor-kappaB (NF-kappaB) activation as revealed by an altered electrophoretic mobility shift. In agreement with this, silencing of the RON expression resulted in a decrease in the nuclear level of the p65 subunit of NF-kappaB. The transfection of siRNA, which blocked the RON expression, also caused a change in the ratio of Bax/Bcl-2 in a manner that favored apoptosis. The siRNA silencing of RON induced cytochrome c release and the activation of caspase-8 and caspase-9. These results indicate that RON-targeted siRNA could be therapeutically efficacious by inducing cell apoptosis through the modulation of the NF-kappaB and Bcl-2 family in gastric cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Receptor Protein-Tyrosine Kinases; RNA Interference; RNA, Messenger; Signal Transduction; Stomach Neoplasms; Time Factors; Transcription Factor RelA; Transfection

Allicin is an active compound derived from garlic that has been shown to have antitumor properties in vitro. The current study was designed to explore the effects and the underlying mechanism of allicin on gastric cancer cells. The MTT assay was used to detect cell viability. Transmission electron microscopy, Rh123 and propidium iodide staining, annexin V/FITC assay and the mitochondrial membrane potential were used to assess for the presence of apoptosis. Immunocytochemistry, western blot analysis, and Q-RT-PCR were used to detect gene expression. We found that allicin reduced cell viability in a dose- and time-dependent manner, partly through induction of apoptosis in gastric cancer cells. At the molecular level, allicin induced cytochrome c release from the mitochondria and increased caspase-3, -8, and -9 activation, with concomitant upregulation of bax and fas expression in the tumor cells. Allicin treatment inhibited proliferation and induced apoptosis in SGC-7901 cancer cells. Both intrinsic mitochondrial and extrinsic Fas/FasL-mediated pathways of apoptosis occur simultaneously in SGC-7901 cells following allicin treatment. Data from the current study demonstrated that allicin should be further investigated as a novel cancer preventive or therapeutic agent in control of gastric cancer, with potential uses in other tumor types.

Topics: Adenocarcinoma; Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Survival; Cytochromes c; Disulfides; Drug Evaluation, Preclinical; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Models, Biological; Signal Transduction; Stomach Neoplasms; Sulfinic Acids; Tumor Cells, Cultured

Human mitochondrial Mrs2 protein (hsaMrs2p) is a magnesium transporter in mitochondria inner membrane. It was identified as an upregulated gene in a multidrug-resistant (MDR) gastric cancer cell line compared to its parental cells by subtractive hybridization. To further explore the role of hsaMrs2p in MDR of gastric cancer cells, the cDNA encoding hsaMrs2p was generated and mouse antisera against hsaMrs2p was raised with recombinant hsaMrs2p as the immunogen. HsaMrs2p expression could positively regulate adriamycin resistance of SGC7901/ADR cells both in vitro and in vivo. Further study showed that hsaMrs2p increased adriamycin-releasing index. Its upregulation inhibited adriamycin-induced apoptosis, probably by suppressing Bax induced cytochrome C release from mitochondria. Additionally, hsaMrs2p promoted cell growth and cells with decreased hsaMrs2p exhibited significant inhibition of cell growth with G(1) cell cycle arrest. By enhanced hsaMrs2p expression, p27 was downregulated whereas cyclinD1 was upregulated. Our results provide new insights into the function of hsaMrs2p that may be a promising target for MDR reversal therapy.

Topics: Animals; Cation Transport Proteins; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitochondrial Proteins; Stomach Neoplasms; Up-Regulation

We investigated the effects of a water-soluble extract of Maitake (Grifola frondosa), a Japanese edible mushroom, on the proliferation and cell death of four human gastric cancer cell lines (TMK-1, MKN28, MKN45 and MKN74). The Maitake extract (ME) inhibited the proliferation of all four cell lines in a time-dependent manner. The inhibition was most pronounced in TMK-1 cells, which exhibited up to 90% inhibition after treatment with 10% ME for 3 days. Staining of ME-treated TMK-1 cells with Hoechst 33258 revealed increased numbers of nuclear condensations and apoptotic bodies. Induction of apoptosis was confirmed by fluorescence-activated cell sorting analyses. Western blot analyses of TMK-1 cells after ME treatment revealed increases in intracytoplasmic cytochrome c and cleavage of caspase-3 and poly(ADP-ribose) polymerase, but no expression of p21 or Bax. The caspase-3 protease activities in lysates of TMK-1 cells treated with 1% or 10% ME were about three times higher than those in control cells. The proliferation of TMK-1 cells was hardly affected by the caspase-3 inhibitor z-DEVD-fmk. Taken together, these results suggest that ME induces apoptosis of TMK-1 cells by caspase-3-dependent and -independent pathways, resulting in potential antitumor effects on gastric cancer.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; Grifola; Humans; Stomach Neoplasms

Cancer is one of the leading causes of death in the world. The triterpenoid compound asiatic acid derived from the tropical medicinal plant Centella asiatica displays cytotoxic activity on fibroblast cells and several other kinds of cells. The present work studies asiatic acid-mediated growth inhibition of cancer cells and the underlying mechanism. Asiatic acid markedly inhibited cancer cell proliferation. Apoptosis of SW480 human colon cancer cells was induced by asiatic acid as shown by flow cytometry, DNA fragmentation and nuclear chromatin condensation experiments. Through increasing mitochondrial membrane permeability and cytochrome c release from mitochondria into cytosol, asiatic acid induced caspase-9 activity, which further activated caspase-3 and poly(ADP-ribose) polymerase cleavage resulting in irreversible apoptotic death in the tumor cells. Taken together, these results suggest that mitochondrial death apoptosis cascade plays very important roles in asiatic acid-induced cancer apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Centella; Chromatin; Colonic Neoplasms; Colorectal Neoplasms; Cytochromes c; DNA Fragmentation; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Molecular Structure; Pentacyclic Triterpenes; Poly Adenosine Diphosphate Ribose; Stomach Neoplasms; Triterpenes

To investigate the effect of parthenolide (PAR) on proliferation and apoptosis in gastric cancer cell line SGC7901.. 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.. 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.. 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.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; NF-kappa B; Sesquiterpenes; Stomach Neoplasms

Docosahexaenoic acid (DHA) shows anti-inflammatory and/or anticancer effects in some cells. Activator protein-1 (AP-1) regulates cellular proliferation and apoptosis. Although recent studies demonstrate the association between gastric cancer risk and DHA, the exact molecular mechanism has not been clarified. We investigated whether AP-1 mediates DHA-induced apoptosis of gastric cancer cells. We found that DHA induced cell death and DNA fragmentation in parallel with the activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) as well as AP-1. DHA increased the protein levels of p53, cytochrome c, and Bax in gastric cancer cells. DHA-induced DNA fragmentation and protein levels of p53, cytochrome c, and Bax were inhibited in the cells transfected with c-jun dominant-negative mutant (TAM67). Because JNK and ERK are upstream signaling for AP-1 activation, we suggest that DHA-induced activation of AP-1 may mediate apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Docosahexaenoic Acids; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Peptide Fragments; Proto-Oncogene Proteins c-jun; Stomach Neoplasms; Transcription Factor AP-1; Transfection; Tumor Suppressor Protein p53

Photodynamic therapy (PDT) shows a limited antitumor effect in treating gastrointestinal tumors because of improper light penetration or insufficient photosensitizer uptake. The aim of this study was to evaluate the cytotoxic effect of PDT combined with paclitaxel on in vitro cancer cells. In vitro photodynamic therapy was performed in gastric cancer cells (NCI-N87) and bile duct cancer cells (YGIC-6B) using verteporfin (2 ug mL(-1)) and a PTH light source (1 000 W, Oriel Co.) with 665-675 nm narrow band pass filter. Cytotoxicity was compared using the MTT assay between cancer cells treated with PDT alone or pretreated with paclitaxel (IC(25)). Apoptotic changes were evaluated using DAPI staining, DNA fragmentation analysis, Annexin V-FITC apoptosis assay, cell cycle analysis, and western blots for cytochrome c, Bax, and Bid. The PDT-induced cytotoxicity was potentiated by pretreating with low dose paclitaxel (P < 0.001). The enhanced cytotoxicity was due to an augmented apoptotic response mediated by exaggerated cytochrome c released from mitochondria, without Bax or Bid activation. These results show that paclitaxel pretreatment enhances PDT-mediated cancer therapy.

Topics: Apoptosis; Bile Duct Neoplasms; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Humans; Mitochondria; Paclitaxel; Photochemotherapy; Photosensitizing Agents; Porphyrins; Stomach Neoplasms; Verteporfin

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.

Topics: Algorithms; Animals; Caseins; Cattle; Cell Line, Tumor; Chickens; Computational Biology; Cytochromes c; Horses; Humans; Mass Spectrometry; Muramidase; Myoglobin; Ovalbumin; Proteomics; Sensitivity and Specificity; Serum Albumin, Bovine; Stomach Neoplasms

To explore changes in structure and function of the mitochondria of human gastric carcinoma BGC-823 cells after Newcastle disease virus (NDV) infection.. Electron microscopy was applied to observe the structure of mitochondria; Rhodamine 123 staining was used to determine the mitochondrial membrane potential; the activity of Na(+)-K(+)-ATPase and Ca(2+)-ATPase were also determined and the release of cytochrome C was detected by Western blotting.. The structure of mitochondria in the tumor cells infected with NDV changed distinctly. In the infected group the activity of mitochondrial Na(+)-K(+)-ATPase and Ca(2+)-ATPase significantly declined (P < 0.01), and compared with control cells, mitochondrial trans-membrane potential was decreased. NDV infection induced the decrease of cytochrome C levels.. The effects of NDV infection on the structure and functions of mitochondria of human gastric carcinoma BGC-823 cells might play a role in the oncolysis of NDV.

Topics: Animals; Carcinoma; Cell Line, Tumor; Chick Embryo; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Newcastle Disease; Newcastle disease virus; Sodium-Potassium-Exchanging ATPase; Stomach Neoplasms

t-Darpp is a cancer-related truncated isoform of Darpp-32 (dopamine and cyclic-AMP-regulated phosphoprotein of M(r) 32,000). We detected overexpression of t-Darpp mRNA in two thirds of gastric cancers compared with normal samples (P = 0.004). Using 20 micromol/L ceramide treatment as a model for induction of apoptosis in AGS cancer cells, we found that expression of t-Darpp led to an increase in Bcl2 protein levels and blocked the activation of caspase-3 and caspase-9. The MitoCapture mitochondrial apoptosis and cytochrome c release assays indicated that t-Darpp expression enforces the mitochondrial transmembrane potential and protects against ceramide-induced apoptosis. Interestingly, the expression of t-Darpp in AGS cells led to >or=2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser(473) Akt and p-Ser(9) GSK3 beta. These findings were further confirmed using tetracycline-inducible AGS cells stably expressing t-Darpp. We also showed transcriptional up-regulation of Bcl2 using the luciferase assay with Bcl2 reporter containing P1 full promoter, quantitative reverse transcription-PCR, and t-Darpp small interfering RNA. The Bcl2 promoter contains binding sites for cyclic AMP-responsive element binding protein CREB/ATF1 transcription factors and using the electrophoretic mobility shift assay with a CREB response element, we detected a stronger binding in t-Darpp-expressing cells. The t-Darpp expression led to an increase in expression and phosphorylation of CREB and ATF-1 transcription factors that were required for up-regulating Bcl2 levels. Indeed, knockdown of Akt, CREB, or ATF1 in t-Darpp-expressing cells reduced Bcl2 protein levels. In conclusion, the t-Darpp/Akt axis underscores a novel oncogenic potential of t-Darpp in gastric carcinogenesis and resistance to drug-induced apoptosis.

Topics: Activating Transcription Factor 1; Carcinoma; Caspase 3; Caspase 9; Cell Survival; Cyclic AMP Response Element-Binding Protein; Cytochromes c; DNA-Binding Proteins; Dopamine and cAMP-Regulated Phosphoprotein 32; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Membrane Potential, Mitochondrial; Nuclear Proteins; Oncogene Protein v-akt; Phosphorylation; Protein Isoforms; Regulatory Factor X Transcription Factors; RNA, Messenger; Signal Transduction; Stomach Neoplasms; Transcription Factors; Tumor Cells, Cultured; Up-Regulation

Upper gastrointestinal adenocarcinomas are a common cause of cancer-related deaths. In this study, the authors investigated the prevalence and biological significance of Aurora Kinase A (AURKA) overexpression in upper gastrointestinal adenocarcinomas.. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining on tumor tissue microarrays (TMA) were used to study the expression of AURKA in upper gastrointestinal adenocarcinomas. To investigate the biological and signaling impact of AURKA, the authors used multiple in vitro assays that included 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling), cytochrome C release, flow cytometry, luciferase reporter, and Western blot analysis.. Frequent overexpression of AURKA transcript in upper gastrointestinal adenocarcinomas was detected compared with normal samples (47%; P= .001). The immunohistochemical analysis of 130 tumors demonstrated moderate-to-strong immunostaining of AURKA in >50% of upper gastrointestinal adenocarcinomas. By using camptothecin as a drug-induced apoptosis in vitro model, the authors demonstrated that the expression of AURKA provided protection against apoptosis to gastrointestinal cancer cells (AGS and RKO) (P= .006) and RIE-1 primary intestinal epithelial cells (P= .001). The AURKA overexpression mediated an increase in phosphorylation of AKT(Ser473) with an increase in HDM2 level. The shRNA-knockdown of AKT in AURKA-overexpressing cells reversed this effect and showed a significant increase in the p53 protein level, indicating a possible nexus of AURKA/AKT/p53. Indeed, overexpression of AURKA led to a remarkable reduction in the transcription activity of p53, with subsequent reductions in transcript and protein levels of its downstream proapoptotic transcription targets (p21, BAX, NOXA, and PUMA).. Study results indicated that AURKA provides potent antiapoptotic properties to gastrointestinal cells by regulating levels of p53 through the AKT/HDM2 axis.

Topics: Adenocarcinoma; Apoptosis; Aurora Kinase A; Aurora Kinases; Biomarkers, Tumor; Blotting, Western; Camptothecin; Coloring Agents; Cytochromes c; Enzyme Inhibitors; Esophageal Neoplasms; Flow Cytometry; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Luciferases; Luminescent Agents; Polymerase Chain Reaction; Protein Array Analysis; Protein Serine-Threonine Kinases; Stomach Neoplasms; Tetrazolium Salts; Thiazoles; Tumor Suppressor Protein p53

Chemically synthesized sugar-cholestanols with mono-, di-, and tri-saccharides attached to cholestanol showed strong inhibiting activity against the proliferation of colorectal and gastric cancer cells. In contrast, cholestanol without sugar moieties was totally ineffective. Furthermore, when cancer cells were exposed to GlcNAcRbetacholestanol (R=(-) or beta1-3Gal), the compound was rapidly taken up via the lipid rafts/microdomains on the cell surface. The uptake of sugar-cholestanol in mitochondria increased gradually and was followed by the release of cytochrome c from mitochondria and the activation of apoptotic signals through the mitochondrial pathway and the caspase cascade, leading to apoptotic cell death, characterized by DNA ladder formation and nuclear fragmentation. Additionally, the examination of GlcNAcRbetacholestanol in a mouse model of peritoneal dissemination showed a dramatic reduction of tumor growth (P < 0.003) and prolonged mouse survival time (P<0.0001). Based on these observations, we believe that the sugar-cholestanols described here have clinical potential as novel anticancer agents.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cholestanols; Chromatography, Liquid; Colorectal Neoplasms; Cytochromes c; DNA Fragmentation; Female; HT29 Cells; Humans; Injections, Intraperitoneal; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Oligosaccharides; Stomach Neoplasms; Tandem Mass Spectrometry

Epidemiological and experimental carcinogenesis studies provide evidence that certain components of garlic have anti-cancer activity. Although the biotransformed garlic derivative S-allylmercapto-L-cysteine (SAMC) has been reported to show an inhibitory effect on tumorigenesis, the mechanisms are poorly understood. The present study investigated the effect of SAMC on the growth of human gastric cancer SNU-1 cells. Upon treatment with SAMC, a concentration-dependent inhibition of cell proliferation was observed and cells developed many of the hallmark features of apoptosis, including DNA fragmentation and an increase in the sub-diploid population. The anti-proliferative and apoptotic effect of SAMC was associated with the induction of Bax, p53, and caspase-9, rather than the induction of Bcl-2 and p21. Mitochondrial cytochrome c activation and an in vitro caspase-3 activity assay demonstrated that the activation of caspases accompanies the apoptotic effect of SAMC, which mediates cell death. These results suggest that the apoptotic effect of SAMC on gastric cancer SNU-1 cells may be connected with caspase-3 activation through the induction of Bax and p53, rather then Bcl-2 and p21.

Topics: Allium; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspases; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cysteine; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Garlic; Humans; Immunoblotting; In Situ Nick-End Labeling; Mitochondria; Oncogene Protein p21(ras); Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms; Tumor Suppressor Protein p53

Nanoscale hydroxyapatite (nano-HAP) has been reported to exhibit anti-cancer effect on several human cancers, but the molecular mechanism of which remains unclear. The aim of this study was to explore the mechanisms by investigating the effects of nano-HAP on human gastric cancer SGC-7901 cells. Our results showed that nano-HAP significantly reduced cell viability, and induced apoptosis in SGC-7901 cells characterized by hypodiploid DNA contents, morphological changes and DNA fragmentation. The increase in apoptosis was accompanied with the increased expression of Bax, a pro-apoptotic protein, and decreased expression of Bcl-2, an anti-apoptotic protein, the decrease of mitochondrial membrane potential and the release of cytochrome c from mitochondria into cytosol. Furthermore, the activation of caspases-3, and -9, but not activation of caspases-8 was induced by nano-HAP. Z-VAD-fmk, a universal caspase inhibitor, dose-dependently inhibited nano-HAP-induced apoptosis. This study demonstrates that nano-HAP inhibits the proliferation of SGC-7901 cells by inducing apoptosis, and the apoptotic pathway of nano-HAP-induced apoptosis is mediated through the mitochondrial-dependent and caspase-dependent pathway.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Durapatite; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Nanoparticles; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms; Time Factors

Solanum nigrum L. (SN) has been used in traditional folk medicine to treat different cancers. It is also used as a hepatoprotective and anti-inflammatory agent. In this study, we demonstrated that the extract of SN (SNE) induced a strong cytotoxic effect toward HepG2 cells but much less to Chang liver and WRL-68 cells. The mechanisms of the cytotoxic effect were concentration-dependent. High doses of SNE (2 and 5 mg/mL) induced apoptotic cell death in HepG2 cells, as evidenced by increases in the expressions of p-JNK and Bax, mitochodrial release of cytochrome c, and caspase activation. On the other hand, cells treated with low concentrations of SNE (50-1000 microg/mL) revealed morphological and ultrastructural changes of autophagocytic death under electron microscopic observation. Furthermore, these cells showed increased levels of autophagic vacuoles and LC3-I and LC3-II proteins, specific markers of autophagy. The levels of Bcl-2 and Akt that have been implicated in the down-regulation of autophagy were decreased upon SNE treatment. Taken together, these findings indicate that SNE induced cell death in hepatoma cells via two distinct antineoplastic activities of SNE, the ability to induce apoptosis and autophagocytosis, therefore suggesting that it may provide leverage to treat liver cancer.

Topics: Apoptosis; Autophagy; Caspase 3; Cell Line; Cell Line, Tumor; Cytochromes c; Fetus; Humans; Liver; Liver Neoplasms; Plant Extracts; Solanum nigrum; Stomach Neoplasms

The purpose of this study was to investigate the anticancer effect of aloe-emodin, an anthraquinone compound present in the leaves of Aloe vera, on two distinct human gastric carcinoma cell lines, AGS and NCI-N87. We demonstrate that aloe-emodin induced cell death in a dose- and time-dependent manner. Noteworthy is that the AGS cells were generally more sensitive than the NCI-N87 cells. Aloe-emodin caused the release of apoptosis-inducing factor and cytochrome c from mitochondria, followed by the activation of caspase-3, leading to nuclear shrinkage and apoptosis. In addition, exposure to aloe-emodin suppressed the casein kinase II activity in a time-dependent manner and was accompanied by a reduced phosphorylation of Bid, a downstream substrate of casein kinase II and a pro-apoptotic molecule. These preclinical studies suggest that aloe-emodin represents a suitable and novel chemotherapeutic drug candidate for the treatment of human gastric carcinoma.

Topics: Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Casein Kinase II; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Humans; Phosphorylation; Stomach Neoplasms; Time Factors

Midkine (MK), a heparin-binding growth factor, is expressed highly in various malignant tumors, so it acts as attractive therapeutic target. In the present study, we used siRNA targeting MK to downregulate human MK expression in human gastric cancer cell line BGC823 and SGC7901 so as to determine the advantages of this anticancer therapeutic. The cell proliferation was evaluated by a WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate sodium salt) assay and colony formation assay. Apoptosis was determined by flow cytometer analysis and colorimetric assay. Our results showed that the BGC823 and SGC7901 cell growth were significantly inhibited by knockdown of MK gene. The loss of mitochondrial membrane potential, release of cytochrome c from the mitochondria into cytosol and increased activity of caspase-3, 8 and 9 occurred concomitantly with inhibition of MK gene. These results indicated that siRNA targeting MK gene can inhibit gastric cancer cells growth and induce apoptosis via mitochondrial depolarization and caspase-3 activation. MK siRNA may be a promising novel and potential therapeutic strategy for the treatment of gastric cancers.

Topics: Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Proliferation; Cytochromes c; Cytokines; Down-Regulation; Genes, bcl-2; Humans; Membrane Potential, Mitochondrial; Midkine; Mitochondria; RNA Interference; RNA, Messenger; RNA, Small Interfering; Stomach Neoplasms; Tumor Cells, Cultured

Pterostilbene, an active constituent of blueberries, is known to possess anti-inflammatory activity and also induces apoptosis in various types of cancer cells. Here, the effects of pterostilbene on cell viability in human gastric carcinoma AGS cells were investigated. This study demonstrated that pterostilbene was able to inhibit cell proliferation and induce apoptosis in a concentration- and time-dependent manner. Pterostilbene-induced cell death was characterized with changes in nuclear morphology, DNA fragmentation, and cell morphology. The molecular mechanism of pterostilbene-induced apoptosis was also investigated. The results show the caspase-2, -3, -8, and -9 are all activated by pterostilbene, together with cleavage of the downstream caspase-3 target DNA fragmentation factor (DFF-45) and poly(ADP-riobse) polymerase. Moreover, the results indicate that the Bcl-family of proteins, the mitochondrial pathway, and activation of the caspase cascade are responsible for pterostilbene-induced apoptosis. Pterostilbene markedly enhanced the expression of growth arrest DNA damage-inducible gene 45 and 153 (GADD45 and GADD153) in a time-dependent manner. Flow cytometric analysis indicated that pterostilbene blocked cell cycle progression at G1 phase in a dose- and time-dependent manner. Pterostilbene increased the p53, p21, p27, and p16 proteins and decreased levels of cyclin A, cyclin E, cyclin-dependent kinase 2 (Cdk2), Cdk4, and Cdk6, but the expression of cyclin D1 was not affected. Over a 24 h exposure to pterostilbene, the degree of phosphorylation of Rb was decreased after 6 h. In summary, pterostilbene induced apoptosis in AGS cells through activating the caspase cascade via the mitochondrial and Fas/FasL pathway, GADD expression, and by modifying cell cycle progress and changes in several cycle-regulating proteins. The induction of apoptosis by pterostilbene may provide a pivotal mechanism of the antitumor effects and for treatment of human gastric cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blueberry Plants; Cell Cycle; Cell Division; Cell Line, Tumor; Cytochromes c; Humans; Male; Mitochondria; Stilbenes; Stomach Neoplasms

To investigate the effect of omega-3 polyunsaturated fatty acids(omega-3PUFAs) on the apoptosis of human gastric cancer cell line SGC-7901 and to explore the potential mechanisms.. Cells were treated with eicosapentaenoic acid (20:5 omega-3,EPA) or docosahexaenoic acid (22:6 omega-3, DHA) at concentrations of 10, 20 and 40 microg/ml. Cell growth and apoptosis were analyzed with MTT assay, cell morphology, DNA electrophoresis and flow cytometry. Mitochondrial membrane potential ( triangle right psi mt) was measured by fluorescent probe rhodamine 123. The distribution of cytochrome C in mitochondria and cytosol was determined by enzyme-linked immunoadsorbent assay. The composition of mitochondrial membrane phospholipid(MMP)was examined by gas chromatography.. Both EPA and DHA markedly inhibited the SGC-7901 cell growth and induced apoptosis in a time- and dose-dependent manner. After incubation of the cells with 40 microg/ml EPA or DHA for 24 hours, the level of Deltapsimt siginificantly decreased (P<0.001), and cytochrome C largely released into cytosol from mitochondria. The proportions of EPA and DHA in MMP rapidly elevated while that of arachidonic acid sharply decreased.. omega-3PUFAs inhibit the growth of gastric cancer cells through promoting apoptosis. Compositional and functional alterations in mitochondrial membrane may be an important initiator of apoptosis induced by omega-3PUFAs.

Topics: Apoptosis; Cell Line, Tumor; Cytochromes c; Fatty Acids, Omega-3; Humans; Membrane Potential, Mitochondrial; Mitochondria; Stomach Neoplasms

Cellular prion protein (PrP(C)), a glycosylphosphatidylinositol-anchored membrane protein, was found in our lab to be widely expressed in gastric cancer cell lines. In order to evaluate its biological significance in human gastric cancer, we investigated its expression in a large series of gastric tissue samples (n = 124) by immuno histochemical staining with the monoclonal antibody 3F4. Compared with normal tissues, gastric adenocarcinoma showed increased PrP(C) expression, correlated with the histopathological differentiation (according to the WHO and Lauren classifications) and tumor progression (as documented by pTNM staging). To better understand the underlying mechanism, we introduced the PrP(C) and two pairs of RNAi into the poorly differentiated gastric cancer cell line AGS and found that PrP(C) suppressed ROS and slowed down apoptosis in transfected cells. Further study proved that the apoptosis-related protein Bcl-2 was upregulated whereas p53 and Bax were downregulated in the PrP(C)-transfected cells. A reverse effect was observed in PrP(C) siRNA-transfected cells. These results strongly suggested that PrP(C) might play a role as an effective antiapoptotic protein through Bcl-2-dependent apoptotic pathways in gastric cancer cells. Further study into the mechanism of these relationships might enrich the knowledge of PrP, better our understanding of the nature of gastric carcinoma, and further develop possible strategies to block or reverse the development of gastric carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; bcl-2-Associated X Protein; Case-Control Studies; Cell Line, Tumor; Cytochromes c; DNA, Complementary; DNA, Neoplasm; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; PrPC Proteins; Reactive Oxygen Species; RNA, Small Interfering; Stomach Neoplasms; Transfection; Tumor Suppressor Protein p53

Inhibition of cyclooxygenase-2 has been proposed to be a potential mechanism for the chemoprevention of gastrointestinal tumors by nonsteroidal anti-inflammatory drugs. This study investigates the mechanisms by which the cyclooxygenase-2 inhibitor SC236 induces apoptosis of gastric cancer cell lines and its downstream signaling pathway.. Two gastric cancer cell lines, AGS and MKN28, were treated with SC236 and assessed for cell growth and apoptosis. The involvement of mitogen-activated protein kinase and Akt kinase/protein kinase B (Akt/PKB) pathways and their downstream signalings were studied in the AGS cell line.. SC236 treatment induced apoptosis in gastric cancer cells and caused activation of p38 and stress-activated protein kinase/jun kinase, but down-regulated Akt/PKB. The specific p38 inhibitor SB203580 and the dominant-negative stress-activated protein kinase/jun kinase both failed, while the constitutively active form of Akt/PKB was able to block SC236-induced apoptosis. SC236-induced apoptosis was coupled with release of cytochrome c and activation of caspases.. One of the pathways involved in SC-236-induced apoptosis in gastric cancer cells is through downregulation of Akt and then release of cytochrome c.

Topics: Acridine Orange; Adenocarcinoma; Apoptosis; Blotting, Western; Caspases; Cyclooxygenase 2 Inhibitors; Cytochromes c; Down-Regulation; Enzyme Activation; Humans; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stomach Neoplasms; Sulfonamides; Transfection; Tumor Cells, Cultured

FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.

Topics: Apoptosis; Caspase 3; Caspase Inhibitors; Cell Survival; Colforsin; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Stomach Neoplasms; Time Factors

Aspirin-induced apoptosis is one of the important mechanisms for its antitumour effect against gastric cancer. We aimed at investigating the involvement of bcl-2 family members in the apoptotic pathway in gastric cancer. Gastric cancer cell line AGS and MKN-45 were observed as to cell growth inhibition and induction of apoptosis in response to treatment with aspirin. Cell proliferation was measured by MTT assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole staining. Protein expression was determined by western blotting. We showed that aspirin activated caspase-8, caspase-9 and capase-3, cleaved and translocated Bid, induced a conformational change in and translocation of Bax and cytochrome c release. In addition, suppression of caspase-8 with the specific inhibitor z-IETD-fmk, as well as the pan-caspase inhibitor z-VAD-fmk, prevented Bid cleavage and subsequent apoptosis. The caspase inhibitors failed to abolish the effects on Bax activation. In conclusion, our results identify a role of caspase-8/Bid and activation of Bax as a novel mechanism for aspirin-induced apoptosis in gastric cancer.

Topics: Apoptosis; Aspirin; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase 8; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Humans; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.

Topics: Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Diterpenes; DNA; DNA Fragmentation; Enzyme Activation; Flavoproteins; Glutathione; Humans; Membrane Potentials; Membrane Proteins; Mitochondria; Necrosis; Oxidants; Oxidative Stress; Plant Extracts; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Pteris; Reactive Oxygen Species; Stomach Neoplasms; Tumor Suppressor Protein p53; Up-Regulation

We have previously reported that an ethanol extract of the dried ripe fruit of Vitex agnus-castus (Vitex) displays cytotoxic activity against certain kinds of human cancer cell line resulting in the induction of apoptosis. In this paper, we investigate the molecular mechanism of apoptosis induced by Vitex using a human gastric signet ring carcinoma cell line, KATO-III. DNA fragmentation was observed in Vitex-treated KATO-III cells in a time- and dose-dependent manner. DNA fragmentation was accompanied by the following phenomena: elevation in the level of hemeoxygenase-1 protein and thioredoxin reductase mRNA; repression of Mn-superoxide dismutase and catalase mRNAs; release of cytochrome c from mitochondria into the cytosol; activation of caspases-8, -9 and -3; decrease in the level of Bcl-2, Bcl-XL and Bid protein; increase in the level of Bad protein. The intracellular oxidized state, measured using 2',7'-dichlorofluorescin diacetate, increased after Vitex treatment. While the amount of intracellular GSH decreased significantly after treatment with Vitex, the level of GSSG was unaffected. Furthermore, no significant perturbation in the amount of proteins/mRNAs related to glutathione metabolism could be detected. These apoptotic alterations induced by exposure to Vitex were blocked by the presence of an anti-oxidative reagent, N-acetyl-l-cysteine, or the addition of exogenous GSH. Our results demonstrate that intracellular oxidative stress and mitochondrial membrane damage is responsible for Vitex-induced apoptosis, which may be mediated by a diminution of reduced type glutathione within the cell.

Topics: Acetylcysteine; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Signet Ring Cell; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Fruit; Glutathione; Humans; Mitochondria; Oxidative Stress; Plant Extracts; Stomach Neoplasms; Vitex

In order to investigate the roles of mitochondria in vitamin E succinate (VES)-induced apoptosis in human gastric cancer SGC-7901 cells.. Mitochondrial transmembrane potential (deltapsi(m)) was observed by confocal laser scanning microscopy, while the expression of cytochrome c in cytosol and caspase-3 was measured by western blotting after the cells were treated with VES at 5, 10, 20 microg/ml.. VES obviously decreased deltapsi(m) with dose- and time-dependent relationship, increased the expression of cytochrome c in cytosol and caspase-3 and activated caspase-3 as well.. VES-induced apoptosis in SGC-7901 cells might involve mitochondrial permeability transition, release of cytochrome c and activation of caspase-3 downstream.

Topics: Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Stomach Neoplasms; Vitamin E

Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear.. We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests.. Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006).. Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cardiolipins; Caspases; Coenzyme A Ligases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Female; Flavoproteins; Gene Transfer Techniques; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Neoplasms, Experimental; Proteins; RNA, Small Interfering; Sequence Analysis, DNA; Stomach Neoplasms; Transfection; Transplantation, Heterologous; Triazenes; Tumor Suppressor Protein p53

To investigate the underlying mechanism of apoptosis-inducing effect of a specific COX-2 inhibitor SC236 in gastric cancer cells.. Western blot analysis was used to measure apoptosis-related proteins, cytochrome c, and caspase-3. The catalytic activity of the caspases was measured using a colorimetric assay.. Treatment of AGS gastric cancer cells with SC236 caused a significant elevation of the pro-apoptotic protein Bak, release of cytochrome c to the cytosol, and activation of caspase-3. A specific caspase-3 inhibitor, z-DEVD-fmk, blocked SC236-induced apoptosis.. SC236 inhibits cell growth and induces apoptosis in gastric cancer cells at least partly through the up-regulation of Bak, stimulation of cytochrome c release, and activation of caspase-3.

Topics: Adenocarcinoma; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Caspase 3; Caspases; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Cytochromes c; Humans; Oligopeptides; Poly(ADP-ribose) Polymerases; Pyrazoles; Stomach Neoplasms; Sulfonamides

To date, two major apoptotic pathways, the death receptor and the mitochondrial pathway, have been well documented in mammalian cells. However, the involvement of these two apoptotic pathways, particularly the death receptor pathway, in transforming growth factor-beta 1 (TGF-beta 1)-induced apoptosis is not well understood. Herein, we report that apoptosis of human gastric SNU-620 carcinoma cells induced by TGF-beta 1 is caused by the Fas death pathway in a Fas ligand-independent manner, and that the Fas death pathway activated by TGF-beta 1 is linked to the mitochondrial apoptotic pathway via Bid mediation. We showed that TGF-beta 1 induced the expression and activation of Fas and the subsequent caspase-8-mediated Bid cleavage. Interestingly, expression of dominant negative FADD and treatment with caspase-8 inhibitor efficiently prevented TGF-beta 1-induced apoptosis, whereas the treatment with an activating CH11 or a neutralizing ZB4 anti-Fas antibody, recombinant Fas ligand, or Fas-Fc chimera did not affect activation of Fas and the subsequent induction of apoptosis by TGF-beta 1. We further demonstrated that TGF-beta 1 also activates the mitochondrial pathway showing Bid-mediated loss of mitochondrial membrane potential and subsequent cytochrome c release associated with the activations of caspase-9 and the effector caspases. Moreover, all these apoptotic events induced by TGF-beta 1 were found to be effectively inhibited by Smad3 knockdown and also completely abrogated by Smad7 expression, suggesting the involvement of the Smad3 pathway upstream of the Fas death pathway by TGF-beta 1.

Topics: Apoptosis; Arabidopsis Proteins; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Carrier Proteins; Caspase Inhibitors; Caspases; Cytochromes c; DNA-Binding Proteins; Enzyme Inhibitors; fas Receptor; Fatty Acid Desaturases; Flow Cytometry; Humans; Jurkat Cells; Mitochondria; Smad3 Protein; Stomach Neoplasms; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Products of the arachidonic acid metabolizing enzyme, 5-lipoxygenase (5-LOX), stimulate the growth of several cancer types. Inhibitors of 5-LOX and 5-LOX-activating protein (FLAP) induce apoptosis in some cancer cells. Here, the authors investigated the effect of a FLAP inhibitor, MK-886, on the inhibition of proliferation and induction of apoptosis in gastric cancer.. Cell proliferation in gastric cancer cells was measured using an 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was measured using acridine orange staining and flow cytometry. Protein expression of apoptosis-related genes p53, p21waf1, p27kip1, bcl-2 families, cytochrome c, and the caspases were examined using Western blotting. Caspase-3 activity was measured using colorimetric assay of substrate cleavage.. MK-886 inhibited cell growth in a dose- and time-dependent manner. Apoptosis was induced in gastric cancer cells and was characterized by upregulation of p27kip1 and bax, with release of cytochrome c from mitochondria into cytosol, which initiated caspase-3 activation. Specific caspase-3 inhibitors partially blocked MK-886-induced apoptosis.. The present results suggest that MK-886 induces apoptosis in gastric cancer cells through upregulation of p27kip1 and bax, and that MK-886 is a potentially useful drug in gastric cancer prevention and therapy.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Flow Cytometry; Humans; Indoles; Lipoxygenase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

The fragile histidine triad (FHIT) tumor suppressor gene incorporates the common human chromosomal fragile site at 3p14.2. The structure and expression of the FHIT gene are frequently altered in many cancers. The tumor suppressor activity of the FHIT gene has been previously demonstrated as potentially involving apoptotic induction. Here, mitochondria are implicated as being involved in the apoptotic activity of the FHIT gene. A number of morphological and biochemical events, including the disruption of the inner mitochondrial transmembrane potential (Delta Psi(m)) and the release of apoptogenic cytochrome c protein into the cytoplasm, are characteristic features of the apoptotic program. The proapoptotic activity of the FHIT gene is studied by investigating the loss of Delta Psi(m) in mitochondria and translocation of cytochrome c. Synchronous luminescence (SL) spectroscopy is applied to measure mitochondrial incorporation of rhodamine 123 for direct analysis of alterations in the mitochondrial Delta Psi(m). The SL methodology is based on synchronous excitation in which the excitation and emission wavelengths are scanned simultaneously while a constant wavelength interval is maintained between the excitation and emission monochromators. An enhanced collapse of Delta Psi(m) in apoptotically induced FHIT expressing cells compared to FHIT negative cells is observed. The loss of Delta Psi(m) is greatly restricted in the presence of the apoptotic inhibitor, cyclosporin A. Cytoplasmic translocation of cytochrome c in the FHIT expressing cells as an early event in apoptosis is also demonstrated. It is concluded that Fhit protein expression maintained apoptotic function by altering the Delta Psi(m) and by enhancing cytochrome c efflux from the mitochondria.

Topics: Acid Anhydride Hydrolases; Apoptosis; Carcinoma; Cell Division; Cell Line, Tumor; Chromosomes, Human, Pair 3; Cyclosporine; Cytochromes c; Cytoplasm; Enzyme Inhibitors; Fluorescent Dyes; Genes, Tumor Suppressor; Humans; Luminescent Measurements; Membrane Potentials; Mitochondria; Neoplasm Proteins; Rhodamine 123; Spectrum Analysis; Stomach Neoplasms; Time Factors

Staurosporine (STS), a non- specific protein kinase inhibitor, can extensively induce cell apoptosis. Orphan receptor TR3, an immediate-early response gene, belongs to the steroid/thyroid receptor superfamily. After stimulated by apoptosis-inducing agents, TR3 is expressed rapidly and translocates from nuclei to mitochondria,which finally induces cell apoptosis. This study was to investigate the mechanism by which STS induces apoptosis of gastric cancer cell line BGC-823,and its correlation with translocation of TR3 in BGC-823 cells.. Fluorescent microscopy was used to observe apoptotic morphology of BGC-823 cells after treated with STS for 3 and 24 h and stained by DAPI, and determine cell apoptotic rate. Laser scan confocal microscopy and Western blot were performed to investigate translocation of orphan receptor TR3 and the release of cytochrome c. Flow cytometry and confocal microscopy were used to examine mitochondrial membrane potential.. After treated for 3 h,and 24 h,apoptosis rates of BGC-823 cells in STS-treatment groups were higher than those of cells in control groups (15.7% vs. 4.0%, and 39.4% vs. 4.7%). After BGC-823 cells exposed to STS for 3 h,TR3 translocated from nuclei to mitochondria, mitochondrial membrane potential was decreased, and cytochrome c released from mitochondria to the cytoplasm.. TR3, in response to STS, translocates from nuclei to cytoplasm, where it targets to mitochondria to induce cytochrome c release, finally results in apoptosis of gastric cancer cells.

Topics: Active Transport, Cell Nucleus; Apoptosis; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cytochromes c; Enzyme Inhibitors; Humans; Membrane Potentials; Mitochondria; Nuclear Receptor Subfamily 4, Group A, Member 1; Receptors, Steroid; Receptors, Thyroid Hormone; Staurosporine; Stomach Neoplasms

The Fas-Fas ligand system is one of the factors involved in cell death signaling. Aberrations in the signaling pathways leading to Fas-mediated apoptosis in tumor cells have been reported in a variety of human malignant tumors. However, the Fas-mediated apoptotic pathway has not been sufficiently elucidated in human gastric carcinomas. We examined the apoptotic pathway induced by anti-Fas antibody using seven human gastric carcinoma cell lines. Apoptosis was induced in a delayed fashion and the apoptotic indices (AI) after 48 h were approximately 30-40% in MKN-45 and KATO-III cells, which both showed cleavage of the Bid protein and release of Cytochrome c from the mitochondria. Our data also demonstrated no significant relationship between the expressions of various apoptosis-related proteins and the sensitivity or resistance to anti-Fas antibody-induced apoptosis, as far as we examined. Furthermore, the apoptosis signal was inhibited by treatment with Caspase-9 and -3 inhibitors in MKN-45 and KATO-III. These findings suggest that anti-Fas antibody induced apoptosis through the type II signaling pathway in the human gastric carcinoma cell lines, MKN-45 and KATO-III.

Topics: Apoptosis; Blotting, Western; Carcinoma; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Line; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Inhibitors; fas Receptor; Humans; Signal Transduction; Stomach Neoplasms; Time Factors

Flavopiridol is a cyclin-dependent kinase inhibitor currently under development by the National Cancer Institute both as a single agent and in combination with chemotherapy. There have been numerous reports that flavopiridol potently enhances the induction of apoptosis by chemotherapy. However, the effect of flavopiridol on radiotherapy (RT)-induced apoptosis has been largely untested. RT has become the cornerstone of adjuvant treatment of colorectal and gastric cancer. In view of this, we elected to evaluate the effect of flavopiridol on potentiating RT-induced apoptosis in the human colon cancer cell line HCT-116 and the gastric cancer cell line MKN-74.. The efficacy of combination of gamma-irradiation and flavopiridol was tested in vitro in MKN-74 and HCT-116 cells and correlated to changes in p21 expression. HCT-116 cells were also established as tumors in nude mice and treated with gamma-irradiation and flavopiridol either as single agents or in sequential combinations such that flavopiridol was either given 7 h before, concomitantly, or 3 and 7 h after gamma-irradiation.. Flavopiridol significantly enhanced the induction of apoptosis by gamma-irradiation in both cell lines as measured by quantitative fluorescent microscopy, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release. To achieve the best effect, it was important to expose the tumor cells to gamma-irradiation before the flavopiridol. This sequence dependence was confirmed in vivo. When gamma-irradiation was administered 7 h before flavopiridol, 42% of the tumor-bearing animals were rendered disease free, compared with no animals treated with either gamma-irradiation or flavopiridol alone. Examination of the p21 status of HCT-116 and MKN-74 cells, after treatment with sequential gamma-irradiation and flavopiridol, indicated a loss of p21 protein expression. Loss of p21 was mainly due to cleavage by caspases. HCT-116 cells that lack p21 (p21(-/-)) also exhibited sensitization to gamma-irradiation and showed an even greater enhancement of gamma-irradiation-induced apoptosis by flavopiridol when compared with the parental HCT-116 cells.. These studies indicate that gamma-irradiation followed by flavopiridol enhances apoptosis and yields significantly increased tumor regressions and cures that are not achievable with radiation alone. These results indicate that flavopiridol can potently enhance the effect of gamma-radiation both in vitro and in vivo and may provide a new means to treat patients with locally advanced gastrointestinal cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gamma Rays; Male; Mice; Mice, Nude; Microscopy, Fluorescence; Piperidines; Poly(ADP-ribose) Polymerases; Stomach Neoplasms; Transplantation, Heterologous; Tumor Cells, Cultured