leupeptins and Stomach-Neoplasms

Topics: Animals; Antineoplastic Agents; Antioxidants; Carcinogens; Dactinomycin; Leupeptins; Mice; Neoplasms, Experimental; Protease Inhibitors; Rabbits; Skin Neoplasms; Stomach Neoplasms

EBV-associated gastric carcinoma (EBVaGC) is a specific subgroup of gastric carcinoma, and the multifunctional transcriptional factor NF-κB may contribute to its tumorigenesis. In this study, we comprehensively characterized NF-κB signaling in EBVaGC using qRT-PCR, western blot, immunofluorescence assays, ELISA, and immunohistochemistry staining. NF-κB-signaling inhibitors may inhibit the growth of EBVaGC cells and induce significant apoptosis. IκBα is a key regulatory molecule, and repression of IκBα can contribute to aberrant NF-κB activation. Overexpression of LMP1 and LMP2A in the EBV-negative GC cell line SGC7901 could inhibit the expression of IκBα and induce NF-κB activation. These findings indicate that the canonical NF-κB signal is constitutively activated and plays an important role in EBVaGC tumorigenesis.

Topics: Carcinogenesis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Epstein-Barr Virus Infections; Epstein-Barr Virus Nuclear Antigens; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Humans; Leupeptins; NF-kappa B; NF-KappaB Inhibitor alpha; Nitriles; RNA, Small Interfering; Signal Transduction; Stomach Neoplasms; Sulfones; TNF Receptor-Associated Factor 1; Viral Matrix Proteins

Here, we found that both SAHA and MG132 synergistically inhibited proliferation, glycolysis and mitochondrial oxidization, induced cell cycle arrest and apoptosis in MGC-803 and MKN28 cells. SAHA increased cell migration and invasionat a low concentration. SAHA induced the overexpression of acetyl histone 3 and 4, which were recruited to p21, p27, Cyclin D1, c-myc and nanog promoters to transcriptionally up-regulate the former two and down-regulate the latter three. The expression of acetyl-histone 3 and 4 was increased during gastric carcinogenesis and positively correlated with cancer differentiation. SAHA and MG132 exposure suppressed tumor growth by inhibiting proliferation and inducing apoptosis in nude mice, increased serum ALT and AST levels and decreased hemaglobin level, white blood cell and neutrophil numbers. These data indicated that SAHA and MG132 in vivo and vitro synergistically induced cytotoxicity and apoptosis, suppressed proliferation, growth, migration and invasion of gastric cancer cells. Therefore, they might potentially be employed as chemotherapeutic agents if the hepatic injury and the killing effects of peripheral blood cells are avoided or ameliorated.

Topics: Adult; Aged; Aged, 80 and over; Alanine Transaminase; Animals; Antineoplastic Agents; Apoptosis; Aspartate Aminotransferases; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neutrophils; Oxygen Consumption; Phenotype; Stomach Neoplasms; Vorinostat; Young Adult

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased β-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of β-catenin induced by 1. The 1-induced degradation of β-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of β-catenin, which resulted in the inhibition of TCF/β-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

Topics: Abietanes; Adenocarcinoma; Apoptosis; Benzothiazoles; beta Catenin; Down-Regulation; Humans; Leupeptins; Molecular Structure; Receptors, TNF-Related Apoptosis-Inducing Ligand; Scoparia; Stomach Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Toluene; Tumor Necrosis Factor-alpha

To investigate the effect of RhoA to VEGF, HIF-1alpha and MVD (microvascular density) and the effect of MG132 to RhoA.. The constitutively-active mutant vectors of RhoA (pCEFL-GST-V14RhoA) were transfected into gastric cancer cell line MKN-45 by Lipofectamine 2000, single clones were selected by G418 and identified with western blot. The content of VEGF in the conditioned media was detected by ELISA. Constitutively-active RhoA nude mice models were established and treated with MG132. The effect of RhoA and MG132 on expression of HIF-1alpha, VEGF and CD31 were detected by immunohistochemistry.. Cell line of stable-transfected constitutively-active RhoA was established and constitutively-active RhoA could stimulate secretion of VEGF but MG132 inhibited that. Constitutively-active RhoA could obviously induce growth of tumor (P < 0.05), but MG132 inhibited it (P < 0.05). Constitutively-active RhoA could promote protein of HIF-1alpha, VEGF and CD31 but MG132 inhibited the function of RhoA (P < 0.05).. Our studies indicates that MG132 could affect angiogenesis of tumors through inhibition the regulating function of RhoA on HIF-1alpha, VEGF and CD31.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Proteasome Inhibitors; rhoA GTP-Binding Protein; Stomach Neoplasms; Transfection; Vascular Endothelial Growth Factor A

The ubiquitin-proteasome system and macroautophagy are two complementary pathways for protein degradation. Emerging evidence suggests that proteasome inhibition might be a promising approach for the treatment of cancer. In this study, we show that proteasome inhibitor MG-132 suppressed gastric cancer cell proliferation and induced macroautophagy. The induction of macroautophagy was evidenced by the formation of LC3(+) autophagosomes and the accumulation of acidic vesicular organelles and autolysosomes and was accompanied by the suppression of mammalian target of rapamycin complex 1 activity. Abolition of macroautophagy by knockdown of Class III phosphatidylinositol-3 kinase Vps34 or ATG5/7 sensitized gastric cancer cells to the antiproliferative effect of MG-132 by promoting G(2)/M cell cycle arrest. In addition, MG-132 increased ERK phosphorylation whose inhibition by MEK inhibitor significantly enhanced the antiproliferative effect of proteasome inhibition. To conclude, this study demonstrates that macroautophagy and ERK phosphorylation serve as protective mechanisms to counteract the antiproliferative effect of proteasome inhibition. This discovery may have implications for the application of proteasome-directed therapy for the treatment of cancer.

Topics: Animals; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Proteinase Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Lysosomes; Microtubule-Associated Proteins; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Serine-Threonine Kinases; RNA, Small Interfering; Signal Transduction; Stomach Neoplasms; TOR Serine-Threonine Kinases; Vacuoles

The ubiquitously expressed serine-threonine kinase Akt and the transcription factor NF-kappaB both are involved in cell proliferation and apoptosis. Furthermore, the activation of Akt or NF-kappaB has been suggested to associate with chemo-resistance of human tumors. The exact mechanism and interreaction of Akt and NF-kappaB pathway on chemoresistance in gastric cancer is still unknown. We explored the function of Akt and NF-kappaB pathway on chemoresistance in human gastric cancer cells. MTT method was used to analyze the influence of chemotherapeutics and the combined use of wortmannin or MG-132 on the growth of SGC-7901 cells. Apoptosis of SGC-7901 was detected by TUNEL and Annexin V/PI methods. The protein level of NF-kappaB was analyzed by immunocytochemical staining. EMSA was used to confirm the increased nuclear translocation of RelA. The protein level of p-Akt and p-IkappaBalpha were analyzed by Western blotting. Etoposide and doxorubicin suppressed the growth of SGC-7901 time and dose-dependently. Combined use of wortmannin or MG-132 can suppress growth further. Chemotherapeutics induced apoptosis of SGC-7901 and activated Akt and NF-kappaB, combined use of wortmannin or MG-132 induced apoptosis further and attenuated the activation of NF-kappaB. The combined use of wortmannin attenuated the activation of Akt, but combined use of MG-132 did not attenuate the activation of Akt. The activation of NF-kappaB is a branch mechanism of Akt anti-apoptosis effects. The chemotherapeutics induced apoptosis and induced the activation of Akt and NF-kappaB in SGC-7901 cell, suppression the activation of Akt or NF-kappaB can increase the effects of chemotherapeutics. NF-kappaB is a downstream target of Akt.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Cell Proliferation; DNA; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; I-kappa B Proteins; Leupeptins; NF-kappa B; NF-KappaB Inhibitor alpha; Oncogene Protein v-akt; Protein Binding; Stomach Neoplasms; Tumor Cells, Cultured; Wortmannin

Ubiquitin-proteasome pathway (UPP) is the major system for the selective degradation of cellular proteins that play key roles in cellular processes. Previous study indicated that ubiquitin-proteasome inhibitor MG-132 could inhibit growth of some carcinoma. However, anti-carcinoma mechanism of MG-132 is unclear. Our objective was to investigate mechanisms of growth inhibitory effect of MG-132 on gastric carcinoma cells. Gastric carcinoma cell SGC-7901 was treated with ubiquitin-proteasome inhibitor MG-132. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by (3)H-thymidine ((3)H-TdR) incorporation. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) PCR-ELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27kip1 and survivin was detected using the western blot method. After exposed to MG-132, the growth and value of (3)H-TdR incorporation of gastric carcinoma cells were obviously inhibited. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 microM of MG-132 for 24, 48, 72 and 96 h (P < 0.01). The percentage of cells at G(0)/G(1) phase was increased and that at S and G(2)/M phase was decreased (P < 0.01). The ratio of apoptotic cells treated with 5 microM MG-132 for 96 h was 53.7 +/- 6.4%. Agarose electrophoresis showed marked ladders. Moreover, expression of p27kip1 of cells was increased and expression of survivin was decreased. Our results suggest that MG-132 inhibits telomerase activity, induces apoptosis and G(1) arrest which is associated with upregulated p27kip1 expression and downregulated survivin expression in gastric carcinoma cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; DNA Replication; Down-Regulation; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Leupeptins; Microtubule-Associated Proteins; Stomach Neoplasms; Survivin; Telomerase; Tumor Cells, Cultured

Multidrug resistance (MDR) is a major impediment to the effective chemotherapy of many human malignancies. Although much effort has been devoted to develop new drugs for overcoming MDR, until now, still no useful method of reversing MDR, suitable for clinical use, has emerged from this large quantity of work. Some researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells. In the present study, we found that, in vincristine-resistant human gastric cancer cell line SGC7901/VCR, proteasome inhibitor MG132 was an effective inducer of apoptosis, and also had the capacity of downregulating the expression of anti-apoptotic Bcl-2 and MDR1 (P-gp), by which MG132 resensitized tumor cells to the apoptosis induced by anticancer drugs. Data presented by drug sensitivity assay further demonstrated that MG132 could reverse the resistant phenotype of gastric cancer cells effectively through both enhancing drug-induced apoptosis and inhibiting P-gp. The further study of the effectiveness and safety of proteasome inhibitor in vivo may be helpful for developing a new possible strategy to treat gastric cancer MDR.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cysteine Proteinase Inhibitors; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Leupeptins; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1alpha (HIF-1alpha) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1alpha protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1alpha protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl(2) treatment, both well known for induction of HIF-1alpha. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1alpha up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1alpha protein accumulation. Blockage of HIF-1alpha activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1alpha strongly inhibited their invasion ability, suggesting that elevation in HIF-1alpha protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1alpha-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1alpha to block HIF-1alpha activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1alpha-dependent up-regulation of PAI-1.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cobalt; Cycloheximide; Cysteine-Rich Protein 61; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Kinetics; Leupeptins; MAP Kinase Signaling System; Neoplasm Invasiveness; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Protein Biosynthesis; Protein Synthesis Inhibitors; Recombinant Proteins; RNA, Messenger; Stomach Neoplasms; Up-Regulation

Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21(Waf1/Cip1) mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21(Waf1/Cip1) induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.

Topics: Adenocarcinoma; Antineoplastic Agents; Bone Morphogenetic Protein 1; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Metalloendopeptidases; Neoplastic Stem Cells; Proteasome Inhibitors; RNA, Messenger; Signal Transduction; Smad6 Protein; Stomach Neoplasms

Phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (pChk2) induced by DNA double-strand breaks is required for inhibition of cell cycle progression in the G(2) phase. The purpose of the present paper was to investigate the expression of wild-type p53-induced phosphatase 1 (Wip1 or PPM1D), a negative regulator of Chk2, to better understand its role in human gastric cancer. In non-neoplastic gastric mucosa, most epithelial cells exhibited Wip1-positive and pChk2-negative immunoreactivity, whereas an inverse pattern of protein expression was detected at the surface of the foveolar epithelium. In tumor tissues, 74% of 53 gastric cancers had intense Wip1 immunoreactivity and close correlation with both tumor size (P = 0.0497) and Chk2 dephosphorylation (P = 0.0213). In MKN-74 gastric cancer cells, ionizing radiation (IR)-induced Wip1 upregulation was detected at protein levels, but the Chk2-mediated cell cycle regulatory mechanism was disrupted. In addition, protease inhibitor Z-Leu-Leu-Leu (ZLLL) effectively upregulated Wip1 levels in the presence or absence of IR, suggesting that Wip1 expression can be modulated post-transcriptionally. Understanding the Wip1-mediated signaling pathway in gastric cancer may provide useful information for the development of new chemo- and radiotherapies.

Topics: Adenocarcinoma; Cell Cycle; Cell Line, Tumor; Cell Survival; Checkpoint Kinase 2; Cysteine Proteinase Inhibitors; Enzyme Induction; Female; Gastric Mucosa; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Leupeptins; Male; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2C; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Radiation, Ionizing; Stomach Neoplasms; Tumor Suppressor Protein p53

To investigate the reversing effect of NF-kappaB inhibitor MG-132 on chemoresistance of gastric cancer cells to vincristine.. In vincristine-resistant human gastric cancer cells (SGC7901/VCR) and the parental sensitive clone (SGC7901), NF-kappaB-DNA binding activity was determined by electrophoreses mobility shift assay (EMSA). The inhibition level of kappaB (IkappaB-alpha) expression was measured by cellular-ELISA. Immunocytochemistry was used to detect the translocation of p65 and chemosensitivity of the cells was determined by MTT assay.. Compared with the parental SGC7901 cells, both the baseline and VCR-induced NF-kappaB-DNA binding activities in various concentrations were all higher in the SGC7901/VCR cells. Pretreatment with MG-132, the NF-kappaB inhibitor, for 30 minutes remarkably reduced the NF-kappaB activation, IkappaB-alpha degradation and nuclear translocation of p65. As to the SGC7901/VCR cells and the parental sensitive SGC7901 cells, the IC(50) values for VCR were 40.03 mg/L and 0.26 mg/L, respectively. MG-132 (2.5 micromol/L) significantly enhanced the toxicity of VCR in SGC7901/VCR cells and decreased the resistance index from 154.0 to 16.5. However, MG-132 did not show an obvious effect on the VCR sensitivity in sensitive SGC7901 cells.. Our data indicate that inhibition of NF-kappaB activation in gastric cancer cells may reverse the drug resistance to VCR in the cancer cells and increase the efficiency of chemotherapy.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Leupeptins; NF-kappa B; Stomach Neoplasms; Vincristine

To investigate the role of Epstein-Barr virus (EBV) in epithelial tumors, we compared the expression pattern of cellular genes in the EBV-infected gastric carcinoma cell line, NU-GC-3, and its uninfected control. Subtractive suppression hybridization (SSH) was combined with high-density DNA array screening to identify differentially expressed genes. We have discovered that EBV infection upregulated a truncated variant of human basic hair keratin 1 (hHb1-DeltaN), a gene that had previously been identified in metastatic breast carcinoma. We verified the differential expression of hHb1-DeltaN in 3 independent EBV-positive and -negative NU-GC-3 clones by Northern blotting. We further verified the EBV-dependent upregulation of hHb1-DeltaN in 3 other carcinoma cell lines (AGS, TWO3 and DLD1) by RT-PCR. Inhibition of CpG methylation by 5-Aza-CdR induced hHb1-DeltaN mRNA expression in the EBV-negative clones but did not alter the expression in the EBV-positive clones. The expression of hHb1-DeltaN protein was detectable by immunofluorescence and Western blotting in EBV-positive but not in EBV-negative NU-GC-3 clones after proteasome inhibitor (MG132) treatment. hHb1-DeltaN protein formed fibrous structures in the cytoplasm and accumulated in distinct nuclear bodies in the euchromatic areas of the cell nucleus. We suggest that the unstable hHb1-DeltaN protein may inhibit some of the functions of the keratin cytoskeleton and/or interfere with transcription regulation. It also may establish a link between EBV and the low differentiated or anaplastic status of the carcinomas that carry the virus.

Topics: Azacitidine; Blotting, Northern; Blotting, Western; CpG Islands; Cysteine Proteinase Inhibitors; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Humans; Keratins; Leupeptins; Oligonucleotide Array Sequence Analysis; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms; Tumor Cells, Cultured; Up-Regulation

The ubiquitin-proteasome pathway plays a critical role in the degradation of cellular proteins and cell cycle control. Dysregulating the degradation of such proteins should have profound effects on tumor growth and causes cells to undergo apoptosis. The aims of this study are to evaluate the ubiquitin-proteasome pathway in gastric cancer and the potential role of pharmacological inhibition of proteasome on induction of apoptosis in gastric cancer cells. Gastric cancer cell lines AGS (p53 wild-type) and MKN-28 (p53 mutant) were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in AGS and MKN-28 cells in a time- and dose-dependent manner. The inhibition of cell proliferation was caused by apoptosis which was also time- and dose-dependent. AGS cells were more responsive to MG132 than MKN-28 cells. Induction of apoptosis was preceded by the activation of caspase-3, as measured by a colorimetric caspase-3 cellular activity and Western blotting of the cleavage of caspase-3 and its substrate PARP. Activation of caspase-7 was also exhibited. In addition, z-VAD-fmk, a broad spectrum caspase inhibitor, reversed apoptosis induced by MG132 in AGS and MKN28 cells. Although z-DEVD-fmk, a specific caspase-3 inhibitor, suppressed MG132-induced apoptosis in MKN28 cells, it only partially rescued the apoptotic effect in AGS cells. Caspase-3 activation was the result of release of cytochrome c from mitochondria into the cytosol, as a consequence of upregulation of bax. There were overexpressions of all the proteasome-related proteins p53, p21(waf1) and p27(kip1) at 4 hr after proteasome inhibition which was identified by the accumulation of ubiquitin-tagged proteins. This was accompanied by accumulation of cells at G(1) phase. Our present study suggests that inhibition of proteasome function in gastric cancer cells induces apoptosis and proteasomal inhibitors have potential use as novel anticancer drugs in gastric cancer.

Topics: Adenocarcinoma; Apoptosis; Caspase 3; Caspase 7; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; Stomach Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ubiquitins; Up-Regulation