benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Stomach-Neoplasms

Curcumin, the biologically active compound from the rhizome of Curcuma longa, could inhibit cell growth and induce apoptosis in gastric carcinoma. However, the underlying mechanism of curcumin on gastric carcinoma cells still needs further investigation. In this study, morphological observation indicated that curcumin inhibited the proliferation of AGS cells in a dose-dependent manner. According to the flow cytometric analysis, curcumin treatment resulted in G2/M arrest in AGS cells, accompanied with an increased expression of cyclin B1 and a decreased expression of cyclin D1. In addition, DNA ladders were observed by gel electrophoresis. Meanwhile, the activities of caspase-3, -8, and -9 were also enhanced in curcumin-treated AGS cells. Nevertheless, the increased activities could be inhibited by benzyloxycarbonyl-Val-Ala-Asp (OME)-fluoromethylketone (z-VAD-fmk), which suggested that the apoptosis was caspase-dependent. Furthermore, downregulation of rat sarcoma (Ras) and upregulation of extracellular-signal-regulated kinase (ERK) were also observed in AGS cells treated with curcumin by Western blot. U0126, an ERK inhibitor, blocked curcumin-induced apoptosis. The results suggested that curcumin inhibited the growth of the AGS cells and induced apoptosis through the activation of Ras/ERK signaling pathway and downstream caspase cascade, and curcumin might be a potential target for the treatment of gastric carcinoma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspases; Cell Cycle; Cell Cycle Checkpoints; Curcuma; Curcumin; Extracellular Signal-Regulated MAP Kinases; Humans; Molecular Structure; Oligopeptides; Signal Transduction; Stomach Neoplasms

Thioridazine, an antipsychotic drug, has been reported to induce apoptosis in various types of cancer cells, with specificity on targeting cancer stem cells (CSCs). However, whether it elicits anticancer effects in gastric cancer has never been reported. In the present study, we examined the ability of thioridazine to induce cell death in the gastric cancer cell lines NCI-N87 and AGS, and detected its in vivo tumor inhibition capacity. Thioridazine elicited cytotoxic effects on NCI-N87 and AGS cells in a dose-dependent manner, and inhibited the colony formation abilitiy of the NCI-N87 and AGS cells. Thioridazine treatment induced nuclear fragmentation, increased the proportion of sub-G1 phase cells, and elevated the percentage of Annexin V-positive cells, suggesting the occurrence of apoptosis. Moreover, thioridazine induced gastric cancer cell apoptosis in a caspase-dependent manner, as shown by a decrease in the precursors of casapse-9, caspase-8 and caspase-3, and by the ability of the caspase inhibitor Z-VAD-FMK to reverse the cytotoxic effect of thioridazine. JC-1 staining further revealed that thioridazine induced gastric cancer cell apoptosis via the mitochondrial pathway. In addition, thioridazine pretreatment inhibited the growth of NCI-N87 cell-derived tumors. The present study demonstrated that the antipsychotic drug thioridazine possesses anti-gastric cancer ability through in vitro and in vivo experiments, suggesting thioridazine as a potential drug in gastric cancer therapy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Antipsychotic Agents; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Stomach Neoplasms; Thioridazine

Our results demonstrate that the addition of cisplatin after paclitaxel-induced mitotic arrest was more effective than individual treatment on gastric adenocarcinoma cells (MKN45). However, the treatment did not induce benefits in cells derived from lymph node metastasis (ST2957). Time-lapse microscopy revealed that cell death was caused by mitotic catastrophe and apoptosis induction, as the use of the caspase inhibitor z-VAD-fmk decreased cell death. We propose that the molecular mechanism mediating this cell fate is a slippage suffered by these cells, given that our Western blot (WB) analysis revealed premature cyclin B degradation. This resulted in the cell exiting from mitosis without undergoing DNA damage repair, as demonstrated by the strong phosphorylation of H2AX. A comet assay indicated that DNA repair was impaired, and Western blotting showed that the Chk2 protein was degraded after sequential treatment (paclitaxel-cisplatin). Based on these results, the modulation of cell death during mitosis may be an effective strategy for gastric cancer therapy.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Checkpoint Kinase 2; Cisplatin; Cyclin B; DNA Repair; Humans; Lymphatic Metastasis; Mitosis; Paclitaxel; Protein Serine-Threonine Kinases; Stomach Neoplasms; Taxoids

Human olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.. OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H2O2) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.. The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H2O2 or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H(2)O(2) or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).. Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H2O2 or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; G1 Phase; Gene Deletion; Granulocyte Colony-Stimulating Factor; Humans; Hydrogen Peroxide; Oxidants; RNA Interference; Stomach Neoplasms; Tumor Necrosis Factor-alpha

Cancer rates are increasing dramatically, and there is currently a strong emphasis on identifying biologically active substances with anti-cancer activity from traditional herbs, as these are thought to have less adverse side-effects than conventional chemotherapy. Here, we examined the effects of extracts of Orostachys japonicus A. BERGER (O. japonicus) on cancer cell proliferation and apoptosis, and investigated the underlying signaling pathways. Dried powdered O. japonicus was extracted with 95% ethyl alcohol and fractionated with a series of organic solvents, including n-hexane (hexane), dichloromethane (DCM), ethylacetate (EtOAc), n-butanol (BuOH), and water (H(2)O). These extracts were tested for anti-cancer activity on a range of cancer cells; of all these, the EtOAc soluble fraction showed the highest anti-cancer activity, which was most marked in AGS human gastric cancer cells. The EtOAc fraction inhibited the proliferation of AGS cells in a dose-dependent and time-dependent manner, by inducing apoptosis and cell cycle arrest, as evidenced by 4,6-diamidino-2-phenylindole (DAPI) staining, annexin V-fluorescein isothiocyanate staining, propidium iodide-labeling, and DNA fragmentation assays. Western blot analysis revealed that p53 and cleaved caspase-3 proteins were up-regulated, and B cell lymphoma-2 (bcl-2) protein and pro-caspase-3 were down-regulated, but bcl-2 associated x protein (bax) protein was not regulated, in response to treatment of AGS cells with the EtOAc fraction. However, the changes of pro-caspase-3 and cleaved caspase-3 could be abolished by the pan-caspase inhibitor Z-VAD-FMK. These results suggest the EtOAc fraction from O. japonicus has substantial anti-cancer activity in human gastric cancer cells.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Crassulaceae; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Humans; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms; Tumor Suppressor Protein p53

The aim of this study is to clarify the benefit of combination chemotherapy in gastric cancer based on a cell-signal inhibitor and an anticancer drug. Two scirrhous gastric cancer cell lines and two non-scirrhous gastric cancer cell lines were used. Five anticancer drugs (5-fluorouracil [5FU], paclitaxel, oxaliplatin, irinotecan, and gemcitabine) and four cell-signal inhibitors, mammalian target of rapamycin (mTOR) inhibitor, glycogen synthase kinase 3beta, p38alphabetaMAPK, and cyclin-dependent kinase, were used. The proliferation of cancer cells was examined by MTT assay and in vivo study. The apoptosis of cancer cells and the expression of apoptosis-related molecules were examined by flow cytometry, real-time PCR, and immunostaining. mTOR inhibitors with 5FU showed a synergistic antiproliferative effect in scirrhous gastric cancer, whereas the other signal inhibitors showed no synergistic effect with any anticancer drugs. mTOR inhibitor decreased the IC(50) of 5FU and increased the apoptosis rate in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. The pan-caspase inhibitor, zVAD-fmk, inhibits apoptosis induced in combination with 5FU and mTOR inhibitor. mTOR inhibitor decreased dihydropyrimidine dehydrogenase, thymidylatesynthase, and bcl-2 expression, and increased caspase-3 and p21 expression of scirrhous gastric cancer cells, but did not affect those of non-scirrhous gastric cancer cells. In an in vivo study, mTOR inhibitor significantly enhanced the therapeutic efficacy of S1, an analog of 5FU. These findings suggest that mTOR inhibitor interacts with 5FU in a synergistic manner in scirrhous gastric cancer cells by the activation of the apoptosis signal. Therefore, mTOR inhibitor is a promising therapeutic agent in combination with 5FU in scirrhous gastric cancer.

Topics: Adenocarcinoma, Scirrhous; Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Fluorouracil; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Protein Serine-Threonine Kinases; Stomach Neoplasms; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Although cisplatin has been shown to induce both apoptosis and necrosis in cancer cells, the potential interconnections between these modes of cell death induced by the drug remain unknown. We studied this phenomenon in gastric cancer cell lines and identified one cell line (SGC-7901) that underwent apoptosis, and another cell line (BGC-823) that primarily underwent nonapoptotic cell death, in response to cisplatin. Apoptosis in cisplatin-treated SGC-7901 cells seemed to be caspase dependent and was mediated, at least in part, by the BH3-only protein, Noxa. This was evidenced by the rapid upregulation of Noxa and inhibition of apoptosis by small interfering RNA knockdown of Noxa. Nonapoptotic cell death induced by cisplatin in BGC-823 cells was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase independence, plasma membrane disruption, and intracellular vacuole formation, indicative of necrosis. Surprisingly, blockage of apoptosis induction by a general caspase inhibitor or by Noxa small interfering RNA in SGC-7901 failed to protect against cisplatin-induced cell death. Under such conditions, SGC-7901 cells displayed cellular features associated with necrosis. Cisplatin-induced apoptosis, thus, seems to precede necrosis when the apoptotic machinery is operative. When the apoptosis program is defective, necrotic cell death takes place as an alternative pathway leading to cell demise. Induction of different modes of cell death that are interrelated in the same cells by cisplatin has the potential to be exploited in formulating new adjuvant cancer therapies.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Blotting, Western; Butylated Hydroxytoluene; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Cisplatin; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Intracellular Fluid; Microscopy, Electron, Transmission; Necrosis; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Stomach Neoplasms; Transfection

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

To investigate the relationship between the inhibited growth (cytotoxic activity) of berberine and apoptotic pathway with its molecular mechanism of action.. The in vitro cytotoxic techniques were complemented by cell cycle analysis and determination of sub-G1 for apoptosis in human gastric carcinoma SNU-5 cells. Percentage of viable cells, cell cycle, and sub-G1 group (apoptosis) were examined and determined by the flow cytometric methods. The associated proteins for cell cycle arrest and apoptosis were examined by Western blotting.. For SNU-5 cell line, the IC50 was found to be 48 micromol/L of berberine. In SNU-5 cells treated with 25-200 micromol/L berberine, G2/M cell cycle arrest was observed which was associated with a marked increment of the expression of p53, Wee1 and CDk1 proteins and decreased cyclin B. A concentration-dependent decrease of cells in G0/G1 phase and an increase in G2/M phase were detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved in 25-200 micromol/L berberine-treated cells by monitoring the apoptotic pathway. Apoptosis was identified by sub-G0 cell population, and upregulation of Bax, downregulation of Bcl-2, release of Ca2+, decreased the mitochondrial membrane potential and then led to the release of mitochondrial cytochrome C into the cytoplasm and caused the activation of caspase-3, and finally led to the occurrence of apoptosis.. Berberine induces p53 expression and leads to the decrease of the mitochondrial membrane potential, Cytochrome C release and activation of caspase-3 for the induction of apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Berberine; Calcium; Caspase 3; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Humans; Membrane Potentials; Mitochondria; Reactive Oxygen Species; Stomach Neoplasms; Tumor Suppressor Protein p53

We have investigated the effects of glycyrrhizin (GL) on cell proliferations of human stomach cancer KATO III and promyelotic leukemia HL-60 cells, and on DNA of those cell lines. GL displayed growth inhibitory effect against KATO III and HL-60 cells. Morphological change showing apoptotic bodies was observed in the KATO III and HL-60 cells treated with GL. The fragmentation of DNA by GL to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in both cell lines. Caspase inhibitors such as Z-VAD-FMK and Z-Asp-CH2-DCB suppressed the DNA fragmentation induced by GL. The data of the present study show that the suppression of KATO III and HL-60 cell-growth by GL results from the induction of apoptosis by GL, and that caspase is involved in the induction of apoptosis by GL in these cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Aspartic Acid; Caspase Inhibitors; Cell Line, Tumor; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Glycyrrhizic Acid; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Oligopeptides; Stomach Neoplasms; Time Factors

Camptothecin, a topoisomerase I inhibitor, is a well-known anticancer drug. However, its mechanism has not been well studied in human gastric cancer cell lines. Camptothecin induced apoptotic cell death in human gastric cancer cell line AGS. Z-VAD-fmk, pan-caspase inhibitor, blocked apoptotic phenotypes induced by camptothecin suggesting that caspases are involved in camptothecin-induced cell death. An inhibitor of caspase-6 or -8 or -9 did not prevent cell death by camptothecin. Various protease inhibitors failed to prevent camptothecin-induced cell death. These results suggest that only few caspases are involved in camptothecin-induced cell death. Camptothecin induced phosphorylation of ERK1/2, JNK, and p38 MAPK, in a dose and time-dependent manner in AGS. Z-VAD-fmk did not affect MAPK signaling induced by camptothecin suggesting that caspase signaling occurs downstream of MAPK signaling. Blocking of p38 MAPK, but not ERK1/2, resulted in partial inhibition of cell death and PARP cleavage by camptothecin in AGS. Taken together, MAPK signaling is associated with apoptotic cell death by camptothecin.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Camptothecin; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Signal Transduction; Stomach Neoplasms; Tumor Cells, Cultured

To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim).. Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism.. JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO.. The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; Benzenesulfonates; Caspase Inhibitors; Caspases; Cyclooxygenase Inhibitors; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Activation; Humans; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Oxazoles; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms; Tumor Cells, Cultured

Arsenic trioxide (As(2)O(3)) can induce clinical remission in patients suffering from acute promyelocytic leukemia, through induction of apoptosis and activation of caspases. We investigated the potential use of As(2)O(3) in human gastric cancer and its possible mechanisms. Human gastric cancer cell lines AGS and MKN-28 were treated with various concentrations (0.1 to 100 microM) of As(2)O(3) for 24 to 72 hr. Apoptosis was determined by acridine orange staining, flow cytometry and DNA fragmentation. Protein levels of p53, p21(waf1/cip1), c-myc, bcl-2 and bax were detected by Western blotting. Effects of As(2)O(3) on caspase-3 protease activity, its protein concentration and cleavage of poly(ADP)-ribose polymerase (PARP) were also studied. As(2)O(3) inhibited cell growth and induced apoptosis in both cell lines, though AGS cells were more sensitive. As(2)O(3) induced apoptosis in AGS cells in a concentration- and time-dependent manner. Treatment resulted in a marked increase in p53 protein levels as early as 4 hr. Co-incubation with p53 anti-sense oligo-nucleotide suppressed As(2)O(3)-induced intracellular p53 over-expression and apoptosis. As(2)O(3) increased the activity of caspase-3, with appearance of its 17 kDa peptide fragment, and cleavage of PARP, with appearance of the 85 kDa cleavage product, both in parallel with the induction of apoptosis. Both the tripeptide caspase inhibitor zVAD-fmk and the specific caspase-3 inhibitor DEVD-fmk partially suppressed As(2)O(3)-induced caspase-3 activation and apoptosis. As(2)O(3) inhibits cell growth and induces apoptosis in gastric cancer cells, involving p53 over-expression and activation of caspase-3. The potential use of this compound in the treatment of gastric cancer is worth further investigation.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Arsenic Trioxide; Arsenicals; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Division; DNA Damage; Enzyme Activation; Humans; Oligonucleotides, Antisense; Oxides; Poly(ADP-ribose) Polymerases; Stomach Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Up-Regulation

To evaluate whether overexpression of Bax, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic agents, three stable cell lines of KATOIII transfected with Bax (KATOIII-Bax), Bcl-2 (KATOIII-Bcl-2), or control pCI-neo expression vector (KATOIII-pCI-neo) were established. The cells were treated with paclitaxel, 5-fluorouracil, or doxorubicin, and the apoptotic response was measured. Our results showed that the sensitivity of the KATOIII-Bax cells to chemotherapeutic agents was enhanced compared with that of the KATOIII-pCI-neo cells, and the KATOIII-Bcl-2 cells were more resistant to these agents. Western blotting revealed that cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells. Significant increase of cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was detected 24 h after exposure to chemotherapeutic agents, when apoptotic cells were less than 10%. The cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells at all time points examined after exposure to chemotherapeutic agents. Marked activation of caspase-3 in the KATOIII-Bax cells was observed 48 h and 72 h after exposure to chemotherapeutic agents compared with that in the KATOIII-pCI-neo cells. Consistently, zVAD-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c release from mitochondria and caspase-3 activation. These findings suggest that cytochrome c release is a major mechanism of apoptotic response and Bax overexpression sensitizes KATOIII cells to chemotherapeutic agent-induced apoptosis through enhancing the release of cytochrome c from mitochondria.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cytochrome c Group; Humans; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms; Tumor Cells, Cultured

We investigated the mechanism of mitomycin C (MMC)-induced apoptosis in SNU-16 human gastric adenocarcinoma cells. Caspase-8 and caspase-3 were activated in MMC-treated cells whereas caspase-1 was not activated, and cytochrome c was released from mitochondrial membrane to cytosol suggesting that caspase-9 was activated during the MMC-induced apoptotic process. Protein kinase C (PKC) delta was cleaved to its characteristic 40 kDa fragment in a caspase-3-dependent manner; on the other hand PKC zeta was cleaved to approximately 40 kDa independently of caspase-3 in the drug-induced apoptosis of the cells. Incubation with z-DEVD-fmk and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) almost completely abrogated MMC-induced DNA fragmentation, indicating that activation of these caspases was crucially involved in MMC-induced apoptosis. Activation of caspase-8 in response to Fas triggering by recruitment of caspase-8 to the Fas has also been found, however, MMC did not induce FasL and Fas expression, as evidenced by reverse transcriptase-polymerase chain reaction and Western blotting. Taken together, these findings indicate that MMC-induced apoptosis in SNU-16 cells was mediated by caspase-8, caspase-9, and caspase-3 activation independently of FasL/Fas interactions.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antibiotics, Antineoplastic; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Death; Cysteine Proteinase Inhibitors; Enzyme Activation; fas Receptor; Humans; Mitomycin; Oligopeptides; Stomach Neoplasms; Time Factors; Tumor Cells, Cultured

Most solid tumors, including gastric cancers, respond poorly to non-surgical treatments which are expected to induce an apoptosis-dependent involution. We hypothesize that the apoptotic machinery in solid tumors is either defective or in a suppressed condition. Overcoming the ineffective induction of apoptosis may improve the responsiveness of solid tumors to non-surgical treatments. Recently, sorbitol, a kind of hexose, has been found to be an effective inducer of apoptosis in HEp-2 cells. Therefore, it is of particular interest to examine the effect of sorbitol-treatment on gastric cancer cells. in the present study, we selected 4 gastric cancer cell lines which have been reported to exhibit different abilities in regard to apoptosis induction, and examined the effect of sorbitol-treatment on apoptosis induction. Within 3 hr after sorbitol-treatment, apoptosis was induced comparably in all cell lines examined. Cell death in MKN-1, MKN-28 or MKN-74 proceeded in a biphasic manner, while cell death in KATO-III was monophasic. The cell death partially depended on caspase activity. Treatments with sorbitol in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly suppressed the apoptotic cell death, suggesting a role of protein kinase-C-dependent process. To our knowledge, this is the most rapid induction of apoptosis in human gastric cancer cells reported to date.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Cysteine Proteinase Inhibitors; DNA Fragmentation; Humans; Sorbitol; Stomach Neoplasms; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured