cytochrome-c-t and acetyl-aspartyl-glutamyl-valyl-aspartal

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling contributes to apoptosis resistance in cholangiocarcinoma. The aim of this study is to check whether matrine, an alkaloid isolated from traditional Chinese herb Sophora flavescens ait, can exert cytotoxic effects against cholangiocarcinoma cells via inactivation of STAT3 signaling.. Mz-ChA-1 and KMCH-1 cholangiocarcinoma cells were treated with matrine at 0.25-2.0 g/L for 48 h and cell viability and apoptosis were assessed. Apoptosis-related molecular changes and STAT3 phosphorylation and transcriptional activities were measured after matrine treatment for 48 h. The effect of expression of a constitutively active STAT3 mutant on matrine-induced apoptosis was determined.. Matrine significantly inhibited the viability and induced apoptosis in cholangiocarcinoma cells. Matrine treatment caused loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-9 and -3. Matrine-induced apoptosis was inhibited in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Matrine reduced the phosphorylation levels of Janus kinase 2 (JAK2) and STAT3, inhibited STAT3-dependent transcriptional activity, and downregulated STAT3 target gene Mcl-1. Notably, expression of the constitutively active form of STAT3 significantly antagonized matrine-induced apoptosis of cholangiocarcinoma cells.. Matrine can trigger mitochondrial apoptotic death of cholangiocarcinoma cells largely through inhibition of JAK2/STAT3 signaling. Therefore, matrine represents a potentially effective anticancer agent for cholangiocarcinoma.

Topics: Alkaloids; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Cytochromes c; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Humans; Janus Kinase 2; Matrines; Membrane Potential, Mitochondrial; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Oligopeptides; Quinolizines; Signal Transduction; STAT3 Transcription Factor

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

The antitumor activity of 7-piperazinethylchrysin (7-PEC) was investigated in HCT-116 human colon cancer cells. MTT assay revealed that the IC50 of 7-PEC in HCT-116 cells was 1.5 µM after 72 h of treatment, much lower than that of chrysin (>100 µM). The data showed that 7-PEC was able to inhibit the growth of HCT-116 cells in a concentration- and time-dependent manner. Topical morphological changes of apoptotic body formation after 7-PEC treatment were observed by Hoechst 33258 staining. 7-PEC reduced mitochondrial membrane potential (∆Ψm) of cells in a concentration-dependent manner and increased the production of intracellular reactive oxygen species (ROS). After treatment with 7-PEC, a significant increase of Bax protein expression and decrease of Bcl-2 protein expression were observed at the same time. These events paralleled with activation of p53, caspase-3 and -9 and the release of cytochrome c (cyt‑c), as well as poly(ADP-ribose) polymerase-1 (PARP1) cleavage and downregulation of p-Akt. However, the apoptosis induced by 7-PEC was blocked by Ac-DEVD-CHO, a caspase-3 inhibitor. These results demonstrate that 7-PEC-induced mitochondrial dysfunction in HCT-116 human colon cancer cells triggers events responsible for caspase-dependent apoptosis pathways, and the elevated ratio of Bax/Bcl-2 is likely involved in this effect.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Survival; Colorectal Neoplasms; Cytochromes c; Down-Regulation; Flavonoids; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oligopeptides; Piperazines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Tumor Suppressor Protein p53

Yersinia species during infection adhere to host immune cells primarily to macrophages and employ its secretary proteins known as Yersinia outer proteins to trigger death in infected cells. In the present study, it is shown that recombinant Yersinia outer protein J (rYopJ) could induce apoptosis in murine peritoneal macrophages in vitro as assessed by morphological features, internucleosomal DNA fragmentation, change in mitochondrial membrane potential (MMP) (Deltapsim), activation of caspases and Annexin V binding. rYopJ-induced cell death was dose and time dependent. Pre-treatment with broad-spectrum caspase inhibitor Z-VAD-FMK, caspase-3 inhibitor Ac-DEVD-CHO and caspase-8 inhibitor Z-IETD-FMK prevented the change in MMP and DNA fragmentation, suggesting caspase-dependent apoptosis of rYopJ-treated macrophages. Blocking the endocytosis by pre-treatment of cells with cytochalasin B did not prevent the rYopJ-induced macrophages apoptosis. The data further suggest that rYopJ-induced apoptosis is mediated by molecules upstream of caspase-8 and relay through mitochondrial pathway involving Bax, Bcl-2, activation of caspase-8 and caspase-3, Bid and polyadenosine diphosphate-ribose polymerase cleavage, cytochrome c release and DNA fragmentation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Apoptosis; Apoptotic Protease-Activating Factor 1; Bacterial Proteins; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytochalasin B; Cytochromes c; Macrophages, Peritoneal; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mitochondria; Oligopeptides; Plague; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Yersinia

DNA fragmentation into internucleosomal fragments is the best recognized biochemical event of apoptosis. Two major caspase pathways have been identified in the signal transduction leading to DNA fragmentation: the receptor pathway and the mitochondrial pathway. DNA fragmentation factor (DFF) has been identified as a major apoptotic endonuclease in the internucleosomal DNA fragmentation process. However, the potential roles of caspases and DFF in internucleosomal DNA fragmentation induced by specific stimuli still need to be investigated since caspase-independent pathways and nuclease(s) other than DFF also play important roles during this process. In the present study, we investigated the activity of GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university, to induce apoptosis of the human leukemia cell line NB4. GP7 induced the release of cytochrome-c from mitochondria, activations of caspase-3, -8, and -9, cleavage of DFF45/inhibitor of caspase-activated DNase, activation of DFF40/caspase-activated DNase, and apoptotic DNA fragmentation in NB4 cells. The broad-spectrum caspase inhibitor zVAD-fmk abrogated GP7-induced caspase-3, -8, and -9 activations but could not inhibit GP7-induced apoptotic DNA fragmentation in NB4 cells. Our findings suggest that GP7-induced apoptotic DNA fragmentation in NB4 cells is independent of caspase activation and DFF, although they are closely involved in this process.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Caspase 3; Caspase 7; Caspase 9; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Leukemia, Promyelocytic, Acute; Nucleosomes; Oligopeptides; Podophyllotoxin; Proteins; Tumor Cells, Cultured

Previous studies reported that the neurotoxin, Crotoxin, isolated from the venom of South American rattlesnake had potent anti-tumor activity. Here, we investigated the involvement of autophagy and apoptosis in the Crotoxin-induced death of chronic myeloid leukemia cell line K562 cells. The neurotoxin dose dependently inhibited the viability of K562 cells. Crotoxin stimulated the autophagic activity as evidenced by the appearance of punctuate monodansylcadaverine (MDC) fluorescence staining in the cytoplasm and increased the formation of autophagosomes. Crotoxin caused the collapse of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-3. Caspase inhibitors attenuated Crotoxin-induced K562 cell death, while blockage of autophagy maturation with 3-methyladenine (3-MA) and NH4Cl potentiated the neurotoxin's cytotoxicity. These results suggest that an apoptotic mechanism contributes to the Crotoxin-induced death of K562 cells, while the activation of autophagy delays neurotoxin-induced apoptosis.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Ammonium Chloride; Apoptosis; Autophagy; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Crotoxin; Cytochromes c; Enzyme Activation; Humans; K562 Cells; Lysosomes; Mitochondria; Oligopeptides; Vacuoles

The goal of this study was to examine the effect of ursolic acid, a pentacyclic triterpenoid compound, on growth of the endometrial cancer cell line SNG-II. We found that ursolic acid strongly inhibited the growth of SNG-II cells in a dose- and time-dependent manner. Morpholgical changes characteristic of apoptosis were observed in treated cells, such as the presence of apoptotic bodies and fragmentation of DNA into oligonucleosomal-sized fragments. We also investigated the active forms of caspase-3, -8 and -9 in ursolic acid-treated SNG-II cells. At 25 and 50 microM strength, ursolic acid induced marked increases in caspase-3 activity to approximately 5-fold that of control cells. Levels of cleaved caspase-3 increased in a time- and dose-dependent manner. Activation of caspases also led to the cleavage of target proteins, such as PARP. Ursolic acid treatment also resulted in a cleavage of poly (ADP-ribose) polymerase in a dose-dependent manner. Testing whether caspase-3 activation and DNA polymerase activity were inhibited by addition of Ac-DEDV-HCO during ursolic acid treatment showed that 50 microM Ac-DEDV-HCO inhibited caspase-3 activity in treated cells. Although DNA fragmentation was observed after ursolic acid treatment, DNA fragmentation did not occur in SNG II cells treated with both Ac-DEDV-HCO and ursolic acid. Because some researchers have suggested that mitochondrial pathways are involved in ursolic acid-induced apoptosis secondary to induction of mitochondrial cytochrome c release, we studied mitochondrial events in ursolic acid-induced apoptosis in these cell lines. After ursolic acid treatment, the anti-apoptotic Bcl-2 protein decreased and Bax expression was enhanced. Our results indicated that ursolic acid induced apoptotic processes in the endometrial cancer SNG-II cell line through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cytochromes c; DNA; DNA Fragmentation; Endometrial Neoplasms; Female; Humans; Mitochondria; Oligopeptides; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Triterpenes; Up-Regulation; Ursolic Acid

To clarify the mechanism underlying the anti-mutagenic and anti-cancer activities of Scorpio water extract (SWE).. Human hepatoma HepG2 cells were incubated with various concentrations of SWE. After 24-h incubation, cytotoxicity and apoptosis evaluations were determined by MTT and DNA fragmentation assay, respectively. After treatment with SWE, mitochondrial membrane potential (MMP) was determined by measuring the retention of the dye 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)) and the protein expression including cytochrome C and poly-(ADP-ribose) polymerase (PARP) were measured by Western blotting. Caspase-3 and -9 enzyme activities were measured using specific fluorescence dyes such as Ac-DEVD-AFC and Ac-LEHD-AFC.. We found that treatment with SWE induced apoptosis as confirmed by discontinuous DNA fragmentation in cultured human hepatoma HepG2 cells. Our investigation also showed that SWE-induced apoptosis of HepG2 cells were associated with intracellular events including disruption of MMP, increased translocation of cytochrome C from mitochondria to cytosol, activation of caspase-3, and PARP. Pre-treatment of N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), a caspase-3 specific inhibitor, or cyclosporin A (CsA), an inhibitor of MMP disruption, completely abolished SWE-induced DNA fragmentation.. These results suggest that SWE possibly causes mitochondrial damage, leading to cytochrome C release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HepG2 cells. These results further suggest that Scorpio may be a valuable agent of therapeutic intervention of human hepatomas.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochromes c; Drugs, Chinese Herbal; Enzyme Inhibitors; Humans; Liver Neoplasms; Membrane Potentials; Mitochondria; Oligopeptides; Poly(ADP-ribose) Polymerases

We studied the effect of ursolic acid, a pentacyclic triterpene acid, on the growth of poorly differentiated type endometrial cancer HEC108 cells in vitro. Ursolic acid strongly inhibited the growth of HEC108 cells in a dose- and time-dependent manner. Morphological changes characteristic of apoptosis were observed in ursolic acid-treated cells, such as the presence of apoptotic bodies and fragmentation of DNA to oligonucleosomal-sized fragments. Investigation of caspase activity in ursolic acid-treated HEC108 cells showed that exposure at 50, 75 or 100 microM induced marked increases in caspase-3 activity (after 24 h) to 5.00, 11.76 or 12.75 times that of control levels, while cleaved caspase-3 levels increased in dose-dependent manner after 24 h. Activation of caspase was shown to lead to the cleavage of target proteins such as PARP. Ursolic acid treatment also resulted in a cleavage of poly(ADP-ribose) polymerase in a dose-dependent manner. Testing whether caspase-3 activation and DNA polymerase activity were inhibited by the addition of Ac-DEDV-HOC during ursolic acid treatment showed that 50 microM Ac-DEDV-HOC inhibited caspase-3 activity in treated cells. A mitochondrial pathway has been suggested to be involved in ursolic acid-induced apoptosis because the treatment induces mitochondria cytochrome c release. Experimentally, we found that anti-apoptotic Bcl-2 protein levels decreased after ursolic acid treatment, while Bax expression increased. Our results indicated that ursolic acid induced apoptotic processes in these poorly differentiated endometrial cancer cells occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Endometrial Neoplasms; Female; Humans; Oligopeptides; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins c-bcl-2; Time Factors; Triterpenes; Ursolic Acid

This study was carried out to investigate apoptotic effects of the glycoprotein (SNL glycoprotein, 150 kDa) isolated from Solanum nigrum Linne, which has been used as an anti-pyretic and anti-cancer agent in folk medicine. We found that the SNL glycoprotein consists of carbohydrate (69.74%) and protein content (30.26%), which has >50% hydrophobic amino acids containing glycine and proline. LDH assay indicated that the SNL glycoprotein has obvious cytotoxic and apoptotic effects (>50% cell death) at 40 microg/ml SNL glycoprotein for 2 h in HT-29 cells. The results showed that the SNL glycoprotein has a stimulatory effect on the release of mitochondrial cytochrome c, cleavages of pro-caspase-9, pro-caspase-3, and poly(ADP-ribose) polymerase (PARP) proteins in HT-29 cells. However, the SNL glycoprotein did not significantly stimulate or change the levels of intracellular reactive oxygen species (ROS). The results of this experiment suggest that the SNL glycoprotein activates caspase-3 in HT-29 cells, independent of ROS.

Topics: Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Glycine; Glycoproteins; HT29 Cells; Humans; Intracellular Space; L-Lactate Dehydrogenase; Oligopeptides; Plant Proteins; Poly(ADP-ribose) Polymerases; Proline; Reactive Oxygen Species; Solanum nigrum; Time Factors

Mahanine, a carbazole alkaloid occurs in the edible part of Micromelum minutum, Murraya koenigii and related species has been found to induce apoptosis in human myeloid cancer cell (HL-60). Concentration of 10 microM mahanine caused a complete inhibition of cell proliferation and the induction of apoptosis in a time dependent manner. Mahanine-induced cell death was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase like activities, poly(ADP-ribose) polymerase cleavage, release of cytochrome c into cytosol and stimulation of reactive oxygen species generation. The cell death was completely prevented by a pancaspase inhibitor benzyloxycarbonyl-L-aspart-1-yl-[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH(2)-DCB). Mahanine activated various caspases such as caspase-3, -6, -8 and -9 (like) activities but not caspase-1 like activity. More than 70% cell survival was observed in the presence of a caspase-3 inhibitor. In addition, co-treatment of cyclosporin A markedly increased the survival of mahanine-treated HL-60 cells. Flow cytometric analysis revealed that mahanine decreased the mitochondrial membrane potential of intact cells, and disrupted cell cycle progression by increasing the number of cells in sub-diploid region, concomitantly with the decrease of cells in diploid phases, particularly at late hours of apoptosis. The overall results suggest that mahanine down regulates cell survival factors by activation of caspase-3 through mitochondrial dependent pathway, and disrupts cell cycle progression.

Topics: Antineoplastic Agents; Apoptosis; Aspartic Acid; Carbazoles; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Division; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; DNA; DNA Fragmentation; HL-60 Cells; Humans; Leukemia; Mitochondria; Oligopeptides; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Proteins; Reactive Oxygen Species