cytochrome-c-t and Carcinoma

SERPINB3 (Squamous Cell Carcinoma Antigen, SCCA1) is a member of the ov-serpins, a serine protease inhibitors family expressed in many cell types including normal epithelium, leukocytes, tumors of epithelial origin and primary liver cancer. Several studies, carried out in vitro and in vivo, have documented an important role of SERPINB3 in the modulation of programmed cell death by different mechanisms, both in inflammatory processes and in cancer. SERPINB3 significantly attenuates apoptosis by contrasting cytochrome c release from the mitochondria and by antichemotactic effect for NK cells. Mechanisms involved in apoptosis induction and regulation play a key role in the balance between cell proliferation and death. Imbalance of this equilibrium may contribute to the development of autoimmune diseases, as defective apoptosis of immune cells leads to deregulated autoreactive cell proliferation. Since defective programmed cell death represents a critical feature of autoimmunity, the involvement of SERPINB3 in this pathological field deserves further studies.

Topics: Animals; Antigens, Neoplasm; Apoptosis; Autoimmunity; Biomarkers, Tumor; Carcinoma; Cell Migration Inhibition; Cell Proliferation; Cytochromes c; Humans; Killer Cells, Natural; Mitochondria; Serpins

Ehrlich ascites carcinoma and its subcutaneous inoculated solid tumour form (SEC) are reliable models for chemotherapeutic molecular targets exploration. Novel chemotherapeutic approaches are identified as molecular targets for intrinsic apoptosis, like the modulation of the second mitochondria-derived activator of caspases (SMAC). SMAC is a physiological substrate of mitogen-activated protein kinases (MAPKs). Glutathione-S-transferase P1 (GSTP1) and its close association with MAPKs play an important role in malignant cell proliferation, metastasis, and resistance to chemotherapeutics. Nitazoxanide (NTZ) is an emerging cancer therapy and its targeted GSTP1 evidence remains a knowledge need.. In the present mice-established SEC, the chemotherapeutic roles of oral NTZ (200 mg/kg/day) and 5-fluorouracil (5-FU; 20 mg/kg/day, intraperitoneally) regimens were evaluated by measuring changes in tumour mass, the tumour MAPKs, cytochrome c, Bcl-2 interacting mediator of cell death (BIM), and SMAC signalling pathway in addition to its molecular downstream; caspases 3 and 9.. Computational analysis for these target protein interactions showed direct-ordered interactions. After individual therapy with NTZ and 5-FU regimens, the histological architecture of the extracted tumour discs revealed decreases in viable tumour regions with significant necrosis surrounds. These findings were consistent with gross tumour sizes. Each separate regimen lowered the remarkable GSTP1 and elevated the low MAPKs expressions, cytochrome c, BIM, SMAC, and caspases 3, and 9 in EST tissues.. The chemotherapeutic activity of NTZ in SEC was proven. Additionally, NTZ possesses a SMAC modulatory activity that, following thorough research, should be taken into consideration as a chemotherapeutic approach in solid tumours.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma; Caspases; Cell Line, Tumor; Computational Biology; Cytochromes c; Fluorouracil; Glutathione Transferase; Mice; Mitochondria; Mitochondrial Proteins; Mitogen-Activated Protein Kinases

Topics: Antineoplastic Agents; Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Coordination Complexes; Copper; Cytochromes c; DNA; Fluoroquinolones; Humans; Reactive Oxygen Species

BACKGROUND Nasopharyngeal carcinoma results in high patient morbidity and mortality, due to early metastasis, and toxicity due to chemotherapy. Mukonal is plant-derived carbazole alkaloid that has been used in traditional Chinese medicine to treat several types of cancer. This study aimed to investigate the effects of mukonal on cell proliferation, apoptosis, autophagy, and the mitochondrial membrane potential of nasopharyngeal carcinoma cells in vitro. MATERIAL AND METHODS CNE1 human nasopharyngeal carcinoma cells and NP69 normal nasopharyngeal epithelial cells were cultured with and without treatment with increasing doses of mukonal. Cell viability was determined by the MTT assay. Fluorescence microscopy was used to detect reactive oxygen species (ROS), mitochondrial membrane potential, and the release of cytochrome C. Flow cytometry was used to examine changes in the cell cycle, electron microscopy examined cell autophagy, and Western blot was performed to measure levels of proteins associated with autophagy and apoptosis. RESULTS Mukonal had an antiproliferative effect on CNE1 cells, with an IC₅₀ of 9 µM and there were effects of toxicity on normal NP69 cells. Mukonal triggered ROS-mediated changes in mitochondrial membrane potential which was also accompanied by the discharge of cytochrome C in the CNE1 cells. Mukonal activated autophagy and apoptosis in CNE1 cells, which was also associated with upregulation of the autophagy-related proteins, LC3 II and beclin-1, as well as apoptosis-associated proteins, Bax, cleaved caspase-3 and -9. Mukonal treatment also resulted in CNE1 cells cycle arrest at G₂/M. CONCLUSIONS Mukonal inhibited the growth of human CNE1 nasopharyngeal carcinoma cells in vitro.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Beclin-1; Carbazoles; Carcinoma; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; China; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Nasopharyngeal Carcinoma; Plant Extracts; Reactive Oxygen Species

Heat shock protein-70kDa (Hsp70) is a member of molecular chaperone family, involved in the proper folding of various proteins. Hsp70 is important for tumor cell survival and is also reported to be involved in enhancing the drug resistance of various cancer types. Hsp70 controls apoptosis both upstream and downstream of the mitochondria by regulating the mitochondrial membrane permeabilization (MMP) and apoptosome formation respectively. In the present study, we have elucidated the role of Hsp70 in Gambogic acid (GA) induced apoptosis in bladder cancer cells. We observed that functional inhibition of Hsp70 by Pifithrin-μ switches GA induced caspase dependent (apoptotic) cell death to caspase independent cell death. However, this cell death was not essentially necrotic in nature, as shown by the observations like intact plasma membranes, cytochrome-c release and no significant effect on nuclear condensation/fragmentation. Inhibition of Hsp70 by Pifithrin-μ shows differential effect on MMP. GA induced MMP and cytochrome-c release was inhibited by Pifithrin-μ at 12h but enhanced at 24h. Pifithrin-μ also reverted back GA inhibited autophagy which resulted in the degradation of accumulated ubiquitinated proteins. Our results demonstrate that Hsp70 plays an important role in GA induced apoptosis by regulating caspase activation. Therefore, inhibition of Hsp70 may hamper with the caspase dependent apoptotic pathways induced by most anti-cancer drugs and reduce their efficacy. However, the combination therapy with Pifithrin-μ may be particularly useful in targeting apoptotic resistant cancer cells as Pifithrin-μ may initiate alternative cell death program in these resistant cells.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Carcinoma; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; Female; HSP70 Heat-Shock Proteins; Humans; Male; Membrane Potential, Mitochondrial; Middle Aged; Necrosis; Proteolysis; Reactive Oxygen Species; Signal Transduction; Sulfonamides; Tumor Cells, Cultured; Ubiquitination; Urinary Bladder Neoplasms; Xanthones

The therapeutic strategy for advanced nasopharyngeal carcinoma (NPC) is still challenging. It is an urgent need to uncover novel treatment targets for NPC. Therefore, understanding the mechanisms underlying NPC tumorigenesis and progression is essential for the development of new therapeutic strategies. Here, we showed that TP53-regulated inhibitor of apoptosis (TRIAP1) was aberrantly overexpressed and associated with poor survival in NPC patients. TRIAP1 overexpression promoted NPC cell proliferation and suppressed cell death in vitro and in vivo, whereas TRIAP1 knockdown inhibited cell tumorigenesis and enhanced apoptosis through the induction of mitochondrial fragmentation, membrane potential alteration and release of cytochrome c from mitochondria into the cytosol. Intersecting with our previous miRNA data and available bioinformatic algorithms, miR-320b was identified and validated as a negative regulator of TRIAP1. Further studies showed that overexpression of miR-320b suppressed NPC cell proliferation and enhanced mitochondrial fragmentation and apoptosis both in vitro and in vivo, while silencing of miR-320b promoted tumor growth and suppressed apoptosis. Additionally, TRIAP1 restoration abrogated the proliferation inhibition and apoptosis induced by miR-320b. Moreover, the loss of miR-320b expression was inversely correlated with TRIAP1 overexpression in NPC patients. This newly identified miR-320b/TRIAP1 pathway provides insights into the mechanisms leading to NPC tumorigenesis and unfavorable clinical outcomes, which may represent prognostic markers and potential therapeutic targets for NPC treatment.

Topics: Adult; Aged; Animals; Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cytochromes c; Cytosol; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Membrane Potentials; Mice; Mice, Inbred BALB C; MicroRNAs; Middle Aged; Mitochondria; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Proportional Hazards Models; Signal Transduction

Rabdosia serra has been widely used for the treatment of the various human diseases. However, the antiproliferative effects and underlying mechanisms of the compounds in this herb remain largely unknown. In this study, an antiproliferative compound against human nasopharyngeal carcinoma (NPC) cells from Rabdosia serra was purified and identified as lasiodin (a diterpenoid). The treatment with lasiodin inhibited cell viability and migration. Lasiodin also mediated the cell morphology change and induced apoptosis in NPC cells. The treatment with lasiodin induced the Apaf-1 expression, triggered the cytochrome-C release, and stimulated the PARP, caspase-3 and caspase-9 cleavages, thereby activating the apoptotic pathways. The treatment with lasiodin also significantly inhibited the phosphorylations of the AKT, ERK1/2, p38 and JNK proteins. The pretreatment with the AKT or MAPK-selective inhibitors considerably blocked the lasiodin-mediated inhibition of cell proliferation. Moreover, the treatment with lasiodin inhibited the COX-2 expression, abrogated NF-κB binding to the COX-2 promoter, and promoted the NF-κB translocation from cell nuclei to cytosol. The pretreatment with a COX-2-selective inhibitor abrogated the lasiodin-induced inhibition of cell proliferation. These results indicated that lasiodin simultaneously activated the Apaf-1/caspase-dependent apoptotic pathways and suppressed the AKT/MAPK and COX-2/NF-κB signaling pathways. This study also suggested that lasiodin could be a promising natural compound for the prevention and treatment of NPC.

Topics: Antineoplastic Agents; Apoptosis; Apoptotic Protease-Activating Factor 1; Carcinoma; Caspases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclooxygenase 2; Cytochromes c; Diterpenes, Kaurane; Humans; Mitogen-Activated Protein Kinases; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; NF-kappa B; Protein Binding; Proto-Oncogene Proteins c-akt; Signal Transduction; Tracheophyta

Different plant parts of Flacourtia indica have long been used in Ayurvedic medicine. Previous studies have demonstrated that the methanolic extract of F. indica possess anti-inflammatory properties. The present study was aimed at investigating the anticancer effects of methanol extract of Flacourtia indica (FIM) aerial parts in human colon cancer (HCT116) cells. Treatment of cells with FIM at a concentration of 500 μg/ml for 24 hours significantly reduced cell viability and induced apoptosis, which was associated with the increased cytoplasmic expression of cytochrome c, activation of caspase-3, and the cleavage of poly-(ADP-ribose) polymerase. Incubation with FIM also inhibited the levels of Bcl-2, Bcl-xl and survivin, which are the markers of cell proliferation, whereas the expression of Bax remained unchanged. Treatment with FIM led to the generation of reactive oxygen species (ROS) in a concentration-dependent manner. Pharmacological inhibition of ROS generation by pretreatment of cells with N-acetyl cysteine abrogated FIM-induced apoptosis in HCT116 cells. Thus, these results demonstrate that FIM has anti-proliferative and pro-apoptotic effects in HCT116 cells and the effects are, at least in part, due to the ROS dependent activation of caspases.

Topics: Apoptosis; bcl-X Protein; Carcinoma; Caspase 3; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Drug Screening Assays, Antitumor; HCT116 Cells; Humans; Inhibitor of Apoptosis Proteins; Methanol; Plant Components, Aerial; Plant Extracts; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Salicaceae; Survivin

One water-soluble polysaccharide fraction (WACP) was purified from the Artemisia capillaris by DEAE-cellulose anion-exchange and Sephacryl S-400 gel-permeation chromatography. Based on gas chromatography (GC) analysis, WACP was an arabinogalactan (AG), consisting of arabinose and galactose in the ratio of 4:2. Its molecular weight was about 5.8×10(4) Da. The present study demonstrated the anticancer potential of WACP on human nasopharyngeal carcinoma CNE-2 cells and elucidated its possible mechanisms. MTT and flow cytometric analysis identified that WACP had a potent anti-proliferation activity on CNE-2 cells by inducing apoptosis, which was parallel with the morphological changes of CNE-2 cells under inverted microscope. In addition, WACP triggered the apoptosis via the mitochondrial apoptotic pathway, which included the loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-3/9. Taken together, these results suggest that WACP has anticancer potential in the treatment of human nasopharyngeal carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Artemisia; Carcinoma; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Enzyme Activation; Humans; Mitochondria; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Polysaccharides; Solubility; Water

Vitexicarpin (3', 5-dihydroxy-3, 4', 6, 7-tetramethoxyflavone), a polymethoxyflavone isolated from Viticis Fructus (Vitex rotundifolia Linne fil.), has long been used as an anti-inflammatory herb in traditional Chinese medicine. It has also been reported that vitexicarpin can inhibit the growth of various cancer cells. However, there is no report elucidating its effect on human prostate carcinoma cells. The aim of the present study was to examine the apoptotic induction activity of vitexicarpin on PC-3 cells and molecular mechanisms involved. MTT studies showed that vitexicarpin dose-dependently inhibited growth of PC-3 cells with an IC50~28.8 μM. Hoechst 33258 staining further revealed that vitexicarpin induced apoptotic cell death. The effect of vitexicarpin on PC-3 cells apoptosis was tested using prodium iodide (PI)/Annexin V-FITC double staining and flow cytometry. The results indicated that vitexicarpin induction of apoptotic cell death in PC-3 cells was accompanied by cell cycle arrest in the G2/M phase. Furthermore, our study demonstrated that vitexicarpin induction of PC-3 cell apoptosis was associated with upregulation of the proapoptotic protein Bax, and downregulation of antiapoptotic protein Bcl-2, release of Cytochrome c from mitochondria and decrease in mitochondrial membrane potential. Our findings suggested that vitexicarpin may become a potential leading drug in the therapy of prostate carcinoma.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma; Cell Cycle Checkpoints; Cell Death; Cell Division; Cell Line, Tumor; Cytochromes c; Down-Regulation; Flavonoids; G2 Phase; Humans; Male; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria; Plant Extracts; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Up-Regulation

Sessile marine animals such as sponges, ascidians, and bryozoans are a rich source of bioactive natural products, many of which exhibit potent anticancer activity.. We extracted and purified a polypeptide with potent antitumor activity from Ciona savignyi by acetone fractionation, ultrafiltration, ion exchange chromatography, gel chromatography, and high-performance liquid chromatography. An MTT assay was used to study the cytotoxicity of the isolated fraction and the purified polypeptide. Cell cycle and Western blot analysis were performed to study the mode of action of the purified polypeptide.. A novel polypeptide with potent antitumor activity was purified. The molecular weight of the polypeptide, designated CS5931, was 5931 Da, and use of the genome basic local alignment search tool (BLAST) revealed that the N-terminal sequence of CS5931 is identical to that of granulin A from C savignyi. CS5931 exhibited significant cytotoxicity for several cancer cell types and induced apoptotic death in HCT-8 cells in a dose- and time-dependent manner. Cell cycle analysis demonstrated that CS5931 caused cell cycle arrest at the G(2)/M phase, and a sub-G(1) peak appeared after treating the cells with CS5931 for 12 hours. The mitochondrial-mediated pathway was implicated in CS5931-induced apoptosis.. Our observations clearly demonstrate the antiproliferative and proapoptotic activities of the polypeptide CS5931 from C savignyi and the mitochondrial-mediated pathway involved in the polypeptide-induced cell death.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma; Caspase 3; Caspase 9; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; G2 Phase Cell Cycle Checkpoints; Granulins; Humans; Intercellular Signaling Peptides and Proteins; Mitochondria; Peptides; Proto-Oncogene Proteins c-bcl-2; Urochordata

EBV (Epstein-Barr virus) is considered to be a major factor that causes NPC (nasopharyngeal carcinoma), which is one of the sneakiest cancers frequently occurring in Southeast Asia and Southern China. Apoptosis and pro-apoptotic signals have been studied for decades; however, few have extended the prevailing view of EBV to its impact on NPC in perspective of apoptosis. One of the important proteins named VDAC1 (voltage-dependent anion protein 1) on the mitochondrial outer membrane controls the pro-apoptotic signals in mammalian cells. The impact of EBV infection on VDAC1 and related apoptotic signals remains unclear. In order to study the VDAC1's role in EBV-infected NPC cells, we employ siRNA (small interfering RNA) inhibition to analyse the release of Ca2+ and Cyto c (cytochrome c) signals in the cytoplasm, as they are important pro-apoptotic signals. The results show a decrease of Ca2+ release and up-regulation of Cyto c with EBV infection. After siRNA transfection, the dysregulation of Cyto c is neutralized, which is evidence that the level of Cyto c release in virus-infected NPC cells is the as same as that of non-infected NPC cells. This result indicates that EBV infection changes the cytoplasmic level of Cyto c through regulating VDAC1. In summary, this study reports that EBV changes the release of Ca2+ and Cyto c in the cytoplasm of NPC cells, and that Cyto c changes are mediated by VDAC1 regulation.

Topics: Calcium; Carcinoma; Cell Line, Tumor; Cytochromes c; Cytoplasm; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Ions; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; RNA Interference; RNA, Small Interfering; Signal Transduction; Up-Regulation; Voltage-Dependent Anion Channel 1

Betulinic acid (BetA) is an effective and potential anticancer chemical derived from plants. BetA can kill a broad range of tumor cell lines, but has no effect on untransformed cells. The chemical also kills melanoma, leukemia, lung, colon, breast, prostate and ovarian cancer cells via induction of apoptosis, which depends on caspase activation. However, no reports are yet available about the effects of BetA on nasopharyngeal carcinoma (NPC), a widely spread malignancy in the world, especially in East Asia. In this study, we first showed that BetA can effectively kill CNE2 cells, a cell line derived from NPC. BetA-induced CNE2 apoptosis was characterized by typical apoptosis hallmarks: caspase activation, DNA fragmentation, and cytochrome c release. Overexpression of Bcl-2 and Bcl-xL could partially prevent apoptosis caused by BetA. Moreover, Bax was not activated during the induction of apoptosis. Bax/Bak knockdown and wild-type CNE2 cells showed the same kinetics of cytochrome c release. We then showed that BetA may impair mitochondrial permeability transition pores (mPTPs), which may partially contribute to cytochrome c release. These observations suggest that BetA may serve as a potent and effective anticancer agent in NPC treatment. Further exploration of the mechanism of action of BetA could yield novel breakthroughs in anti-cancer drug discovery.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Betulinic Acid; Carcinoma; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Humans; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Pentacyclic Triterpenes; Triterpenes

It has been previously shown that the naturally occurring antioxidant (-)-epigallocatechin-3-gallate (EGCG), found in green tea, and pterostilbene, a stilbenoid derived from blueberries, inhibit pancreatic cancer in vitro when used individually. We hypothesized that the combination of EGCG and pterostilbene would reveal additive effects in vitro.. Using the pancreatic cancer cell lines MIA PaCa-2 and PANC-1, efficacy and synergism were evaluated for cell proliferation and viability (3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays, cell cycle analysis) and mitochondrial apoptosis (mitochondrial depolarization, cytochrome C release, caspase-3/7 activity, cell death detection using enzyme-linked immunosorbent assay).. Cell proliferation assays revealed significant additive antiproliferative effects with pterostilbene and EGCG in both cell lines at the later, 72-h, point (P < 0.05). MIA underwent S-phase arrest with the combination (10-12% increase); however, cell cycle arrest was not observed in PANC. The combination induced mitochondrial depolarization and upregulated cytochrome C (P < 0.05) in MIA, but these effects were not observed in PANC. EGCG increased caspase-3/7 in MIA; however, the combination did not significantly increase the activity in either cell line (P < 0.05). Apoptosis was only observed in PANC (P < 0.05). The reduction in proliferation in MIA in the 3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays with the combination indicated that cell death occurs, possibly through another mechanism.. Our results are encouraging regarding the future use of EGCG and pterostilbene to improve traditional pancreatic cancer therapies. In conclusion, EGCG and pterostilbene have additive, antiproliferative effects in vitro and alter the apoptotic mechanisms in both cell lines by modulation at different points in the mechanism.

Topics: Anticarcinogenic Agents; Carcinoma; Caspases; Catechin; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Drug Evaluation, Preclinical; Humans; Pancreatic Neoplasms; Stilbenes

Resveratrol shows chemopreventive activity in a variety of human cancers by targeting mitochondria and triggering apoptosis. The purpose of this study was to investigate the antitumor action of resveratrol in bladder cancer and its underlying mechanism.. Using two different bladder cell lines, BTT739 and T24, the cytotoxicity of resveratrol were determined by MTT assay. The apoptosis induced by resveratrol was assayed by transferase dUTP nick end labeling staining. To show whether the mitochondrial dysfunction involved in the effects of resveratrol, mitochondrial function was detected by mitochondrial membrane potential, reactive oxygen species production and adenosine 5'-triphosphate content. In addition, the markers of apoptosis in the intrinsic mitochondrial-dependent pathway were analyzed by the release of cytochrome c and the activities of caspase-9 and caspase-3.. Resveratrol effectively decreased cell viability and induced apoptosis in a concentration- and time-dependent manner. In addition, resveratrol significantly disrupted the mitochondrial membrane potential in both intact cells and isolated mitochondria. Resveratrol also increased reactive oxygen species production and reduced adenosine 5'-triphosphate concentrations. Western blot analysis showed that resveratrol provoked the release of cytochrome c from mitochondria to the cytosol. Furthermore, resveratrol significantly promoted the activation of caspase-9 and caspase-3.. These findings suggest that resveratrol efficiently triggers apoptosis in bladder cancer cells through the intrinsic mitochondrial-dependent pathway, which is associated with mitochondrial dysfunction. Resveratrol might have great pharmacological promise in the treatment of bladder cancer.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Mice; Reactive Oxygen Species; Resveratrol; Stilbenes; Urinary Bladder Neoplasms

The anti-tumor effect of Icariside II (IcaS), a natural prenylated flavonol glycoside, was studied on human breast cancer MCF7 cells to unveil the underlying mechanisms involved. IcaS in MCF7 cells produced a loss of mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9 revealed the involvement of the intrinsic apoptosis pathway. In contrast, IcaS enhanced the expression level of Fas and the Fas-associated death domain (FADD), and activated caspase-8, suggesting the involvement of the extrinsic apoptosis pathway. IcaS also increased the expression of Bax and BimL without affecting the expression status of Bcl-2 and Bid, suggesting that the apoptosis induced by IcaS was related to Bcl-2 family protein regulation. IcaS thus induced apoptosis in MCF7 cells involving both the intrinsic and extrinsic signaling pathways. Its potential as a candidate for an anti-cancer agent warrants further investigation.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Carcinoma; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; fas Receptor; Fas-Associated Death Domain Protein; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

In this work, the effects of a pair of positional isomer of ganoderic acids (GAs), namely ganoderic acid Mf (GA-Mf) and ganoderic acid S (GA-S) purified from the fermented mycelia of Ganoderma lucidum, on induction of cell apoptosis and the apoptotic pathway in HeLa cells were investigated. The results demonstrate that both isomers decreased cell population growth on various human carcinoma cell lines by MTT assay, while GA-Mf had better selectivity between normal and cancer cells. The flow cytometry analysis indicated that treatment of HeLa cells with GA-S caused cell cycle arrest in the S phase, while GA-Mf caused cell cycle arrest in the G1 phase. Compared with GA-S, GA-Mf had more potent increase in the number of early and late apoptotic cells. Treatment of HeLa cells with each isomer decreased the mitochondria membrane potential and caused the release of cytochrome c from mitochondria into the cytosol. In addition, stimulation of caspase-3 and caspase-9 activity was observed. The Bax/Bcl-2 ratio was also increased in GA-treated HeLa cells. The results demonstrated that both isomers GA-Mf and GA-S induced apoptosis of human HeLa cells through a mitochondria mediated pathway, but they had the different cell cycle arrest specificity. The findings will be helpful to the development of useful cancer chemopreventive compounds from G. lucidum.

Topics: Apoptosis; Carcinoma; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line; Cytochromes c; Dose-Response Relationship, Drug; Female; G1 Phase; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mycelium; Reishi; S Phase; Triterpenes; Uterine Cervical Neoplasms

A key player in prostate cancer development and progression is the androgen receptor (AR). Tumor-associated lipogenesis can protect cancer cells from carcinogenic- and therapeutic-associated treatments. Increased synthesis of fatty acids and cholesterol is regulated by androgens through induction of several genes in androgen-responsive cancer cells. Acetyl-CoA-carboxylase-α (ACCA) is a key enzyme in the regulation of fatty acids synthesis. Here we show that AR binds in vivo to intron regions of human ACCA gene. We also show that the level of ACCA protein in LNCaP depends on AR expression and that DHT treatment increases ACCA expression and fatty acid synthesis. Inhibition of ACCA by TOFA (5-tetradecyl-oxy-2-furoic acid) decreases fatty acid synthesis and induces caspase activation and cell death in most PCa cell lines. Our data suggest that TOFA can kill cells via the mitochondrial pathway since we found cytochrome c release after TOFA treatment in androgen sensitive cell lines. The results also imply that the pro-apoptotic effect of TOFA may be mediated via a decrease of neuropilin-1(NRP1) and Mcl-1expression. We have previously reported that Mcl-1 is under AR regulation and plays an important role in resistance to drug-induced apoptosis in prostate cancer cells, and NRP1 is known to regulate Mcl-1 expression. Here, we show for the first time that NRP1 expression is under AR control. Taken together, our data suggest that TOFA is a potent cell death inducing agent in prostate cancer cells.

Topics: Acetyl-CoA Carboxylase; Androgen Receptor Antagonists; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Death; Cell Line, Tumor; Cytochromes c; Dihydrotestosterone; Fatty Acids; Furans; Genes, p53; Humans; Introns; Lipids; Male; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neuropilin-1; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Androgen

Cinnamomum subavenium has long been used as a traditional Chinese medicine to treat carcinomatous swelling, abdominal pain and other diseases.. The goal of this work was to study the cytotoxic effect of subamolide A, a constituent isolated from the stems of Cinnamomum subavenium Miq., and to extend its traditional use for clinical applications in treating human urothelial carcinoma.. Cytotoxic effect of subamolide A was determined by the MTT assay in NTUB1, T24, PC3 and SV-HUC-1 cells treated with various concentrations of subamolide A for three days. Apoptosis was detected by the change of cell morphology and flow cytometry analysis. The reactive oxygen species (ROS) level and mitochondria membrane potential (Δψm) were determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related and stress-induced signaling molecules.. Subamolide A selectively induced apoptosis in two cancerous human urothelial carcinoma cell lines (NTUB1 and T24) in comparison with normal immortalized uroepithelial cells (SV-HUC-1). Subamolide A reduced mitochondrial membrane potential (Δψm) and caused apoptosis of NTUB1 cells. Subamolide A increased Bax/Bcl-2 ratios, the amount of cytochrome c released from the mitochondria, caspase-3 and PARP cleavage, activated p53 and ERK1/2 and ultimately led to apoptosis in NTUB1 cells. Furthermore, a higher dose (10μM) of subamolide A synergistically enhanced the cytotoxicity of cisplatin and gemcitabine in NTUB1 cells.. The current study demonstrated that subamolide A triggered the mitochondria-dependent apoptotic pathways and p53 and ERK1/2 activation in the human urothelial carcinoma cell line NTUB1. In addition, subamolide A synergistically enhanced cytotoxic effect of CDDP and Gem in NTUB1. These data suggested that subamolide A exhibited a potent anti-proliferation activity. This study supports the traditional use of Cinnamomum subavenium stems with a therapeutic potential for the treatment of human urothelial carcinoma.

Topics: 4-Butyrolactone; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cell Survival; Cinnamomum; Cisplatin; Cytochromes c; Deoxycytidine; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; Gemcitabine; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Plant Preparations; Plant Stems; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Time Factors; Tumor Suppressor Protein p53; Urologic Neoplasms; Urothelium

A traditional Chinese medicine Selaginella doederleinii Hieron has been combined with radiotherapy for the treatment of human nasopharyngeal carcinoma in clinic in China. However, the detailed mechanism of anti-tumor effect of Selaginella doederleinii remains elusive.. This study was designed to investigate the anti-tumor effect of ethanol extract of Selaginella doederleinii (SDE) on human nasopharyngeal carcinoma and its possible mechanisms.. Viability, apoptosis and protein expression of tumor cells were analyzed by MTT, Annexin V staining and Western blot, respectively. Formation of intracellular reactive oxygen species was determined using dichlorofluorescin fluorescence. The in vivo anti-tumor effect was evaluated by measuring tumor volume changes and TUNEL staining in nude mice.. SDE significantly inhibited the growth and induced apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, SDE triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade. Moreover, SDE provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked SDE-induced disruption of mitochondrial membrane potential, caspases activation and apoptosis. Furthermore, a transplantable nude mice model was utilized to estimate the effectiveness of SDE in vivo. The treated mice displayed decreased tumor size, which was associated with enhanced apoptotic cell death.. These results, offering solid evidence of the induction of mitochondria-related apoptosis in tumor cells, provide the molecular theoretical basis of clinical application of Selaginella doederleinii for the treatment of human nasopharyngeal carcinoma.

Topics: Acetylcysteine; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Disease Models, Animal; Female; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Selaginellaceae

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

A synthetic amphiphilic block copolymer, Pluronic, is a potent chemosensitizer of multidrug resistant (MDR) cancers that has shown promise in clinical trials. It has unique activities in MDR cells, which include a decrease in ATP pools and inhibition of P-glycoprotein (Pgp) resulting in increased drug accumulation in cells. This work demonstrates that Pluronic rapidly (15min) translocates into MDR cells and co-localizes with the mitochondria. It inhibits complex I and complex IV of the mitochondria respiratory chain, decreases oxygen consumption and causes ATP depletion in MDR cells. These effects are selective and pronounced for MDR cells compared to non-MDR counterparts and demonstrated for both drug-selected and Pgp-transfected cell models. Furthermore, inhibition of Pgp functional activity also abolishes the effects of Pluronic on intracellular ATP levels in MDR cells suggesting that Pgp contributes to increased responsiveness of molecular "targets" of Pluronic in the mitochondria of MDR cells. The Pluronic-caused impairment of respiration in mitochondria of MDR cells is accompanied with a decrease in mitochondria membrane potential, production of ROS, and release of cytochrome c. Altogether these effects eventually enhance drug-induced apoptosis and contribute to potent chemosensitization of MDR tumors by Pluronic.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cytochromes c; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Mitochondria; Nitrogen Oxides; Oxygen Consumption; Phosphorylation; Poloxamer; Reactive Oxygen Species

CMTM5 (CKLF-like MARVEL transmembrane domain-containing member 5) exhibits tumor inhibition activity with frequent epigenetic inactivation in various tumor cell lines including cervical carcinoma (CC) cells. In this paper, we examined the function of CMTM5-v1 (the primary RNA splicing form) in both HeLa and SiHa cells. Overexpression of CMTM5-v1 in both cells can induce apoptosis, but the effects are more obvious in SiHa than that in HeLa. In SiHa cells, restoration of CMTM5-v1 caused disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase3 and cleavage of PARP. General caspase inhibitor almost prevented apoptosis of SiHa cells, suggesting that CMTM5-v1 induces apoptosis mainly through caspase-dependent pathway. These findings verify that CMTM5-v1 inhibits the growth of CC cell lines via inducing apoptosis.

Topics: Apoptosis; Carcinoma; Caspase 3; Cell Proliferation; Chemokines; Cytochromes c; Female; HeLa Cells; Humans; MARVEL Domain-Containing Proteins; Tumor Suppressor Proteins; Uterine Cervical Neoplasms

Topics: Carcinoma; Cytochromes c; Humans; Leukemia; Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational

MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription-independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function. Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46(P)) and (iii) promotes binding between p53Ser46(P) and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription. Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46(P) to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Line, Tumor; Cells, Cultured; Cisplatin; Cytochromes c; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitochondria; Ovarian Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Although N(1)-guanyl-1,7,-diamineoheptane (GC7), an inhibitor of deoxyhypusine synthase, has been shown to inhibit cell growth, the mechanism of its action is not completely understood. In this study, we investigated the mechanisms of the effects of GC7 on cell growth, differentiation and apoptosis in relation to adenosine monophosphate-activated protein kinase (AMPK) activation, as AMPK is known to be a possible target for cancer treatment.. The effects of GC7 on the growth of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4), was investigated using MTT assay, Western blotting, cell cycle analysis, DNA fragmentation and expression of apoptotic pathway proteins.. N(1)-guanyl-1,7,-diamineoheptane inhibited cell proliferation in a time- and dose-dependent manner in IHOK and HN4 cells. GC7 treatment decreased the expression of differentiation markers, such as involucrin, CK13 and CK19. The major mechanism of growth inhibition by GC7 treatment was induction of apoptosis, which is supported by sub-G(1) phase arrest, annexin V-FITC staining and DNA fragmentation analysis. GC7 treatment increased the cytosolic level of cytochrome c and resulted in caspase-3 activation. GC7 treatment also resulted in a strong activation of AMPK. Furthermore, specific AMPK activator blocked the GC7-induced growth inhibition effect, as well as apoptosis.. These results demonstrate that GC7 blocks immortalized and malignant keratinocyte cell proliferation and differentiation by inducing apoptosis through the mitochondrial and AMPK pathways. On the basis of these observations, we propose that a strategy combining GC7 and AMPK inhibition could be developed into a novel chemotherapeutic modality in oral cancer.

Topics: AMP-Activated Protein Kinases; Annexin A5; Apoptosis; Carcinoma; Caspase 3; Cell Cycle Proteins; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; G1 Phase; Guanine; Humans; Keratin-13; Keratin-19; Keratinocytes; Mitochondria; Mouth Mucosa; Mouth Neoplasms; Oxidoreductases Acting on CH-NH Group Donors; Protein Precursors

Alpinia pricei Hayata (A. pricei) is well known in Taiwan as a traditional Chinese medicine. In this study, the ability of ethanol (70%) extracts of A. pricei rhizome (AP extracts) to induce apoptosis in cultured human carcinoma KB cells was investigated. Treatment of KB cells with various concentrations of AP extracts (25-200 microg/mL) resulted in sequences of events marked by apoptosis, such as loss of cell viability, morphology change, and internucleosomal DNA fragmentation. AP extract-induced apoptotic cell-death was associated with loss of mitochondrial membrane potential, cytochrome c translocation, caspase-3 and -9 activation, and poly ADP-ribose polymerase (PARP) degradation in KB cells. This increase in AP extract-induced apoptosis was also associated with a reduction in the levels of Bcl-2, a potent cell-death inhibitor, and an increase in levels of the Bax protein, which heterodimerizes with and thereby inhibits Bcl-2. Furthermore, AP extracts induced a dose-dependent elevation of reactive oxygen species (ROS) in KB cells. Our findings suggest that A. pricei exerts antiproliferative action and growth inhibition on human carcinoma KB cells through a mitochondria-dependent apoptotic pathway. A. pricei may, therefore, have anticancer properties valuable for application in food and drug products.

Topics: Alpinia; Apoptosis; bcl-2-Associated X Protein; Carcinoma; Caspases; Cell Cycle; Cell Line; Collagen Type XI; Cytochromes c; Fibroblasts; Humans; Membrane Potential, Mitochondrial; Mitochondria; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Rhizome

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

Oestrogen (E2) is known to promote the proliferation of thyroid papillary carcinoma cells (KAT5). However, the molecular mechanism responsible is not well understood. In the study reported herein, the localization of ER alpha (ERalpha) and beta (ERbeta) in KAT5 and anaplastic carcinoma cells (FRO) was studied by immunofluorescence staining and by immunoblotting the proteins in subcellular fractions. Cell proliferation and apoptosis were also determined together with the expression of relevant proteins. The pattern of the subcellular localization of ERalpha and ERbeta differed between papillary and anaplastic cancer. Upon E2 treatment, the level of ERalpha increased in the nuclei of papillary cancer cells but ERbeta remained unchanged. The level of mitochondrial ERbeta surpassed that of ERalpha in anaplastic cancer cells. The different locations of ERalpha and ERbeta in KAT5 and FRO agreed with the finding that E2 promoted the proliferation of KAT5 but inhibited or did not affect that of FRO cells, and with the proposed functions of these two receptors. E2 inhibited the level of Bax in the mitochondria of papillary cancer, followed by a decrease of cytochrome c and/or apoptosis-inducing factor (AIF) release from the mitochondria into the cytosol. However, in anaplastic cancer, E2 promoted the expression of Bax in the mitochondria and the release of cytochrome c and/or AIF from mitochondria into the cytosol. Our results may explain the differences in epidemiology and responses to anti-tumour therapy between papillary and anaplastic cancer in terms of the subcellular localization of ER isoforms. In conclusion, the findings provide evidence to support the observation that E2 is an important factor in the development of thyroid cancer. The subcellular localization of ERalpha and ERbeta may account for the different pathogenesis of thyroid papillary and anaplastic cancers.

Topics: Apoptosis Inducing Factor; bcl-2-Associated X Protein; Carcinoma; Carcinoma, Papillary; Cell Death; Cell Proliferation; Cytochromes c; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Fulvestrant; Humans; Neoplasm Proteins; Receptors, Estrogen; Subcellular Fractions; Thyroid Neoplasms; Tumor Cells, Cultured

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

The biological functions and reaction pathways of lipocalins in mammalian system were sought. Mouse uterine 24p3 protein is a secreted lipocalin from mouse uterus. To evaluate the effect of uterine 24p3 protein on the reproductive system, endometrial carcinoma cell line (RL95-2) was an experimental target for achieving the in vitro study. The cells were treated with 0.75 microM dexamethasone (DEX) or under serum-free medium to mimic the stress environment for various time periods, then employing Western blot to measure the 24p3 protein secretion. It showed the time-dependent induction effect on 24p3 protein and suggested the level of protein secretion correlating to environmental stress. Furthermore, the supplementation of 24p3 protein to the medium accompanied the reduction of cell viability. It showed that the 24p3 protein may be a death factor under conditional media via PI and annexinV-FITC assay. Based on the autocrine hypothesis, we investigated the effect of 24p3 protein on cultured RL95-2 cells upon the 24p3 protein interaction. We have demonstrated significant increase in intracellular reactive oxygen species upon 24p3 protein interaction. While the changes of mitochondrial membrane potential and cytochrome c release from mitochondria occurred, the activities of caspase-8, -9 and -3 were found to have increased. The condensation of DNA was occurred suggesting that 24p3 protein induced irreparable DNA damage, which in turn triggered the process of apoptosis. It shows evidence for the direct effect of this protein on endometrial cells. These findings suggest that 24p3 protein creates an intracellular oxidative environment that induces apoptosis in RL95-2 cells.

Topics: Acute-Phase Proteins; Apoptosis; Blotting, Western; Carcinoma; Caspases; Cell Line, Tumor; Cell Survival; Coloring Agents; Culture Media; Cytochromes c; Dexamethasone; DNA, Neoplasm; Endometrial Neoplasms; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; HeLa Cells; Humans; Lipocalin-2; Lipocalins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Proto-Oncogene Proteins; Reactive Oxygen Species; Trypan Blue; Uterus

Galectin-3 (GAL3), a beta-galactoside-binding lectin, confers chemoresistance to a wide variety of cancer cell types. It may exhibit anti- or pro-apoptotic activity depending on the nature of the stimulus. We report here that introducing phosphorylated galectin-3 (P-GAL3) into GAL3-null, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant human breast carcinoma cells promotes TRAIL-induced apoptotic cell death by stimulating the phosphorylation/inactivation of the pro-apoptotic molecule Bad resulting in the inhibition of mitochondrial depolarization and the release of cytochrome c. Exposure of the transfectant cells to TRAIL leads to the recruitment of the initiator capase-8 followed by activation of the effector caspase-9, independent of cytochrome c, and subsequently the processing of the executioner caspase-3. P-GAL3 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) were coordinately expressed, with concomitant dephosphorylation of Akt in TRAIL-sensitive cells. In contrast, overexpression of phospho-mutant GAL3 (incapable of phosphorylation) failed to elicit similar responses. Depletion of PTEN using small interference RNAs reinstated Akt phosphorylation and conferred TRAIL resistance. In addition phosphatidylinositol 3-kinase inhibitors rendered the phospho-mutant GAL3-resistant cells sensitive to TRAIL. These findings suggest a pivotal role for P-GAL3 in promoting TRAIL sensitivity through activation of a nonclassic apoptotic pathway and identify P-GAL3 as a novel regulator of PTEN.

Topics: Apoptosis; Breast Neoplasms; Carcinoma; Chromosomes, Human, Pair 10; Cytochromes c; Enzyme Inhibitors; Galectin 3; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Models, Biological; Phosphorylation; PTEN Phosphohydrolase; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

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

Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.

Topics: Amaryllidaceae Alkaloids; Antineoplastic Agents, Phytogenic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carcinoma; Caspases; Cytochromes c; DNA Fragmentation; Drug Resistance, Neoplasm; Enzyme Activation; fas Receptor; Female; Fibroblasts; Humans; Male; Mitochondria; Narcissus; Neoplasm Proteins; Phenanthridines; Prostatic Neoplasms; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor

Berberine, a naturally occurring isoquinoline alkaloid, has been shown to possess anti-inflammatory and antitumor properties in some in vitro systems. Here, we report that in vitro treatment of androgen-insensitive (DU145 and PC-3) and androgen-sensitive (LNCaP) prostate cancer cells with berberine inhibited cell proliferation and induced cell death in a dose-dependent (10-100 micromol/L) and time-dependent (24-72 hours) manner. Treatment of nonneoplastic human prostate epithelial cells (PWR-1E) with berberine under identical conditions did not significantly affect their viability. The berberine-induced inhibition of proliferation of DU145, PC-3, and LNCaP cells was associated with G1-phase arrest, which in DU145 cells was associated with inhibition of expression of cyclins D1, D2, and E and cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 proteins, increased expression of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27), and enhanced binding of Cdk inhibitors to Cdk. Berberine also significantly (P < 0.05-0.001) enhanced apoptosis of DU145 and LNCaP cells with induction of a higher ratio of Bax/Bcl-2 proteins, disruption of mitochondrial membrane potential, and activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase. Pretreatment with the pan-caspase inhibitor z-VAD-fmk partially, but significantly, blocked the berberine-induced apoptosis, as also confirmed by the comet assay analysis of DNA fragmentation, suggesting that berberine-induced apoptosis of human prostate cancer cells is mediated primarily through the caspase-dependent pathway. The effectiveness of berberine in checking the growth of androgen-insensitive, as well as androgen-sensitive, prostate cancer cells without affecting the growth of normal prostate epithelial cells indicates that it may be a promising candidate for prostate cancer therapy.

Topics: Androgens; Antineoplastic Agents, Phytogenic; Apoptosis; Berberine; Carcinoma; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Epithelial Cells; G1 Phase; Humans; Male; Membrane Potentials; Mitochondria; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2

Photodynamic therapy (PDT) typically involves systemic or topical administration of a tumor-localizing photosensitizer or prodrug and its subsequent activation by visible light. This results primarily in singlet oxygen-induced photodamage to the tumor. 5-Aminolevulinic acid (ALA) and its derivatives have recently been widely used for PDT due to their selective induction in tumor of endogenous protoporphyrin IX (PpIX), a potent photosensitizer. Although ALA-PDT has achieved successful results in the treatment of several clinical oncological and nononcological diseases, the mechanisms of this modality are still not fully elucidated. In the present study, the human colon carcinoma cell line 320DM was treated in vitro with PDT using hexaminolevulinate (HAL), a hexylester of ALA known to be 50 to 100 times more efficient at producing PpIX formation than ALA itself. PpIX production increased with increasing HAL concentrations in the cells and phototoxicity of the cells was enhanced with increasing light (450 nm) doses. HAL-PDT induced apoptotic cell death, as measured by nuclear staining of Hoechst 33342 for fluorescence microscopy, DNA electrophoresis and TdT staining for flow cytometry. PDT with 5 muM of HAL and a light dose of 640 mJ/cm2 produced a 75% apoptotic cell population 40 hr after the treatment. Furthermore, the loss of mitochondrial membrane potential coincident with the release of cytochrome c from the mitochondria into the cytosol led to a rapid activation of caspase-9 and caspase-3 (an executioner), indicating that the selective damage to the mitochondria by HAL-PDT can induce a cytochrome-c-mediated apoptotic response in the 320DM cells.

Topics: Aminolevulinic Acid; Apoptosis; Carcinoma; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Humans; Membrane Potentials; Mitochondria; Photochemotherapy; Photosensitizing Agents; Protoporphyrins

Sensitivity of tumor cells to treatment with anticancer drugs depends on expression and function of antiapoptotic and antioxidant proteins. The goal of our study was to determine the functional role of the novel antioxidant protein Peroxiredoxin V (PrxV), in protection of human lung carcinoma cell lines against apoptosis. Analysis of expression of PrxV in multiple lung carcinoma cell lines revealed a positive correlation between the expression of PrxV and radioresistance in vitro. Clones of the lung carcinoma cells U1810 with down-regulated expression of PrxV, or with its impaired enzymatic function (expression of redox-negative PrxV), demonstrated increased sensitivity to treatment with anticancer drugs etoposide and adriamycin. Pre-treatment of these clones with antioxidant N-acetyl-cysteine did not change their sensitivity to adriamycin, suggesting the involvement of a non-redox activity of PrxV. Expression of the redox-negative PrxV mainly affected the mitochondrial pathway of apoptosis, as assessed by cytochrome c release assay. Impairment of the PrxV enzymatic function also affected transmembrane potential and calcium loading capacity of mitochondria, as well as mitochondrial morphology. Altogether, these findings suggest that PrxV is a multifunctional protein, which is essential for protection against apoptosis induced by anticancer drugs.

Topics: Apoptosis; Calcium; Carcinoma; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cytochromes c; Down-Regulation; Humans; Lung Neoplasms; Membrane Potentials; Mitochondria; Peroxidases; Peroxiredoxins

Amooranin (AMR), a natural triterpenoid drug isolated and characterized from Amoora rohituka stem bark, is cytotoxic to SW620 human colon carcinoma cell line with an IC(50) value of 2.9 microg/ml. This novel compound caused depolarization of mitochondrial membrane and decrease of membrane potential, indicating initial signal of apoptosis induction. The percentage of cells with decreased mitochondrial potential ranged from 7.4% at 1 microg/ml to 60.5% at 100 microg/ml AMR. Flow cytometric analysis of apoptosis using Annexin-V-FITC staining showed that the percentage of apoptotic cells ranged from 7.5% at 1 microg/ml to 59.2% at 100 microg/ml AMR. AMR-induced apoptosis was accompanied by redistribution of cytochrome c from mitochondria to cytosol as well as down regulation of Bcl-2 and Bcl-X(L) proteins in a dose-dependent manner. SW620 human colon carcinoma xenograft mice treated with AMR showed significant reduction in tumor growth rates compared to saline- and doxorubicin-treated groups. The reduction in tumor growth rate was better in xenografts treated with 2 mg/kg AMR than 5 and 10 mg/kg treated mice. The analysis of global gene expression changes induced by AMR in xenograft tumors by microarray hybridization revealed that several genes involved in energy pathways, transport, apoptosis, immune response, nucleic acid metabolism, protein metabolism, cell growth and/or maintenance, signal transduction and cell communication, were affected by this natural cancer drug. These results suggest that the anticancer properties of AMR in SW620 human colon carcinoma cell line are mediated through its effects on functional genomics, targeting the apoptotic process.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous; Triterpenes

alpha-Santalol, an active component of sandalwood oil, has been studied in detail in recent years for its skin cancer preventive efficacy in murine models of skin carcinogenesis; however, the mechanism of its efficacy is not defined. Two major biological events responsible for the clonal expansion of transformed/initiated cells into tumors are uncontrolled growth and loss of apoptotic death. Accordingly, in the present study, employing human epidermoid carcinoma A431 cells, we assessed whether alpha-santalol causes cell growth inhibition and/or cell death by apoptosis. Treatment of cells with alpha-santalol at concentrations of 25-75 microM resulted in a concentration- and a time-dependent decrease in cell number, which was largely due to cell death. Fluorescence-activated cell sorting analysis of Annexin V/propidium iodide (PI) stained cells revealed that alpha-santalol induces a strong apoptosis as early as 3 h post-treatment, which increases further in a concentration- and a time-dependent manner up to 12 h. Mechanistic studies showed an involvement of caspase-3 activation and poly(ADP-ribose) polymerase cleavage through activation of upstream caspase-8 and -9. Further, the treatment of cells with alpha-santalol also led to disruption of the mitochondrial membrane potential and cytochrome c release into the cytosol, thereby implicating the involvement of the mitochondrial pathway. Pre-treatment of cells with caspase-8 or -9 inhibitor, pan caspase inhibitor or cycloheximide totally blocked alpha-santalol-caused caspase-3 activity and cleavage, but only partially reversed apoptotic cell death. This suggests involvement of both caspase-dependent and -independent pathways, at least under caspase inhibiting conditions, in alpha-santalol-caused apoptosis. Together, this study for the first time identifies the apoptotic effect of alpha-santalol, and defines the mechanism of apoptotic cascade activated by this agent in A431 cells, which might be contributing to its overall cancer preventive efficacy in mouse skin cancer models.

Topics: Apoptosis; Carcinoma; Caspase Inhibitors; Caspases; Cell Proliferation; Cycloheximide; Cytochromes c; Enzyme Inhibitors; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Plant Oils; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Polycyclic Sesquiterpenes; Sesquiterpenes; Skin Neoplasms; Tumor Cells, Cultured

The chemical compound ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), isolated from the Chinese herbal medicine plant Pteris semipinnata L, has been known to exert antitumor activity. However, the molecular mechanism of the action is not understood. In this study we demonstrated that apoptotic cell death induced by 5F in FRO cells was concentration- and time-dependent. The rapid increase in intracellular reactive oxygen species (ROS) levels was involved in the mechanism of cell death. c-Jun N-terminal kinase (JNK) activation and G2 block were related to cell death induced by 5F. Extracellular signal-related kinase (ERK) and p38 were also activated, but as survival signals in response to 5F treatment to counteract the induction of cell death. In the process of the induction of apoptotic cell death, Bax translocated into mitochondria, a reduction in Delta psi(m) was observed and a release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria into the cytosol occurred, indicating that cell death induced by 5F was through a mitochondrial-mediated pathway.

Topics: Apoptosis Inducing Factor; Carcinoma; Caspase 3; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Diterpenes; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; G2 Phase; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Mitochondria; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Protein Transport; Reactive Oxygen Species; Thyroid Neoplasms; Time Factors

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

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L), a member of the tumor necrosis factor (TNF) gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively kill tumor cells. Although microenvironments of solid tumors (hypoxia, nutrient deprivation, and low pH) often affect the effectiveness of chemotherapy, few studies have been reported on the relationship between tumor microenvironments and TRAIL. In this study, we investigated whether low extracellular pH affects TRAIL-induced apoptotic death. When human prostate carcinoma DU145 cells were treated with 200 ng/ml His-tagged TRAIL for 4 h, the survival was approximately 10% at pH 6.3-6.6 and 61.3% at pH 7.4. Similar results were observed in human colorectal carcinoma CX-1 cell line. The TRAIL-mediated activation of caspase, cytochrome c release, and poly (ADP-ribose) polymerase (PARP) cleavage was promoted at low extracellular pH. Immunoprecipitation followed by western blot analysis shows that low extracellular pH enhances the association of truncated Bid with Bax during treatment with TRAIL. Western blot analysis also shows that the low extracellular pH-enhanced TRAIL cytotoxicity does not involve modulation of the levels of TRAIL receptors (DR4, DR5, and DcR2), FLIP, inhibitor of apoptosis (IAP), and Bcl-2. Overexpression of Bcl-2 effectively prevented low extracellular pH-augmented TRAIL cytotoxicity. Taken together, we propose that TRAIL-mediated cytotoxicity is greatly enhanced in low pH environments by promoting caspase activation.

Topics: Adenocarcinoma; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Carrier Proteins; Caspases; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Male; Membrane Glycoproteins; Mitochondria; Models, Biological; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

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

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.

Topics: Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Carcinoma; Carrier Proteins; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Mutation; Oligopeptides; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Photodynamic treatment (PDT) can elicit a diverse range of cellular responses, including apoptotic cell death. Previously, we showed that PDT stimulates caspase-3 activation and subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. Curcumin, the yellow pigment of Curcuma longa, is known to have anti-oxidant and anti-inflammatory properties. In the present study, using Rose Bengal (RB) as the photosensitizer, we investigated the effect of curcumin on PDT-induced apoptotic events in human epidermal carcinoma A431 cells. We report that curcumin prevented PDT-induced JNK activation, mitochondrial release of cytochrome c, caspase-3 activation, and cleavage of PAK2. Using the cell permeable dye DCF-DA as an indicator of reactive oxygen species (ROS) generation, we found that both curcumin and ROS scavengers (i.e., l-histidine, a-tocopherol, mannitol) abolished PDT-stimulated intracellular oxidative stress. Moreover, all these PDT-induced apoptotic changes in cells could be blocked by singlet oxygen scavengers (i.e., l-histidine, a-tocopherol), but were not affected by the hydroxyl radical scavenger mannitol. In addition, we found that SP600125, a JNK-specific inhibitor, reduced PDT-induced JNK activation as well as caspase-3 activation, indicating that JNK activity is required for PDT-induced caspase activation. Collectively, these results demonstrate that singlet oxygen triggers JNK activation, cytochrome c release, caspase activation and subsequent apoptotic biochemical changes during PDT and show that curcumin is a potent inhibitor for this process.

Topics: alpha-Tocopherol; Apoptosis; Carcinoma; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Curcumin; Cytochromes c; Enzyme Activation; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Oxidative Stress; p21-Activated Kinases; Photochemotherapy; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Reactive Oxygen Species

The purpose of this study was to determine whether expression of tissue transglutaminase (TG2) and caspase-3 proteins in drug-resistant breast carcinoma MCF-7/DOX cells would render these cells selectively susceptible to apoptotic stimuli. Despite high resistance to multidrug resistance (MDR)-related drug, doxorubicin (> or =150-fold), the MCF-7/DOX cells were extremely sensitive to apoptotic stimuli. Thus, calcium ionophore, A23187 (A23187) and the protein kinase C inhibitor staurosporine (STS) each induced rapid and time-dependent apoptosis in MCF-7/DOX cells. The apoptosis induced by either agent was accompanied by caspase-3 activation and other downstream changes that are typical of cells undergoing apoptosis. The alterations upstream of caspase-3 activation, however, such as loss in mitochondrial membrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-8, and caspase-9, were detected only in STS-treated cells. The A12387 failed to induce any of the caspase-3 upstream changes, implying that A23187-induced apoptosis may utilize one or more novel upstream pathways leading to the activation of caspase 3. In summary, these data demonstrate that MCF-7/DOX cells are much more sensitive to apoptotic stimuli than previously thought and that A23187-induced apoptosis may involve some novel, yet unidentified, upstream pathway that leads to the activation of caspase-3 and other downstream events.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Calcimycin; Carcinoma; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cytochromes c; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Ionophores; Membrane Potentials; Mitochondria; Staurosporine

Cytotoxic agents eliminate tumor cells via different mechanisms including apoptosis, although this process is not equally efficient in all kinds of cancer cells. Thus, small cell lung carcinomas (SCLCs) are more sensitive than non-small cell lung carcinomas (NSCLCs) to therapy-induced killing. During apoptosis, several apoptogenic proteins release from the mitochondria. Among these proteins is Smac/DIABLO. Overexpression of Smac effectively potentiates apoptosis by neutralizing the caspase-inhibitory function of the inhibitors of apoptosis proteins (IAPs). However, the physiological relevance of endogenously released Smac in the promotion of malignant cell death is still unclear. Analysis of a panel of human lung cancer cell lines revealed that there is no altered Smac expression in NSCLC and SCLC that might initially impair the drug-induced cell death. Upon engagement of the mitochondrial pathway of apoptosis, etoposide provoked cytosolic accumulation of Smac along with cytochrome c and loss of the mitochondrial membrane potential. Most of these events as well as nuclear apoptotic changes required caspase activation in SCLC, but not in NSCLC. Unexpectedly, pan-caspase inhibition had no effect on Smac release. Co-treatment of SCLC with the IAP-binding peptide Smac-N7 enhanced etoposide-induced apoptosis in a concentration-dependent manner, whereas Smac downregulation by small interfering RNA (siRNA) did not influence caspase-3/-7 activities, nuclear morphological changes, DNA fragmentation, and plasma membrane integrity. Release of cytochrome c and mitochondrial protease Omi/HtrA2 is still detectable at these conditions. These data suggest that Smac deficiency may be compensated for by action of redundant determinants to kill cancer cells. Thus, translocation of endogenous Smac into cytosol does not play a critical role in cell death of human lung carcinoma after etoposide treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carrier Proteins; Caspases; Cell Line, Tumor; Cell Membrane; Cytochromes c; DNA Fragmentation; Down-Regulation; Etoposide; HeLa Cells; High-Temperature Requirement A Serine Peptidase 2; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Peptides; Protein Transport; Proteins; RNA, Small Interfering; Serine Endopeptidases

Inhibitors of histone deacetylase (HDAC) are considered as potential anticancer agents. We have previously demonstrated that an inhibitor of HDAC, sodium butyrate (NaB), induces apoptosis of breast cancer cells in a P53-independent and P21(waf1)-dependent manner. In this study, we showed that tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and anti-Fas agonist antibody potentiated NaB-induced growth inhibition through synergistic induction of apoptosis in breast cancer cell lines (MCF-7, T47-D, and BT-20). In MCF-7 cells, NaB increased the expression of death receptors; NaB alone or in combination with TNF-alpha, TRAIL, and anti-Fas agonist antibody increased the levels of Bid, tBid, and that of cytosolic cytochrome c. Synergistic induction of apoptosis was strongly inhibited by dominant-negative Fas-associated death domain (FADD) and inhibitors of caspases-8 and -9, indicating that potentiation of apoptosis involved key elements of death receptors' signaling pathways. Moreover, cotreatment of NaB and ligands of death receptors up-regulated the levels of P21(waf1) and that of proliferating cell nuclear antigen (PCNA) associated with P21(waf1). Transient transfections of p21(waf1) antisense or p21(waf1) deficient for its interaction with PCNA abolished synergistic induction of apoptosis. This suggested that potentiation of apoptosis by cotreatments required P21(waf1) and its interaction with PCNA. Since breast tumors contain rarely p21 mutations, our results may open interesting prospects in the fight against breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Antibodies; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Butyrates; Carcinoma; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytochromes c; Drug Synergism; fas Receptor; Fas-Associated Death Domain Protein; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Membrane Glycoproteins; Proliferating Cell Nuclear Antigen; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Up-Regulation

We are attempting to elucidate the mechanism of apoptotic cell death induced by hypoxia in oral cancer cells. Since hypoxia can render solid tumors more resistant to radiation and chemotherapy, understanding the pathways involved in hypoxia-induced apoptosis of oral cancer cells would be of significant therapeutic value.. Here we showed that oral cancer cells from primary tumor and lymph node metastasis undergo apoptosis after 24 to 48 h of hypoxia. During hypoxic growth, an increase in caspase-3 proteolytic activity was observed, accompanied by the cleavage of PARP (poly (ADP-ribose) polymerase) indicative of caspase activity. In addition, hypoxic stress also lead to activation of caspase-8, -9, and -10 but not -1, elicited the release of cytochrome C into the cytosol, and resulted in internucleosomal DNA fragmentation.. These results show that hypoxia-induced apoptosis in oral carcinoma cell lines relies on both intrinsic (mitochondrial) and extrinsic (cell death receptor mediated) pathways. This novel evidence will assist in designing more efficient combination chemotherapy approaches as promising strategy for the treatment of oral cancers.

Topics: Aged; Apoptosis; Carcinoma; Caspases; Cell Hypoxia; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Humans; Male; Middle Aged; Mouth Neoplasms; Poly(ADP-ribose) Polymerases; Signal Transduction

Antiandrogens such as Casodex (Bicalutamide) are designed to treat advance stage prostate cancer by interfering with androgen receptor-mediated cell survival and by initiating cell death. Treatment of androgen sensitive, non-metastatic LNCaP human prostate cancer cells with 0-100 microM Casodex or 0-10 ng/ml TNF-alpha induces cell death in 20-60% of the cells by 48 h in a dose-dependent manner. In cells treated with TNF-alpha, this is accompanied by the loss of mitochondrial membrane potential (DeltaPsim) and cell adhesion. In contrast, cells treated with Casodex display loss of cell adhesion, but sustained mitochondrial dehydrogenase activity. Overexpression of Bcl-2 in LNCaP cells attenuates the induction of cell death by TNF-alpha but not Casodex, suggesting that mitochondria depolarization is not required for the induction of cell death by Casodex. While both TNF-alpha and Casodex-induced release of cytochrome c in LNCaP cell is predominantely associated with the translocation and cleavage of Bax, our data also suggest that Casodex induces cell death by acting on components downstream of decline of DeltaPsim and upstream of cytochrome c release. Furthermore, while induction of both caspase-3 and caspase-8 activities are observed in TNF-alpha and Casodex-treated cells, a novel cleavage product of procaspase-8 is seen in Casodex-treated cells. Taken together, these data support the hypothesis that Casodex induces cell death by a pathway that is independent of changes in DeltaPsim and Bcl-2 actions and results in an extended lag phase of cell survival that may promote the induction of an invasive phenotype after treatment.

Topics: Androgen Antagonists; Anilides; bcl-2-Associated X Protein; Carcinoma; Caspases; Cell Adhesion; Cell Death; Cell Nucleus; Cell Survival; Cytochromes c; Humans; Male; Membrane Potentials; Mitochondria; Nitriles; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tosyl Compounds; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

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