cytochrome-c-t and Nasopharyngeal-Neoplasms

Bacterial pore-forming toxins, BinA and BinB together known as the binary toxin are potent insecticidal proteins, that share structural homology with antitumor bacterial parasporin-2 protein. The underlying molecular mechanism of Bin toxin-induced cancer cell cytotoxicity requires more knowledge to understand whether the toxin induced human cytotoxic effects occur in the same way as that of parasporin-2 or not.. In this study, anticancer properties of Lysinibacillus sphaericus derived Bin toxin on HK1 were evaluated through MTT assay, morphological analysis and lactate dehydrogenase efflux assay. Induction of apoptosis was determined from RT-qPCR, caspase activity and cytochrome c release assay. Internalization pattern of Bin toxin in HK1 cells was studied by confocal laser-scanning microscopic analysis.. Activated Bin toxin had strong cytocidal activity to HK1 cancer cell line at 24 h postinoculation. Both BinA and BinB treated HK1 cells showed significant inhibition of cell viability at 12 μM. Induction of apoptotic mediators from RT-qPCR and caspase activity analyses indicated the activation of programmed cell death in HK1 cells in response to Bin toxin treatment. Internalization pattern of Bin toxin studied by using confocal microscopy indicated the localization of BinA on cell surface and internalization of BinB in the cytoplasm of cancer cells as well as colocalization of BinA with BinB. Evaluation of cytochrome c release also showed the association of BinB and BinA+BinB with mitochondria.. Bin toxin is a cytotoxic protein that induces cytotoxic and apoptotic events in HK1 cells, and may have high therapeutic potential as an anti-cancer agent.

Topics: Apoptosis; Bacterial Toxins; Caspases; Cytochromes c; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms

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

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

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

Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cells are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential (ΔΨm), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).

Topics: Antineoplastic Agents; Apoptosis; Asteraceae; Caspases; Cell Cycle; Cell Line, Tumor; Cytochromes c; Humans; Lactones; Nasopharyngeal Neoplasms; Proto-Oncogene Proteins c-akt; Sesquiterpenes

Previous studies revealed that polydatin, a natural small compound, possessed protective effect against ischemia/reperfusion injury and inflammation. However, the action and molecular mechanism of its potent anti-cancer activity remain poorly understood. In the present study, polydatin significantly killed several human tumor cell lines in a dose- and time-dependent manner. The compound also dose-dependently caused mitochondrial apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, polydatin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt in CNE cells, while knock-down of CCAAT/enhancer-binding protein homologous protein (CHOP) dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of polydatin. Furthermore, polydatin provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting polydatin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in CNE cells. Taken together, these findings strongly suggest that polydatin might be a promising anti-tumor drug and our data provide the molecular theoretical basis for clinical application of polydatin.

Topics: Apoptosis; Base Sequence; Caspases; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum; Glucosides; Humans; Mitochondria; Nasopharyngeal Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA, Small Interfering; Stilbenes; Stress, Physiological

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

Previous studies have shown that Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) enhances etoposide-induced apoptosis in epithelial cells. Our study was undertaken to further dissect the modulation of tumor cell apoptosis by this viral protein. Using an inducible system of LMP1 expression in HeLa cells, we show herein that etoposide-triggered apoptosis, as evidenced by nuclear condensation and caspase-3 activation, is enhanced by LMP1. LMP1 also potentiates etoposide-induced processing and activation of caspase-2 in this model and enhances the dissipation of mitochondrial transmembrane potential and the release of cytochrome c in response to etoposide. Moreover, cisplatin-triggered activation of caspases 2 and 3 is potentiated upon expression of LMP1. A similar LMP1-mediated enhancement of cisplatin-induced caspase activation was seen upon stable transfection of wild-type LMP1 into the nasopharyngeal carcinoma cell line, TW03. Finally, using deletion mutants of LMP1 to determine the region of LMP1 required for apoptosis potentiation, we found that amino acids 350-386 (located within the CTAR2 domain) were responsible for sensitizing cells to cisplatin. We conclude that LMP1-dependent potentiation of stress-induced apoptosis occurs at an early step in the apoptosis cascade, upstream of the activation of caspase-2, and involves the C-terminal signaling domain of LMP1. These findings could have important ramifications for the treatment of EBV-associated malignancies of epithelial origin, including nasopharyngeal carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Caspase 2; Caspases; Cisplatin; Cytochromes c; Enzyme Activation; Etoposide; HeLa Cells; Herpesvirus 4, Human; Humans; Membrane Potentials; Mitochondria; Nasopharyngeal Neoplasms; Protein Structure, Tertiary; Stress, Physiological; Transfection; Tumor Cells, Cultured; Viral Matrix Proteins

The mechanism of action, leakage of cytochrome c from mitochondria into cytosol, for the antineoplastic compound glyfoline was examined. Additionally, our current studies revealed that glyfoline induced apoptotic changes and arrested cell cycle procession at the G2/M phase in nasopharyngeal carcinoma (NPC). A reverse transcriptase polymerase chain reaction (RT-PCR) showed no specific changes of apoptosis-related gene expression (i. e., bax, ICE-alpha,beta, bcl-2, and c-myc). However, no similar changes were detected in fibroblasts and peripheral lymphocytes after glyfoline treatment suggesting that glyfoline has a higher affinity for tumor cells than for normal cells.

Topics: Acridines; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cytochromes c; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Mitochondria; Nasopharyngeal Neoplasms; Phytotherapy; Plant Extracts; Reverse Transcriptase Polymerase Chain Reaction; Rutaceae

Apoptosis is a tightly controlled multistep mechanism of cell death, and mitochondria are considered to play a central role in this process. Mitochondria initiate two distinct apoptosis pathways, one caspase-dependent and the other caspase-independent. In addition, mitochondrial production of reactive oxygen species (ROS) seems to play a role in cell death. Most chemotherapeutic agents induce apoptosis through at least one of these pathways. The post-initiation mechanisms of gold(III) porphyrin 1a were investigated in this study. HONE1 cells exposed to gold(III) porphyrin 1a underwent apoptosis after 24 hours. Functional proteomic studies revealed the alteration of several cytoplasmic protein expressions in HONE1 cells after treatment with the drug. These proteins include enzymes participating in energy production and proteins involved in cellular redox balance. There was a quick attenuation of mitochondrial membrane potential (DeltaPsi(m)) with the alterations of Bcl-2 family proteins, the release of cytochrome c, and apoptosis-inducing factor (AIF) following gold(III) porphyrin 1a treatment. Cytochrome c in turn activated caspase-9 and caspase-3. Cotreatment with caspase inhibitor (zVAD-fmk) showed that the activated caspases worked in conjunction with AIF-initiated apoptosis pathways. Further study showed that ROS played a part in gold(III) porphyrin 1a-induced apoptosis by regulating DeltaPsi(m). In summary, gold(III) porphyrin 1a induced apoptosis through both caspase-dependent and caspase-independent mitochondrial pathways, and intracellular oxidation affected gold(III) porphyrin 1a-induced apoptosis. These results support a role for gold(III) porphyrin 1a as a promising anticancer drug lead and as a possible novel therapeutic agent directed toward the mitochondria.

Topics: Apoptosis; Apoptosis Inducing Factor; Caspases; Cytochromes c; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Gold Compounds; Humans; Mass Spectrometry; Membrane Potentials; Mitochondria; Nasopharyngeal Neoplasms; Oxidation-Reduction; Porphyrins; Proteomics; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured