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1.
Brain Res ; 1766: 147515, 2021 09 01.
Article in English | MEDLINE | ID: mdl-33984327

ABSTRACT

OBJECTIVE: This work was designed to explore whether the combination of Tanshinone IIA (T-IIA) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has a direct anti-cancer effect in glioblastoma (GBM) and the possible mechanisms. METHODS: GBM cells (U-87 and U-251 MG) were treated with T-IIA or/and TRAIL, or the expression of death receptors (DRs), DR4 and DR5, was suppressed in GBM cells. The activity of GBM cells was determined by MTT, and the apoptosis was assessed by Hoechst33342 staining and flow cytometry. The expression levels of cleaved caspase-3/8/9, phosphorylated (p)-STAT3 as well as DR4 and DR5 in GBM cells were assessed by Western blotting. A nude mouse xenograft model was constructed to evaluate the effects of T-IIA and TRAIL cotreatment on tumor growth and apoptosis in vivo. RESULTS: After T-IIA treatment, GBM cells resumed the sensitivity to TRAIL-induced apoptosis dependent on inhibition of p-STAT3 and activation of DR4, DR5 and caspases. DR4 or/and DR5 knockdown significantly abated the co-effect of T-IIA and TRAIL on GBM cell apoptosis and proliferation. Furthermore, T-IIA and TRAIL cotreatment markedly inhibited the growth of transplanted tumor and activated U87 cell apoptosis in nude mice. CONCLUSION: T-IIA increases TRAIL-induced apoptosis by downregulating STAT3 and upregulating DR4 and DR5, indicating T-IIA therapy as a novel treatment strategy for TRAIL-resistant GBM.


Subject(s)
Abietanes/administration & dosage , Apoptosis/drug effects , Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , Receptors, Death Domain/biosynthesis , STAT3 Transcription Factor/antagonists & inhibitors , TNF-Related Apoptosis-Inducing Ligand/administration & dosage , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Apoptosis/physiology , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Drug Synergism , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Receptors, Death Domain/genetics , STAT3 Transcription Factor/metabolism , Xenograft Model Antitumor Assays/methods
2.
Tissue Cell ; 48(3): 160-7, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27105607

ABSTRACT

Disruption of the adhesive role of beta-catenin by caspases has been reported; however, the relationship between the Wnt/beta-catenin signaling pathway and apoptosis remains unclear. Therefore, we aimed to evaluate squamous epithelial cells in cervicovaginal smears by using cytological and immunocytochemical methods to observe changes in the presence and localization of beta-catenin during apoptosis, death receptor-, and mitochondria-mediated apoptosis. We investigated 224 cervicovaginal smears using the Papanicolaou method. Anti-beta-catenin and anti-cleaved caspase 3, 8, and 9 antibodies were used for immunocytochemical staining. Apoptotic cells were negative for beta-catenin. This showed that the Wnt/beta-catenin signaling pathway was inactive in apoptotic cells. However, beta-catenin showed intense positivity in the membrane, cytoplasm, and nucleus of non-apoptotic epithelial cells around these apoptotic cells. Therefore, the Wnt/beta-catenin signaling pathway was active in non-apoptotic epithelial cells, and this activity in non-apoptotic cells may have been induced by apoptotic cells. A highly significant association between the presence of death receptor-mediated apoptosis and the activity of the Wnt/beta-catenin signaling pathway was also found (P<0.001). In conclusion, the Wnt/beta-catenin signaling pathway was found to be inactive in apoptotic cells, but apoptotic cells may induce the Wnt/beta-catenin signaling pathway in non-apoptotic cells to compensate for a decrease in epithelial cells because of apoptosis in order to maintain epithelial tissue integrity.


Subject(s)
Apoptosis/genetics , Receptors, Death Domain/biosynthesis , Wnt Signaling Pathway , beta Catenin/biosynthesis , Caspase 3/metabolism , Epithelial Cells/metabolism , Female , Gene Expression Regulation , Humans , Receptors, Death Domain/isolation & purification , Vaginal Smears/methods , beta Catenin/genetics
3.
Biotechniques ; 59(4): 231-8, 240, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26458551

ABSTRACT

We developed a strategy for identifying modulators of juxtacrine signaling, triggered by a cell-surface ligand displayed on synthetic lipid bilayers, via cognate receptors on apposed cells. Using readouts for receptor lateral transport and intracellular signaling, we screened a small interfering RNA (siRNA) library and identified specific receptor tyrosine kinases (RTKs) that directly or indirectly modulate apoptosis signaling by a model death ligand through its cognate death receptors. This approach may be broadly useful for studying juxtacrine cell-cell signaling systems.


Subject(s)
Apoptosis/genetics , Cell Communication/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , Cell Line, Tumor , Gene Expression Regulation , Humans , Ligands , Lipid Bilayers/chemical synthesis , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Receptors, Death Domain/biosynthesis , Receptors, Death Domain/genetics , Signal Transduction/genetics , TNF-Related Apoptosis-Inducing Ligand/chemistry , TNF-Related Apoptosis-Inducing Ligand/genetics
4.
Arch Pharm Res ; 37(4): 501-11, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24395529

ABSTRACT

It has been known that myriocin inhibits melanoma growth. However, the effects and action mechanisms of myriocin on lung cancer cell growth have not been reported. In this study, we examined whether myriocin isolated from Mycelia sterilia inhibits cell growth of lung cancer cells (A549 and NCI-H460) as well as possible signaling pathways involved in cell growth inhibition. Different concentrations of myriocin inhibited the growth of lung cancer cells through the induction of apoptotic cell death. Consistent with cancer cell growth inhibition, myriocin induced the expression of death receptors (DRs) as well as p-JNK and p-p38 in both cell lines. Moreover, the combination of myriocin with DR4 ligand TRAIL, and other well known anti-tumor drugs (docetaxel and cisplatin) synergistically inhibited cancer cell growth, and induced DR4 expression. These results showed that myriocin inhibits lung cancer cells growth through apoptosis via the activation of DR4 pathways, and enhanced anti-cancer effects with well known drugs. Thus, our study indicates that myriocin could be effective for lung cancer cells as an anti-cancer drug and/or a conjunction agent with well known anti-cancers.


Subject(s)
Apoptosis/drug effects , Fatty Acids, Monounsaturated/pharmacology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Receptors, Death Domain/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Docetaxel , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Drug Synergism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Receptors, Death Domain/biosynthesis , Signal Transduction/drug effects , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Taxoids/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism
5.
Z Naturforsch C J Biosci ; 68(1-2): 29-38, 2013.
Article in English | MEDLINE | ID: mdl-23659170

ABSTRACT

A new naturally occurring compound based on the dammarane skeleton, i.e. cabralealactone 3-acetate-24-methyl ether, was isolated from the aqueous methanolic extract of Forsythia koreana fruits, along with eight known compounds: cabralealactone 3-acetate, ursolic acid, arctigenin, arctiin, phillyrin, rutin, caffeic acid, and rosmarinic acid. The identification of the isolated compounds was based on their spectral analysis including: HREI-MS, 1D and 2D NMR spectroscopy. The selected compounds and the aqueous methanolic extract were evaluated for their cytotoxic activity against human solid tumour cell lines. Cabralealactone 3-acetate-24-methyl ether and ursolic acid were found to be active against human breast cancer cells (MCF-7). The cytotoxicity was associated with the activation of caspase-8, the induction of the death receptors DR4 and DR5, as well as DNA fragmentation, and was thus due to apoptosis rather than necrosis.


Subject(s)
Caspase 8/biosynthesis , Forsythia/chemistry , Receptors, Death Domain/biosynthesis , Triterpenes/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Enzyme Induction , Humans , Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization , Triterpenes/chemistry , Dammaranes
6.
J Clin Pathol ; 64(1): 75-82, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21045235

ABSTRACT

BACKGROUND: Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. AIMS: To evaluate expression of death receptors' family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. SUBJECTS AND METHODS: Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-ΔΔCt). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. RESULTS: In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. CONCLUSIONS: The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.


Subject(s)
Apoptosis/genetics , Myeloproliferative Disorders/metabolism , Receptors, Death Domain/biosynthesis , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, CD34/analysis , Apoptosis/physiology , Cells, Cultured , Female , Gene Expression Regulation , Humans , Janus Kinase 2/genetics , Leukocytes/metabolism , Male , Middle Aged , Mutation , Myeloproliferative Disorders/genetics , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Receptors, Death Domain/genetics , Young Adult
7.
Cancer Res ; 71(2): 538-49, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21127198

ABSTRACT

Because tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells, it is being tested in cancer patients. Unfortunately, patients develop resistance to the cytokine, therefore, agents that can sensitize cells to TRAIL are urgently needed. In this study, we investigated whether dibenzylideneacetone (DBA) can sensitize cancer cells to TRAIL and potentiates TRAIL-induced apoptosis. As indicated by accumulation of the membrane phospholipid phosphatidylserine, DNA breaks, intracellular esterase activity, and activation of caspase-8, -9, and -3, we concluded that DBA potentiated TRAIL-induced apoptosis in colon cancer cells. DBA also converted TRAIL resistant-cells to TRAIL-sensitive. When examined for the mechanism, we found that DBA decreased the expression of antiapoptotic proteins and decoy receptor-2 and increased proapoptotic proteins. DBA also induced both death receptor (DR)-5 and DR4. Knockdown of DR5 and DR4 by small interfering RNA (SiRNA) reduced the sensitizing effect of DBA on TRAIL-induced apoptosis. In addition, DBA increased the expression of CHOP proteins. Knockdown of CHOP by siRNA decreased the induction of DBA-induced DR5 expression and apoptosis. Induction of receptors by DBA, however, was p53-independent, as deletion of p53 had no effect on receptor induction. We observed that DBA-induced induction of DR5 and DR4 was mediated through generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of death receptors and suppression of cell survival proteins by DBA. Overall, our results show that DBA potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of death receptors via activation of ROS and CHOP mediated pathways.


Subject(s)
Pentanones/pharmacology , Reactive Oxygen Species/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Transcription Factor CHOP/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Down-Regulation/drug effects , Drug Synergism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , HCT116 Cells , HT29 Cells , Humans , Mitogen-Activated Protein Kinases/metabolism , PPAR gamma/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Death Domain/biosynthesis , Receptors, Death Domain/genetics , Tumor Necrosis Factor Decoy Receptors/biosynthesis , Tumor Necrosis Factor Decoy Receptors/genetics , Up-Regulation/drug effects
8.
Biochem Pharmacol ; 80(11): 1650-61, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20804743

ABSTRACT

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising anticancer agent but cutaneous T lymphoma cells (CTCL) are less sensitive to TRAIL-induced apoptosis. Here, we report that pentoxifylline (PTX), a phosphodiesterase inhibitor, augments TRAIL-mediated apoptosis in HuT-78 and MyLa cells through modulating extrinsic death receptors and intrinsic mitochondria dependent pathways. Our results clearly show that PTX augments TRAIL-mediated activation of caspase-8 and induces cleavage of Bid, although PTX alone cannot activate caspase-8. This is followed by cytochrome c release and subsequent, activation of caspase-9 and caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). Combined treatment downregulates the expression of various antiapoptotic proteins including c-FLIP, Bcl-xl, cIAP-1, cIAP-2 and XIAP. PTX induces the expression of death receptors DR4 and DR5 on cell surface of both the cell types where c-Jun NH2-terminal kinase (JNK) pathway plays an important role. Moreover, combined silencing of DR4 and DR5 by small interfering RNA abrogates the ability of PTX to induce TRAIL-mediated apoptosis. Thus, this is the first demonstration that PTX can potentiate TRAIL-mediated apoptosis through downregulation of cell survival gene products and upregulation of death receptors.


Subject(s)
Adjuvants, Pharmaceutic/pharmacology , Apoptosis Regulatory Proteins/biosynthesis , Apoptosis/physiology , Gene Expression Regulation, Neoplastic/drug effects , Lymphoma, T-Cell, Cutaneous/pathology , Pentoxifylline/pharmacology , Receptors, Death Domain/biosynthesis , TNF-Related Apoptosis-Inducing Ligand/physiology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/physiology , Cell Line, Tumor , Drug Synergism , Gene Expression Regulation, Neoplastic/physiology , Humans , Lymphoma, T-Cell, Cutaneous/metabolism , Receptors, Death Domain/physiology
9.
Mol Aspects Med ; 31(1): 93-112, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19995571

ABSTRACT

The observation that certain types of cancer express death receptors on their cell surface has triggered heightened interest in exploring the potential of receptor ligation as a novel anti-cancer modality, and since the expression is somewhat restricted to cancer cells the therapeutic implications are very promising. One such death receptor ligand belonging to the tumor necrosis receptor (TNF) superfamily, TNF-related apoptosis-inducing ligand (TRAIL), has been in the limelight as a tumor selective molecule that transmits death signal via ligation to its receptors (TRAIL-R1 and TRAIL-R2 or death receptors 4 and 5; DR4 and DR5). Interestingly, TRAIL-induced apoptosis exhibits hallmarks of extrinsic as well as intrinsic death pathways, and, therefore, is subject to regulation both at the cell surface receptor level as well as more downstream at the post-mitochondrial level. Despite the remarkable selectivity of DR expression on cancer cell surface, development of resistance to TRAIL-induced apoptosis remains a major challenge. Therefore, unraveling the cellular and molecular mechanisms of TRAIL resistance as well as identifying strategies to overcome this problem for an effective therapeutic response remains the cornerstone of many research endeavors. This review aims at presenting an overview of the biology, function and translational relevance of TRAIL with a specific view to discussing the various regulatory mechanisms and the current trends in reverting TRAIL resistance of cancer cells with the obvious implication of an improved clinical outcome.


Subject(s)
Neoplasms/drug therapy , Neoplasms/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Humans , Mitochondria/drug effects , Mitochondria/physiology , Neoplasms/pathology , Receptors, Death Domain/biosynthesis , TNF-Related Apoptosis-Inducing Ligand/metabolism
10.
Biotechnol Lett ; 28(18): 1445-52, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16858509

ABSTRACT

Glutamine and glucose are often controlled at low levels in fed-batch strategies to limit ammonia and lactate accumulation and improve productivity of mammalian cell cultures. However, this risks triggering apoptosis if cells are depleted of glutamine or glucose. To examine the apoptosis cascade during glutamine or glucose limitation, the transcriptional profile of FAS, FASL, FADD, FLIP, BAX, p53 and PEG3 in CRL 1606 hybridoma culture was investigated using quantitative real-time PCR. Activities of caspases 2, 3, 8 and 9 were also analyzed. Increase in the activities of the caspases was observed with up-regulation in the expression of FAS (6-8-fold) and PEG3 (2.5-fold), suggesting that the cells experienced apoptotic cell death via both the death receptor and mitochondrial pathways.


Subject(s)
Apoptosis/physiology , Glucose/deficiency , Glutamine/deficiency , Hybridomas/drug effects , Receptors, Death Domain/biosynthesis , Animals , Apoptosis/drug effects , Bioreactors , Caspases/metabolism , Cell Culture Techniques , Cell Line , Hybridomas/metabolism , Kruppel-Like Transcription Factors , Mice , Mitochondria/metabolism , Protein Kinases/biosynthesis , Transcription Factors/biosynthesis , fas Receptor/biosynthesis
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