OBJECTIVETumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem.

METHODSNuclear and cytoplasmic protein extraction and immuno？uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR.

RESULTSPreviously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3β phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells.

CONCLUSIONTaken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.

Apoptosis ; physiology ; Cell Line, Tumor ; Gene Expression Regulation ; physiology ; Heterogeneous-Nuclear Ribonucleoprotein K ; genetics ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Up-Regulation ; physiology ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism

The Raf kinase inhibitory protein (RKIP) has been demonstrated to modulate different intracellular signaling pathways in cancers. Studies have shown that RKIP is frequently downregulated in cancers; therefore, attempts have been made to upregulate the expression of RKIP using natural and synthetic agents for the treatment of human malignancies. Moreover, various regulators such as specific proteins and microRNAs (miRNAs) that are involved in the regulation of RKIP expression have also been identified. RKIP mechanistically modulates the apoptotic regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. Because of its critical role in human cancers, RKIP has drawn much research attention, and our understanding is expanding rapidly. Here, we summarize some of the biological complexities of RKIP regulation. However, we restrict our discussion to selected tumors by focusing on TRAIL, miRNAs and natural agents. Emerging evidence suggests a role for natural agents in RKIP regulation in cancer cells; therefore, naturally occurring agents may serve as cancer-targeting agents for cancer treatment. Although the literature suggests some advancement in our knowledge of RKIP biology, it is incomplete with regard to its preclinical and clinical efficacy; thus, further research is warranted. Furthermore, the mechanism by which chemotherapeutic drugs and novel compounds modulate RKIP and how nanotechnologically delivered RKIP can be therapeutically exploited remain to be determined.
Apoptosis ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics ; Neoplasms/genetics/*metabolism ; Phosphatidylethanolamine Binding Protein/genetics/*metabolism ; Protein Interaction Maps ; *Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism

Human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) might be developed as a novel anti-tumor drug due to its selective cytotoxicity in tumor cells. The predicted Macaca mulatta TRAIL (mmTRAIL) is highly homologous to hTRAIL in nucleotide acid as well as amino acid sequence, suggesting that mmTRAIL might induce apoptosis of human cancer cells. However, the cytotoxicity of mmTRAIL in human cancer cells has not been investigated. In this paper, it is reported that the gene encoding mmTRAIL has been cloned by using reverse-transcriptase polymerase chain reaction (RT-PCR) from monkey peripheral blood mononuclear cells (PBMCs) in our laboratory. Subsequently, an expression plasmid was constructed by inserting mmTRAIL gene into pQE30 plasmid. After induction by addition of Isopropyl β-D-1-Thiogalactopyranoside (IPTG), mmTRAIL was expressed. MmTRAIL was recovered from supernatant of sonicated bacteria by Ni-NTA agarose affinity chromatography. SDS-PAGE and gel filtration chromatography demonstrated that mmTRAIL forms trimer in solution. In vitro assays indicated that mmTRAIL was cytotoxic to human COLO205 tumor cells but not to normal cells at low concentration of nanomole. In addition, antitumor effect of mmTRAIL was evaluated in mice bearing human COLO205 tumor xenografts. Intratumorally injected mmTRAIL significantly inhibited growth of tumor grafts. These results suggested that mmTRAIL was valuable as candidate drug for cancer-targeted therapy.
Animals ; Antineoplastic Agents ; Apoptosis ; Cell Line, Tumor ; Cloning, Molecular ; Humans ; Leukocytes, Mononuclear ; Macaca mulatta ; Mice ; Plasmids ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Xenograft Model Antitumor Assays

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) is potentially a novel anti-cancer drug due to its superior selective cytotoxicity in a wide variety of tumor cells. Zinc ion (Zn2+) insufficiency might be an important cause for weak cytotoxicity of sTRAIL prepared by gene engineering. In this paper, the sTRAIL protein is highly-expressed with insertion of the synthesized gene encoding sTRAIL into pQE30 plasmid. The polymerization and cytotoxicity in tumor cells of sTRAIL prepared in presence of different concentrations of Zinc ions were compared. It was found that the sTRAIL protein prepared in absence of Zinc ions mainly existed as monomer with weak cytotoxicity. However, in the presence of Zinc ions, sTRAIL formed homotrimer and showed strong cytotoxicity in tumor cells. These results demonstrate that Zinc ion is very important for cytotoxicity of sTRAIL. It is necessary for keeping stable activity of sTRAIL by addition of proper concentration of Zinc ion in the media.
Antineoplastic Agents ; metabolism ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; Female ; Humans ; Lung Neoplasms ; pathology ; Polymerization ; drug effects ; Recombinant Proteins ; metabolism ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Zinc ; pharmacology

OBJECTIVETo detect the expression profile of NK ligands in acute leukemia cell lines and investigate the differential expression pattern between acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

METHODSUsing quantitative real-time PCR, 23 NK ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, HLA-E, HLA-G, CD48, NBTA, HLA-F, LLT-1, PVR, Nectin2, CD72, CD80, ICAM-1, LFA-3, CRACC, Fas, DR4, DR5, TNFR1) were detected in 6 acute leukemia cell lines, including 3 ALL cell lines (CEM, Jurkat T, Reh) and 3 AML cell lines (HL-60, KG-1a, NB4), respectively. Independent-samples t test analysis was performed to determine statistical significance.

RESULTSUsing β-actin as reference gene, the relative expression results showed that the expression of 4 NK ligands between ALL and AML is significantly different. Specifically, the level of ULBP-2 is higher in ALL (CEM: 1, Jurkat T: 0.617, Reh: 0.246) than that in AML (HL-60: 0.000, KG-1a: 0.003, NB4: 0.000)(P = 0.047). However, the expressions of CD48, PVR(PVR-1, PVR-2) and DR4 is higher in AML (HL-60: 13.987, 4.403, 10.334, 8.711; KG-1a: 5.387, 2.900, 7.315, 4.512; NB4: 7.763, 3.248, 7.049, 6.127) than that in ALL (CEM: 1, 1, 1, 1; Jurkat T: 2.035, 1.553, 3.888, 0.449; Reh: 1.559, 0.000, 0.000, 1.304) (P = 0.044, 0.014, 0.014, 0.011). And there're no significant differences between the rest 19 NK ligands.

CONCLUSIONSULBP-2, CD48, PVR and DR4 might play an important role in the distinct mechanisms in leukemogenesis between ALL and AML and could be potential targets for diagnosis and treatment.

Acute Disease ; Antigens, CD ; genetics ; metabolism ; CD48 Antigen ; Cell Line, Tumor ; GPI-Linked Proteins ; genetics ; metabolism ; HL-60 Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Leukemia ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Ligands ; Membrane Proteins ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Receptors, Virus ; genetics ; metabolism

This study was designed to investigate the effects of the prenylated flavonoid kurarinone on TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis and its underlying mechanism. A low dose of kurarinone had no significant effect on apoptosis, but this compound markedly promoted tumor cell death through elevation of Bid cleavage, cytochrome c release and caspase activation in HeLa cells treated with TRAIL. Caspase inhibitors inhibited kurarinone-mediated cell death, which indicates that the cytotoxic effect of this compound is mediated by caspase-dependent apoptosis. The cytotoxic effect of kurarinone was not associated with expression levels of Bcl-2 and IAP family proteins, such as Bcl-2, Bcl-xL, Bid, Bad, Bax, XIAP, cIAP-1 and cIAP-2. In addition, this compound did not regulate the death-inducing receptors DR4 and DR5. On the other hand, kurarinone significantly inhibited TRAIL-induced IKK activation, IkappaB degradation and nuclear translocation of NF-kappaB, as well as effectively suppressed cellular FLICE-inhibitory protein long form (cFLIPL) expression. The synergistic effects of kurarinone on TRAIL-induced apoptosis were mimicked when kurarinone was replaced by the NF-kappaB inhibitor withaferin A or following siRNA-mediated knockdown of cFLIPL. Moreover, cFLIP overexpression effectively antagonized kurarinone-mediated TRAIL sensitization. These data suggest that kurarinone sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-kappaB-dependent cFLIP expression, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; CASP8 and FADD-Like Apoptosis Regulating Protein/*genetics/metabolism ; Caspase 3/metabolism ; Caspase 8/metabolism ; Drug Synergism ; Enzyme Activation/drug effects ; Flavonoids/*pharmacology ; Gene Expression/drug effects ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; NF-kappa B/antagonists & inhibitors/*metabolism ; Protein Transport/drug effects ; RNA, Small Interfering/genetics ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand/*physiology ; Up-Regulation/drug effects

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be regulated by the epidermal growth factor (EGF) signaling pathway. In this study, recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter (AdTRAIL) was combined with drugs including gefitinib, elotinib, and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer (NSCLC) cell lines to investigate their antitumor activity. In vitro, compared to single reagent, AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460, A549, and SW1573 cell lines. Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT. In vivo, AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice. Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL. These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.
Adenoviridae ; genetics ; Animals ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cetuximab ; Drug Synergism ; Erlotinib Hydrochloride ; Female ; Genetic Therapy ; Genetic Vectors ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Mice ; Mice, Nude ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; physiology ; Transfection ; Tumor Burden ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPS) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; Astrocytes/metabolism ; CASP8 and FADD-Like Apoptosis Regulating Protein/genetics/*metabolism ; Caspase 3/metabolism ; Cell Line, Tumor ; Drug Discovery ; Flow Cytometry ; Glioma/*metabolism/pathology ; Humans ; Indoles/*pharmacology ; Inhibitor of Apoptosis Proteins/metabolism ; RNA, Small Interfering ; Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand/metabolism/*pharmacology ; Transcription Factor CHOP/analysis

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.
Adenoviridae/genetics ; Animals ; Blood-Brain Barrier ; Brain/drug effects/*metabolism/pathology ; Brain Neoplasms/genetics/metabolism/pathology/*therapy ; Clinical Trials, Phase I as Topic ; DNA, Viral/metabolism ; Disease Models, Animal ; Drug Delivery Systems ; Drug Evaluation, Preclinical ; *Gene Therapy ; Glioma/genetics/metabolism/pathology/*therapy ; Humans ; Liver/drug effects/metabolism/pathology ; Protein Multimerization/genetics ; Rats ; Spleen/drug effects/metabolism/pathology ; TNF-Related Apoptosis-Inducing Ligand/genetics/*pharmacokinetics

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.
Adenoviridae/genetics ; Animals ; Blood-Brain Barrier ; Brain/drug effects/*metabolism/pathology ; Brain Neoplasms/genetics/metabolism/pathology/*therapy ; Clinical Trials, Phase I as Topic ; DNA, Viral/metabolism ; Disease Models, Animal ; Drug Delivery Systems ; Drug Evaluation, Preclinical ; *Gene Therapy ; Glioma/genetics/metabolism/pathology/*therapy ; Humans ; Liver/drug effects/metabolism/pathology ; Protein Multimerization/genetics ; Rats ; Spleen/drug effects/metabolism/pathology ; TNF-Related Apoptosis-Inducing Ligand/genetics/*pharmacokinetics