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1.
J Cell Biochem ; 120(4): 6264-6276, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30378157

RESUMO

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC50 s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 + T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Triterpenos/farmacologia , Calpaína/metabolismo , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
2.
J Cell Biochem ; 119(2): 2212-2221, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28857256

RESUMO

Melanoma represents an ever-increasing problem in the western world as incidence rates continue to climb. Though manageable during early stages, late stage metastatic disease is highly resistant to current intervention. We have previously shown that gamma-interferon-inducible lysosomal thiol-reductase (GILT) enhances HLA class II antigen processing and immune detection of human melanoma cells. Here we report that GILT expression inhibits a potential target, paired box-3 (PAX-3) protein, in late stage human metastatic melanoma. We also show that GILT transfection or induction by IFN-γ, decreases PAX-3 protein expression while upregulating the expression of Daxx, which is also a repressor of PAX-3. Confocal microscopic analysis demonstrated that GILT co-localizes with PAX-3 protein, but not with Daxx within melanoma cells. Immunoprecipitation and immunoblotting studies suggest that GILT expression negatively regulates PAX-3 through the autophagy pathway, potentially resulting in increased susceptibility to conventional treatment in the form of chemotherapy or radiotherapy. While high-dose radiation is a common treatment for melanoma patients, our data suggest that GILT expression significantly increased the susceptibility of melanoma cells to low-dose radiation therapy via upregulation of tumor suppressor protein p53. Overall, these data suggest that GILT has multiple roles in inducing human melanoma cells as better targets for radiation and immunotherapy.


Assuntos
Melanoma/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Fator de Transcrição PAX3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Linhagem Celular Tumoral , Proteínas Correpressoras , Regulação Neoplásica da Expressão Gênica , Humanos , Lisossomos/metabolismo , Melanoma/patologia , Melanoma/radioterapia , Chaperonas Moleculares , Estadiamento de Neoplasias , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
3.
J Cell Biochem ; 116(1): 102-14, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25142864

RESUMO

Lymphoma is a potentially life threatening disease. The goal of this study was to investigate the therapeutic potential of a natural triterpenoid, Ganoderic acid A (GA-A) in controlling lymphoma growth both in vitro and in vivo. Here, we show that GA-A treatment induces caspase-dependent apoptotic cell death characterized by a dose-dependent increase in active caspases 9 and 3, up-regulation of pro-apoptotic BIM and BAX proteins, and a subsequent loss of mitochondrial membrane potential with release of cytochrome c. In addition to GA-A's anti-growth activity, we show that lower doses of GA-A enhance HLA class II-mediated antigen (Ag) presentation and CD4+ T cell recognition of lymphoma cells in vitro. The therapeutic relevance of GA-A treatment was also tested in vivo using the EL4 syngeneic mouse model of metastatic lymphoma. GA-A-treatment significantly prolonged survival of EL4 challenged mice and decreased tumor metastasis to the liver, an outcome accompanied by a marked down-regulation of STAT3 phosphorylation, reduction myeloid-derived suppressor cells (MDSCs), and enhancement of cytotoxic CD8+ T cells in the host. Thus, GA-A not only selectively induces apoptosis in lymphoma cells, but also enhances cell-mediated immune responses by attenuating MDSCs, and elevating Ag presentation and T cell recognition. The demonstrated therapeutic benefit indicates that GA-A is a candidate for future drug design for the treatment of lymphoma.


Assuntos
Linfoma/tratamento farmacológico , Triterpenos/uso terapêutico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Linfoma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Triterpenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Apoptosis ; 17(10): 1066-78, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22847295

RESUMO

Melanoma is the most aggressive form of skin cancer, responsible for the majority of skin cancer related deaths. Thus, the search for natural molecules which can effectively destroy tumors while promoting immune activation is essential for designing novel therapies against metastatic melanoma. Here, we report for the first time that a natural triterpenoid, Ganoderic acid DM (GA-DM), induces an orchestrated autophagic and apoptotic cell death, as well as enhanced immunological responses via increased HLA class II presentation in melanoma cells. Annexin V staining and flow cytometry showed that GA-DM treatment induced apoptosis of melanoma cells, which was supported by a detection of increased Bax proteins, co-localization and elevation of Apaf-1 and cytochrome c, and a subsequent cleavage of caspases 9 and 3. Furthermore, GA-DM treatment initiated a possible cross-talk between autophagy and apoptosis as evidenced by increased levels of Beclin-1 and LC3 proteins, and their timely interplay with apoptotic and/or anti-apoptotic molecules in melanoma cells. Despite GA-DM's moderate cytotoxicity, viable cells expressed high levels of HLA class II proteins with improved antigen presentation and CD4+ T cell recognition. The antitumor efficacy of GA-DM was also investigated in vivo in murine B16 melanoma model, where GA-DM treatment slowed tumor formation with a significant reduction in tumor volume. Taken together, these findings demonstrate the potential of GA-DM as a natural chemo-immunotherapeutic capable of inducing a possible cross-talk between autophagy and apoptosis, as well as improved immune recognition for sustained melanoma tumor clearance.


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Antígenos HLA-D/imunologia , Melanoma/imunologia , Melanoma/patologia , Triterpenos/uso terapêutico , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Humanos , Melanoma/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Camundongos
5.
Leuk Lymphoma ; 53(2): 305-14, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21854084

RESUMO

Malignant B-cells express measurable levels of human leukocyte antigen (HLA) class II proteins, but often escape immune recognition by CD4 + T cells. Resveratrol (Resv) has been the focus of numerous investigations due to its potential chemopreventive and anti-cancer effects, but it has never been tested in the regulation of immune components in B-cell tumors. Here, we show for the first time that Resv treatment enhances HLA class II-mediated immune detection of B-cell lymphomas by altering immune components and class II presentation in tumor cells. Resv treatment induced an up-regulation of both classical and non-classical HLA class II proteins (DR and DM) in B-lymphoma cells. Resv also altered endolysosomal cathepsins (Cat S, B and D) and a thiol reductase (GILT), increasing HLA class II-mediated antigen (Ag) processing in B-cell lymphomas and their subsequent recognition by CD4 + T cells. Mechanistic study demonstrated that Resv treatment activated the recycling class II pathway of Ag presentation through up-regulation of Rab 4B protein expression in B-lymphoma cells. These findings suggest that HLA class II-mediated immune recognition of malignant B-cells can be improved by Resv treatment, thus encouraging its potential use in chemoimmunotherapy of B-cell lymphoma.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Linfócitos T CD4-Positivos/imunologia , Antígenos HLA-D/metabolismo , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/imunologia , Estilbenos/farmacologia , Apresentação de Antígeno , Western Blotting , Catepsinas/metabolismo , Proliferação de Células , Endocitose , Citometria de Fluxo , Antígenos HLA-D/imunologia , Humanos , Linfoma de Células B/metabolismo , Resveratrol , Células Tumorais Cultivadas
6.
Clin Dev Immunol ; 2011: 780839, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22162713

RESUMO

While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL) have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s) have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.


Assuntos
Apresentação de Antígeno , Antineoplásicos/farmacologia , Briostatinas/farmacologia , Linfoma de Burkitt/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Antígenos HLA-D/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Interleucina-2/imunologia , Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Transdução de Sinais
7.
J Clin Cell Immunol ; S3: 4, 2011 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23336088

RESUMO

Considering the fact that a key factor in tumor development is the evasion of immune detection, the search for natural products, which have reduced toxicity towards normal tissues as well as immunostimulatory capabilities has received growing interest. One attractive source of antitumor products is the Ganoderma lucidum mushroom, which has been used for centuries as an herbal medicine for the prevention and treatment of a variety of diseases, including cancer, and has been shown to improve immune function. Interestingly, its methanol soluble triterpenoid extracts, namely Ganoderic Acids (GAs), have been the subject of several recent investigations on their chemotherapeutic effects. While current research has revealed GAs' role in inducing apoptosis of cancer cells with a much lower toxicity to healthy cells, little information is available on their in vitro and/or in vivo immune activities. In this review, we aim to discuss the current knowledge on GAs, and their potential as apoptosis inducing as well as immune activating molecules that could be a potential alternative approach for designing novel chemoimmunotherapeutics against malignant diseases. We also discuss other new approaches for exploiting the advantages of using a nanoparticle polymer-GA conjugate as a tool for a sustained and targeted delivery of drug in vivo.

8.
Environ Health Perspect ; 116(7): 930-6, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18629316

RESUMO

BACKGROUND: Human exposure to brevetoxins produced by the red tide organism, Karenia brevis, is an increasing public health concern. Using in vitro exposure of rat liver cells to brevetoxin B (PbTx-2), the primary toxin product of K. brevis, we previously showed that it formed C(27,28)-epoxy brevetoxin metabolites capable of covalently binding to nucleic acids, a common initiation step for carcinogenesis. OBJECTIVE: This study was undertaken to evaluate nucleic acid adduction in lung following in vitro and in vivo brevetoxin exposures. METHODS: To clarify reactions of brevetoxin epoxide with DNA, we analyzed reaction products of PbTx-6 (a C(27,28) epoxide metabolite of brevetoxin B) with nucleosides. We also analyzed adducts from nucleic acid hydrolysates of isolated rat lung cells treated with PbTx-2 or PbTx-6 in vitro and lung tissue from rats after intratracheal exposure to PbTx-2 or PbTx-6 at 45 microg toxin/kg body weight. RESULTS: Our results indicate that PbTx-2 forms DNA adducts with cytidine after treatment of isolated lung cells, and forms DNA adducts with adenosine and guanosine after intratracheal exposure. CONCLUSIONS: These results are consistent with metabolic activation of highly reactive brevetoxin intermediates that bind to nucleic acid. These findings provide a basis for monitoring exposure and assessing the hazard associated with depurination of brevetoxin-nucleotide adducts in lung tissue.


Assuntos
Adutos de DNA/metabolismo , DNA/metabolismo , Dinoflagellida , Pulmão/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Oxocinas/toxicidade , Animais , Dano ao DNA , Técnicas In Vitro , Pulmão/citologia , Pulmão/metabolismo , Ratos
9.
Toxicol Sci ; 89(1): 57-65, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16221966

RESUMO

Brevetoxins are potent marine toxins produced by the dinoflagellate Karenia brevis, the causative organism of Florida red tides. An in vitro metabolism of PbTx-2 was performed using purified cDNA-expressed rat liver cytochrome P-450 (CYP) enzymes and freshly isolated rat hepatocytes. The metabolic activities of six CYP enzymes, CYP1A2, CYP2A2, CYP2C11, CYP2D1, CYP2E1, and CYP3A1, were examined by incubation with PbTx-2 for up to 4 h in the presence of a NADPH-generating system. Further identification of the metabolites produced by CYP1A2 and CYP3A1 was preformed using high performance liquid chromatography-mass spectrometry (LC/MS). Both CYP1A2 and CYP3A1 metabolized PbTx-2 to PbTx-3 (MH+: m/z 897), PbTx-9 (MH+: m/z 899), and a newly recorded diol brevetoxin-2 metabolite (MH+: m/z 929). CYP3A1 also produced a considerably higher amount of BTX-B5 (MH+: m/z 911). Subsequent incubation of PbTx-2 with rat hepatocytes produced additional phase 1 metabolites of MH+: m/z 911, 913, 915, 917, and 931, indicating a CYP-catalyzed epoxidation at H-ring (C27,C28-double bond) and a subsequent A-ring hydrolysis of PbTx-2 metabolic products. A conjugation metabolism was identified by the production of a glutathione-brevetoxin conjugate (MH+: m/z 1222) and a cysteine-brevetoxin conjugate (MH+: m/z 1018). Structures of the new metabolites are postulated, and a likely CYP-catalyzed metabolism pathway of PbTx-2 metabolism are discussed.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Dinoflagellida , Hepatócitos/enzimologia , Toxinas Marinhas/metabolismo , Desintoxicação Metabólica Fase II , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Hepatócitos/efeitos dos fármacos , Masculino , Toxinas Marinhas/toxicidade , Oxirredução , Oxocinas , Ratos , Ratos Sprague-Dawley
10.
Toxicon ; 46(3): 243-51, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15979117

RESUMO

Ciguatera is a human food poisoning caused by consumption of tropical and subtropical fish that have, through their diet, accumulated ciguatoxins in their tissues. This study used laboratory mice to investigate the potential to apply blood collection cards to biomonitor ciguatoxin exposure. Quantitation by the neuroblastoma cytotoxicity assay of Caribbean ciguatoxin (C-CTX-1) spiked into mice blood was made with good precision and recovery. The blood collected from mice exposed to a sublethal dose of Caribbean ciguatoxic extract (0.59 ng/g C-CTX-1 equivalents) was analyzed and found to contain detectable toxin levels at least 12 h post-exposure. Calculated concentration varied from 0.25 ng/ml at 30 min post-exposure to 0.12 ng/ml at 12 h. A dose response mice exposure revealed a linear dose-dependent increase of ciguatoxin activity in mice blood, with more polar ciguatoxin congeners contributing to 89% of the total toxicity. Finally, the toxin measurement in mice blood exposed to toxic extracts from the Indian Ocean or from the Pacific Ocean showed that the blood collection card method could be extended to each of the three known ciguatoxin families (C-CTX, I-CTX and P-CTX). The low matrix effect of extracted dried-blood samples (used at 1:10 or 1:20 dilution) and the high sensitivity of the neuroblastoma assay (limit of detection 0.006 ng/ml C-CTX-1), determined that the blood collection card method is suitable to monitor ciguatoxin at sublethal doses in mice and opens the potential to be a useful procedure for fish screening, environmental risk assessment or clinical diagnosis of ciguatera fish poisoning in humans or marine mammals.


Assuntos
Ciguatera/sangue , Ciguatoxinas/sangue , Exposição Ambiental , Animais , Ciguatera/epidemiologia , Ciguatoxinas/toxicidade , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Modelos Animais , Neuroblastoma/metabolismo , Estados do Pacífico/epidemiologia , Medição de Risco , Fatores de Tempo
11.
Toxicol Sci ; 85(2): 839-46, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15746006

RESUMO

We examined detoxification of brevetoxin in rats through metabolic activities and key elimination routes by analyzing samples from individual rats exposed to two brevetoxin congeners (PbTx-2 and PbTx-3). Brevetoxins were detected by radioimmunoassay in methanolic extracts of blood within 1 h post intraperitoneal (ip) administration. The toxin assay response was about three times higher in PbTx-2-treated rats versus the same dose (180 microg/kg) of PbTx-3. This difference persisted for up to 8 h postexposure. When the blood samples were reextracted with 20% methanol to enhance recovery of potential polar brevetoxin metabolites, 25-fold higher assay activity was present in the PbTx-2-treated rats. Analysis of urine from the same animals identified 7-fold more activity in the PbTx-2-treated rats that accumulated over the course of 24 h. Radioimmunoassay-guided high performance liquid chromatographic analysis of urine from PbTx-2-treated rats yielded three major peaks of activity. The first peak was attributed to the two cysteine adducts, cysteine-PbTx sulfoxide and cysteine-PbTx (MH(+): m/z 1034 and 1018). The second peak was attributed to the oxidized form of PbTx-2 (MH(+): m/z 911) and its reduction product PbTx-3. The third peak remains unidentified. Brevetoxin cysteine conjugate and its sulfoxide product contributed nearly three-quarters of the brevetoxin immunoactivity. Our findings indicate the most commonly occurring PbTx-2 is rapidly transformed to a polar metabolite of a reduced biological activity that appears in blood and remains for up to 8 h, yet is cleared mostly to the urine within 24 h.


Assuntos
Inativação Metabólica , Toxinas Marinhas/metabolismo , Toxinas Marinhas/toxicidade , Oxocinas/metabolismo , Oxocinas/toxicidade , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Cisteína/urina , Indicadores e Reagentes , Injeções Intraperitoneais , Masculino , Toxinas Marinhas/química , Espectrometria de Massas , Dados de Sequência Molecular , Oxocinas/química , Radioimunoensaio , Ratos , Receptores de Droga/metabolismo
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