RESUMO
Glioblastoma (GBM) is the most frequent malignant primary tumor of the CNS in adults, with a median survival of 14.6 months after diagnosis. The effectiveness of GBM therapies remains poor, highlighting the need for new therapeutic alternatives. In this work, we evaluated the effect of 4-methylumbelliferone (4MU), a coumarin derivative without adverse effects reported, in combination with temozolomide (TMZ) or vincristine (VCR) on U251, LN229, U251-TMZ resistant (U251-R) and LN229-TMZ resistant (LN229-R) human GBM cells. We determined cell proliferation by BrdU incorporation, migration through wound healing assay, metabolic and MMP activity by XTT and zymography assays, respectively, and cell death by PI staining and flow cytometry. 4MU sensitizes GBM cell lines to the effect of TMZ and VCR and inhibits metabolic activity and cell proliferation on U251-R cells. Interestingly, the lowest doses of TMZ enhance U251-R and LN229-R cell proliferation, while 4MU reverts this and even sensitizes both cell lines to TMZ and VCR effects. We showed a marked antitumor effect of 4MU on GBM cells alone and in combination with chemotherapy and proved, for the first time, the effect of 4MU on TMZ-resistant models, demonstrating that 4MU would be a potential therapeutic alternative for improving GBM therapy even on TMZ-refractory patients.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/uso terapêutico , Glioblastoma/patologia , Himecromona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Encefálicas/metabolismo , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Apoptose , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma (GBM), the most frequent primary tumor of the central nervous system, has a median survival of 14.6 months. 4-Methylumbelliferone (4MU) is a coumarin derivative widely used as a hyaluronan synthesis inhibitor with proven antitumor activity and without toxic effects reported. We aim to evaluate the antitumor effect of 4MU alone or combined with temozolomide (TMZ) on a GBM cell line, its absence of toxicity on brain cells and its selectivity for tumor cells. The antitumor effect of 4MU alone or combined with TMZ was evaluated on GL26 cells by assessing the metabolic activity through the XTT assay, cell proliferation by BrdU incorporation assay, migration by the wound healing assay, cell death by fluorescein diacetate/propidium iodide (FDA/PI) staining, apoptosis by membrane asymmetry and DNA fragmentation and metalloproteinase activity by zymography. The levels of hyaluronan and its capacity to counteract the effects of 4MU and the expression of RHAMM and CD44 were also determined. The toxicity and selectivity of 4MU were determined by XTT assay and PI staining on normal brain primary cell culture (NBPC-GFP) and GL26/NBPC-GFP cocultures. The GL26 cells expressed RHAMM but not CD44 while synthetized hyaluronan. 4MU decreased hyaluronan synthesis, diminished proliferation and induced apoptosis while reducing cell migration and the activity of metalloproteinases, which was restored by addition of hyaluronic acid. Furthermore, 4MU sensitized GL26 cells to the TMZ effect and showed selective toxicity on tumor cells without exhibiting neurotoxic effects. We demonstrated for the first time the cytotoxic effect of 4MU on GBM cells, highlighting its potential usefulness to improve GBM treatment.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Himecromona/farmacologia , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Glioblastoma/metabolismo , Glioblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais CultivadasRESUMO
Hyaluronan (HA) is the major glycosaminoglycan present in the extracellular matrix. It is produced by some tumours and promotes proliferation, differentiation and migration among others cellular processes. Gestational trophoblastic disease (GTD) is composed by non-tumour entities, such as hydatidiform mole (HM), which is the most common type of GTD and also malignant entities such as choriocarcinoma (CC) and placental site trophoblastic tumour (PSTT), being CC the most aggressive tumour. Although there is a growing understanding of GTD biology, the role of HA in the pathogenesis of this group of diseases remains largely unknown. The aim of this work was to study the role of HA in the pathogenesis of GTD by defining the expression pattern of HA and its receptors CD44 and RHAMM, as well as to determine if HA can modulate proliferation, differentiation and migration of CC cells. Receptors and signalling pathways involved were also analyzed. We demonstrated that HA and RHAMM are differently expressed among GTD entities and even among trophoblast subtypes. We also showed that HA is able to enhance the expression of extravillous trophoblast markers and also to induce migration of JEG-3 cells, the latter mediated by RHAMM as well as PI3K and MAPK pathways. These findings indicate a novel regulatory mechanism for CC cell biology and also contribute to the understanding of GTD pathophysiology.
Assuntos
Movimento Celular/efeitos dos fármacos , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Peso Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.
Assuntos
Doenças dos Bovinos/imunologia , Paratuberculose/imunologia , Animais , Argentina , Bovinos , Interações Hospedeiro-Patógeno , Interleucina-10/biossíntese , Ativação Linfocitária , Macrófagos/imunologia , Linfócitos T/imunologiaRESUMO
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention.
Assuntos
Anticorpos Antibacterianos/imunologia , Apoptose , Macrófagos/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Doenças dos Bovinos/imunologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Feminino , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD50 of this phospholipase in CD-1 mice was around 6 µg/g of mouse body weight (more exactly 117 µg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 µg/g. In contrast, the LD50 of the venom was circa 2 µg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Fosfolipases A2/isolamento & purificação , Sequência de Aminoácidos , Animais , Creatina Quinase/sangue , Camundongos , Dados de Sequência Molecular , Fosfolipases A2/química , Fosfolipases A2/toxicidade , Filogenia , Ratos , Ratos WistarRESUMO
OBJECTIVE: To analyze the effect of corticosteroid administration on the concentration of hyaluronan (HA) in bronchoalveolar lavage (BAL) in a murine model of eosinophilic airway inflammation and to study the mechanisms involved. MATERIALS AND METHODS: Untreated-mice or mice treated with 1 µg/g/day betamethasone (Bm) or 0.25 µg/g/day(-1) budesonide (Bd) were sensitized and challenged with Dermatophagoides pteronyssinus (Dp) or saline (control group). The concentration of HA in BAL was determined by ELISA. In vitro migration assays were performed using a Boyden chamber and the expression of HA synthases (HAS) was analyzed by RT-PCR. RESULTS: We found a significant increase (P < 0.01) in the levels of HA in BAL from Dp-treated mice that was prevented by Bm or Bd. Corticosteroids also inhibited the increase in HAS expression, and the phosphorylation of Akt and ERK in the lungs of challenged mice. Finally, we found that low molecular weight HA induces the chemotaxis of BAL cells in vitro through a mechanism mediated by CD44. CONCLUSION: We conclude that corticosteroids prevent the increase in HA in BAL from Dp-challenged mice. This effect is associated with reduced expression of HAS and reduced phosphorylation of Akt and ERK in the lungs of challenged mice.
Assuntos
Betametasona/farmacologia , Budesonida/farmacologia , Eosinofilia/imunologia , Glucocorticoides/farmacologia , Ácido Hialurônico/imunologia , Pneumonia/imunologia , Alérgenos , Animais , Anti-Inflamatórios/farmacologia , Antígenos de Dermatophagoides , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Feminino , Glucuronosiltransferase/genética , Hialuronan Sintases , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-akt/imunologiaRESUMO
Hyaluronan (HA) modulates multidrug resistance (MDR) as well as cell migration. Tiam1 is involved in cytoskeleton reorganization during tumor invasion. In this report we show the relationship among HA, Tiam1, migration and MDR in murine lymphoma cell lines. We observed that MDR cells presented higher migratory capacity towards HA in vitro as well as higher constitutive active Tiam1 expression than the sensitive cell line. Besides, HA treatment induced migration towards HA of MDR cell lines through Tiam1 activation by a PI3K-dependent mechanism, showing that disruption of HA signaling would be useful in treatment of MDR hematological malignancies.
Assuntos
Movimento Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ácido Hialurônico/farmacologia , Linfoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Linfoma/patologia , Camundongos , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor kappaB (NF-kappaB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-kappaB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-kappaB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.
RESUMO
HLA-G is a nonclassical MHC class I antigen that displays tolerogenic functions; MICA is a stress-regulated molecule recognized by NKG2D cytotoxicity-activating receptor expressed by NK and T cells subsets. We evaluated HLA-G isoforms and MICA mRNA levels in peripheral blood mononuclear cells (PBMCs) and in biopsies from kidney allograft recipients with acute rejection (AR), chronic rejection (CR), and stable graft evolution (SE). HLA-G1 was the only transcript resulted from amplification, both in PBMCs as in biopsy samples. HLA-G1 mRNA levels in PBMCs from 9/10 patients with CR, 7/9 with AR and 8/10 healthy volunteers were below the median value of SE patients. The analysis of biopsies revealed that patients with AR (n=6), who overcame rejection had a tendency towards higher HLA-G1 levels than those with nephrotoxic acute tubular necrosis (ATN) (n=3). Similar levels of MICA expression were observed in PBMCs from AR, CR, SE and C groups; MICA expression levels were similar also in biopsy specimens from AR and nephrotoxic ATN patients. No correlation was found between MICA expression and the graft state. These preliminary results suggest that HLA-G1 isoforms, but not MICA mRNA levels, may provide a marker for measuring the state of kidney allograft, and be the basis for further studies that may establish the influence of these molecules in renal allograft rejection or acceptance.
Assuntos
Rejeição de Enxerto/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe I/genética , Transplante de Rim , Adulto , Feminino , Rejeição de Enxerto/metabolismo , Antígenos HLA/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas , RNA Mensageiro/análise , Transplante HomólogoRESUMO
Upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been described in some tumors related to multidrug resistance (MDR). The aim of this work was to analyze the relationship between PI3K/Akt, MDR and NF-kappaB in murine lymphoma cell lines resistant to vincristine (LBR-V160) and doxorubicin (LBR-D160) as well as in the sensitive line (LBR-). PI3K/Akt activity, analyzed by phosphatidylinositol trisphosphate production and phosphorylated Akt (p-Akt) expression, was higher in the resistant cell lines than in the sensitive one and inhibition with wortmannin or LY294002 improved apoptosis in the resistant cell lines. Vincristine but not doxorubicin increased p-Akt expression whereas co-treatment with PI3K inhibitors and vincristine increased apoptosis in the three cell lines. Wortmannin and LY294002 inhibited P-glycoprotein (Pgp) function and also increased NF-kappaB activity. We concluded that the PI3K/Akt pathway is involved in MDR in lymphoma cell lines and PI3K/Akt inhibition correlates down-regulation of NF-kappaB activity and inhibition Pgp function.
Assuntos
Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Linfoma/metabolismo , NF-kappa B/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Doxorrubicina/farmacologia , Linfoma/patologia , Camundongos , Vincristina/farmacologiaRESUMO
Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteric disease in cattle. Among molecular components of Map, protein p34 was identified as specific and immunodominant for bovine B cells. In order to determine if specific antibodies could influence the course of Map pathogenesis, the interaction between bacteria and bovine macrophages was studied. Bovine polyclonal antibodies from 3 calves vaccinated with protein p34-cx, 6 calves vaccinated with heat-killed Map, 8 naturally infected, and 3 healthy calves -as negative controls- were used. Specific anti-Map, -p34-cx and -PPA-3 antibodies were evaluated and isotype characterized. Infected and Map vaccinated animals showed similar IgG1 and IgG2 response against Map whole bacteria. When p34-cx was used as the antigen, mainly IgG1 and IgG3 were detected in infected and only IgG1 in p34-cx vaccinated animals. Bovine polyclonal antibodies from three animals of each category were isolated and affinity purified through Map and p34-cx columns. The effect of these antibodies in association with Map and a transformed bovine peritoneal macrophage's cell line (Bov-Mac) as well as activation of NF-kappaB transcription factor was studied. Our results show that association of Map significantly increased in vitro after pretreatment with bovine anti-Map or anti-p34-cx antibodies obtained from vaccinated or infected cattle when compared with those of controls. Improved activation of NF-kappaB was detected in macrophages that ingested Map opsonized with either anti-Map or anti-p34-cx specific antibodies of infected or vaccinated calves, suggesting that both anti-Map and IgG1 anti-p34-cx antibodies support Map-macrophage interactions.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Macrófagos/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Especificidade de Anticorpos , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas , Macrófagos/microbiologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , NF-kappa B/metabolismo , Paratuberculose/sangue , Paratuberculose/imunologiaRESUMO
Multidrug resistance (MDR) is one of the main reasons for failure of cancer therapy. It may be mediated by overexpression of ATP-dependent efflux pumps or by alterations in survival or apoptotic pathways. Fragments generated by enzymatic degradation of hyaluronan (oHA) were able to modulate growth and cell survival and sensitize MDR breast cancer cells to cytotoxic drugs. In this work the relationship between oHA and MDR in lymphoid malignancies was analyzed using murine lymphoma cell lines resistant to doxorubicin (LBR-D160) or vincristine (LBR-V160) and a sensitive line (LBR-). After oHA treatment, higher apoptosis levels were observed in the resistant cell lines than in the sensitive one. Besides, oHA sensitized LBR-D160 and LBR-V160 to vincristine showing increased apoptosis induction when used in combination with vincristine. Native hyaluronan failed to increase apoptosis levels. As different survival factors could be modulated by hyaluronan, we investigated the PI3K/Akt pathway through PIP3 production and phosphorylated Akt (p-Akt) and survivin expression was also evaluated. Our results showed that oHA decreased p-Akt in the 3 cell lines while anti-CD44 treatment abolished this effect. Besides, survivin was downregulated only in LBR-V160 by oHA. When Pgp function was evaluated, we observed that oHA were able to inhibit Pgp efflux in murine and human resistant cell lines in a CD44-dependent way. In summary, we report for the first time that oHA per se modulate MDR in lymphoma cells by decreasing p-Akt as well as Pgp activity, thus suggesting that oHA could be useful in combination with classical chemotherapy in MDR hematological malignancies.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Ácido Hialurônico/farmacologia , Linfoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vincristina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Camundongos , Oligossacarídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Several studies indicate that hyaluronan oligosaccharides (oHA) are able to modulate growth and cell survival in solid tumors; however, no studies have been undertaken to analyze the effect of oHA on T-lymphoid disorders. In this work we showed that oHA were able to induce apoptosis in lymphoma cell lines. Since PI3-K/Akt and nuclear factor-kappaB (NF-kappaB) are major factors involved in cell survival and anti-apoptotic pathways in lymphoma cells, we hypothesized that oHA could induce apoptosis through inhibition of these pathways. oHA were identified by a method which allows characterization of length using a high pH anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). oHA inhibited PIP(3) production (principal product of PI3-K activity) and reduced Akt phosphorylation levels, similarly to the specific inhibitor wortmannin. However, treatment with either oHA or wortmannin failed to inhibit constitutive NF-kappaB activity and modulate IkappaBalpha protein levels, suggesting that PI3-K and NF-kappaB signaling pathways are not related in the cell lines used. Cell behavior differed using native hyaluronan (HA), which induced PIP(3) production, Akt phosphorylation, and NF-kappaB activation, although not related with cell survival since treatment with native HA showed no effect on apoptosis. Our results suggest that oHA induce apoptosis by suppression of PI3-K/Akt cell survival pathway without involving NF-kappaB activation, through a mechanism that differs from the one mediated by native HA.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Hialurônico/farmacologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ácido Hialurônico/química , Linfoma/patologia , Camundongos , Peso Molecular , Transdução de SinaisRESUMO
Apoptosis is an active form of cell death that plays a critical role in physiological and pathological conditions of multicellular organisms. These conditions include development, organogenesis, and elimination of infected, mutated, or damaged cells. Sipunculan cells may respond to changes in environmental exposure to oxidative stress by induction of apoptotic cell death. In coelomocytes of the sipunculan worm Themiste petricola, we evaluated morphological and biochemical changes that were induced by hydrogen peroxide (H2O2) and that could be compatible with an apoptotic-like phenotype. At an exposure of 100 mM H2O2, coelomocytes exhibited several morphological hallmarks of apoptosis such as chromatin condensation, nuclear segmentation, cell volume decrease, membrane blebbing, and formation of apoptotic bodies. Biochemical evidences of apoptotic-like cell death included exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane and oligonucleosomal DNA fragmentation. In addition, exposure of coelomocytes to H2O2 induced a rapid massive loss of mitochondrial membrane potential and of the acidic pH of lysosomes. Overall, our results showed that, in sipunculan coelomocytes, H2O2 can induce changes compatible with an apoptotic-like phenotype. The finding of an oxidative-stress-induced apoptotic-like phenotype in a sipunculan worm may indicate that this kind of cell death process participates in regulation of cell number during physiological and pathological situations, including immune responses.
Assuntos
Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Nematoides/citologia , Laranja de Acridina , Animais , Fragmentação do DNA/efeitos dos fármacos , Etídio , Técnicas In Vitro , Lisossomos/efeitos dos fármacos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Nematoides/fisiologia , FosfolipídeosRESUMO
Multidrug resistance (MDR) is the main reason for failure of cancer therapy with resistance to apoptosis being one of the mechanisms involved. Constitutive NF-kappaB activity has been detected in many tumors contributing to oncogenesis and tumor survival whereas inhibition of NF-kappaB activity has proved to enhance cell death induced by chemotherapeutic agents. Consequently, the use of BAY 11-7082, an irreversible inhibitor of IkappaB-alpha phosphorylation, could be beneficial in the treatment of certain tumors. Although there are several reports which demonstrate a transient activation of NF-kappaB by cytotoxic drugs, little is known about the role of NF-kappaB activation in the development of a chemoresistant phenotype in leukemic cells. In this study, we analyzed the relationship between NF-kappaB and the survival of murine leukemic drug resistant cell lines. The modulation of this transcription factor by BAY 11-7082 and the chemotherapeutic agents vincristine and doxorubicin was evaluated. The effect of BAY 11-7082 on the expression of genes containing NF-kappaB-binding sites was also studied. We found that the cell lines LBR-V160 and LBR-D160 (resistant to vincristine and doxorubicin, respectively) presented higher constitutive NF-kappaB activity than the sensitive LBR- and the active complex contained both p50 and p65 subunits. BAY 11-7082 (3.5 microM) inhibited constitutive NF-kappaB activity in the three cell lines whereas the anticancer agents did not. Treatment with BAY 11-7082 induced a higher percentage of apoptosis in LBR-V160 and LBR-D160 than in LBR-. Cells treated with BAY 11-7082 displayed modulation of NF-kappaB-inducible genes such as IL-10, IL-15, TNF-alpha and TGF-beta. Taken together, these data suggest that suppression of constitutive NF-kappaB activity by BAY 11-7082 may be a useful treatment for MDR leukemias.
Assuntos
Apoptose/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Leucemia de Células T/patologia , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Sulfonas/farmacologia , Animais , Linhagem Celular Tumoral , Citocinas/genética , Doxorrubicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Leucemia de Células T/tratamento farmacológico , Camundongos , Subunidade p50 de NF-kappa B/fisiologia , Vincristina/farmacologiaRESUMO
Previous findings from our laboratory demonstrated that when used at low concentration (0.1 microg ml(-1)), CsA as well as its analog PSC 833 were able to revert the MDR phenotype, while at high concentration (1 microg ml(-1)) were able to induce apoptosis. CsA induced apoptosis in leukemia cell lines sensitive (LBR-) and resistant to vincristine (LBR-V160), and doxorubicin (LBR-D160), while PSC 833 only induced apoptosis in vincristine-resistant cell line (LBR-V160). In this work, we investigated mitochondrial-associated mechanisms during CsA- and PSC 833-induced apoptosis. Mitochondrial function was evaluated by recording changes in its transmembrane potential, cytochrome c release, and caspase activation cascade. Results showed that CsA- and PSC 833-induced apoptosis was associated with mitochondrial depolarization, through potentiometric measurements with JC-1 and DiOC(6) probes. Collapse of mitochondrial potential in these cell lines after CsA treatment was followed by cytochrome c release to the cytosol, reaching an increase of 2.61-fold in LBR-, 1.98-fold in LBR-V160, and 3.01-fold in the case of LBR-D160. However, in the case of PSC 833 treatment, induction of apoptosis in LBR-V160 was associated with mitochondrial depolarization followed by a lower cytochrome c release of 1.15-fold as compared with untreated cells. Caspase 3 activation was clearly observed in LBR-, LBR-V160, and LBR-D160 after CsA treatment, while in LBR-V160, PSC 833 was less effective inducing activation of this caspase. Neither caspase 6 nor 8 activity was observed in these three cell lines. Our results suggest that during CsA- and PSC 833-induced apoptosis, mitochondrial dysfunction occurs. This is mediated through mitochondrial events, associated with an evident decrease in DeltaPsi(m), cytochrome c release and caspase 3 activation.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Ciclosporina/farmacologia , Ciclosporinas/farmacologia , Imunossupressores/farmacologia , Leucemia Linfoide/tratamento farmacológico , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Caspases/metabolismo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Leucemia Linfoide/enzimologia , Leucemia Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais CultivadasRESUMO
Phoradendron liga (Gill. ex H. et A.) Eichl. is a Viscaceae widely distributed in Argentina. It has been commonly used in folk medicine as a substitute of the European mistletoe (Viscum album L.) to decrease high blood pressure due to their external similarity. In this study, the anatomical features as well as micromolecular and macromolecular analysis of this species are reported. Anatomical study has shown that Phoradendron liga presents as anatomic features: papillous cuticle, clusters in leaves and stems, and isodiametric stone cells only in stems. The analysis of flavonoids showed that this species produces C-glycosylflavones and 3-desoxyproanthocyanidins. Protein study showed a protein pattern with components ranging from 14 to 90 kDa and the presence of related epitopes between the species was demonstrated by cross recognition using anti-Phoradendron and anti-Viscum antisera of both species by Western blot assay. In addition, a galactose specific lectin (L-Phl) was isolated form Phoradendron liga extracts. These results are part of a comprehensive project on Argentine hemiparasite species destinated to be applied to quality control of commercial samples and disclosed their potential use as a potential source for immunomodulatory compounds.
Assuntos
Phoradendron/anatomia & histologia , Phoradendron/química , Argentina , Western Blotting , Flavonoides/isolamento & purificação , Medicina Tradicional , Extratos Vegetais/químicaRESUMO
En las últimas décadas la medicina evolucionó hacia un enfoque molecular en la búsqueda de blancos específicos que permitan seguir adecuadamente la progresión de las patologías neoplásicas y desarrollar terapias exitosas. El conocimiento y comprensión de que la matriz extracelular (MEC) que rodea a un tumor influye sobre el comportamiento del mismo, reveló la importancia que cumplen las metaloproteinasas (MMPs) en la regulación de los componentes de la MEC, y por lo tanto en el desarrollo tumoral. Es por ello que actualmente se está evaluando la eficacia de inhibidores sintéticos de estas enzimas en ensayos clínicos, algunos ya en fase clínica III de investigación. También se propone que estas MMPs podrían ser utilizadas como marcadores bioquímicos, permitiendo evaluar la progresión de la enfermedad en pacientes que sufren patologías neoplásicas, e incluso dar un valor pronóstico. Es en este punto en que el laboratorio de análisis clínicos cumplirá un papel fundamental y deberá contar con los conocimientos y las herramientas necesarias para evaluar la presencia y actividad de estas enzimas. En este trabajo se expone el conocimiento actual de la estructura y funciones biológicas de las MMPs así como los antecedentes que muestran el papel que cumplen en las enfermedades neoplásicas, en especial en leucemias y linfomas
Assuntos
Humanos , Fatores de Crescimento Endotelial , Leucemia , Linfoma , Biomarcadores Tumorais , Neoplasias , Neoplasias Experimentais , Neovascularização Patológica/etiologia , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica/fisiopatologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Leucemia , Leucemia Mieloide Aguda , Linfoma , Transtornos Linfoproliferativos , Metástase Neoplásica , Neoplasias , Neoplasias Experimentais , Neovascularização Patológica/fisiopatologia , Progressão da DoençaRESUMO
En las últimas décadas la medicina evolucionó hacia un enfoque molecular en la búsqueda de blancos específicos que permitan seguir adecuadamente la progresión de las patologías neoplásicas y desarrollar terapias exitosas. El conocimiento y comprensión de que la matriz extracelular (MEC) que rodea a un tumor influye sobre el comportamiento del mismo, reveló la importancia que cumplen las metaloproteinasas (MMPs) en la regulación de los componentes de la MEC, y por lo tanto en el desarrollo tumoral. Es por ello que actualmente se está evaluando la eficacia de inhibidores sintéticos de estas enzimas en ensayos clínicos, algunos ya en fase clínica III de investigación. También se propone que estas MMPs podrían ser utilizadas como marcadores bioquímicos, permitiendo evaluar la progresión de la enfermedad en pacientes que sufren patologías neoplásicas, e incluso dar un valor pronóstico. Es en este punto en que el laboratorio de análisis clínicos cumplirá un papel fundamental y deberá contar con los conocimientos y las herramientas necesarias para evaluar la presencia y actividad de estas enzimas. En este trabajo se expone el conocimiento actual de la estructura y funciones biológicas de las MMPs así como los antecedentes que muestran el papel que cumplen en las enfermedades neoplásicas, en especial en leucemias y linfomas (AU)