RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a survival rate of 4-6 months from diagnosis. PDAC is the fourth leading cause of cancer-related death in the Western world, with a mortality rate of 10 cases per 100,000 population. Chemotherapy constitutes only a palliative strategy, with limited effects on life expectancy. AIMS: To investigate the biological response of PDAC to mitogen-activated protein kinase (MAPK) and NF-kappaB (NF-kB) inhibitors and the role of autophagy in the modulation of these signaling pathways in order to address the challenge of developing improved medical protocols for patients with PDAC. METHODS: Two ATCC cell lines, MIAPaCa-2 and PANC-1, were used as PDAC models. Cells were exposed to inhibitors of MAPK or NF-kB survival pathways alone or after autophagy inhibition. Several aspects were analyzed, as follows: cell proliferation, by [(3)H]TdR incorporation; cell death, by TUNEL assay, regulation of autophagy by LC3-II expression level and modulation of pro-and anti-apoptotic proteins by Western blot. RESULTS: We demonstrated that the inhibition of the MAPK and NF-kB survival pathways with U0126 and caffeic acid phenethyl ester (CAPE), respectively, produced strong inhibition of pancreatic tumor cell growth without inducing apoptotic death. Interestingly, U0126 and CAPE induced apoptosis after autophagy inhibition in a caspase-dependent manner in MIA PaCa-2 cells and in a caspase-independent manner in PANC-1 cells. CONCLUSIONS: Here we present evidence that allows us to consider a combined therapy regimen comprising an autophagy inhibitor and a MAPK or NF-kB pathway inhibitor as a possible treatment strategy for pancreatic cancer.

Assuntos

Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Butadienos/farmacologia , Ácidos Cafeicos/farmacologia , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Processos de Crescimento Celular , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Nitrilas/farmacologia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Taxa de Sobrevida

RESUMO

We previously demonstrated that arsenic trioxide (ATO) and proteasome inhibitor MG132 synergistically induced cell death in promonocytic leukaemia cell line U937 but were antagonistic in Burkitt's lymphoma cell line Raji. Here we explore the role of autophagy, expression of BNIP3, and mitochondrial mass, in determining whether ATO and MG132 interaction can be shifted from antagonism to synergism in Raji cells. Treatment with ATO+MG132 increased the percentage of cells with collapsed mitochondrial membrane potential (MMP) in U937 cells, but had no effect in Raji cells. Mitochondria were found in cytoplasmic marginal location in U937 cells but at perinuclear location in Raji cells. ATO+MG132 increased mitochondrial mass in U937 cells but decreased it in Raji cells, while autophagy was increased in both cell lines. BNIP3 was expressed in U937 cells at cytoplasmic marginal locations and was hardly detected in Raji cells. Histone deacetylase (HDAC) inhibitor valproic acid (VPA) increased expression of BNIP3 in Raji cells at perinuclear locations. However antagonism between ATO and MG132 was increased in the presence of low doses of VPA. Addition of vincristine (VCR) blocked autophagy, while VPA+VCR treatment of Raji cells at sub-cytotoxic doses caused BNIP3 and mitochondria to redistribute to cytoplasmic peripheral location and increased mitochondrial mass. ATO+MG132 in the presence of subcytotoxic doses of VPA+VCR caused collapse of MMP in Raji cells, while interaction between ATO and MG132 shifted from antagonism to synergism. We conclude that synergism between ATO and MG132 was attained in Raji cells by disruption of the perinuclear mitochondrial cluster, blockage of selective autophagy of mitochondria (mitophagy) by VCR, increased mitochondrial mass, and upregulation of BNIP3 by VPA.

Assuntos

Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Trióxido de Arsênio , Arsenicais/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Humanos , Leupeptinas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Óxidos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Células U937 , Regulação para Cima , Ácido Valproico/administração & dosagem , Ácido Valproico/farmacologia , Vincristina/administração & dosagem , Vincristina/farmacologia

RESUMO

Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav.) Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm) and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

RESUMO

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou-Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O2⁻) levels. Our results showed that combined arsenite+MG132 produced low levels of O2⁻ at 6h and 24h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80µM hydrogen peroxide together with arsenite+MG132 changed synergism on cell death to antagonism at all effect levels while increasing O2⁻ levels. Arsenite+hydrogen peroxide also resulted in antagonism with increased O2⁻ levels in U937 cells. In Raji cells, arsenite+MG132 also produced low levels of O2⁻ at 6h and 24h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite+CAPE showed high levels of O2⁻ production at 6h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O2⁻ levels at early time points after exposure.

Assuntos

Antineoplásicos/farmacologia , Arsenitos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leupeptinas/farmacologia , Compostos de Sódio/farmacologia , Antineoplásicos/administração & dosagem , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Peróxido de Hidrogênio/farmacologia , Leucemia Mieloide Aguda/patologia , Leupeptinas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Fatores de Tempo , Células U937

RESUMO

Sipunculans, a small phylum of coelomated marine worms closely related to polychaete annelids, lack a true circulatory system. We have previously shown that the sipunculan Themiste petricola can form a cellular clot, without congealing, of cell-free coelomic fluid. The clot is formed by the aggregation of large granular leukocytes (LGLs) and may serve not only haemostatic but immune functions, since dissimilar particles may become entrapped within it. We have now evaluated the capacity of a massive clot, induced in vitro by sea water contact, to stop coelomic fluid flow. We have further studied smaller clots induced on glass-slides either with or without the presence of bacteria placed for entrapment within the clot. The fate of clotting LGLs is cell death while forming a cohesive mass, although cytoplasmic and nuclear remnants are shed from the clot. These remnants and any bacteria that avoid clot entrapment or are detached from the clot are engulfed by non-clotting cells that include small granular leukocytes (SGLs) and large hyaline amebocytes (LHAs). Both cell types can be found other than in the clot but SGLs also occur around the clot edges heavily loaded with engulfed material. The cytoskeletal arrangement of SGLs evaluated with phalloidin-rhodamine correspond to motile cells and contrast with that of clotting LGLs that form a massive network of F-actin. Thus, the complementary roles between clotting LGLs and non-clotting SGLs and LHAs act a central immune strategy of Themiste petricola to deal with body wall injury and pathogen intrusion into the coelomic cavity.

Assuntos

Coagulação Sanguínea/imunologia , Hemostasia/imunologia , Nematoides/citologia , Nematoides/imunologia , Animais , Bactérias/metabolismo , Morte Celular , Linhagem Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Fragmentação do DNA , Citometria de Fluxo , Hemorreologia , Humanos , Leucócitos/citologia , Leucócitos/microbiologia , Fagócitos , Fagocitose , Água do Mar , Leveduras/metabolismo

RESUMO

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

Expression and activity of matrix metalloproteinase-9 (MMP-9) as well as its relationship with hyaluronan (HA) and NF-kappaB activity were analyzed in two murine lymphoma cell lines with dissimilar migration and invasive behavior. MMP activity was evaluated by zymograms in supernatants, membrane extracts of tumor cells, and in the organs invaded by these cells. The more aggressive LBLa cell line showed MMP-9 activity in vitro, which increased after HA treatment and was blocked by anti-CD44 mAb. Such activity was not found in the less aggressive LBLc. MMP-9 and MMP-2 activity was found in organs invaded by both cell lines, although differential MMP-9 activity was observed in lung infiltrated only by LBLa cell line. NF-kappaB activation was evaluated to determine whether differential activity of MMP-9 was dependent on downstream signaling pathway, showing higher NF-kappaB activity in the more aggressive LBLa cell line. Our results showed that MMP-9 activity modulated by HA through NF-kappaB signaling pathway may be involved in the aggressive behavior of LBLa.

Assuntos

Ácido Hialurônico/farmacologia , Linfoma/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais