Derivados 1,3,4 - Tiadiazóis Mesoiônicos : Disfunção Mitocondrial e Toxidade em células HEPG2
Resumo
Resumo: As celulas cancerosas sao tolerantes a muitos tratamentos devido a aquisicao de mecanismos de resistencia a apoptose e por apresentarem um programa proliferativo anormal. Neste contexto, o desenvolvimento de novos farmacos com atividade antitumoral e o entendimento de seu mecanismo de acao e essencial. Os compostos mesoionicos tem mostrado atividade contra diferentes linhagens tumorais, a qual tem sido atribuida as suas caracteristicas fisico-quimicas. Dentre estes compostos, os derivados 1,3,4-tiadiazois-2-fenilamina tem merecido destaque devido a sua importante atividade antimelanoma. No presente estudo, os efeitos de derivados desta classe, o MI-J, MI-4F, MI-2,4diF e MI-D, foram avaliados sobre mitocondrias isoladas e cultura de celulas de hepatocarcinoma humano (HepG2). Em experimentos utilizando mitocondrias isoladas, o MI-J, MI-4F e MI-2,4diF inibiram de forma dose-dependente (6,5 . 130 nmol.mg-1 de proteina) o inchamento mitocondrial em presenca de acetato sodio pela adicao de glutamato mais malato e succinato. A contracao da organela apos adicao de inibidores especificos da cadeia respiratoria tambem foi comprometida. Experimentos de polarizacao de fluorescencia das sondas DPH e DPH-PA em lipossomas de dimiristoil-fosfatidilcolina (DMPC), demonstraram que o derivado MI-J (5 ƒÊmol.L-1) diminuiu a fluidez da membrana em regioes hidrofilicas e hidrofobicas, enquanto que os derivados fluorados (5 e 15 ƒÊmol.L-1) exerceram este efeito predominantemente em regioes hidrofobicas. Todos os derivados, de forma dose-dependente (5, 25 e 80 nmol.mg-1 de proteina), inibiram a lipoperoxidacao induzida por AAPH e Fe3+/ADP/2-oxoglutarato. Estes derivados, nessas mesmas concentracoes, tambem foram capazes de sequestrar radicais superoxido e inibir a atividade da superoxido dismutase mitocondrial. Alem disso, em concentracoes de 6,5 e 32,5 nmol.mg-1 de proteina preveniram tanto a oxidacao espontanea, como a induzida por calcio, dos nucleotideos de piridina. Todos os derivados (65 e 130 nmol.mg-1 de proteina) inibiram a captacao de calcio, enquanto que o efluxo do cation foi inibido somente pelo MI-J (~52%) e MI-4F (~50%), possivelmente devido a inibicao da formacao do poro de transicao de permeabilidade mitocondrial (PTPM) em 100% e 50%, respectivamente. Todos os derivados (MI-J, MI-4F, MI-2,4diF e MI-D) foram toxicos para celulas HepG2 em cultura. Na concentracao de 50 ƒÊmol.L-1, os derivados reduziram em ~50% a viabilidade destas celulas, tratadas durante 24 horas. A analise da morfologia celular mostrou alteracoes sugestivas de formacao de blebs, corpos apoptoticos e retracao celular, causadas pelo tratamento com os derivados (5 ƒÊmol.L-1 por 3 horas). Os derivados MI-J, MI-4F e MI-2,4diF (25 ƒÊmol.L-1 por 24 horas) promoveram o aumento da fragmentacao de DNA, enquanto que o MI-D nao causou alteracoes significativas neste parametro. Os resultados obtidos neste estudo sugerem que os derivados exercem efeito antioxidante em mitocondrias isoladas, o qual e dependente de sua interacao com a membrana mitocondrial interna. No entanto, apesar disto, sao toxicos para celulas HepG2 em cultura, indicando que o mecanismo responsavel pela atividade antitumoral destes compostos e complexo e exige maiores investigacoes. Abstract: Cancer cells are tolerant to many treatments since they possess mechanisms of resistance to apoptosis and an unusual proliferation program. In this context, the development of new drugs with antitumor activity and the understanding of their action mechanism are essential. Mesoionic compounds have shown antitumoral activity against different tumor cell lines, which has been attributed to its physical and chemical characteristics. Among these compounds, 1,3,4-thiadiazolium-2-phenylamine derivatives have received special attention due to their antimelanoma activity. In this study, the effects of derivatives belongs to this class, MI-J, MI-4F, MI-2,4diF and MI-D, were evaluated on isolated mitochondria and on culture of human hepatocarcinoma cells (HepG2). In experiments using isolated mitochondria, MI-J, MI-4F and MI-2,4diF inhibited, in a dose-dependent way (6.5 to 130 nmol.mg-1 protein), mitochondrial swelling in the presence of sodium acetate by addition of glutamate plus malate and succinate. The organelle shrinkage after addition of specific respiratory chain inhibitors was also impaired. Experiments of fluorescence polarization of DPH and DPH-PA probes in dimyristoyl-phosphatidylcholine (DMPC) liposomes, showed that the derived MI-J (5 ìmol.L-1) decreased the fluidity of hydrophilic and hydrophobic regions of membrane, whereas fluorine derivatives (5 and 15 ìmol.L-1) decreased it predominantly in hydrophobic regions. All derivatives, in a dose-dependent way (5, 25 and 80 nmol.mg-1 protein), inhibited lipid peroxidation induced by AAPH and Fe3+/ADP/2-oxoglutarato. At the same concentrations, these derivatives were also able to scavenger superoxide radicals and to inhibit the activity of mitochondrial superoxide dismutase. In addition, at concentrations of 6.5 and 32.5 nmol.mg-1 protein, they prevented either the spontaneous and calcium induced oxidation of pyridine nucleotides. All derivatives inhibited the uptake of calcium (65 and 130 nmol.mg-1 protein), whereas the cation efflux was inhibited only by the MI-J (~ 52%) and MI-4F (~50%), possibly due to inhibition of the mitochondrial permeability transition pore (MPTP) in 100% and 50%, respectively. All derivatives (MI-J, MI-4F, MI-2,4diF and MI-D) were toxic to HepG2 cells. Derivatives at concentration of 50 ìmol.L-1 reduced by ~50% the viability of cells treated for 24 hours. The analysis of cell morphology showed changes suggestive of blebs formation, apoptotic bodies and cell shrinkage caused by treatment with the derivatives (5 ìmol.L-1 for 3 hours). MI-J, MI-4F and MI-2,4diF (25 ìmol.L-1 for 24 h) promoted the increase of DNA fragmentation, while MI-D did not cause significant changes in this parameter. The results of this study suggest that derivatives exert antioxidant effects in isolated mitochondria, which are dependent on their interaction with the inner mitochondrial membrane. However, in despite of this, they are toxic to HepG2 cells indicating that the mechanism responsible for their antitumor activity is complex and requires additional investigations.
Collections
- Teses [197]