Derivados 1,3,4 - Tiadiazóis Mesoiônicos

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

Derivados 1,3,4 - Tiadiazóis Mesoiônicos : Disfunção Mitocondrial e Toxidade em células HEPG2

Orientadora : Profª Drª Silvia Maria Suter Correia CadenaCo-Orientadora: Profa. Dra. Guilhermina Rodrigues NoletoTese (doutorado) - Universidade Federal do Paraná, Setor de Ciencias Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba,2011Bibliografia: fls.147-157Resumo: 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

Cytotoxic effect of Agaricus bisporus and Lactarius rufus β-d-glucans on HepG2 cells

AbstractThe cytotoxic activity of β-d-glucans isolated from Agaricus bisporus and Lactarius rufus fruiting bodies was evaluated on human hepatocellular carcinoma cells (HepG2). NMR and methylation analysis suggest that these β-d-glucans were composed of a linear (1→6)-linked and a branched (1→3), (1→6)-linked backbone, respectively. They both decreased cell viability at concentrations of up to 100μgmL−1, as shown by MTT assay. The amount of LDH released and the analysis of cell morphology corroborated these values and also showed that the β-d-glucan of L. rufus was more cytotoxic to HepG2 cells than that of A. bisporus. The treatment of HepG2 cells with L. rufus and A. bisporus β-d-glucans at a dose of 200μgmL−1 for 24h promoted an increase of cytochrome c release and a decrease of ATP content, suggesting that these polysaccharides could promote cell death by apoptosis. Both β-d-glucans were tested against murine primary hepatocytes at a dose of 200μgmL−1. The results suggest that the L. rufus β-d-glucan was as cytotoxic for hepatocytes as for HepG2 cells, whereas the A. bisporus β-d-glucan, under the same conditions, was cytotoxic only for HepG2 cells, suggesting cell selectivity. These results open new possibilities for use of mushroom β-d-glucans in cancer therapy

Derivados 1,3,4 - Tiadiazóis Mesoiônicos : Disfunção Mitocondrial e Toxidade em células HEPG2

Orientadora : Profª Drª Silvia Maria Suter Correia CadenaCo-Orientadora: Profa. Dra. Guilhermina Rodrigues NoletoTese (doutorado) - Universidade Federal do Paraná, Setor de Ciencias Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba,2011Bibliografia: fls.147-157Resumo: 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

Derivados 1,3,4 - Tiadiazóis Mesoiônicos

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

Celecoxib prevents tumor growth in an animal model by a COX-2 independent mechanism

Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to reduce cell growth in several tumors. Among these possible antineoplastic drugs are cyclooxygenase-2 (COX-2)-selective drugs, such as celecoxib, in which antitumoral mechanisms were evaluated in rats bearing Walker-256 (W256) tumor. W256 carcinosarcoma cells were inoculated subcutaneously (10(7) cells/rat) in rats submitted to treatment with celecoxib (25 mg kg(-1)) or vehicle for 14 days. Tumor growth, body-weight gain, and survival data were evaluated. The mechanisms, such as COX-2 expression and activity, oxidative stress, by means of enzymes and lipoperoxidation levels, and apoptosis mediators were also investigated. A reduction in tumor growth and an increased weight gain were observed. Celecoxib provided a higher incidence of survival compared with the control group. Cellular effects are probably COX-2 independent, because neither enzyme expression nor its activity, measured by tumoral PGE(2), showed significant difference between groups. It is probable that this antitumor action is dependent on an apoptotic way, which has been evaluated by the expression of the antiapoptotic protein Bcl-xL, in addition to the cellular changes observed by electronic microscopy. Celecoxib has also a possible involvement with redox homeostasis, because its administration caused significant changes in the activity of oxidative enzymes, such as catalase and superoxide dismutase. These results confirm the antitumor effects of celecoxib in W256 cancer model, contributing to elucidating its antitumoral mechanism and corroborating scientific literature about its effect on other types of cancer.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ)[402414/2005-5]UFPRUFP

Uncompetitive nanomolar dimeric indenoindole inhibitors of the human breast cancer resistance pump ABCG2

International audienceMultidrug resistance membrane pumps reduce the efficacy of chemotherapies by exporting a wide panel of structurally-divergent drugs. Here, to take advantage of the polyspecificity of the human Breast Cancer Resistance Protein (BCRP/ABCG2) and the dimeric nature of this pump, new dimeric indenoindole-based inhibitors from the monomeric α,β-unsaturated ketone 4b and phenolic derivative 5a were designed. A library of 18 homo/hetero-dimers was synthesised. Homo-dimerization shifted the inhibition efficacy from sub-micromolar to nanomolar range, correlated with the presence of 5a, linked by a 2-6 methylene-long linker. Non-toxic, the best dimers displayed a therapeutic ratio as high as 70,000. It has been found that the high potency of the best compound 7b that displays a KI of 17 nM is due to an uncompetitive behavior toward mitoxantrone efflux and specific for that drug, compared to Hoechst 33342 efflux. Such property may be useful to target such anticancer drug efflux mediated by ABCG2. Finally, at a molecular level, an uncompetitive mechanism by which substrate promotes inhibitor binding implies that at least 2 ligands should bind simultaneously to the drug-binding pocket of ABCG2

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma