RESUMO
A new series of amphiphilic η6-areneruthenium(II) compounds containing phenylazo ligands (group I: compounds 1a, 1b, 2a and 2b) and phenyloxadiazole ligands (group II: compounds 3a, 3b, 4a and 4b) were synthesized and characterized for their anti-glioblastoma activity. The effects of the amphiphilic η6-areneruthenium(II) complexes on the viability of three human glioblastoma cell lines, U251, U87MG and T98G, were evaluated. The azo-derivative ruthenium complexes (group I) showed high cytotoxicity to all cell lines, whilst most oxadiazole-derivative complexes (group II) were less cytotoxic, except for compound 4a. The cationic complexes 2a, 2b and 4b were more cytotoxic than the neutral complexes. Compounds 2a and 2b caused a significant reduction in the percentage of cells in the G0/G1 phase, with concomitant increases in the G2/M phase and fragmented DNA in the T98G cell line. The η6-areneruthenium(II) compounds were also tested in cell lines that overexpress the multidrug ABC transporters P-gp, MRP1 and ABCG2. Compounds 2b and 4a were substrates for the P-gp protein, with resistance indexes of 8.6 and 1.9, respectively. Compound 2b was also a substrate for ABCG2 and MRP1 proteins, with lower resistance indexes (1.8 and 1.6, respectively). The contribution of multidrug ABC transporters to the cytotoxicity of compound 2b in T98G cells was evidenced, since verapamil (a characteristic inhibitor of MRP1) increased the cytotoxicity of compound 2b at concentrations up to 20⯵molâ¯L-1, whilst GF120918 and Ko143 (specific inhibitors of P-gp and ABCG2, respectively) had no significant effect. In addition, we showed that compound 2b interacts with glutathione (GSH), which could explain its cellular efflux by MRP1. Our results showed that the amphiphilic η6-areneruthenium(II) complexes are promising anti-glioblastoma compounds, especially compound 2b, which was cytotoxic for all three cell lines, although it is transported by the three main multidrug ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos , Glioblastoma/tratamento farmacológico , Rutênio/farmacologia , Compostos Azo , Transporte Biológico , Linhagem Celular Tumoral , Glioblastoma/patologia , Humanos , Ligantes , Compostos Organometálicos/farmacologia , OxidiazóisRESUMO
Hepatocellular carcinoma is the third most common cause of cancer-related deaths worldwide. Furthermore, the existing pharmacological-based treatments are insufficiently effective and generate many side effects. Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) is a flavonoid found in various medicinal herbs that present antineoplastic properties. Here we evaluated how modulation of reactive oxygen species (ROS) and alterations of antioxidant defenses could be associated to the antiproliferative effects of hispidulin in HepG2 cells. In addition, we studied the inhibitory activity of hispidulin on the efflux of drugs mediated by ABC transporters involved in multidrug resistance. In order to understand the increase of intracellular ROS promoted by hispidulin, we investigated the mRNA expression levels and activities of antioxidant enzymes, and the GSH/GSSG ratio. We showed that hispidulin significantly down-regulated the transcription levels of catalase, leading to reduction of enzyme activity and decrease of the GSH content. We also observed that, in the presence of N-acetylcysteine or exogenous catalase, the proliferation was lowered back to the control levels. These data clearly indicate a strong involvement of intracellular ROS levels for triggering the antiproliferative effects. We also demonstrated that the inhibition produced by hispidulin on drug efflux was specific for ABCG2, since no effects were observed with ABCB1 and ABCC1. Furthermore, HepG2 cells were more sensitive to hispidulin-mediated cell death than immortalized L929 fibroblasts, suggesting a differential toxicity of this compound between tumor and non-tumor cell lines. Our results suggest that hispidulin constitutes a promising candidate to sensitize chemoresistant cancer cells overexpressing ABCG2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antioxidantes/farmacologia , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Flavonas/farmacologia , Neoplasias Hepáticas/patologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Catalase/biossíntese , Catalase/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Células L , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Mitoxantrona/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Plantas Medicinais/metabolismo , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismoRESUMO
Flavones have received considerable attention because of their antiproliferative properties and selective effects on cancer cells, making them good candidates for use in cancer therapy. In contrast to other flavones, little is known about the effects of the flavone core structure (2-phenyl-4H-1-benzopyran-4one) on cancer cells. Here, we report that flavone induces cell death in human hepatoma HepG2 cells. Furthermore, annexin-V+/PI- and SubG1 populations of HepG2 cells increased after flavone treatment. Exposure of HepG2 to flavone did not result in either cytochrome c release into the cytosol or changes in the mitochondrial membrane potential. Treatment of HepG2 cells with flavone for 24 h reduced the accumulation of intracellular ROS, which correlated with upregulation of Gred, CuZnSOD and MnSOD mRNA levels. Taken together, our results provided useful insights into the mechanism of cell death caused by flavones, in order to evaluate their future application in hepatocarcinoma therapy.
Assuntos
Morte Celular/efeitos dos fármacos , Flavonas/farmacologia , Citocromos c/metabolismo , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoAssuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Cromonas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Triptaminas/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/síntese química , Cromonas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Estrutura Molecular , Proteínas de Neoplasias/metabolismo , Relação Estrutura-AtividadeRESUMO
ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC(50) values below 0.5 µM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6'-hydroxyl-2',4'-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/síntese química , Chalconas/síntese química , Proteínas de Neoplasias/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Chalconas/química , Chalconas/farmacologia , Células HEK293 , Humanos , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Proteínas de Neoplasias/genética , Relação Quantitativa Estrutura-Atividade , TransfecçãoRESUMO
The ABCG2 multidrug transporter is known to confer cancer cell multidrug resistance by causing the efflux of anticancer drugs; therefore, selective inhibitors have the potential to improve chemotherapeutic treatments. Here, various methoxy derivatives of resveratrol are shown to be potent inhibitors of mitoxantrone efflux by ABCG2: among a series of 11 derivatives, compound 9 (3,5,3',4'-tetramethoxy trans-stilbene) had an IC(50) of 0.16 µM and showed a maximal inhibition of 75%, as measured by flow cytometry. It was not transported, as shown by HPLC fractionation and mass spectrometry titration and the lack of any cross-resistance in cell survival experiments. Compound 9 had a very low intrinsic cytotoxicity and was able to chemosensitize the growth of resistant ABCG2-transfected HEK293 cells at submicromolar concentrations. Drug-efflux inhibition was specific for ABCG2 since very low effects were observed with ABCB1 and ABCC1. The action mechanism of compound 9 was different from that of GF120918, which produced a complete and partly competitive but not ABCG2-specific inhibition, since ABCB1 was even more strongly inhibited. The two inhibitors also displayed different effects on the ABCG2 vanadate-sensitive ATPase activity, suggesting that they either bound to distinct sites or induced different conformational changes. Mitoxantrone efflux was fully inhibited by combining low concentrations of compound 9 with either GF120918 or a transport substrate such as prazosin or nilotinib. We conclude that methoxy derivatives of stilbene are good candidates for investigating future in vivo modulation of ABCG2 drug-efflux activity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mitoxantrona/metabolismo , Proteínas de Neoplasias/metabolismo , Estilbenos/química , Estilbenos/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , ResveratrolRESUMO
A series of 13 disubstituted chromones was synthesized. Two types of substituents, on each side of the scaffold, contributed to both the potency of ABCG2 inhibition and the cytotoxicity. The best compound, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one (6g), displayed high-affinity inhibition and low cytotoxicity, giving a markedly high therapeutic index. The chromone derivative specifically inhibited ABCG2 versus other multidrug ABC transporters and was not transported. It constitutes a highly promising candidate for in vivo chemosensitization of ABCG2-expressing tumors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/síntese química , Cromonas/síntese química , Indóis/síntese química , Proteínas de Neoplasias/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromonas/química , Cromonas/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Humanos , Indóis/química , Indóis/farmacologia , Mitoxantrona/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H(2)O(2)-dependent pathway via reduction of the antioxidant defenses.
Assuntos
Apigenina/farmacologia , Apoptose/fisiologia , Catalase/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Biocatálise/efeitos dos fármacos , Catalase/genética , Catalase/farmacologia , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Glutationa Peroxidase GPX1RESUMO
The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM) decreased approximately 30 percent the rate and total amplitude of valinomycin induced swelling and 60-100 percent the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM) reduced 35 percent the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM) promoted a reduction of 40 percent in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.
O objetivo deste estudo foi avaliar se eupafolina e hispidulina, flavonas extraídas do Eupatorium littorale Cabrera, Asteraceae, possuíam a capacidade de alterar propriedades das membranas biológicas e promover efeitos citotóxicos. Eupafolina (50-200 µM) reduziu em aproximadamente 30 por cento a velocidade e amplitude do inchamento mitocondrial induzido por valinomicina e 60-100 por cento o inchamento mitocondrial dependente de substrato. Além disso, eupafolina na dose de 200 µM reduziu a transição de permeabilidade mitocondrial em 35 por cento entretanto, a hispidulina não alterou este parâmetro em todas as doses testadas. A avaliação da transição de fase dos lipossomas de DMPC com a sonda DPH demonstrou que ambas as flavonas afetam a fase gel e fluida. Quando lipossomas de membranas mitocondriais e a sonda DPH-PA foram utilizados, houve aumento da polarização de fluorescência promovido pela hispidulina. Eupafolina e hispidulina, na dose de 100 µM, promoveram 40 por cento de redução da viabilidade de células HeLa em 24 h. Nossos resultados sugerem que eupafolina e hispidulina têm efeitos citotóxicos que podem ser explicados em parte pelas alterações promovidas por estas flavonas sobre propriedades de membranas biológicas e sobre a bioenergética mitocondrial.
RESUMO
Antioxidant potential is generally investigated by assaying the ability of a compound to protect biological systems from free radicals. However, non-radical reactive oxygen species can also be harmful. Singlet molecular oxygen ((1)O(2)) is generated by energy transfer to molecular oxygen. The resulting (1)O(2) is able to oxidize the nucleoside 2'-deoxyguanosine (dGuo), which leads to the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and spiroiminodihydantoin 2'-deoxyribonucleoside diastereomers (dSp) in an aqueous solution. The main objective of the present study was to verify whether the presence of flavonoids (flavone, apigenin, quercetin, morin and catechin) at different concentrations could protect dGuo from (1)O(2) damage. Of the tested flavonoids, flavone possessed antioxidant activity, as determined by a decrease in the formation of both products. Apigenin, morin, quercetin and catechin all increased the formation of 8-oxodGuo at a concentration of 100 microM. The quantification of plasmid strand breaks after treatment with formamidopyrimidine-DNA glycosylase showed that flavone protected and quercetin and catechin enhanced DNA oxidation. Our results show that compounds, such as flavonoids, may affect the product distribution of (1)O(2)-mediated oxidation of dGuo, and, in particular, high concentrations of flavonoids with hydroxyl groups in their structure lead to an increase in the formation of the mutagenic lesion 8-oxodGuo.
Assuntos
Antioxidantes/farmacologia , DNA/efeitos dos fármacos , Desoxiguanosina/metabolismo , Flavonoides/farmacologia , Espécies Reativas de Oxigênio/química , Apigenina/química , Catequina/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Flavonas/química , Flavonoides/química , Naftalenos/química , Oxirredução , Peróxidos/química , Quercetina/química , Espectrofotometria UltravioletaRESUMO
This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3',4'-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25-200microM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated approximately 30% by eupafolin (100microM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA-Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.
Assuntos
Respiração Celular/efeitos dos fármacos , Flavonas/farmacologia , Mitocôndrias/efeitos dos fármacos , Adenosina Trifosfatases/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Flavonas/química , Flavonas/isolamento & purificação , Flavonas/toxicidade , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Estrutura Molecular , Oxirredução , Relação Estrutura-AtividadeRESUMO
Plant mitochondria differ from those of mammals, since they incorporate an alternative electron transport pathway, which branches at ubiquinol to an alternative oxidase (AOX), characteristically inhibited by salicylhydroxamic acid (SHAM). Another feature of plant mitochondria is that besides complex I (EC 1.6.5.3) they possess alternative NAD(P)H-dehydrogenases insensitive to rotenone. Many stress conditions are known to alter the expression of the alternative electron transport pathway in plant mitochondria. In the present study we investigated the effects of some thiol reagents and Ca(2+) on potato mitochondrial respiratory chain presenting different activities of the alternative respiratory components AOX and external NADH dehydrogenase, a condition induced by previous treatment of potato tubers (Solanum tuberosum L., cv. Bintje) to cold stress. The results showed that Ca(2+) presented an inhibitory effect on AOX pathway in potato mitochondria energized with NADH or succinate, which was only now observed when the cytochrome pathway was inhibited by cyanide. When the cytochrome pathway was functional, Ca(2+) stimulated the external NADH dehydrogenase. Diamide was a potent AOX inhibitor and this effect was only now observed when the cytochrome pathway was inactive, as was the case for the calcium ion. Mersalyl inhibited the externally located NADH dehydrogenase and had no effect on AOX activity. The results may represent an important function of Ca(2+) on the alternative mitochondrial enzymes NADH-DH(ext) and AOX.
Assuntos
Cálcio/farmacologia , Mitocôndrias/fisiologia , NADH Desidrogenase/metabolismo , Oxirredutases/metabolismo , Solanum tuberosum/fisiologia , Reagentes de Sulfidrila/farmacologia , Ubiquinona/análogos & derivados , Membrana Celular/enzimologia , Membrana Celular/fisiologia , Diamida/toxicidade , Potenciais da Membrana/fisiologia , Mersalil/toxicidade , Mitocôndrias/enzimologia , Proteínas Mitocondriais , Oxirredução/efeitos dos fármacos , Oxigênio/metabolismo , Proteínas de Plantas , Solanum tuberosum/enzimologia , Ácido Succínico/metabolismo , Ubiquinona/metabolismoRESUMO
5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.