RESUMO
Toxicity of the SYD-1 mesoionic compound (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) was evaluated on human liver cancer cells (HepG2) grown in either high glucose (HG) or galactose (GAL) medium, and also on suspended cells kept in HG medium. SYD-1 was able to decrease the viability of cultured HepG2 cells in a dose-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays, but no effect was observed on suspended cells after 1-40 min of treatment. Respiration analysis was performed after 2 min (suspended cells) or 24 h (cultured cells) of treatment: no change was observed in suspended cells, whereas SYD-1 inhibited as well basal, leak and uncoupled states of the respiration in cultured cells with HG medium. These inhibitions were consistent with the decrease in pyruvate level and increase in lactate level. Even more extended results were obtained with HepG2 cells grown in GAL medium where, additionally, the ATP amount was reduced. Furthermore, SYD-1 appears not to be transported by the main ABC multidrug transporters. These results show that SYD-1 is able to change the metabolism of HepG2 cells, and suggest that its cytotoxicity is related to impairment of mitochondrial metabolism. Therefore, we may propose that SYD-1 is a potential candidate for hepatocarcinoma treatment.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Oxidiazóis/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Carcinoma Hepatocelular/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Ácido Láctico/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Ácido Pirúvico/metabolismoRESUMO
Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N (5)-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Desenho de Fármacos , Indenos/farmacologia , Indóis/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Fenóis/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Sítios de Ligação , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indenos/síntese química , Indenos/metabolismo , Indóis/síntese química , Indóis/metabolismo , Camundongos , Mitoxantrona/metabolismo , Modelos Moleculares , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células NIH 3T3 , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fenóis/síntese química , Fenóis/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , TransfecçãoRESUMO
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
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.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Tiadiazóis/química , Tiadiazóis/farmacologia , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Ratos , Ratos Wistar , Tiadiazóis/efeitos adversosRESUMO
A series of chalcones substituted by a quinoxaline unit at the B-ring were synthesized and tested as inhibitors of breast cancer resistance protein-mediated mitoxantrone efflux. These compounds appeared more efficient than analogs containing other B-ring substituents such as 2-naphthyl or 3,4-methylenedioxyphenyl while an intermediate inhibitory activity was obtained with a 1-naphthyl group. In all cases, two or three methoxy groups had to be present on the phenyl A-ring to produce a maximal inhibition. Molecular modeling indicated both electrostatic and steric positive contributions. A higher potency was observed when the 2-naphthyl or 3,4-methylenedioxyphenyl group was shifted to the A-ring and methoxy substituents were shifted to the phenyl B-ring, indicating preferences among polyspecificity of inhibition.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Chalconas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Quinoxalinas/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos/síntese química , Antineoplásicos/química , Células Cultivadas , Chalconas/síntese química , Chalconas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HEK293 , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) plays a major role in cancer cell multidrug resistance, which contributes to low efficacy of chemotherapy. Chalcones were recently found to be potent and specific inhibitors, but unfortunately display a significant cytotoxicity. A cellular screening against ABCG2-mediated mitoxantrone efflux was performed here by flow cytometry on 54 chalcone derivatives from three different series with a wide panel of substituents. The identified leads, with submicromolar IC50 (half maximal inhibitory concentration) values, showed that the previously identified 2'-OH-4',6'-dimethoxyphenyl, as A-ring, could be efficiently replaced by a 2'-naphthyl group, or a 3',4'-methylenedioxyphenyl with lower affinity. Such a structural variability indicates 3polyspecificity of the multidrug transporter for inhibitors. At least two methoxyl groups were necessary on B-ring for optimal inhibition, but substitution at positions 3, 4, and 5 induced cytotoxicity. The presence of a large O-benzyl substituent at position 4 and a 2'-naphthyl as A-ring markedly decreased the cytotoxicity, giving a high therapeutic ratio, which constitutes a critical requirement for future in-vivo assays in animal models.