RESUMO
BACKGROUND/AIM: Metal-containing compounds (e.g., platinum complexes) belong to the standard armamentarium of cancer chemotherapy. Copper N-(2-hydroxy acetophenone) glycinate (CuNG) exerts anticancer activity in vitro and in vivo and modulates drug resistance related to glutathione or P-glycoprotein. The potential of CuNG to interact with ATP-binding cassette (ABC) transporters has not been fully explored yet. This study focused on the modulatory effects of CuNG on four ABC transporters (MRP1, MRP1, BCRP, and P-glycoprotein). MATERIALS AND METHODS: Cell viability, drug uptake and ABC transporter expression were measured by resazurin assays, flow cytometry, and ELISA in HL60AR, MDCKII-hBCRP, and Caco-2 cells. RESULTS: CuNG increased doxorubicin sensitivity of MRP1-over-expressing HL60AR with a similar efficacy as the control MRP1 inhibitor MK571. CuNG also increased MRP1's efflux activity. Comparable results were obtained with MDCKII cells over-expressing hBCRP. ELISA assays revealed that the expression of MRP1 in HL60AR cells and BCRP in MDCKII- cells was predominant but other ABC-transporters were also expressed at lower levels. Caco-2 cells expressed high levels of MRP2, but MRP1, BCRP, and P-glycoprotein were also expressed. In contrast to the two former cell lines, CuNG increased doxorubicin resistance and decreased efflux activity in Caco-2 cells. CONCLUSION: CuNG exerted different modulatory activities towards ABC-transporter-expressing cells. While CuNG-mediated ABC-transporter inhibition may improve tumor chemotherapy (like in HL60AR and MDCKII-hBCRP cells), CuNG-mediated enhanced ABC-transport (like in Caco-2 cells) may be a new strategy to ameliorate inflammatory diseases associated with decreased ABC-transporter expression such as ulcerative colitis.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Acetofenonas , Compostos de Cobre Orgânico , Humanos , Acetofenonas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Células CACO-2/efeitos dos fármacos , Cobre/farmacologia , Doxorrubicina/farmacologia , Proteínas de Neoplasias , Compostos de Cobre Orgânico/farmacologiaRESUMO
Two Cu(II) (C1) and Ni(II) (C2) complexes were designed through the one-pot reaction of pyridine-2,6-dicarboxylic acid and 2-aminobenzimidazole respectively with copper(II) nitrate hexahydrate and nickel(II) nitrate hexahydrate. Both complexes were characterized by single-crystal X-ray diffraction and the distorted octahedral geometry was recognized for them. The MTT assay indicated that the complexes have a significant antiproliferative effect on BEL-7404 cells. IC50 values confirmed that C1 (IC50 = 0.56 µM) is several times more potent than C2 (IC50 = 5.13 µM). The similar cellular uptake of the complexes in mentioned cells led to this proposal that the production of ROS with different values can be the main reason for different cytotoxicity of the complexes. In this study, C1 and C2 caused BEL-7404 cells arrest at the G2/M and S phases, respectively. The expression of p53, Bax up-regulation, and Bcl-2 down-regulation and also activation of procaspase-9, and 3 indicated that apoptosis through a caspase-dependent mitochondrion pathway is a remarkable pathway in BEL-7404 cells treated by C1 while mechanistic studies proved that C2 induce death of BEL-7404 cells through the activation of RAGE/PI3KC3/Beclin 1 autophagic cell signaling pathway, more specifically. The cytostatic effect of the complexes in the BEL-7404 3D spheroid model was depicted.
Assuntos
Antineoplásicos , Níquel , Compostos de Cobre Orgânico , Piridinas , Antineoplásicos/farmacologia , Antineoplásicos/química , Apoptose , Ligantes , Piridinas/química , Piridinas/farmacologia , Níquel/química , Níquel/farmacologia , Linhagem Celular Tumoral , Humanos , Compostos de Cobre Orgânico/química , Compostos de Cobre Orgânico/farmacologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons progressively and rapidly degenerate, eventually leading to death. The first protein found to contain ALS-associated mutations was copper/zinc superoxide dismutase 1 (SOD1), which is conformationally stable when it contains its metal ligands and has formed its native intramolecular disulfide. Mutations in SOD1 reduce protein folding stability via disruption of metal binding and/or disulfide formation, resulting in misfolding, aggregation, and ultimately cellular toxicity. A great deal of effort has focused on preventing the misfolding and aggregation of SOD1 as a potential therapy for ALS; however, the results have been mixed. Here, we utilize a small-molecule polytherapy of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuATSM) and ebselen to mimic the metal delivery and disulfide bond promoting activity of the cellular chaperone of SOD1, the "copper chaperone for SOD1." Using microscopy with automated image analysis, we find that polytherapy using CuATSM and ebselen is highly effective and acts in synergy to reduce inclusion formation in a cell model of SOD1 aggregation for multiple ALS-associated mutants. Polytherapy reduces mutant SOD1-associated cell death, as measured by live-cell microscopy. Measuring dismutase activity via zymography and immunoblotting for disulfide formation showed that polytherapy promoted more effective maturation of transfected SOD1 variants beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may merit more study as an effective method of treating SOD1-associated ALS.
Assuntos
Esclerose Lateral Amiotrófica , Compostos de Cobre Orgânico , Superóxido Dismutase-1 , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Materiais Biomiméticos/farmacologia , Cobre/metabolismo , Dissulfetos/química , Humanos , Isoindóis/farmacologia , Chaperonas Moleculares/metabolismo , Mutação , Compostos de Cobre Orgânico/farmacologia , Compostos Organosselênicos/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Despite the availability of a myriad targeted treatments, resistance and treatment failures remains common in cancer treatment. Moreover, the high cost of targeted antibodies excludes a large cohort of patients from their benefits. In this context, copper-imidazo[1,2-a]pyridines were evaluated as alternative drug candidates against two common leukaemias, represented by HL-60 and K562 cells. A previous study identified JD88(21), JD47(29) and JD49(28) to be active against these cell lines with IC50 values between 1.9 and 6 µM and low leukocyte toxicity. To better understand their mechanism of action, their mode of cell death, effects on expression of apoptotic regulatory proteins and their respective genes were investigated. In both cell lines, the copper-imidazo[1,2-a]pyridines, at IC75 concentrations, caused membrane blebbing, raised phosphatidyl-serine levels on cell membranes and increased caspase-3 activity. A loss of mitochondrial membrane potential and activation of caspase-9, combined with poor caspase-8 activity indicated activation of intrinsic apoptosis. Apoptotic proteome analysis showed that the copper-imidazo[1,2-a] pyridines elevated protein levels of pro-apoptotic Bax and Smac/DIABLO in both cell lines, confirming their importance in apoptotic cell death. Conversely, though survivin was increased, this was counteracted by high levels of HTRA2/Omi expression. Effects on apoptotic regulatory proteins Bad, Bcl-2, XIAP and cIAP-1 was inconsistent between the copper-imidazo[1,2-a]pyridines and between the two cell lines, suggesting that the effect of the complexes was modulated by the molecular signature of each cell line. Analysis of mRNA transcripts showed a poor correlation between mRNA levels and associated proteins, implying that copper-imidazo[1,2-a]pyridines compromised protein synthesis and degradation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Compostos de Cobre Orgânico/farmacologia , Proteínas Reguladoras de Apoptose/genética , Humanos , Imidazóis/química , Células K562 , Compostos de Cobre Orgânico/química , Piridinas/químicaRESUMO
The synthetic copper-containing compound, CuATSM, has emerged as one of the most promising drug candidates developed for the treatment of amyotrophic lateral sclerosis (ALS). Multiple studies have reported CuATSM treatment provides therapeutic efficacy in various mouse models of ALS without any observable adverse effects. Moreover, recent results from an open label clinical study suggested that daily oral dosing with CuATSM slows disease progression in patients with both sporadic and familial ALS, providing encouraging support for CuATSM in the treatment of ALS. Here, we assessed CuATSM in high copy SOD1G93A mice on the congenic C57BL/6 background, treating at 100 mg/kg/day by gavage, starting at 70 days of age. This dose in this specific model has not been assessed previously. Unexpectedly, we report a subset of mice initially administered CuATSM exhibited signs of clinical toxicity, that necessitated euthanasia in extremis after 3-51 days of treatment. Following a 1-week washout period, the remaining mice resumed treatment at the reduced dose of 60 mg/kg/day. At this revised dose, treatment with CuATSM slowed disease progression and increased survival relative to vehicle-treated littermates. This work provides the first evidence that CuATSM produces positive disease-modifying outcomes in high copy SOD1G93A mice on a congenic C57BL/6 background. Furthermore, results from the 100 mg/kg/day phase of the study support dose escalation determination of tolerability as a prudent step when assessing treatments in previously unassessed models or genetic backgrounds.