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1.
Toxicol In Vitro ; 50: 172-178, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29574240

RESUMO

We previously showed that heterocyclic organobismuth compounds have excellent antimicrobial and antitumor potential. These compounds structurally consist of either six- or eight-membered rings. Previous research has shown that bi-chlorodibenzo[c,f][1,5]thiabismocine (Compound 3), an eight-membered ring, induced G2/M arrest via inhibition of tubulin polymerization in HeLa cells. Additionally, N-tert-butyl-bi-chlorodi-benzo[c,f][1,5]azabismocine (Compound 1), another eight-membered ring, exhibited higher cytotoxicity than Compound 3 against several cancer cell lines, including HeLa and K562. Finally, bi-chlorophenothiabismin-S,S-dioxide (Compound 5), a six-membered ring, exhibited lower antitumor activity than eight-membered ring compounds. In this study, we investigated the antimitotic activity of Compounds 1 and 5 in HeLa cells. At low concentrations, (0.1 and 0.25 µM), Compound 1 inhibited cell growth and arrested the cell cycle in mitosis. However, 0.5 µM Compound 1 exhibited no antimitotic activity. Conversely, Compound 5 weakly inhibited cell growth and did not markedly arrest the cell cycle. Flow cytometry showed that Compound 1 arrested the cell cycle at G2/M, resulting in apoptosis. Compound 1 inhibited tubulin polymerization as revealed by a cell-free assay, and both Compounds 1 and 3 inhibited microtubule spindle formation and chromosome alignment during prometaphase. These results suggest that eight-membered ring-containing organobismuth compounds can induce mitotic arrest by perturbing spindle dynamics.


Assuntos
Bismuto/toxicidade , Ciclo Celular/efeitos dos fármacos , Compostos Heterocíclicos/toxicidade , Compostos Organometálicos/toxicidade , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos
2.
Exp Cell Res ; 342(2): 135-44, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26976509

RESUMO

Ku protein is a heterodimer composed of two subunits, and is capable of both sequence-independent and sequence-specific DNA binding. The former mode of DNA binding plays a crucial role in DNA repair. The biological role of Ku protein during apoptosis remains unclear. Here, we show characterization of Ku protein during apoptosis. In order to study the DNA binding properties of Ku, we used two methods for the electrophoresis mobility shift assay (EMSA). One method, RI-EMSA, which is commonly used, employed radiolabeled DNA probes. The other method, WB-EMSA, employed unlabeled DNA followed by western blot and detection with anti-Ku antiserum. In this study, Ku-DNA probe binding activity was found to dramatically decrease upon etoposide treatment, when examined by the RI-EMSA method. In addition, pre-treatment with apoptotic cell extracts inhibited Ku-DNA probe binding activity in the non-treated cell extract. The inhibitory effect of the apoptotic cell extract was reduced by DNase I treatment. WB-EMSA showed that the Ku in the apoptotic cell extract bound to fragmented endogenous DNA. Interestingly, Ku in the apoptotic cell extract purified by the Resource Q column bound 15-bp DNA in both RI-EMSA and WB-EMSA, whereas Ku in unpurified apoptotic cell extracts did not bind additional DNA. These results suggest that Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. In conclusion, Ku is intact and retains DNA binding activity in early apoptotic cells.


Assuntos
Antígenos Nucleares/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Antígenos Nucleares/química , Antineoplásicos/química , DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Etoposídeo/química , Células HL-60 , Humanos , Autoantígeno Ku , Ligação Proteica , Transporte Proteico
3.
J Inorg Biochem ; 117: 77-84, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23078777

RESUMO

A novel organobismuth compound, 1-[(2-di-p-tolylbismuthanophenyl)diazenyl]pyrrolidine (4), which has 1-(phenyldiazenyl)pyrrolidine (1) substituent in a benzene ring of tri(p-tolyl)bismuthane (2), was synthesized and tested for biological activity toward human tumor cell lines. 4 had a potent anti-proliferative effect on human cancer cell lines, although both 1 and 2 exhibited only weak activity. The sensitivity of leukemic cell lines to 4 was relatively high; IC(50) values for the human leukemia cell line NB4 and cervical cancer cell line HeLa were 0.88 µM and 5.36 µM, respectively. Treatment of NB4 cells with 4 induced apoptosis, loss of mitochondrial membrane potential (ΔΨ(mt)) and the generation of cellular reactive oxygen species (ROS). 1 and 2 did not induce apoptosis and had only a marginal effect on ΔΨ(mt) and the generation of ROS. N-acetyl cysteine (NAC) reduced the generation of ROS and conferred protection against 4-induced apoptosis, indicating a role for oxidative stress. 4 did not inhibit the polymerization of tubulin in vitro. 1-[2-(di-p-tolylstibanophenyl)diazenyl]pyrrolidine (3), which has the same chemical structure as 4 but contains antimony in place of bismuth, did not show any cytotoxic activity. The results suggest that the conjugated structure of the diazenylpyrrolidine moiety and bismuth center are key to the bioactivity of 4.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Leucemia Promielocítica Aguda/metabolismo , Compostos Organometálicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/química , Divisão Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Leucemia Promielocítica Aguda/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos Organometálicos/química , Tubulina (Proteína)/metabolismo
4.
J Pharmacol Sci ; 109(4): 573-82, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19352070

RESUMO

Our previous study showed that organobismuth compounds induce apoptosis in human promyelocytic leukemia cells, although solid tumor cell lines were relatively resistant. Herein, we investigated the primary cellular target of these compounds in HeLa cells. One organobismuth compound, bi-chlorodibenzo[c,f][1,5]thiabismocine (compound 3), arrested the cell cycle at G(2)/M as assessed by flow cytometry and by upregulating the expression of cyclin B1. At a low concentration (0.5 microM), compound 3 caused cell cycle arrest at the mitotic phase and induced apoptosis. At a higher concentration (>1.0 microM), it induced an arrest in the G(2)/M phase, leading to apoptosis. In many cells blocked at the M phase, the organization of microtubules was affected, indicating depolymerization of the microtubule network. Western blotting demonstrated that compound 3 depolymerized microtubules similar to colchicine and nocodazole. Experiments in vitro also showed that compound 3 inhibited the assembly of purified tubulin in a concentration-dependent manner by interacting with the colchicine-binding site of tubulin through its SH groups. Heterocyclic organobismuth compounds are novel tubulin ligands.


Assuntos
Bismuto/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Tubulina (Proteína)/efeitos dos fármacos , Anexina A5/metabolismo , Western Blotting , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colchicina/farmacologia , Corantes , Citometria de Fluxo , Células HeLa , Humanos , Imuno-Histoquímica , Microtúbulos/efeitos dos fármacos , Índice Mitótico , Propídio , Compostos de Sulfidrila/farmacologia , Tubulina (Proteína)/metabolismo , Ensaio Tumoral de Célula-Tronco
5.
Biochem Pharmacol ; 76(8): 974-86, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18760261

RESUMO

We have synthesized novel heterocyclic organobismuth compounds that have potent antibacterial properties. In this study, we examined their anticancer activity and addressed the cellular mechanisms involved. Heterocyclic organobismuth compounds showed anticancer activities in various human cancer cell lines. These compounds have particularly potent anticancer activities against leukemia cell lines. One of them, bi-chlorodibenzo [c,f][1,5] thiabismocine (compound 3), inhibited the growth of the human promyelocytic leukemia cell line HL-60 at a concentration of 0.22 microM. Low concentrations of compound 3 (0.22-0.44 microM) induced apoptosis, whereas at a higher concentration (>1.1 microM) it causes acute necrosis. During the apoptosis, caspase-3, -8, and -9 were activated but caspase-12 was not. A broad caspase inhibitor (z-VAD-fmk), and caspase-3 (z-DEVD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors suppressed the compound 3-induced apoptosis, but a caspase-8 inhibitor (z-IETD-fmk) was less effective, suggesting that the caspase-8 activity only partially participates in the apoptosis. In the apoptotic cells, cytochrome c was released from mitochondria to cytosol and a loss of mitochondrial transmembrane potential (DeltaPsi(m)) was detected. Compound 3-induced apoptosis was associated with enhanced generation of intracellular reactive oxygen species (ROS). Pretreatment of the cells with N-acetyl-L-cysteine or catalase suppressed the apoptosis. On the other hand, buthionine sulfoximine enhanced the compound 3-induced collapse of DeltaPsi(m) and apoptosis. Taken together, these results indicate that compound 3 is a potent inducer of apoptosis, triggering a caspase-3-mediated mechanism via the generation of ROS and release of cytochrome c from mitochondria, suggesting a potential mechanism for the anticancer activity of compound 3.


Assuntos
Apoptose/efeitos dos fármacos , Bismuto/farmacologia , Células HL-60/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Compostos Organometálicos/farmacologia , Trifosfato de Adenosina/metabolismo , Anexina A5/metabolismo , Inibidores de Caspase , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Células HL-60/ultraestrutura , Humanos
6.
Antimicrob Agents Chemother ; 49(7): 2729-34, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15980343

RESUMO

Bismuth compounds are known for their low levels of toxicity in mammals, and various types of bismuth salts have been used to treat medical disorders. As part of our program to probe this aspect of bismuth chemistry, cyclic organobismuth compounds 1 to 8 bearing a nitrogen or sulfur atom as an additional ring member have been synthesized, and their antimicrobial activities against five standard strains of gram-negative and gram-positive bacteria were assessed. The eight-membered-ring compounds, compounds 1 to 3, exhibited MICs of less than 0.5 microg/ml against Staphylococcus aureus and were more active than the six-membered ones, compounds 5 to 8 (MICs, 4.0 to 16 microg/ml). The gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis) were more susceptible to both types of ring compounds than the gram-negative ones (Escherichia coli and Pseudomonas aeruginosa). Treatment with polymyxin B nonapeptide increased the susceptibility of E. coli to cyclic organobismuth compounds, indicating the low permeability of the outer membrane of gram-negative bacteria to the compounds. Compound 1 also had activity against methicillin-resistant S. aureus, which had an MIC for 90% of the hospital stock strains of 1.25 microg/ml. The killing curves for S. aureus treated with compound 1 or 3 revealed a static effect at a low dose (2x the MIC). However, when S. aureus was treated with 10x the MIC of compound 1 or 3, there was an approximately 3-log reduction in the viable cell number after 48 h of treatment. Electron microscopic inspection demonstrated a considerable increase in the size of S. aureus and the proportion of cells undergoing cell division after treatment with compound 1 at 0.5x the MIC.


Assuntos
Antibacterianos/farmacologia , Bismuto/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Bismuto/química , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura , Relação Estrutura-Atividade
7.
Life Sci ; 73(18): 2335-44, 2003 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-12941435

RESUMO

Mechanisms for hyperleptinemia elicited by a serotonin (5-hydroxytryptamine, 5-HT) precursor, 5-hydroxytryptophan (5-HTP), were investigated. 5-HTP elicited apparent increases in serum leptin levels of mice. Administration of 5-HTP did not alter expression of leptin mRNA in white adipose tissues. Furthermore, neither 5-HTP nor 5-HT increased leptin secretion from isolated fat pads of mice. Since insulin is known to enhance leptin release, involvement of insulin in 5-HTP-induced hyperleptinemia was examined. 5-HTP significantly elevated serum insulin levels. In mice treated with streptozotocin, which depletes insulin, 5-HTP did not increase serum leptin levels. These results suggest that hyperinsulinemia participates the elevation of serum leptin levels elicited by 5-HTP.


Assuntos
5-Hidroxitriptofano/farmacologia , Insulina/fisiologia , Leptina/sangue , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Benserazida/farmacologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Leptina/genética , Masculino , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serotonina/farmacologia , Serotoninérgicos/farmacologia , Fatores de Tempo
8.
Biol Pharm Bull ; 26(1): 105-7, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12520184

RESUMO

Effects of leptin on food intake and nitric oxide (NO) metabolites (nitrite and nitrate, NOx) levels of brain were investigated in mice. Leptin dose-dependently decreased milk intake in food-deprived mice. Administration of leptin at a dose of 1 mg/kg, which induces an apparent hypophagia, did not affect NOx levels in the hypothalamus and frontal cortex. These results suggest that leptin reduces food intake in food-deprived mice without altering NO production in the hypothalamus, which plays an important role in regulation of feeding.


Assuntos
Encéfalo/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Privação de Alimentos/fisiologia , Leptina/farmacologia , Óxido Nítrico/metabolismo , Animais , Encéfalo/metabolismo , Ingestão de Alimentos/fisiologia , Masculino , Camundongos , Nitratos/metabolismo , Nitritos/metabolismo
9.
Cell Struct Funct ; 27(2): 117-25, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12207053

RESUMO

In order to elucidate the mechanism controlling the biogenesis of the Golgi complex, we have studied whether the expression of a resident membrane protein p138 of the Golgi complex is dependent upon the cell cycle. The protein level of p138 in human KB cells was increased during thymidine block to synchronize the cells in the early-S phase, but changed little from S to G2 after release from the block. On the other hand, the mRNA level of the p138 gene was constant during the block. The change in mRNA level in the cells was small with a low peak at S to G2. Both p138 protein and mRNA levels decreased after cell division and then rose rapidly to the same level as those of log-phase cells in the next G1 to S. Thus, translation of p138 protein was upregulated in the cells at the early-S phase. However, we found also that the p138 protein level increased during an arrest at G2/M caused by etoposide. The kinetics of centrosome duplication apparently differ from those of p138 protein production. The duplication occurred mainly at S to G2 after the release from thymidine block, while the ratio of cells containing duplicated centrosomes increased gradually during the block. Taken together, these results show that both the translation and transcription of p138 protein are regulated independent of the cell cycle and dissociated from the duplication of the centrosome. Rather, the expression of p138 protein seems to be coupled with a change in cell size since both thymidine block and etoposide inhibition resulted in an apparent increase in cell size.


Assuntos
Centrossomo/fisiologia , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Ciclo Celular , Tamanho Celular/efeitos dos fármacos , Etoposídeo/farmacologia , Citometria de Fluxo , Fase G2/fisiologia , Complexo de Golgi/efeitos dos fármacos , Humanos , Hibridomas , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitose/fisiologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosfoproteínas/genética , RNA Mensageiro/análise , Fase S/fisiologia , Timidina/farmacologia , Inibidores da Topoisomerase II , Proteínas de Transporte Vesicular
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