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1.
Blood Cells Mol Dis ; 79: 102345, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31351219

RESUMO

Pharmacologic induction of fetal hemoglobin (HbF) is an effective strategy for treating sickle cell disease (SCD) by ameliorating disease severity. Hydroxyurea is the only FDA-approved agent that induces HbF, but significant non-responders and requirement for frequent monitoring of blood counts for drug toxicity limit clinical usefulness. Therefore, we investigated a novel prodrug conjugate of butyric acid (BA) and δ-aminolevulinate (ALA) as a potential HbF inducing agent, using erythroid precursors and a preclinical ß-YAC mouse model. We observed significantly increased γ-globin gene transcription and HbF expression mediated by AN-233 in K562 cells. Moreover, AN-233 stimulated mild heme biosynthesis and inhibited expression of heme-regulated eIF2α kinase involved in silencing γ-globin expression. Studies using primary erythroid precursors generated from sickle peripheral blood mononuclear cells verified the ability of AN-233 to induce HbF, increase histone H3 and H4 acetylation levels at the γ-globin promoter and reduce erythroid precursor sickling by 50%. Subsequent drug treatment of ß-YAC transgenic mice confirmed HbF induction in vivo by AN-233 through an increase in the percentage of HbF positive red blood cells and HbF levels measured by flow cytometry. These data support the potential development of AN-233 for the treatment of SCD.


Assuntos
Anemia Falciforme/terapia , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/efeitos dos fármacos , Ácidos Levulínicos/farmacologia , Pró-Fármacos/farmacologia , Animais , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Humanos , Células K562 , Ácidos Levulínicos/uso terapêutico , Camundongos , Camundongos Transgênicos , Ativação Transcricional , gama-Globinas/genética
2.
Int J Mol Sci ; 20(3)2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736437

RESUMO

: Choroidal neovascularization (CNV) is a complication of age-related macular degeneration and a major contributing factor to vision loss. In this paper, we show that in a mouse model of laser-induced CNV, systemic administration of Butyroyloxymethyl-diethyl phosphate (AN7), a histone deacetylase inhibitor (HDACi), significantly reduced CNV area and vascular leakage, as measured by choroidal flatmounts and fluorescein angiography. CNV area reduction by systemic AN7 treatment was similar to that achieved by intravitreal bevacizumab treatment. The expression of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), and the endothelial cells marker CD31, was lower in the AN7 treated group in comparison to the control group at the laser lesion site. In vitro, AN7 facilitated retinal pigmented epithelium (RPE) cells tight junctions' integrity during hypoxia, by protecting the hexagonal pattern of ZO-1 protein in the cell borders, hence reducing RPE permeability. In conclusion, systemic AN7 should be further investigated as a possible effective treatment for CNV.


Assuntos
Neovascularização de Coroide/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Acetilação , Animais , Biomarcadores , Permeabilidade Capilar , Linhagem Celular , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/química , Histonas/metabolismo , Hipóxia , Imuno-Histoquímica , Masculino , Camundongos , Junções Íntimas
3.
J Cell Biochem ; 119(4): 3417-3428, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29135083

RESUMO

The HDAC inhibitory activity of valproic acid (VPA) has led to on-going evaluation of it as an anticancer agent. The histone deacetylase (HDAC) inhibitor AN446, a prodrug of VPA, releases the acid upon metabolic degradation. AN446 is >60-fold more potent than VPA in killing cancer cells in vitro. Herein, we compare the activities of AN446, as an anticancer agent, to those of representative types from each of the four major classes of HDAC inhibitors (HDACIs): vorinostat, romidepsin, entinostat, and VPA. AN446 exhibited the greatest selectivity and HDAC inhibitory activity against cancer cells. In glioblastoma cells only AN446, and in MDA-MB-231 cells only AN446 and VPA interacted in synergy with doxorubicin (Dox). AN446 was superior to the studied HDACIs in inducing DNA-damage in cancer cells, while in normal astrocytes and cardiomyoblasts AN446 was the least toxic. AN446 was the only HDACI tested that exhibited selective HDAC inhibitory activity that was high in cancer cells and low in noncancerous cells. This discriminating inhibition correlated with the toxicity of the HDACIs, suggesting that their effects could be attributed to HDAC inhibition. In cardiomyoblasts, the HDACIs tested, except for AN446, hampered DNA repair by reducing the level of Rad 51. VPA and AN446 were the most effective HDACIs in inhibiting in vitro migration and invasion. The advantages of AN446 shown here, position it as a potentially improved HDACI for treatment of glioblastoma and triple negative breast cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Pró-Fármacos/farmacologia , Ácido Valproico/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/síntese química , Humanos
4.
Invest New Drugs ; 36(1): 1-9, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28884410

RESUMO

We previously found that the novel histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) had greater selectivity against cutaneous T-cell lymphoma (CTCL) than SAHA. AN-7 synergizes with doxorubicin (Dox), an anthracycline antibiotic that induces DNA breaks. This study aimed to elucidate the mechanism underlying the effect of AN-7 on Dox-induced double-strand DNA breaks (DSBs) in CTCL, MyLa and Hut78 cell lines. The following markers/assays were employed: comet assay; western blot of γH2AX and p-KAP1; immunofluorescence of γH2AX nuclear foci; Western blot of repair protein; quantification of DSBs-repair through homologous recombination. DSB induction by Dox was evidenced by an increase in DSB markers, and DSBs-repair, by their subsequent decrease. The addition of AN-7 slightly increased Dox induction of DSBs in MyLa cells with no effect in Hut78 cells. AN-7 inhibited the repair of Dox-induced DSBs, with a more robust effect in Hut78. Treatment with AN-7 followed by Dox reduced the expression of DSB-repair proteins, with direct interference of AN-7 with the homologous recombination repair. AN-7 sensitizes CTCL cell lines to Dox, and when combined with Dox, sustains unrepaired DSBs by suppressing repair protein expression. Our data provide a mechanistic rationale for combining AN-7 with Dox or other DSB inducers as a therapeutic modality in CTCL.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Butiratos/farmacologia , Doxorrubicina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Humanos , Linfoma Cutâneo de Células T/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico
5.
Invest New Drugs ; 35(4): 412-426, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28315153

RESUMO

The histone deacetylase (HDAC) inhibitory prodrugs of butyric (AN7) and valproic (AN446) acids, which release the active acids upon metabolic degradation, were studied examining their differential effects on the viability, HDAC inhibitory activity and the DNA damage response (DDR), in glioblastoma cell and normal human astrocytes (NHAs). In xenografts of glioblastoma, AN7 or AN446 given or the combination of each of them with Dox augmented the anticancer activity of Dox and protected the heart from its toxicity. In order to determine the processes underlying these opposing effects, the changes induced by these treatments on the epigenetic landscape, the DDR, and fibrosis were compared in tumors and hearts of glioblastoma xenografts. The potency of AN7 and AN446 as HDAC inhibitors was correlated with their effects on the viability of the cancer and non-cancer cells. The prodrugs affected the epigenetic landscape and the DDR in a tissue-specific and context-dependent manner. Findings suggest that the selectivity of the prodrugs could be attributed to their different effects on histone modification patterns in normal vs. transformed tissues. Further studies are warranted to substantiate the potential of AN446 as a new anticancer drug for glioblastoma patients.


Assuntos
Antineoplásicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Glioblastoma/genética , Inibidores de Histona Desacetilases/farmacologia , Pró-Fármacos/farmacologia , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Doxorrubicina/farmacologia , Doxorrubicina/toxicidade , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Metilação/efeitos dos fármacos , Camundongos Nus , Miocárdio/metabolismo , Pró-Fármacos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Invest New Drugs ; 30(3): 1028-38, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21509470

RESUMO

Herein we describe a series of multifunctional 5-aminolevulinic-acid (ALA) prodrugs for photodynamic dependent and independent cancer therapy (PDT). We studied the cell-death mechanisms in glioblastoma U251 cells treated with four ALA-prodrugs: (1) AlaAcBu, that releases ALA, acetaldehyde, and butyric acid; (2) AlaFaBu, that releases ALA, formaldehyde, and butyric acid; (3) AlaFaPi, that releases ALA, formaldehyde and pivalic acid (4) AlaAcPi that releases ALA, acetaldehyde and pivalic acid. We examined the light-activated and dark cell-death mechanisms of the active metabolites released from the prodrugs by unspecific cellular hydrolases. The active moieties accelerated biosynthesis of protoporphyrin IX (PpIX) due to upregulated porphobilinogen deaminase (PBGD) activity. AlaAcBu was found to be the superior prodrug for PDT due to its ability to induce the highest PpIX synthesis. Photo-irradiation of AlaAcBu-treated cells led to dissipation of the mitochondrial membrane potential and reduction in the mitochondria metabolic activities; apoptosis and necrosis. Electron microscopy analyses of these cells revealed mitochondrial and endoplasmic reticulum swelling, membrane blebbing, apoptotic bodies and necrotic cell rupture. The formaldehyde-releasing prodrugs AlaFaBu and AlaFaPi induced low PDT efficacy, moreover sequestering the formaldehyde with semicarbazide resulted in high PpIX synthesis, suggesting that formaldehyde inhibited its synthesis. ALA and AlaAcBu phototherapy resulted in a dramatic accumulation of ubiquitinated proteins due to reduced proteasome activity and expression. In conclusion, the PDT potency of the prodrugs was in the order: AlaAcBu, AlaAcPi > AlaFaBu ≥ ALA > AlaFaPi, and the superiority of AlaAcBu stems from lower molar concentrations of AlaAcBu and lower light intensity needed to activate cell death following PDT.


Assuntos
Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/farmacologia , Morte Celular/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Fotoquimioterapia , Pró-Fármacos/farmacologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/ultraestrutura , Humanos , Hidroximetilbilano Sintase/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Protoporfirinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Invest New Drugs ; 30(1): 130-43, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20862515

RESUMO

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) has been shown to synergize doxorubicin (Dox) anticancer activity while attenuating its cardiotoxicity. In this study we further explored the selectivity of AN-7's action in several cancer and normal cells treated with anticancer agents. The cells studied were murine mammary 4T1, human breast T47D and glioblastoma U251 cancer cell lines, neonatal rat cardiomyocytes, cardiofibroblasts and astrocytes, and immortalized cardiomyocyte H9C2 cells. Cell death, ROS production and changes in protein expression were measured and in vivo effects were evaluated in Balb-c mice. AN-7 synergized Dox and anti-HER2 cytotoxicity against mammary carcinoma cells with combination indices of 0.74 and 0.79, respectively, while it protected cardiomyocytes against their toxicity. Additionally AN-7 protected astrocytes from Dox-cytoxicity. Cell-type specific changes in the expression of proteins controlling survival, angiogenesis and inflammation by AN-7 or AN-7+Dox were observed. In mice, the protective effect of AN-7 against Dox cardiotoxicity was associated with a reduction in inflammatory factors. In summary, AN-7 augmented the anticancer activity of Dox and anti-HER2 and attenuated their toxicity against normal cells. AN-7 modulation of c-Myc, thrombospondin-1, lo-FGF-2 and other proteins were cell type specific. The effects of AN-7, Dox and their combination were preserved in vivo indicating the potential benefit of combining AN-7 and Dox for clinical use.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Astrócitos/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Fibroblastos/efeitos dos fármacos , Glioblastoma/patologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Angiogênicas/metabolismo , Animais , Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Astrócitos/patologia , Neoplasias Encefálicas/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/imunologia , Butiratos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Feminino , Fibroblastos/patologia , Glioblastoma/enzimologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/patologia , Compostos Organofosforados/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-2/imunologia , Fatores de Tempo
8.
Cancer Chemother Pharmacol ; 89(6): 773-784, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35460360

RESUMO

PURPOSE: Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin's lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation. METHODS: In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival. RESULTS: Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10-40 µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10 kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9. CONCLUSIONS: The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties.


Assuntos
Neoplasias , Pró-Fármacos , Adutos de DNA , DNA Topoisomerases Tipo II/metabolismo , Formaldeído/farmacologia , Humanos , Isoquinolinas , Pró-Fármacos/farmacologia
9.
Photochem Photobiol Sci ; 10(12): 1926-33, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22020364

RESUMO

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.


Assuntos
Ácido Aminolevulínico/farmacologia , Ácidos Levulínicos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Pró-Fármacos/farmacologia , Ácido Aminolevulínico/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Ácidos Levulínicos/química , Ácidos Levulínicos/uso terapêutico , Microscopia de Fluorescência , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Pró-Fármacos/uso terapêutico , Protoporfirinas/metabolismo
10.
Pharmaceuticals (Basel) ; 14(12)2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34959644

RESUMO

We studied the unique inhibitor of the histone deacetylases (HDAC) valproate-valpromide of acyclovir (AN446) that upon metabolic degradation release the HDAC inhibitor (HDACI) valproic acid (VPA). Among the HDAC inhibitors that we have tested, only AN446, and to a lesser extent VPA, synergized with doxorubicin (Dox) anti-cancer activity. Romidepsin (Rom) was additive and the other HDACIs tested were antagonistic. These findings led us to test and compare the anticancer activities of AN446, VPA, and Rom with and without Dox in the 4T1 triple-negative breast cancer murine model. A dose of 4 mg/kg once a week of Dox had no significant effect on tumor growth. Rom was toxic, and when added to Dox the toxicity intensified. AN446, AN446 + Dox, and VPA + Dox suppressed tumor growth. AN446 and AN446 + Dox were the best inhibitory treatments for tumor fibrosis, which promotes tumor growth and metastasis. Dox increased fibrosis in the heart and kidneys, disrupting their function. AN446 most effectively suppressed Dox-induced fibrosis in these organs and protected their function. AN446 and AN446 + Dox treatments were the most effective inhibitors of metastasis to the lungs, as measured by the gap area. Genes that control and regulate tumor growth, DNA damage and repair, reactive oxygen production, and generation of inflammation were examined as potential therapeutic targets. AN446 affected their expression in a tissue-dependent manner, resulting in augmenting the anticancer effect of Dox while reducing its toxicity. The specific therapeutic targets that emerged from this study are discussed.

11.
Biochem Pharmacol ; 185: 114410, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33428897

RESUMO

Cancer patients treated with doxorubicin are at risk of congestive heart failure due to doxorubicin-mediated cardiotoxicity via topoisomerase IIß poisoning. Acute cardiac muscle damage occurs in response to the very first dose of doxorubicin, however, cardioprotection has been reported after co-treatment of doxorubicin with acyloxyalkyl ester prodrugs. The aim of this study was to examine the role played by various forms of acute cardiac damage mediated by doxorubicin and determine a mechanism for the cardioprotective effect of formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate). Doxorubicin-induced cardiac damage in BALB/c mice bearing mammary tumours was established with a single dose of doxorubicin (4 or 16 mg/kg) administered alone or in combination with AN-9 (100 mg/kg). AN-9 protected the heart from doxorubicin-induced myocardial apoptosis and also significantly reduced dsDNA breaks, independent from the level of doxorubicin biodistribution to the heart. Covalent incorporation of [14C]doxorubicin into DNA showed that the combination treatment yielded significantly higher levels of formaldehyde-mediated doxorubicin-DNA adducts compared to doxorubicin alone, yet this form of damage was associated with cardioprotection from apoptosis. The cardiac transcriptomic analysis indicates that the combination treatment initiates inflammatory response signalling pathways. Doxorubicin and AN-9 combination treatments were cardioprotective, yet preserved doxorubicin-mediated anti-tumour proliferation and apoptosis in mammary tumours. This was associated with a switch in doxorubicin action from cardiac topoisomerase IIß poisoning to covalent-DNA adduct formation. Co-administration of doxorubicin and formaldehyde-releasing prodrugs, such as AN-9, may be a promising cardioprotective therapy while maintaining doxorubicin activity in primary mammary tumours.


Assuntos
Antibióticos Antineoplásicos/toxicidade , Cardiotoxicidade/patologia , Cardiotoxicidade/prevenção & controle , Doxorrubicina/toxicidade , Miocárdio/patologia , Animais , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Cardiotoxicidade/metabolismo , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/metabolismo
12.
Eur J Pharmacol ; 882: 173255, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32553737

RESUMO

The anticancer prodrug butyroyloxymethyl diethylphosphate (AN-7), upon metabolic hydrolysis, releases the histone deacetylase inhibitor butyric acid and imparts histone hyperacetylation. We have shown previously that AN-7 increases doxorubicin-induced cancer cell death and reduces doxorubicin toxicity and hypoxic damage to the heart and cardiomyocytes. The cardiofibroblasts remain unprotected against both insults. Herein we examined the selective effect of AN-7 on hypoxic cardiomyocytes and cardiofibroblasts and investigated mechanisms underlying the cell specific response. Hypoxic cardiomyocytes and cardiofibroblasts or H2O2-treated H9c2 cardiomyoblasts, were treated with AN-7 and cell damage and death were evaluated as well as cell signaling pathways and the expression levels of heme oxygenase-1 (HO-1). AN-7 diminished hypoxia-induced mitochondrial damage and cell death in hypoxic cardiomyocytes and reduced hydrogen peroxide damage in H9c2 cells while increasing cell injury and death in hypoxic cardiofibroblasts. In the cell line, AN-7 induced Akt and ERK survival pathway activation in a kinase-specific manner including phosphorylation of the respective downstream targets, GSK-3ß and BAD. Hypoxic cardiomyocytes responded to AN-7 treatment by enhanced phosphorylation of Akt, ERK, GSK-3ß and BAD and a significant 6-fold elevation in HO-1 levels. In hypoxic cardiofibroblasts, AN-7 did not activate Akt and ERK beyond the effect of hypoxia alone and induced a limited (~1.5-fold) increase in HO-1. The cell specific differences in kinase activation and in heme oxygenase-1 upregulation may explain, at least in part, the disparate outcome of AN-7 treatment in hypoxic cardiomyocytes and hypoxic cardiofibroblasts.


Assuntos
Antineoplásicos/farmacologia , Butiratos/farmacologia , Cardiotônicos/farmacologia , Fibroblastos/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Animais , Ácido Butírico , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Inibidores de Histona Desacetilases , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos
13.
Oncol Res ; 17(7): 283-99, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19408574

RESUMO

The chemotherapeutic agent doxorubicin forms drug-DNA adducts that are enhanced by formaldehyde-releasing prodrugs such as AN-9. One of the major limitations of doxorubicin is dose-limiting cardiotoxicity; therefore, the use of a targeting strategy that enables drug delivery and release at tumor sites is of great interest. The major aim of this study was to use the Pluronic-ultrasound delivery system to encapsulate doxorubicin and formaldehyde-releasing prodrugs within Pluronic micelles, and then use ultrasound to trigger controlled drug release from micelles. Pluronic micelles themselves were not stable upon dilution and required the use of a stabilizing agent DSPE-PEG2000 to form stable "mixed micelles." Following the separation of free doxorubicin, approximately 60% of doxorubicin remained encapsulated within mixed micelles with a retention half-life of approximately 12 h. The formaldehyde-releasing prodrugs, however, were not retained within mixed micelles, but could potentially be administered separately to doxorubicin-loaded micelles to achieve tumor-localized formation of doxorubicin-DNA adducts. The use of low-frequency, high-power ultrasound (20 kHz, 100 W/cm2) released 7-10% of doxorubicin from mixed micelles. Collectively, these results indicate that the Pluronic-ultrasound system could be used to deliver and release doxorubicin with the potential of forming cytotoxic DNA adducts at tumor sites with coadministrated formaldehyde-releasing prodrugs.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Pró-Fármacos/administração & dosagem , Formaldeído/química , Células HL-60/efeitos dos fármacos , Humanos , Micelas , Ultrassom
14.
Clin Exp Metastasis ; 25(7): 703-16, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18506586

RESUMO

Histone deacetylase inhibitory prodrugs that are metabolized to butyric acid and formaldehyde possess antineoplastic properties and low toxicity. We sought to characterize the antiangiogenic and antimetastatic activities of two lead prodrugs, pivaloyloxymethyl butyrate (AN-9) and butyroyloxymethyl-diethyl phosphate (AN-7) in murine cancer models. In the sc implanted human colon carcinoma HT-29 xenograft model AN-7, exhibited superior anticancer activity compared to AN-9, as was evident by the significantly greater inhibition of tumor growth and reduction of serum CEA. AN-7 was also more effective in reducing mean vessel density (MVD) by 7-fold, bFGF, Ki-67 (7-fold) and HIF-1alpha in immunohistochemically stained tumor sections. Semi-quantitative evaluation of the levels of bFGF, HDAC1 and HIF-1alpha by Western blot analysis showed a decrease in expression only in the tumors of mice treated with AN-7. The level of bFGF was reduced 3-fold in the tumor and that of TIMP1 was elevated (by 3-fold) in the serum of AN-7 treated mice. In a 4T1 metastatic breast carcinoma model, AN-7 inhibited the formation of lung lesions by 76% and AN-9 by 47%, further demonstrating the greater efficacy of AN-7 compared to AN-9 (P<0.02). Both AN-7 and AN-9 exhibited antimetastatic and antiangiogenic activities by reducing vascularization, bFGF expression and HIF-1alpha. Yet, AN-7 was more potent than AN-9.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Butiratos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Metástase Neoplásica/prevenção & controle , Compostos Organofosforados/farmacologia , Animais , Antígenos CD34/análise , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , PTEN Fosfo-Hidrolase/análise , Inibidor Tecidual de Metaloproteinase-1/análise
15.
J Med Chem ; 51(2): 314-23, 2008 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-18163551

RESUMO

New and more potent prodrugs of the 5-fluorouracyl family derived by hydroxymethylation or acyloxymethylation of 5-fluoro-1-(tetrahydro-2-furanyl)-2,4(1H,3H)-pyrimidinedione (tegafur, 1) are described. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity, and anticancer activities in vitro and in vivo, superior to those of tegafur. 5-fluoro-1-(tetrahydro-2-furanyl)-2,4(1 H,3 H)-pyrimidinedione (tegafur, 1), the oral prodrug of 5-FU, has been widely used for treatment of gastrointestinal malignancies with modest efficacy. The aim of this study was to develop and characterize new and more potent prodrugs of the 5-FU family derived by hydroxymethylation or acyloxymethylation of tegafur. Comparison between the effect of tegafur and the new prodrugs on the viability of a variety of cancer cell lines showed that the IC50 and IC90 values of the novel prodrugs were 5-10-fold lower than those of tegafur. While significant differences between the IC50 values of tegafur were observed between the sensitive HT-29 and the resistant LS-1034 colon cancer cell lines, the prodrugs affected them to a similar degree, suggesting that they overcame drug resistance. The increased potency of the prodrugs could be attributed to the antiproliferative contribution imparted by formaldehyde and butyric acid, released upon metabolic degradation. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity and anticancer activities in vitro and in vivo, superior to those of tegafur.


Assuntos
Antineoplásicos/síntese química , Pró-Fármacos/síntese química , Tegafur/análogos & derivados , Tegafur/síntese química , Acetilcisteína/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Formaldeído/agonistas , Formaldeído/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Humanos , Masculino , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Pró-Fármacos/farmacologia , Semicarbazidas/farmacologia , Relação Estrutura-Atividade , Tegafur/farmacologia , Transplante Heterólogo , Cordão Umbilical/citologia
16.
J Med Chem ; 51(9): 2858-62, 2008 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-18363346

RESUMO

The perphenazine and fluphenazine GABA esters 3 and 4 evaluated in rat models for antipsychotic activity displayed a significant decrease of catalepsy associated with increased prolactin blood levels. Efficacy was evaluated in the d-amphetamine-induced hyperactivity model, where perphenazine abolished hyperactivity and induced sedation and catalepsy, whereas 3 reduced hyperactivity without sedation or catalepsy. Thus, 3 (BL-1020) constitutes a prototype of novel antipsychotics possessing GABAergic activity. A phase II study is in progress.


Assuntos
Antipsicóticos/síntese química , Discinesia Induzida por Medicamentos/etiologia , Perfenazina/análogos & derivados , Perfenazina/síntese química , Pró-Fármacos/síntese química , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/síntese química , Administração Oral , Animais , Antipsicóticos/efeitos adversos , Antipsicóticos/farmacologia , Disponibilidade Biológica , Catalepsia/induzido quimicamente , Dextroanfetamina , Ésteres , Flufenazina/efeitos adversos , Flufenazina/análogos & derivados , Flufenazina/síntese química , Flufenazina/farmacologia , Masculino , Perfenazina/efeitos adversos , Perfenazina/farmacologia , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacologia , Prolactina/metabolismo , Ratos , Ratos Wistar , Esquizofrenia/tratamento farmacológico , Ácido gama-Aminobutírico/efeitos adversos , Ácido gama-Aminobutírico/farmacologia
17.
Cancer Chemother Pharmacol ; 61(5): 739-49, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17594094

RESUMO

PURPOSE: The importance of understanding the mechanism of action of anticancer agents is sometimes overlooked in the pursuit of new and therapeutically advantageous compounds. Doxorubicin has long been identified as an inhibitor of the DNA-decatenating enzyme topoisomerase II, this being believed to be the major mechanism of action of this drug. However, the complex nature of cytotoxicity induced by doxorubicin suggests that more than one mechanism of action is responsible for cell kill. Investigation into various other cellular effects has shown that doxorubicin can, in the presence of formaldehyde, form doxorubicin-DNA adducts, resulting in enhanced cell death. METHODS: We have used six catalytic inhibitors of topoisomerase II (aclarubicin, merbarone, suramin, staurosporine, maleimide and sobuzoxane) to investigate the role of topoisomerase II mediated cell effects in doxorubicin-DNA adduct inducing treatments. Adduct levels were determined by scintillation counting of [14C]doxorubicin-DNA lesions and DNA damage responses by Comet analysis and flow cytometry (apoptosis). RESULTS: Here we show that sobuzoxane inhibits topoisomerase II but in the presence of doxorubicin also enhances the production of doxorubicin-DNA adducts resulting in an enhanced cytotoxic response. We show that the formation of doxorubicin-DNA adducts is mediated by formaldehyde released from sobuzoxane when it is metabolised. CONCLUSIONS: Sobuzoxane has also been shown to decrease the normally dose limiting cardiotoxicity commonly exhibited with clinical use of doxorubicin. The potential combination of doxorubicin and sobuzoxane in cancer chemotherapy has two advantages. First, the mechanism of doxorubicin toxicity is shifted away from topoisomerase II inhibition and towards drug-DNA adduct formation which may allow for a lower drug dose to be used and circumvent some drug resistance problems. Second, the addition of a cardioprotecting agent will counteract the commonly dose limiting side effect of cardiac damage resulting from doxorubicin treatment. The importance of the potentiation of cell kill of doxorubicin and sobuzoxane provides a rationalisation of a mechanistic-based combination of anticancer drugs for an improved clinical outcome.


Assuntos
Antineoplásicos/farmacologia , Adutos de DNA/farmacologia , Doxorrubicina/farmacologia , Piperazinas/farmacologia , Inibidores da Topoisomerase II , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Sinergismo Farmacológico , Citometria de Fluxo , Formaldeído/metabolismo , Células HL-60 , Humanos , Piperazinas/farmacocinética , Contagem de Cintilação
18.
Cancer Chemother Pharmacol ; 62(3): 471-82, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18030472

RESUMO

Histone deacetylase inhibitory prodrugs that are metabolized to carboxylic acid(s) and aldehyde(s) possess antineoplastic properties. Formaldehyde-releasing prodrugs were shown to be the most potent. The objective of this study was to gain understanding on the mode of action of these prodrugs in cancer cells. HL-60 and MCF-7 cells in the presence of N-acetylcysteine or glutathione were protected from death induced by formaldehyde-releasing prodrugs but not from death caused by the homologous acetaldehyde-releasing ones. Cell death induced by the former was accompanied by depletion of intracellular glutathione and increased reactive oxygen species that were attenuated by N-acetylcysteine. At fourfold higher concentration, acetaldehyde-releasing prodrugs increased reactive oxygen species that were further augmented by N-acetylcysteine. In HL-60 cells, formaldehyde-releasing prodrugs dissipated the mitochondrial membrane potential and glutathione or N-acetylcysteine restored it. Although acetaldehyde-releasing prodrugs dissipated mitochondrial membrane potential, it occurred at 20-fold greater concentration and was unaffected by the antioxidants. Formaldehyde-releasing prodrugs abrogated c-myc protein expression and elevated c-Jun and H2AX phosphorylation, N-acetylcysteine partially reversed these changes. Herein, we show that formaldehyde-releasing prodrugs diminish the level of glutathione most likely by forming S-formylglutathione adducts resulting in increase of reactive oxygen species followed by signaling events that lead to cancer cells death.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Formaldeído/farmacologia , Glutationa/metabolismo , Pró-Fármacos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetaldeído/química , Acetaldeído/farmacologia , Acetilcisteína/farmacologia , Antineoplásicos/química , Western Blotting , Linhagem Celular Tumoral , Formaldeído/química , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Pró-Fármacos/química , Relação Estrutura-Atividade
19.
Nanomedicine ; 4(2): 121-6, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18482873

RESUMO

We describe a new method for rapid, sensitive, and high-throughput detection of colon cancer cells' response to differentiation therapy, using a novel electrochemical lab-on-a-chip system. Differentiation-inducing agents such as butyric acid and its derivatives were introduced to miniature colon cancer samples within the nanovolume chip chambers. The efficacy of each of the differentiation-inducing agents was evaluated by electrochemical detection of the cellular enzymatic activity level, whereas reappearance of normal enzymatic activity denotes effective therapy. The results demonstrate the ability to evaluate simultaneously multiplex drug effects on miniature tumor samples (approximately 15 cells) rapidly (5 minutes) and sensitively, with quantitative correlation between cancer cells' number and the induced current. The use of miniature analytical devices is of special interest in clinically relevant samples, in that it requires less tissue for diagnosis, and enables high-throughput analysis and comparison of various drug effects on one small tumor sample, while maintaining uniform biological and environmental conditions.


Assuntos
Antineoplásicos/administração & dosagem , Bioensaio/instrumentação , Sobrevivência Celular/efeitos dos fármacos , Eletroquímica/instrumentação , Análise de Injeção de Fluxo/instrumentação , Análise em Microsséries/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Eletroquímica/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/métodos , Células HT29 , Humanos , Análise em Microsséries/métodos , Técnicas Analíticas Microfluídicas/métodos
20.
Cancer Res ; 66(9): 4863-71, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16651442

RESUMO

Doxorubicin (Adriamycin) is one of the most commonly used chemotherapeutic drugs and exhibits a wide spectrum of activity against solid tumors, lymphomas, and leukemias. Doxorubicin is classified as a topoisomerase II poison, although other mechanisms of action have been characterized. Here, we show that doxorubicin-DNA adducts (formed by the coadministration of doxorubicin with non-toxic doses of formaldehyde-releasing prodrugs) induce a more cytotoxic response in HL-60 cells than doxorubicin as a single agent. Doxorubicin-DNA adducts seem to be independent of classic topoisomerase II-mediated cellular responses (as observed by employing topoisomerase II catalytic inhibitors and HL-60/MX2 cells). Apoptosis induced by doxorubicin-DNA adducts initiates a caspase cascade that can be blocked by overexpressed Bcl-2, suggesting that adducts induce a classic mode of apoptosis. A reduction in the level of topoisomerase II-mediated double-strand-breaks was also observed with increasing levels of doxorubicin-DNA adducts and increased levels of apoptosis, further confirming that adducts exhibit a separate mechanism of action compared with the classic topoisomerase II poison mode of cell death by doxorubicin alone. Collectively, these results indicate that the presence of formaldehyde transfers doxorubicin from topoisomerase II-mediated cellular damage to the formation of doxorubicin-DNA adducts, and that these adducts are more cytotoxic than topoisomerase II-mediated lesions. These results also show that doxorubicin can induce apoptosis by a non-topoisomerase II-dependent mechanism, and this provides exciting new prospects for enhancing the clinical use of this agent and for the development of new derivatives and new tumor-targeted therapies.


Assuntos
Adutos de DNA/biossíntese , Dano ao DNA/fisiologia , Doxorrubicina/farmacologia , Inibidores da Topoisomerase II , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Ciclo Celular , Processos de Crescimento Celular , DNA Topoisomerases Tipo II/metabolismo , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Doxorrubicina/biossíntese , Células HL-60 , Humanos
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