RESUMO
Phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) are a new family of antimitotic prodrugs bioactivated in breast cancer cells expressing CYP1A1. In this study, we report that the 14C-labeled prototypical PAIB-SO [14C]CEU-818 and its antimitotic counterpart [14C]CEU-602 are distributed in whole mouse body and they show a short half-life in mice. To circumvent this limitation, we evaluated the effect of the homologation of the alkyl side chain of the imidazolidin-2-one moiety of PAIB-SOs. Our studies evidence that PAIB-SOs bearing an n-pentyl side chain exhibit antiproliferative activity in the nanomolar-to-low-micromolar range and a high selectivity toward CYP1A1-positive breast cancer cells. Moreover, the most potent n-pentyl PAIB-SOs were significantly more stable toward rodent liver microsomes. In addition, PAIB-SOs 10 and 14 show significant antitumor activity and low toxicity in chorioallantoic membrane (CAM) assay. Our study confirms that homologation is a suitable approach to improve the rodent hepatic stability of PAIB-SOs.
Assuntos
Antimitóticos , Neoplasias , Pró-Fármacos , Camundongos , Animais , Antimitóticos/química , Pró-Fármacos/química , Citocromo P-450 CYP1A1 , Roedores , Microssomos Hepáticos , Benzenossulfonatos/químicaRESUMO
We recently discovered a new family of prodrugs deriving from phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) bioactivatable by cytochrome P450 1A1 (CYP1A1) into potent antimitotics referred to as phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs). PAIB-SOs display significant selectivity toward human breast cancer cells based on the N-dealkylation of PAIB-SOs into their corresponding PIB-SOs by CYP1A1. In this study, we have evaluated the molecular mechanism of the bioactivation of PAIB-SOs into PIB-SOs by branching the linear alkyl chain on the imidazolidin-2-one (IMZ) moiety of PAIB-SOs by branched alkyl groups such as isopropyl, isobutyl and sec-butyl. Our results show that PAIB-SOs bearing an isobutyl group on the IMZ moiety and either a methoxy, a chloro or a bromo group at positions 3, 3,5 or 3,4,5 on the aromatic ring B exhibit antiproliferative activity ranging from 0.13 to 6.9 µM and selectivity toward MCF7 and MDA-MB-468 mammary cancer cells comparatively to other cell lines tested. Moreover, the most potent and selective PAIB-SOs bearing an isobutyl group and either a 3,5-Cl (44), 3,5-Br (45) or a 3,4,5-OMe (46) on the IMZ moiety exhibit antiproliferative activity in the sub-micromolar range and high selectivity ratios toward mammary cancer cells. They stop the cell cycle of MCF7 cells in the G2/M phase and disrupt their cytoskeleton. Furthermore, our studies evidenced that PAIB-SOs bearing either an isopropyl, a sec-butyl or an isobutyl group are hydroxylated on the carbon atom adjacent to the IMZ (Cα-OH) but only PAIB-SOs bearing an isobutyl group are bioactivated into PIB-SOs. Finally, PAIB-SOs 45 and 46 exhibit low toxicity toward normal cells and chick embryos and are thus promising antimitotic prodrugs highly selective toward CYP1A1-expressing breast cancer cells.
Assuntos
Antimitóticos/química , Benzenossulfonatos/química , Citocromo P-450 CYP1A1/metabolismo , Pró-Fármacos/química , Animais , Antimitóticos/síntese química , Antimitóticos/farmacologia , Benzenossulfonatos/síntese química , Benzenossulfonatos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Galinhas , Citocromo P-450 CYP1A1/química , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Meia-Vida , Humanos , Microssomos Hepáticos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Triple-negative breast cancer (TNBC) is a major concern among the different subtypes of breast cancer (BC) due to the lack of effective treatment. In a previous study by our group aimed at understanding the difference between TNBC and non-TNBC tumors, we identified the gene TBC1 domain family member 9 (TBC1D9), the expression of which was lower in TNBC as compared to non-TNBC tumors. In the present study, analysis of TBC1D9 expression in TNBC (n = 58) and non-TNBC (n = 25) patient tumor samples validated that TBC1D9 expression can differentiate TNBC (low) from non-TNBC (high) samples and that expression of TBC1D9 was inversely correlated with grade and proliferative index. Moreover, we found that downregulation of the TBC1D9 gene decreases the proliferation marginally in non-TNBC and was associated with increased migratory and tumorigenic potential in both TNBC and luminal BC cell lines. This increase was mediated by the upregulation of ARL8A, ARL8B, PLK1, HIF1α, STAT3, and SPP1 expression in TBC1D9 knockdown cells. Our results suggest that TBC1D9 expression might limit tumor aggressiveness and that it has a differential expression in TNBC vs. non-TNBC tumors.
RESUMO
OBJECTIVES: In this study, the antiproliferative activity of 3 phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) was assessed in a time-dependent manner together with their hepatic stability and metabolism using human, mouse and rat liver microsomes. METHODS: CEU-818, -820 and -913 were selected as promising hit compounds. Their antiproliferative activity on human breast carcinoma MCF-7 cells was evaluated using escalating concentrations of drugs at 24, 36 and 48 h and the sulforhodamine B assay. Their hepatic stability was evaluated by HPLC-UV of extracts obtained from human, mouse and rat liver microsomes. KEY FINDINGS: The antiproliferative activity of PAIB-SOs is concentration and time-dependent and requires between 24 and 36 h of contact with MCF-7 cells to detect a significant antiproliferative activity. PAIB-SOs stability in microsomes usually decreases following this order: human ≈ (rat > mouse). The CEU-913 exhibits the longest half-life in rat and human liver microsomes while the CEU-820 exhibits the longest half-life in mouse liver microsomes. CONCLUSIONS: Our in vitro results suggest that PAIB-SOs should have a minimum contact time of 24 h with the tumour to trigger significant antitumoural activity. The activity of mouse liver microsomes towards PAIB-SOs is higher than rat microsomes and tends to be higher than human liver microsomes.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Microssomos Hepáticos/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/química , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1A1/metabolismo , Feminino , Meia-Vida , Humanos , Células MCF-7 , Camundongos , Pró-Fármacos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Fatores de TempoRESUMO
Prodrug-mediated utilization of the cytochrome P450 (CYP) 1A1 to obtain the selective release of potent anticancer products within cancer tissues is a promising approach in chemotherapy. We herein report the rationale, preparation, biological evaluation, and mechanism of action of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) that are antimicrotubule prodrugs activated by CYP1A1. Although PAIB-SOs are inert in most cells tested, they are highly cytocidal toward several human breast cancer cells, including hormone-independent and chemoresistant types. PAIB-SOs are N-dealkylated into cytotoxic phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) in CYP1A1-positive cancer cells, both in vitro and in vivo. In conclusion, PAIB-SOs are novel chemotherapeutic prodrugs with no equivalent among current antineoplastics and whose selective action toward breast cancer is tailored to the characteristic pattern of CYP1A1 expression observed in a large percentage of human breast tumors.
Assuntos
Antimitóticos/farmacologia , Benzenossulfonatos/química , Neoplasias da Mama/tratamento farmacológico , Citocromo P-450 CYP1A1/metabolismo , Pró-Fármacos/farmacologia , Animais , Antimitóticos/farmacocinética , Benzenossulfonatos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Embrião de Galinha , Citocromo P-450 CYP1A1/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Pró-Fármacos/farmacocinéticaRESUMO
The synthesis of two series of novel substituted pyrimidine derivatives bearing a sulfamide group have been described and their in vitro cancer growth inhibition activities have been evaluated against three human tumour cell lines (HT-29, M21, and MCF7). In general, growth inhibition activity has been enhanced by the introduction of a bulky substituent on the aromatic ring with the best compound having GI50<6µM for all the human tumour cell lines. The MCF7 selective compounds were evaluated on four additional human invasive breast ductal carcinoma cell lines (MDA-MB-231, MDA-MB-468, SKBR3, and T47D) and were selective against T47D cell line in all cases except one, suggesting a potential antiestrogen activity.
Assuntos
Antineoplásicos/farmacologia , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
A series of 7α-linked testosterone dimers were made and tested for biological activity on both androgen-dependent (LNCaP) and androgen-independent (DU-145 and PC3) prostate cancer cell lines. The synthesis proceeds through the formation of a trans-4-(17ß-acetoxy-4-androsten-3-one-7α-yl)-but-2-enoic acid 4-hydroxy-alkyl ester intermediate of various length (7a-d) followed by the final dimerization step. The dimers showed interesting biological activity in comparison to the ω-hydroxyalkyl ester intermediates 7a-d. The most active dimer 8a (n=1) showed IC50 of 3.8, 1.4 and 1.8µM, respectively on LNCaP, DU-145 and PC3 cancer cell lines. On these cell lines, this dimer is about 12, 70 and 47 times more powerful than cyproterone acetate (CPA) the reference antiandrogen. Furthermore, dimers 8b-d (n=2, 3, 4) were less active than 8a but showed selective activity on androgen-dependent LNCaP prostate cancer cells. This indicates possible application for the treatment of androgen-dependent prostate cancer.
Assuntos
Androgênios/metabolismo , Neoplasias da Próstata/metabolismo , Testosterona/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Estrutura Molecular , Testosterona/químicaRESUMO
Islet amyloid polypeptide (IAPP) has been shown to form amyloid deposits in pancreatic islets, thereby furthering type 2 diabetes disease progression. Further discovery of new molecules is needed to create a diverse set of molecules that impede pancreatic amyloidosis. We have recently designed and synthesized N-phenyl-N'-(2-ethyl)ureas (EU) that are non-cytotoxic small molecules, to evaluate the role of the aryl-substituted moiety on the inhibition of hIAPP fibrillization. Several EUs were tested in vitro for their anti-amyloidogenic activity using the fluorometric ThT assay, the photo-induced cross-linking (PIUCP) assay, and cell survival assay in pancreatic MIN-6 cells. EU-362 and EU-418 were able to significantly inhibit the formation of hIAPP fibrils and protected cells from amyloid cytotoxic effects. Our results suggest that increasing the nucleophilic potency of the aryl moiety significantly enhances the anti-amyloidogenic activity of the molecules.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ureia/análogos & derivados , Amiloide/metabolismo , Amiloidose/tratamento farmacológico , Amiloidose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/efeitos adversos , Ilhotas Pancreáticas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ureia/farmacologiaRESUMO
Combretastatin A-4 (CA-4) is a well-studied and attractive molecular template to develop new antimitotics. Several thousand of modifications were performed on the ring B and the ethenyl bridge of CA-4 but only a few involved the trimethoxyphenyl moiety (TMP, ring A) often considered essential to the antiproliferative and antimicrotubule activities. In this study, we described the design, the preparation, the characterization and the biological evaluation of three new series of CA-4 analogs namely styryl-N-phenyl-N'-ethylureas (SEUs), styryl-N-phenyl-N'-(2-chloroethyl)ureas (SCEUs) and styrylphenylimidazolidin-2-ones (SIMZs) bearing a 3-Cl (series a), 3,5-Me (series b) and TMP (series c) substituents, respectively. All SCEU and SIMZ Z-isomers were active in the high and the low nanomolar range, respectively. Conversely to SEUs and their E-isomers that were significantly less active or inactive. Interestingly, the TMP moiety is giving rise to derivatives exhibiting the lowest antiproliferative activity in the SCEU series (10c) and the most active compound in the SIMZ series (12c). Moreover, SIMZ Z-isomers bearing either a 3-Cl (12a) or a 3,5-Me (12b) exhibited antiproliferative activities that are also in the same order of magnitude as 12c. All SCEU and SIMZ Z-isomers also arrested the cell cycle progression in G2/M phase, bound to the colchicine-binding site and disrupted the cytoskeleton of cancer cells. In addition to the promising and innovative microtubule-disrupting properties of SCEUs and SIMZs, these results show that the TMP moiety is not essential for the cytocidal activity of these new CA-4 analogs.
Assuntos
Antineoplásicos/farmacologia , Imidazolidinas/farmacologia , Microtúbulos/efeitos dos fármacos , Estilbenos/farmacologia , Ureia/análogos & derivados , Antineoplásicos/síntese química , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Humanos , Imidazolidinas/síntese química , Imidazolidinas/química , Células MCF-7 , Estrutura Molecular , Estilbenos/química , Relação Estrutura-Atividade , Ureia/síntese química , Ureia/química , Ureia/farmacologiaRESUMO
Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents.
Assuntos
Anticarcinógenos/química , Anticarcinógenos/farmacologia , Benzenossulfonatos/química , Benzenossulfonatos/farmacologia , Proliferação de Células/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Fase S/efeitos dos fármacos , Animais , Anticarcinógenos/síntese química , Benzenossulfonatos/síntese química , Ciclo Celular , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Cisplatino/farmacologia , Células HT29 , Histonas/metabolismo , Humanos , Imidazolidinas/síntese química , Imidazolidinas/química , Imidazolidinas/farmacologia , Células Jurkat , Neoplasias/tratamento farmacológico , Compostos de Fenilureia/síntese química , Fosforilação , Estilbenos/farmacologia , Relação Estrutura-AtividadeRESUMO
Sixty-one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and 13 of their tetrahydro-2-oxopyrimidin-1(2H)-yl analogues (PPB-SOs) were prepared and biologically evaluated. The antiproliferative activities of PIB-SOs on 16 cancer cell lines are in the nanomolar range and unaffected in cancer cells resistant to colchicine, paclitaxel, and vinblastine or overexpressing the P-glycoprotein. None of the PPB-SOs exhibit significant antiproliferative activity. PIB-SOs block the cell cycle progression in the G(2)/M phase and bind to the colchicine-binding site on ß-tubulin leading to cytoskeleton disruption and cell death. Chick chorioallantoic membrane tumor assays show that compounds 36, 44, and 45 efficiently block angiogenesis and tumor growth at least at similar levels as combretastatin A-4 (CA-4) and exhibit low to very low toxicity on the chick embryos. PIB-SOs were subjected to CoMFA and CoMSIA analyses to establish quantitative structure-activity relationships.
Assuntos
Sulfonatos de Arila/síntese química , Derivados de Benzeno/síntese química , Imidazolidinas/síntese química , Estilbenos/química , Moduladores de Tubulina/síntese química , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Sulfonatos de Arila/química , Sulfonatos de Arila/farmacologia , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Colchicina/metabolismo , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazolidinas/química , Imidazolidinas/farmacologia , Modelos Moleculares , Mimetismo Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Estilbenos/farmacologia , Transplante Heterólogo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologiaRESUMO
The cyclization of anticancer drugs into active intermediates has been reported mainly for DNA alkylating molecules including nitrosoureas. We previously defined the original cytotoxic mechanism of anticancerous N-phenyl-N'-(2-chloroethyl)ureas (CEUs) that involves their reactivity towards cellular proteins and not against DNA; two CEU subsets have been shown to alkylate ß-tubulin and prohibitin leading to inhibition of cell proliferation by G2/M or G1/S cell cycle arrest. In this study, we demonstrated that cyclic derivatives of CEUs, N-phenyl-4,5-dihydrooxazol-2-amines (Oxas) are two- to threefold more active than CEUs and share the same cytotoxic properties in B16F0 melanoma cells. Moreover, the CEU original covalent binding by an ester linkage on ß-tubulin Glu198 and prohibitin Asp40 was maintained with Oxas. Surprisingly, we observed that Oxas were spontaneously formed from CEUs in the cell culture medium and were also detected within the cells. Our results suggest that the intramolecular cyclization of CEUs leads to active Oxas that should then be considered as the key intermediates for protein alkylation. These results will be useful for the design of new prodrugs for cancer chemotherapy.
Assuntos
Alquilantes/farmacologia , Aminas/metabolismo , Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Repressoras/metabolismo , Tubulina (Proteína)/metabolismo , Ureia/metabolismo , Aminas/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ciclização , Eletroforese em Gel Bidimensional , Imunofluorescência , Melanoma Experimental/patologia , Camundongos , Proibitinas , Proteínas Repressoras/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tubulina (Proteína)/química , Ureia/químicaRESUMO
N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) are antimicrotubule agents interacting covalently with ß-tubulin near the colchicine-binding site (C-BS). Glutamyl 198 residue in ß-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic residues, Cys239 and Cys354, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). By use of mass spectrometry, we have now identified residues in ß-tubulin that have become modified irreversibly by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU), and N,N'-ethylenebis(iodoacetamide) (EBI). The binding of HPCEU and 4ZCombCEU to ß-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs then were used as molecular probes to assess, in mouse B16F0 and human MDA-MB-231 cells, the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU, and 4ZCombCEU on the acetylation of Lys40 on α-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation, as observed also with other microtubule depolymerizing agents. Therefore, CEU affects the stability and the dynamics of microtubule, likewise a E198G mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular cross-link between Cys239 and Cys354 of ß-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/function and the biological activity of several cellular proteins under physiological conditions.
Assuntos
Antineoplásicos/metabolismo , Microtúbulos/química , Moduladores de Tubulina/metabolismo , Tubulina (Proteína)/metabolismo , Ureia/análogos & derivados , Acetilação , Acilação , Animais , Células Cultivadas , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Relação Estrutura-Atividade , Tubulina (Proteína)/químicaRESUMO
Aryl chloroethyl ureas (CEUs) are new protein alkylating agents exhibiting anticancer activity both in vitro and in vivo. We report herein that 14C-labeled CEU derivatives, designated CEU-025 and CEU-027, covalently bind to thioredoxin-1 (TRX1). Covalent binding of these molecules slightly decreases the disulfide-reducing activity of recombinant TRX1, when compared with the effect of strong thioalkylating agents such as N-ethylmaleimide. Moreover, site-directed mutagenesis and diamide competition assays demonstrated that TRX1 cysteinyl residues are not the prime targets of CEUs. CEU-025 abrogates the nuclear translocation of TRX1 in human cancer cells. In addition, we show that CEU-025 can block TRX1 nuclear translocation induced by cisplatin. Unexpectedly, pretreatment with sublethal CEU-025 concentrations that block TRX1 nuclear translocation protected the cells against cisplatin cytotoxicity. Overexpression of TRX1 in HT1080 fibrosarcoma cells attenuated CEU-025 cytotoxicity, while its suppression using TRX1-specific siRNA increased the effects of CEU-025, suggesting that loss of function of TRX1 is involved, at least in part, in the cytotoxic activity of CEU-025. These results suggest that CEU-025 and CEU-027 exhibit anticancer activity through a novel, unique mechanism of action. The importance of TRX1 and the dependence of the cytotoxicity of CEU-025 and CEU-027 on TRX1 intracellular localization are also discussed.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Tiorredoxinas/metabolismo , Ureia/análogos & derivados , Ureia/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Catálise , Linhagem Celular Tumoral , Cisplatino/farmacologia , Humanos , Transporte Proteico/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
PURPOSE: We investigated the involvement of MAPK signaling in the cell death mechanisms of classical microtubule interfering agents (MIA) and aryl-3-(2-chloroethyl)ureas (CEU) acting as antimitotics, along with CEU that don't affect directly microtubules (non-MIA CEU). METHODS: To ascertain the activated signaling pathway profile of MIA and non-MIA CEU, Western blot, immunoprecipitation and transfection experiments were performed. RESULTS: Non-MIA CEU do not activate p38, as opposed to MIA, and the extent of ERK and JNK activation is lower than in response to MIA. The effect of MIA and non-MIA CEU on focal adhesion associated protein was also studied; MIA were shown to induce focal adhesion dismantlement associated with a sustained increase in paxillin phosphorylation and FAK cleavage, as opposed to non-MIA CEU. In addition, bcl-2 phosphorylation and AKT cleavage, induced by all MIA tested, was not observed in response to non-MIA CEU further emphasizing the differential cell death mechanisms induced by MIA and non-MIA CEU. Pharmacologic and genetic approaches emphasize that the ASK1-p38 pathway activation contributes to the cytotoxic mechanism of MIA, in contrast to non-MIA CEU. ASK1-p38 is important for increased paxillin phosphorylation and FAK cleavage, suggesting that ASK-1-p38 is an upstream event of FA structure dismantlement induced by MIA. Moreover, the endogen inhibitor of ASK-1, thioredoxin, is released from ASK-1 in response to MIA as opposed to non-MIA CEU. CONCLUSION: Our study supports that ASK1-p38 activation is an important signaling event, induced by MIA, which impairs focal adhesion structure and induces anchorage-dependent apoptosis or anoikis.
Assuntos
Antineoplásicos/farmacologia , MAP Quinase Quinase Quinase 5/metabolismo , Moduladores de Tubulina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anoikis/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Humanos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Seven subsets of aromatic urea and amide analogues of N-phenyl-N'-(2-chloroethyl)ureas (CEU) have been synthesized by nucleophilic addition of 3-chloropropylisocyanate, 2-chloroacetylisocyanate, ethylisocyanate, 2-chloroacetyl chloride, 3-chloropropanoyl chloride, 4-chlorobutanoyl chloride, and acryloyl chloride, respectively, to selected anilines or benzylamines to afford 3-chloropropylureas (1, CPU), 2-chloroacetylureas (2, CAU), ethylureas (3, EU), 2-chloroacetamides (4, CA), 3-chloropropionamides (5, CPA), 4-chlorobutyramides (6, CBA) and acrylamides (7, Acr). The molecular structure of these compounds has been confirmed by IR, (1)H and (13)C NMR, and MS spectra and their purity also confirmed by HPLC. The CEU analogues were evaluated for their antiproliferative activity against three human tumor cell lines, namely human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7. CAU (2c to 2g), CA (4a to 4d, 4f and 4 g), CPA (5a) and Acr (7a and 7b) had IC(50) ranging from 1.4 to 25 microM. CAU, CA, CPA and Acr exhibited interesting antiproliferative activity through mechanism(s) of action unrelated to the acylation of glutamic acid at position 198 on beta-tubulin that is characterizing CEU.
Assuntos
Amidas/química , Amidas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Ureia/química , Ureia/farmacologia , Amidas/síntese química , Amidas/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colchicina/metabolismo , Humanos , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Ureia/síntese química , Ureia/metabolismoRESUMO
Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide) (EBI) to crosslink in living cells the cysteine residues at position 239 and 354 of ß-tubulin, residues which are involved in the colchicine-binding site. The ß-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of ß-tubulin that migrates faster than ß-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: ß-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.
RESUMO
Gene and synthetic drug-delivery vectors have been developed and characterized to treat several genetic diseases and cancers. Our study aims at characterizing cationic liposomes containing the zwitterionic phospholipid DMPC and the cationic lipid DOTAP as well as their interactions with two types of DNA and a new class of antineoplastic agents derived from arylchloroethylureas (CEU). Results obtained using FTIR spectroscopy as well as (31)P and (2)H NMR indicate that DMPC and DOTAP form cationic liposomes in a highly disordered fluid phase at a molar ratio of 1:1. In addition, the FTIR results indicate that the presence of DNA or CEUs within the liposomes does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains. Our results therefore provide a detailed characterization of complexes between cationic liposomes and both DNA and drugs and indicate that these complexes are stable and fluid assemblies.
Assuntos
DNA/metabolismo , Dimiristoilfosfatidilcolina/química , Ácidos Graxos Monoinsaturados/química , Lipossomos/química , Lipossomos/metabolismo , Compostos de Fenilureia/metabolismo , Compostos de Amônio Quaternário/química , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Cátions/química , Bovinos , DNA/química , Dimiristoilfosfatidilcolina/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Ressonância Magnética Nuclear Biomolecular , Compostos de Fenilureia/química , Plasmídeos , Compostos de Amônio Quaternário/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Timo/químicaRESUMO
Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure-activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N'-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on beta(II)-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The FlexiDock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-beta198 rather than Cys-beta239. In addition, these studies suggest that Cys-beta239 together with Asn-alpha99, Ser-alpha176, Thr-alpha177, Leu-beta246, Asn-beta247, Ala-beta248, Lys-beta252 and Asn-beta256 are implicated in the stabilization of a C-BS-CEU complex prior to the acylation of Glu-beta198 by CEU. Our molecular models propose the formation of a stabilized C-BS-CEU complex before the completion of the Glu-beta198 acylation; acylation triggering conformational changes of beta-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin.
Assuntos
Colchicina/metabolismo , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Ureia/análogos & derivados , Sítios de Ligação , Linhagem Celular Tumoral , Colchicina/química , Simulação por Computador , Humanos , Modelos Químicos , Modelos Moleculares , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologia , Ureia/química , Ureia/farmacologiaRESUMO
Aryl chloroethylureas (CEUs) are potent anti-neoplastic agents alkylating specific intracellular proteins such as beta(II)-tubulin. Recently we have identified a new subset of CEU derived from compound 36 that alkylates thioredoxin isoform 1 (Trx-1), inhibits the nuclear translocation of Trx-1, and favors the accumulation of cells in G(0)/G(1) phase. We have evaluated the effects of various substituents and their position on the aromatic ring of a series of derivatives of 36 on (i) the anti-proliferative activity, (ii) the cell cycle progression, (iii) the nuclear translocation of Trx-1, and (iv) their covalent binding to beta-tubulin. The same experiments were performed on representative CEU derivatives where the 2-chloroethyl amino moiety is replaced by either an ethyl, a 2-aminooxazolinyl or a 2-chloroacetyl group. On one hand, our results suggest that CEUs substituted on the phenyl ring at position 3 or 4 by cycloalkyl and substituted cycloalkyl or cycloalkoxy groups inhibit the nuclear translocation of Trx-1 and arrest the cell cycle progression in G(0)/G(1). On the other hand, CEUs substituted by a fused aromatic ring, an aliphatic chain, or a fused aliphatic ring are alkylating beta(II)-tubulin but not Trx-1. Beside the expected inactivity of the ethylurea derivatives, none of the modification to the electrophilic moiety led to cross-selectivity of the drugs toward beta-tubulin but increased the anti-proliferative activity and resulted in mitigated effects on Trx-1 translocation.