RESUMEN
DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Carcinoma/tratamiento farmacológico , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas/metabolismo , Antineoplásicos Alquilantes/agonistas , Proteína 7 Relacionada con la Autofagia , Carbamatos/agonistas , Carbamatos/farmacología , Carcinoma/enzimología , Carcinoma/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Femenino , Silenciador del Gen , Células HeLa , Compuestos Heterocíclicos con 3 Anillos/agonistas , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Indenos/agonistas , Indenos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Quinasas/química , Proteínas Quinasas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Fase S/efectos de los fármacos , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
We studied the effects of three newly synthesized steroidal derivatives of nitrogen mustards, alone or in combination with caffeine, on sister chromatid exchange (SCE) frequencies and on human lymphocyte proliferation kinetics. The agents have as alkylator functionalities either P-N,N-bis(2-chloroethyl)aminophenyl-buturate (CHL) or P-N,N-bis(2-chloroethyl)aminophenyl-acetate (PHE), esterified with a modified steroidal nucleus. An enhancement of SCE frequency was seen with compounds which contain either PHE or CHL as alkylators and are esterified with a steroidal nucleus having added a cholestene group in the 17-position of the D-ring. The exocyclic insertion of an -NHCO- group in the D-ring of the steroidal nucleus esterified with PHE (amide ester of PHE) gave a compound showing increased SCE frequency. Enhanced cytogenetic damage was observed when lymphocytes were exposed in vitro to caffeine. The compounds, alone or in combination with caffeine, caused a concentration-dependent increase in SCE frequencies and cell division delays, and caffeine was found to act synergistically with the steroidal alkylators.
Asunto(s)
Antineoplásicos Alquilantes , Cafeína , Núcleo Celular/metabolismo , Estimulantes del Sistema Nervioso Central , Aberraciones Cromosómicas/inducido químicamente , Linfocitos/metabolismo , Compuestos de Mostaza Nitrogenada , Intercambio de Cromátides Hermanas/efectos de los fármacos , Adulto , Antineoplásicos Alquilantes/efectos adversos , Antineoplásicos Alquilantes/agonistas , Antineoplásicos Alquilantes/farmacología , Cafeína/efectos adversos , Cafeína/agonistas , Cafeína/farmacología , Núcleo Celular/genética , Núcleo Celular/patología , Estimulantes del Sistema Nervioso Central/efectos adversos , Estimulantes del Sistema Nervioso Central/agonistas , Estimulantes del Sistema Nervioso Central/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Linfocitos/patología , Masculino , Compuestos de Mostaza Nitrogenada/efectos adversos , Compuestos de Mostaza Nitrogenada/agonistas , Compuestos de Mostaza Nitrogenada/farmacología , Esteroides/efectos adversos , Esteroides/agonistas , Esteroides/farmacologíaRESUMEN
Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ~IC(10) drug concentrations (57 µM B, 1 µM M and 0.02 µM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 µM B, 0.7 µM M and 0.01 µM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 µM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated-CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2'-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2'-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre-hematopoietic stem cell transplantation conditioning for lymphoma.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Azacitidina/análogos & derivados , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Epigénesis Genética/efectos de los fármacos , Linfoma/metabolismo , Linfoma/terapia , Antineoplásicos Alquilantes/agonistas , Proteínas de la Ataxia Telangiectasia Mutada , Azacitidina/agonistas , Azacitidina/farmacología , Busulfano/agonistas , Busulfano/farmacología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa de Punto de Control 2 , Citotoxinas , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/metabolismo , Decitabina , Desoxicitidina/agonistas , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales/métodos , Sinergismo Farmacológico , Trasplante de Células Madre Hematopoyéticas , Humanos , Ácidos Hidroxámicos/agonistas , Ácidos Hidroxámicos/farmacología , Linfoma/patología , Melfalán/agonistas , Melfalán/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo , Proteínas Supresoras de Tumor/metabolismo , Gemcitabina , ADN Metiltransferasa 3BRESUMEN
To improve conventional chemotherapeutic efficacy, a combination use of traditional medicines is effective but detailed mechanisms have been rarely elucidated. In the this study, we attempted to clarify how triptolide (PG490), an oxygenated diterpene derived from a Chinese herb, enhances the cisplatin (CDDP)-induced cytotoxicity in urothelial cancer cells. Our results showed that a combined CDDP/triptolide therapy induced apoptosis in urothelial cancer cell lines with wild-type p53, but not in those with mutant-type p53 or normal human urothelium. As the mechanism, triptolide suppressed CDDP-induced p53 transcriptional activity, leading to p21 attenuation, which promoted apoptosis via the activation of c-Jun N-terminal kinase (JNK) and Bax. We further demonstrated that the functional regulation of p53 by triptolide was mediated by an intranuclear association of p53 with glycogen synthase kinase-3beta (GSK3beta), which was inactivated by protein kinase C (PKC). This modulation of the PKC-GSK3beta axis by triptolide was observed in a cancer-specific manner. A mouse xenograft model also showed that a combined CDDP/triptolide therapy completely suppressed tumor growth without any side effects. We expect that cancer-specific enhancement of CDDP-induced cytotoxicity with triptolide may effectively overcome the resistance to a CDDP-based conventional chemotherapy as a treatment for urothelial cancer.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Cisplatino/farmacología , Diterpenos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/metabolismo , Fenantrenos/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos Alquilantes/agonistas , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/agonistas , Diterpenos/agonistas , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Compuestos Epoxi/agonistas , Compuestos Epoxi/farmacología , Femenino , Glucógeno Sintasa Quinasa 3 beta , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Fenantrenos/agonistas , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAIL-induced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.