RESUMEN
Mutations in the γ-amino butyric acid type A (GABAA) receptor γ2 subunit gene, GABRG2, have been associated with refractory epilepsy. Increasing evidence indicates that suberoylanilide hydroxamic acid (SAHA), a broad-spectrum histone acetyltransferases (HDACs) inhibitor, can inhibit seizure onset. However, the mechanisms involved remains unknown. The present study aimed to explore the anti-epileptic effect and underlying mechanisms of SAHA in the treatment of refractory epilepsy induced by GABRG2 mutation. In the zebrafish line expressing human mutant GABRG2(F343L), Tg(hGABRG2F343L), SAHA was found to reduce seizure onset, swimming activity, and neuronal activity. In both Tg(hGABRG2F343L) zebrafish and HEK293T cells transfected with GABAA receptor subunits, SAHA could improve the pan-acetylation level and reduce the expression of HDAC1/10. The decreased expressions of GABAA receptor subunits could be rescued by SAHA treatment both in vivo and in vitro, which might be the result of increased gene transcription and protein trafficking. The up-regulated acetylation of histone H3 and H4 as well as Bip expression might be involved in the process. Taken together, our data proved that both histone and non-histone acetylation might contribute to the anti-epileptic effect of SAHA in refractory epilepsy caused by GABRG2(F343L) mutation, demonstrating SAHA as a promising therapeutic agent for refractory epilepsy.
Asunto(s)
Mutación , Receptores de GABA-A , Vorinostat , Pez Cebra , Animales , Humanos , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Células HEK293 , Vorinostat/farmacología , Vorinostat/uso terapéutico , Epilepsia Refractaria/tratamiento farmacológico , Epilepsia Refractaria/genética , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Animales Modificados GenéticamenteRESUMEN
Gold nanorods are a promising nanoscale material in clinical diagnosis and treatment. The physicochemical properties of GNRs, including size, shape and surface features, are crucial factors affecting their cytotoxicity. In this study, we investigated the effects of different aspect ratios and surface modifications on the cytotoxicity and cellular uptake of GNRs in cultured cells and in mice. The results indicated that the surface chemistry but not the aspect ratio of GNRs mediates their biological toxicity. CTAB-GNRs with various aspect ratios had similar abilities to induce cell apoptosis and autophagy by damaging mitochondria and activating intracellular reactive oxygen species (ROS). However, GNRs coated with CTAB/PSS, CTAB/PAH, CTAB/PSS/PAH or CTAB/PAH/PSS displayed low toxicity and did not induce cell death. CTAB/PAH-coated GNRs caused minimally abnormal cell morphology compared with CTAB/PSS and CTAB/PSS/PAH coated GNRs. Moreover, the intravenous injection of CTAB/PAH GNRs enabled the GNRs to reach tumor tissues through blood circulation in animals and remained stable, with a longer half-life compared to the other GNRs. Therefore, our results demonstrated that further coating can prevent cytotoxicity and cell death upon CTAB-coated GNR administration, similar to changing the GNR aspect ratio and CTAB/PAH coated GNRs show superior biological properties with better biocompatibility and minimal cytotoxicity.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Compuestos de Cetrimonio/farmacología , Oro/toxicidad , Nanopartículas del Metal/toxicidad , Tensoactivos/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Cetrimonio , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Nanotubos/química , Especies Reactivas de Oxígeno/metabolismo , Propiedades de SuperficieRESUMEN
BACKGROUND AND PURPOSE: Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephaniae tetrandrae, has a long history in Chinese clinical applications to treat diverse diseases. Tetrandrine induced apoptosis or, at low concentrations, autophagy of human hepatocellular carcinoma cells. Here we have tested the effects of inhibitors of autophagy such as chloroquine, on the response to low concentrations of tetrandrine in cancer cells. EXPERIMENTAL APPROACH: Cultures of several cancer cell lines, including Huh7, U251, HCT116 and A549 cells, were exposed to tetrandrine, chloroquine or a combination of these compounds. Cell viability and content of reactive oxygen species (ROS) were measured and synergy assessed by calculation of the combination index. Western blot and RT-PCR assays were also used along with fluorescence microscopy and histochemical techniques. KEY RESULTS: Combinations of tetrandrine and chloroquine were more cytotoxic than the same concentrations used separately and these effects showed synergy. Such effects involved increased ROS generation and were dependent on caspase-3 but independent of Akt activity. Blockade of tetrandrine-induced autophagy with 3-methyladenine or bafilomycin-A1 induced apoptosis in cancer cells. Lack of p21 protein (p21(-/-) HCT116 cells) increased sensitivity to the apoptotic effects of the combination of tetrandrine and chloroquine. In a tumour xenograft model in mice, combined treatment with tetrandrine and chloroquine induced ROS accumulation and cell apoptosis, and decreased tumour growth. CONCLUSIONS AND IMPLICATIONS: The combinations of tetrandrine and chloroquine exhibited synergistic anti-tumour activity, in vitro and in vivo. Our results suggest a novel therapeutic strategy for tumour treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bencilisoquinolinas/farmacología , Cloroquina/farmacología , Neoplasias del Colon/tratamiento farmacológico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Células HCT116 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND PURPOSE: Sorafenib, a potent inhibitor that targets several kinases associated with tumourigenesis and cell survival, has been approved for clinical treatment as a single agent. However, combining sorafenib with other agents improves its anti-tumour efficacy in various preclinical tumour models. ABT-263, a second-generation BH3 mimic, binds to the anti-apoptotic family members Bcl-2, Bcl-xL and Bcl-w, and has been demonstrated to enhance TNFSF10 (TRAIL)-induced apoptosis in human hepatocarcinoma cells. Hence, we investigated the effects of ABT-263 treatment combined with sorafenib. EXPERIMENTAL APPROACH: The effects of ABT-263 combined with sorafenib were investigated in vitro, on cell viability, clone formation and apoptosis, and the mechanism examined using western blot and flow cytometry. This combination was also evaluated in vivo, in a mouse xenograft model; tumour growth, volume and weights were measured and a TUNEL assay performed. KEY RESULTS: ABT-263 enhanced sorafenib-induced apoptosis while sparing non-tumourigenic cells. Although ABT-263 plus sorafenib significantly stimulated intracellular reactive oxygen species production and subsequent mitochondrial depolarization, this was not sufficient to trigger cell apoptosis. ABT-263 plus sorafenib significantly decreased Akt activity, which was, at least partly, involved in its effect on apoptosis. Bax and p21 (CIP1/WAF1) were shown to play a critical role in ABT-263 plus sorafenib-induced apoptosis. Combining sorafenib with ABT-263 dramatically increased its efficacy in vivo. CONCLUSION AND IMPLICATIONS: The anti-tumour activity of ABT-263 plus sorafenib may involve the induction of intrinsic cell apoptosis via inhibition of Akt, and reduced Bax and p21 expression. Our findings offer a novel effective therapeutic strategy for tumour treatment.