RESUMO
Imaging agents that enable direct detection of apoptosis are highly desirable in the field of monitoring chemotherapeutic response as well as early diagnosis and disease monitoring. Previous work demonstrated that the dansyled amino acid DNSBA is used to specifically and selectively detect apoptotic cancer cells at the both early and late stages, but the mechanism remains unclear. In this work, we evaluated DNSBA as a tool for monitoring cell apoptosis in CNE1 tumor cell models both in vitro and ex vivo after its in vivo administration, which was confirmed by other assays. The ability of DNSBA to detect multiple pathways and different stages of apoptosis leading to cell death may be advantageous in the evaluation of cancer treatment indicative of a positive therapeutic outcome. The uptake change of molecular probes DNSBA in CNE1 cells represented the changes of apoptotic rate in a caspase-dependent manner. However, the accumulation of DNSBA in apoptotic cells did not increase with the enhanced membrane permeability. Furthermore, ex vivo study demonstrated DNSBA has a similar pattern as the TUNEL-positive cells. In conclusion, DNSBA cellular imaging is useful for the early assessment of treatment-induced apoptosis, and thus may act as a substitute for Annexin V for assessing treatment response.
Assuntos
Compostos de Dansil/metabolismo , Sondas Moleculares/metabolismo , Neoplasias Nasofaríngeas/genética , Imagem Óptica/métodos , Fenilpropionatos/metabolismo , Animais , Anexina A5/genética , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Transporte Biológico , Carcinoma , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Compostos de Dansil/química , Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Injeções Subcutâneas , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Sondas Moleculares/química , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Transplante de Neoplasias , Paclitaxel/farmacologia , Fenilpropionatos/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We conducted this research to explore the role of latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) in modulating the DNA damage response (DDR) and its regulatory mechanisms in radioresistance. Our results revealed that LMP1 repressed the repair of DNA double strand breaks (DSBs) by inhibiting DNA-dependent protein kinase (DNA-PK) phosphorylation and activity. Moreover, LMP1 reduced the phosphorylation of AMP-activated protein kinase (AMPK) and changed its subcellular location after irradiation, which appeared to occur through a disruption of the physical interaction between AMPK and DNA-PK. The decrease in AMPK activity was associated with LMP1-mediated glycolysis and resistance to apoptosis induced by irradiation. The reactivation of AMPK significantly promoted radiosensitivity both in vivo and in vitro. The AMPKα (Thr172) reduction was associated with a poorer clinical outcome of radiation therapy in NPC patients. Our data revealed a new mechanism of LMP1-mediated radioresistance and provided a mechanistic rationale in support of the use of AMPK activators for facilitating NPC radiotherapy.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Carcinoma/radioterapia , Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/metabolismo , Neoplasias Nasofaríngeas/radioterapia , Proteínas Nucleares/metabolismo , Tolerância a Radiação , Proteínas da Matriz Viral/metabolismo , Animais , Apoptose/efeitos da radiação , Carcinoma/enzimologia , Carcinoma/patologia , Carcinoma/virologia , Linhagem Celular Tumoral , Reparo do DNA , Relação Dose-Resposta à Radiação , Ativação Enzimática , Feminino , Glicólise , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Fosforilação , Transdução de Sinais/efeitos da radiação , Fatores de Tempo , Carga Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Stathmin, a microtubule associated protein (MAP), is an important molecular target for cancer therapy. Paclitaxel is one of the primary antitumor drugs targeting microtubules (MTs). We hypothesized that decreasing the expression level of Stathmin might improve the effectiveness of paclitaxel in the treatment of nasopharyngeal carcinoma (NPC). METHODS: NPC cell lines, CNE1-LMP1 and HNE2, and a CNE1-LMP1 tumor xenograft mouse model were used to test both in vitro and in vivo our siRNA-based Stathmin silencing strategy. The effects of Stathmin silencing on cell proliferation, apoptosis, and viability were investigated using MTT, AO/EB staining, TUNEL, caspase protein detection, and FCM assays. Cell migration and invasion were assayed using a Transwell assay. The combined effects of Stathmin silencing and paclitaxel were investigated using MTT, FCM, Western blot and indirect immunofluorescence assays. The effect of paclitaxel on Stathmin expression in NPC cells and, in addition, A375, MGC and HeLa cells was determined by RT-PCR and Western blotting. RESULTS: We found that siRNA-mediated silencing of Stathmin suppresses proliferation, induces apoptosis through the mitochondrial pathway, and causes G2/M-phase cell cycle arrest in the NPC cell lines CNE1-LMP1 and HNE2. Also, the migration and invasion of the respective NPC cells were found to be inhibited. In addition, we show that a combination of Stathmin silencing and paclitaxel is more effective than either agent alone in inhibiting proliferation and inducing apoptosis, cell cycle arrest, and MT polymerization. Furthermore, we found that Stathmin expression in the tumor cells is down-regulated by paclitaxel treatment. CONCLUSION: siRNA-mediated silencing of Stathmin suppresses the proliferation, invasion and metastasis, and induces the apoptosis of NPC cells. Paclitaxel reduces the expression of Stathmin, and combining Stathmin silencing with paclitaxel treatment enhances MT polymerization. This combined strategy may provide a new approach for clinical NPC treatment.