RESUMEN
The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cells to ionizing radiation were investigated in vitro. Further, the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of X-ray irradiation on A549 cells resulted in a low level of radiosensitivity with a D0 value of 12 Gy. The slopes of the survival curves in the exponential phase were plotted on semilogarithmic paper for radiation combined with AMR (2.5 microg/ml) and AMROH (0.02 microg/ml) treatment, and were shown to be approximately parallel to treatment with irradiation alone. The initial shoulder-shape portion of the survival curve for radiation alone, indicating the repair of sublethal damage, was reduced as compared to that for sequential combined treatment with AMR or AMROH. Sequential treatments with AMR or AMROH prior to ionizing radiation resulted in an additive radio-enhancement effect that reduced not only survival, but also the shoulder width. Fractionated irradiation with 2 Gy per fraction of A549 cells was carried out in vitro similar to that commonly performed in clinical radiotherapy and the radio-resistance of the cells was shown to be inhibited by AMR and AMROH. Similar to AMR and AMROH, adriamycin and etoposide (VP-16) are DNA topoisomerase II inhibitors. The effects of these 4 agents on cells that received X-ray irradiation were compared and all of the agents exhibited comparable radio-enhancement effects. The induction of apoptosis was investigated at 48 and 72 h after administration of AMROH, radiation or combined treatment, and apoptosis was not significantly induced after any of the treatments. We also examined the induction of necrosis, and found that the incidence of necrosis following combined treatment was approximately 2 times higher than that with either of the single treatments.
Asunto(s)
Adenocarcinoma/radioterapia , Antraciclinas/farmacología , Neoplasias Pulmonares/radioterapia , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Inhibidores de Topoisomerasa II , Apoptosis , Línea Celular Tumoral , Humanos , Cinética , Necrosis , Rayos XRESUMEN
The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cell line to hyperthermia at 44 degrees C were investigated. The cell phase response as well as the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of 44 degrees C hyperthermia on A549 cells exhibited low thermosensitivity with a T(0) value of 12 min. The slope of the survival curve in the exponential phase, described semilogarithmically, in 44 degrees C hyperthermia combined treatment with AMROH (0.02 microg/ml) was approximately parallel to 44 degrees C hyperthermia alone. The initial shoulder shape portion of the survival curve from 44 degrees C hyperthermia alone, indicating the repair of sublethal thermal damage (SLTDR), was reduced with the sequential combined treatment of AMR or AMROH. Sequential treatments with AMR or AMROH prior to 44 degrees C hyperthermia resulted in additive thermo-enhancement effect by reducing not only survival but was shoulder wide. Furthermore, like AMR and AMROH, adriamycin (ADM) and etoposide (VP-16) are DNA topoisomerase II inhibitors, and the effects of these 4 agents on 44 degrees C hyperthermia were compared. All 4 agents exhibited comparable thermo-enhancement effects. Using synchronized A549 cells, AMR or AMROH did not elicit cell phase responses, irrespective of the concentration. The induction of apoptosis was investigated at 48 and 72 h after AMROH treatment, 44 degrees C hyperthermia or the combined treatment, in which apoptosis was not significantly induced after any treatment. Furthermore, the incidence of necrosis was examined as well as apoptosis. The incidence of necrosis at 48 and 72 h after AMROH was 2.4 and 4.3%, respectively; after 44 degrees C hyperthermia was 3.3 and 4.0%, respectively; and after the combined treatment it was 10.7 and 8.7%, respectively. The necrosis induced after the combined treatment was circa 3 times higher than that in either of the single treatments.
Asunto(s)
Antraciclinas/farmacología , Apoptosis/efectos de los fármacos , Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Sinergismo Farmacológico , Etopósido/farmacología , Calor , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/fisiopatología , Necrosis/inducido químicamente , Factores de TiempoRESUMEN
PURPOSE: To elucidate the relationship between p53 functions and the interactive effects of the combined treatment with mild hyperthermia and mitomycin C. METHODS AND MATERIALS: p53-deficient human non-small-cell lung cancer H1299 cells were transfected with a vector carrying a neomycin-resistant gene (neo) or together with a wild-type or mutant p53 gene. Sensitivities of these transfectants to mild hyperthermia at 42 degrees C, mitomycin C (0.05 microg/mL) at 37 degrees C, or the combination treatment were determined by colony formation assay. After these treatments, the induction of apoptosis, the changes in cell cycle distribution, and the accumulation of Hsp72 were examined. RESULTS: The combined treatment resulted in an enhanced cell killing effect in H1299 cells in a p53-independent manner, which was partially the result of an enhancement of heat-induced apoptosis. The treatment also caused a marked G(2)/M arrest in the neo and the mutant p53 cells, but not in the wild-type p53 cells. The subsequent release of G(2)/M arrest was accompanied with an increase in the sub-G(1) fractions. Mitomycin C did not affect the accumulation of Hsp72 induced by hyperthermia in H1299 cells regardless of their p53 gene status. CONCLUSION: Our findings demonstrate a p53-independent mechanism for an interactively cytotoxic enhancement by combined treatment with mild hyperthermia and mitomycin C.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/terapia , Ciclo Celular , Genes p53/fisiología , Hipertermia Inducida , Neoplasias Pulmonares/terapia , Mitomicina/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Terapia Combinada , Resistencia a Antineoplásicos/genética , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neomicina/farmacología , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
Our research group has reported the enhanced cytotoxicity of combined treatment with bleomycin (BLM) and low hyperthermia at 40 degrees C, using murine L cells, and suggested that post-heating could inhibit BLM-induced sublethal damage repair. For further understanding of the involved mechanisms, we subsequently investigated the kinetics of the cellular accumulations of inducible 72-kDa heat shock protein (hsp72) after 40 degrees C hyperthermia and/or BLM treatment using the same cell line. Western blot analysis showed significantly enhanced accumulation of hsp72 after a low hyperthermia at 40 degrees C for 40, 105 or 180 min, and no significant enhancement of it after exposure to 10 microg/ml BLM at 37 degrees C for either 40 or 105 min. When the cells were heated in the presence of BLM, the accumulations of hsp72 were markedly suppressed, with the maxima of hsp72 accumulation decreasing to 38% and 63% of those induced by hyperthermia alone for 40 or 105 min, respectively. On the other hand, sequential treatment with hyperthermia either before or after BLM treatment did not show significant alteration of the heat-induced accumulations of hsp72. It was demonstrated that BLM was necessary during heating to effectively suppress the heat-induced accumulation of hsp72. This study indicates that the suppression of heat-induced accumulation of hsp72 by BLM may partially contribute to enhance cytotoxicity of the simultaneous treatment of 40 degrees C hyperthermia and BLM.