RESUMEN
Nitric oxide is known to be a multifunctional physiological substance. Recently, it was suggested that nitric oxide is involved in p53-dependent response to many kinds of stress, such as heat shock and changes in cellular metabolism. To verify this hypothesis, we examined the effect of nitric oxide produced endogenously by heat-shocked cells on nonstressed cells using a human glioblastoma cell line, A-172, and its mutant p53 (mp53) transfectant (A-172/mp53). The accumulation of inducible nitric oxide synthase was caused by heat treatment of the mtp53 cells but not of the wild-type p53 (wtp53) cells. The accumulation of heat shock protein 72 (hsp72) and p53 was observed in nontreated mtp53 cells cocultivated with heated mp53 cells, and the accumulation of these proteins was suppressed by the addition of a specific inducible nitric oxide synthase inhibitor, aminoguanidine, to the medium. Furthermore, the accumulation of these proteins was observed in the wtp53 cells after exposure to the conditioned medium by preculture of the heated mp53 cells, and the accumulation was completely blocked by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. In addition, the accumulation of hsp72 and p53 in the wtp53 cells was induced by the administration of an nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, to the medium. Finally, the thermosensitivity of the wtp53 cells was reduced in the conditioned medium by preculture of the heated mp53 cells as compared with conventional fresh growth medium. Our finding of the accumulation of hsp72 and p53 in nitric oxide-recipient cells cocultivated with heated nitric oxide-donor cells provides the first evidence for an intercellular signal transduction pathway via nitric oxide as intermediate without cell-to-cell interactions such as gap junctions.
Asunto(s)
Genes p53 , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Benzoatos/farmacología , Neoplasias Encefálicas , División Celular , Técnicas de Cocultivo , Glioblastoma , Guanidinas/farmacología , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Calor , Humanos , Hipertermia Inducida , Imidazoles/farmacología , Cinética , Mutagénesis , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Penicilamina/análogos & derivados , Penicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Hyperthermia has been introduced as a new modality of treatment for glioma. In these experiments, the cytotoxicity of hyperthermia in C6 glioma cells was enhanced by increasing the intracellular acidity with amiloride and/or 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS). Intracellular pH (pHi) is regulated mainly by Na+/H+ and HCO3-/Cl- antiports through the cell membrane, and amiloride acts on the former, DIDS on the latter to lower pHi. The cellular thermosensitivity to clinically achievable brain hyperthermia at 42 degrees C was enhanced by 0.5 mM amiloride (Na+/H+ antiport inhibitor). T0 values (T0 = the heating period required to reduce experimental survival rate by 1/e) at 42 degrees C without and with amiloride was 192 and 81 min, respectively. The addition of DIDS (HCO3-/Cl- antiport inhibitor) further enhanced. T0 value was 25 min. Fluorophotometric measurement of pHi was employed using the pH sensitive dye, bis(carboxyethyl)carboxyfluorescein, which is trapped in viable cells. The average pHi in control C6 glioma cells in pH 7.2 media was 7.21. In the untreated cells heated at 42 degrees C for 1 hour, the pHi was 7.12. The pHi of the cells heated in the presence of amiloride was decreased to 6.83. The pHi was further lowered to 6.67 by the treatment with amiloride in combination with DIDS for 2 hours. Hyperthermia with amiloride and DIDS may be a more effective treatment for malignant gliomas.
Asunto(s)
Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/uso terapéutico , Amilorida/uso terapéutico , Neoplasias Encefálicas/terapia , Glioma/terapia , Hipertermia Inducida/métodos , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Ácidos , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Glioma/tratamiento farmacológico , Concentración de Iones de Hidrógeno , Ratas , Células Tumorales CultivadasRESUMEN
PURPOSE: The kinetics of the accumulation of inducible 72-kD heat shock protein (hsp72) and the activation of heat shock transcriptional factor (HSF) after hyperthermia and/or CDDP treatment in two human glioblastoma cell lines, A-172 having the wild-type p53 gene and T98G having the mutated p53 gene were evaluated. METHODS AND MATERIALS: Western blot analysis of hsp72, gel-mobility shift assay of HSF, cell survival, and development of thermotolerance were examined. RESULTS: The prominent suppression of heat-induced hsp72 accumulation by CDDP was seen in A-172 cells, but not in T98G cells. This was due to the p53-dependent inhibition of heat-induced HSF activation by CDDP. The interactive hyperthermic enhancement of CDDP cytotoxicity was observed in A-172 cells, but not in T98G cells. In addition, the heat-induced thermotolerance was suppressed by the presence of CDDP in the pretreatment. CONCLUSION: Suppression of heat-induced hsp72 accumulation by CDDP contributes to an interactive hyperthermic enhancement of CDDP cytotoxicity in the cells bearing the wild-type p53 gene.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Glioblastoma/metabolismo , Proteínas de Choque Térmico/efectos de los fármacos , Hipertermia Inducida , Proteínas de Neoplasias/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Factores de Transcripción/efectos de los fármacos , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Modification effects of low hyperthermia (40 degrees C) on cellular chemosensitivity to bleomycin (BLM), and of BLM on thermosensitivity (40 degrees C) were investigated in cultured murine L cells with simultaneous or sequential treatments of these two agents. The heating of L cells at 40 degrees C up to 6 hours resulted in no remarkable lethal damage. Treatment with low hyperthermia followed by BLM for 4 hours showed no more than additive effect. However, a significant chemical (BLM) enhancement effect on the cellular thermosensitivity was observed by the treatment with BLM for 4 hours prior to the low hyperthermia at 40 degrees C. No appreciable thermal enhancement effect on the cellular chemosensitivity to BLM was shown by preheating at 40 degrees C for 3 hours, while post-heating at 40 degrees C showed a significant thermal enhancement effect as well as in the simultaneous treatments. The cell phase response to BLM was levelled in the clinical range of the doses. It is possible that the repair of sublethal damage (SLDR) from treatment with BLM was inhibited by 40 degrees C post-heating and this SLDR was completed at 3 hours of the interval at 37 degrees C between BLM and 40 degrees C heating.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Hipertermia Inducida/métodos , Animales , Antibióticos Antineoplásicos/toxicidad , Antineoplásicos/farmacología , Bleomicina/toxicidad , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , Ensayos de Selección de Medicamentos Antitumorales , Hidroxiurea/farmacología , Células L , Ratones , Factores de TiempoRESUMEN
We examined the interactive effects of hyperthermia combined with cisplatin (CDDP) (0.5 micrograms/ml) or peplomycin (PEP) (1.0 microgram/ml) on surviving fractions of human maxillary carcinoma IMC-2 cells. Either CDDP or PEP enhanced the 44 degree C thermosensitivity of thermotolerant cells after heating at 42 degrees C for 2 hours. The development of thermotolerance at 42 degrees C with either of the two drugs for 2 hours was not inhibited by CDDP, but it was partially inhibited by PEP. Moreover, for PEP throughout the entire period of 42-44 degrees C step-up heating, the 44 degree C thermosensitivity of thermotolerant cells after heating at 42 degrees C with PEP for 2 hours was enhanced similarly to that at 44 degrees C with PEP. Heating at 42 degrees C combined with either of the two drugs showed a marked interactive effect.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Hipertermia Inducida , Neoplasias Maxilares/patología , Peplomicina/farmacología , Antineoplásicos/uso terapéutico , Recuento de Células/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/uso terapéutico , Terapia Combinada , Humanos , Neoplasias Maxilares/terapia , Peplomicina/uso terapéutico , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The accumulation of the inducible hsp72 (72-kDa heat shock protein) after hyperthermia and/or cisplatin treatment in human glioblastoma cell line (A-172) was studied by Western blot analysis. The level of hsp72 increased to eight-fold 10 h after hyperthermia alone (44 degrees C for 20 min, D50) and to three-fold 10 h after cisplatin treatment (5 microg/ml) at 37 degrees C for 15 min (D50). In contrast, when the cells were simultaneously heated with cisplatin, the accumulation of hsp72 was suppressed. The level of hsp72 increased to about six-fold and two-fold 10 h after hyperthermia (44 degrees C, 15 min) in the presence of 1 and 10 microg/ml (D50 or D10) of cisplatin, respectively. In addition, we found both the enhancement of thermosensitivity and the suppression of thermotolerance by the simultaneously combined treatment of hyperthermia and cisplatin. It has been reported that the enhancement of cisplatin cytotoxicity by hyperthermia is due to increase of both cisplatin uptake and DNA damage by hyperthermia. Our results suggest that the interactive cytotoxic enhancement by the combination of hyperthermia and cisplatin may be also due to the suppression of heat-induced hsp72 accumulation by cisplatin.