The apoptotic effects and synergistic interaction of sodium butyrate and MG132 in human retinoblastoma Y79 cells

This study deals with the apoptotic effect exerted on human retinoblastoma Y79 cells by both sodium butyrate and an inhibitor of 26S proteasome [z-Leu -Leu-Leu-CHO (MG132)] and their synergistic effect. Exposure to sodium buty rate (1-4 mM) induced an accumulation of cells in the G(2)-M phase that was already visible after 24 h of treatment, when morphological and biochemica l signs of apoptosis appeared only in a small number of cells (5-10%). Ther eafter, the apoptotic effects increased progressively with slow kinetics, r eaching a maximum after 72 h of exposure, when they concerned a large fract ion of cells (>75% with 4 mM sodium butyrate). Sodium butyrate stimulated t he conversion of procaspase-3 into caspase-3 and also induced the cleavage of poly-(ADP-ribose) polymerase and lamin a, two hallmarks of apoptosis. Al l of the apoptotic signals were suppressed by benzyloxy carbonyl-Val-Ala-As p-fluoromethylketone (a general inhibitor of caspase activities), whereas a cetyl-Asp-Glu-Val-Asp aldehyde, a specific inhibitor of caspase-3 activity, only induced a partial reversion of the apoptotic effects. Sodium butyrate also decreased the Bcl-2 level, whereas it increased the Bar level and sti mulated the release of cytochrome c from the mitochondria, an event that wa s most likely responsible for the activation of caspase-3. Finally, sodium butyrate activated 26S proteasome, the major extralysosomal degradative mac hinery, which is responsible for the degradation of short-lived proteins. C onsequently, the levels of p53, N-myc, and I kappa B alpha (factors that pl ay regulatory roles in apoptosis) diminished, whereas the nuclear level of nuclear factor KB concomitantly increased, Treatment of Y79 cells with MG13 2 induced apoptosis with more rapid kinetics than with sodium butyrate. The effects appeared after 8 h of incubation, reaching a maximum at 24 h, and they were accompanied by increased levels of N-myc, p53, and I kappa B alph a. MG132 also favored the release of cytochrome c from the mitochondria and Increased the activity of caspase-3. When Y79 cells were exposed to combin ations of sodium butyrate and MG132, the Latter compound suppressed the dec reasing effect induced by sodium butyrate on the levels of p53, N-myc, and IkB alpha and the increasing effect on the nuclear level of nuclear factor kappa B. Moreover, an increase in the level of Bax and an enhancement in th e release of cytochrome c from the mitochondria were observed, Clear synerg istic effects concerning the activation of both caspase-3 and apoptosis wer e induced by a combination of suboptimal doses of sodium butyrate and MG132 . The results support the conclusion that MG132 potentiates the apoptotic e ffect of sodium butyrate by suppressing its stimulatory effect on 26S prote asome activity. Synergistic interactions between butyrate and inhibitors of proteasome could represent a new important tool in tumor therapy and, in p articular, the treatment of retinoblastoma.