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 butyrate (1-4 mM) induced an accumulation of cells in the G2-M phase that was already visible after 24 h of treatment, when morphological and biochemical signs of apoptosis appeared only in a small number of cells (5-10%). Thereafter, the apoptotic effects increased progressively with slow kinetics, reaching a maximum after 72 h of exposure, when they concerned a large fraction of cells (>75% with 4 mM
sodium butyrate).
Sodium butyrate stimulated the conversion of
procaspase-3 into
caspase-3 and also induced the cleavage of
poly-(ADP-ribose) polymerase and
lamin B, two hallmarks of apoptosis. All of the apoptotic signals were suppressed by benzyloxy carbonyl-Val-
Ala-Asp-fluoromethylketone (a general inhibitor of
caspase activities), whereas acetyl-
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 Bax level and stimulated the release of
cytochrome c from the mitochondria, an event that was most likely responsible for the activation of
caspase-3. Finally,
sodium butyrate activated
26S proteasome, the major extralysosomal degradative machinery, which is responsible for the degradation of short-lived
proteins. Consequently, the levels of p53, N-myc, and
IkappaBalpha (factors that play regulatory roles in apoptosis) diminished, whereas the nuclear level of
nuclear factor kappaB concomitantly increased. Treatment of Y79 cells with
MG132 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
IkappaBalpha.
MG132 also favored the release of
cytochrome c from the mitochondria and increased the activity of
caspase-3. When Y79 cells were exposed to combinations of
sodium butyrate and
MG132, the latter compound suppressed the decreasing effect induced by
sodium butyrate on the levels of p53, N-myc, and
IkappaBalpha and the increasing effect on the nuclear level of
nuclear factor kappaB. Moreover, an increase in the level of Bax and an enhancement in the release of
cytochrome c from the mitochondria were observed. Clear synergistic effects concerning the activation of both
caspase-3 and apoptosis were induced by a combination of suboptimal doses of
sodium butyrate and
MG132. The results support the conclusion that
MG132 potentiates the apoptotic effect of
sodium butyrate by suppressing its stimulatory effect on
26S proteasome activity. Synergistic interactions between
butyrate and inhibitors of
proteasome could represent a new important tool in
tumor therapy and, in particular, the treatment of
retinoblastoma.