Inhibition of etoposide-induced apoptosis with peptide aldehyde inhibitors of proteasome

Abstract

Recent investigations have indicated the involvement of proteasome in programmed cell death. The present studies show that although peptide aldehyde inhibitors of proteasome are by themselves weak inducers of apoptosis, they inhibit the apoptotic effect of the anticancer drug etoposide in rat thymocytes. Acetyl-Leu-Leu-norvalinal (LLnV-al) and other related peptide aldehydes inhibited the increase in caspase activity and DNA fragmentation that followed treatment with etoposide and their effect was related to their potency as proteasome inhibitors. To inhibit etoposide-induced apoptosis, LLnV-al must be present within 3 h of treatment with etoposide, in the same way as the inhibitor of protein synthesis cycloheximide must be. Etoposide caused a rapid accumulation of p53 protein that was not inhibited by LLnV-al, which was also a strong inducer of p53. Peptide aldehydes were also weak activators of caspase activity, suggesting that the same mechanism, i.e. the blocking of proteasome function, both triggers apoptosis and inhibits the effect of etoposide. These results are consistent with a model in which proteasome is selectively involved in the pathway used by etoposide to induce cell suicide.

Proteasome Involvement and Accumulation of Ubiquitinated Proteins in Cerebellar Granule Neurons Undergoing Apoptosis

“…This hypothesis is strengthened by the observation that proteasomes can cleave after Asp in synthetic peptide substrates (Rivett et al, 1994;Kisselev et al, 1999) and that proteasomes are the major ICE-like proteinase in P19 cells treated with retinoic acid (Kobayashi et al, 1996). On the basis of such considerations and of previous findings, it is tempting to place the proteasome activity in CGCs undergoing apoptosis upstream of the caspase activation, as already shown in sympathetic neurons deprived of NGF (Sadoul et al, 1996) and in thymocytes treated with dexamethasone, ␥-irradiation, or etoposide (Grimm et al, 1996;Hirsch et al, 1998;Stefanelli et al, 1998). The suggestion that the proteasome complex is acting upstream of the caspases is also based on the finding that the proteasomal inhibitors block caspase-3 activity and prevent the cleavage of tau in granule neurons undergoing apoptosis (Fig.…”

The role of the ubiquitin-proteasome pathway in apoptosis

“…The addition of staurosporine or phorbol ester with the calcium ionophore A23187, however, resulted in apoptosis which could not be antagonized. 49 Similar inhibition of thymocyte apoptosis by proteasome inhibitors was noted for dexamethasone-induced cell death; 50 for etoposide-induced apoptosis, though not for calcium ionophore-or staurosporine-induced cell death; 66 and for both dexamethasone-and etoposide-induced apoptosis. 51 In experiments using rat sympathetic neurons induced to undergo apoptosis by NGF withdrawal, inhibitors of both cysteine proteinases and the proteasome promoted neuron survival.…”

“…Increased p53 levels alone, however, must not be sufficient to induce apoptosis, since such accumulation is seen after proteasome inhibition of thymocytes, yet these cells are prevented from undergoing cell death induced by etoposide. 66 Also, proteasome inhibition is sufficient to induce apoptosis in HL60 cells, which are p53-null, 58 and U937 cells undergo apoptosis without detectable accumulation of p53. 53 p27 and other cell cycle regulatory proteins Due to the impact of proteasome inhibition on cell cycling through the G 1 /S and G 2 /M boundaries, other proteins which impact upon these transition points are being investigated.…”

“…67 Caspase 3-like activity stimulated by etoposide-treated thymocytes was decreased by proteasome inhibition. 66 Recently published work investigated the impact of proteasome inhibition on mitochondrial membrane potentials in thymocytes induced to undergo apoptosis by dexamethasone or etoposide. Several proteasome inhibitors prevented dissipation of the mitochondrial transmembrane potential, post-mitochondrial generation of superoxide anions, loss of nuclear DNA, and exposure of phosphatidyl-serine.…”

The proteasome: a novel target for cancer chemotherapy

“…Precisely where they inhibit apoptosis is not clear but it is most likely upstream of mitochondrial perturbation 41 and effector caspase activation as caspase-3 processing and activity are partially blocked as evidenced by inhibition of processing of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). [42][43][44][45] In cell death pathways like these, where the proteasome has an important role in initiating apoptosis, it may act by degrading anti-apoptotic regulatory proteins or other proteins necessary for cell survival. In some cells proteasome inhibitors appear to induce both pro-and anti-apoptotic signals, the predominant signal determining the fate of the cell.…”