Serine protease inhibitors block neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin

Abstract

To address the role of a plausible protease cascade in daunorubicin-triggered apoptosis, we evaluated the effect of cell-permeant protease inhibitors on its signal transduction pathway. Treatment of U937 and HL-60 cells with 0.5-1 microM of the chemotherapeutic drug daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. DNA fragmentation and the classical morphological features of apoptosis were observed within 4-6 h. Pretreatment of cells with the serine protease inhibitors N-tosyl-L-phenylalanyl chloromethyl ketone (20 microM) or dichloroisocoumarin (20 microM) for 30 min inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. Other cell-permeant protease inhibitors such as pepstatin, leupeptin, and antipain had no such effect. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. Daunorubicin-induced NF-kappaB activation was inhibited by dichloroisocoumarin but not by N-tosyl-L-phenylalanyl chloromethyl ketone, suggesting that this transcription factor can be activated independently of ceramide and is not directly implicated in the apoptotic pathway. These results suggest that inhibitors of serine proteases can act upstream of ceramide in drug-triggered apoptosis and that neutral sphingomyelinase activation is either directly or indirectly serine protease dependent.

The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes

“…49 Generation of ceramides has been already described with various chemotherapeutic agents. 25,27,28,30,38,50 Here we report that ETO and CPT increased the generation of ceramide in sensitive (activated) but not in resistant (resting) PBL. Ceramide production was maximal after 30 and 60 min of treatment with ETO or CPT, respectively, corresponding to the earliest signal observed in ETO-and CPT-induced apoptosis.…”

“…50 Briefly, after 3 days of culture, 2610 7 PBL were starved for 2 h in RPMI containing 2% BSA and then treated with ETO, CPT or a CD95 agonistic mAb (7C11) for indicated time. …”

N-acetylcysteine blocks apoptosis induced by N-α-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

“…1,2,5,45 TPCK and DCI, but not other cell-permeable protease inhibitors, have been shown to inhibit the generation of ceramide and apoptosis, by blocking daunorubicin-induced sphingomyelinase activation. 46 Also internucleosomal DNA fragmentation induced by the topoisomerase I inhibitor camptothecin was suppressed by TPCK in B lymphoma Namalwa cells 47 and HL60 cells, 1,48 but caspase-3-like activity and high molecular weight DNA fragmentation were not affected. 47 On the other hand, our work and that of others 9,10,49 shows that TPCK can clearly also induce apoptosis.…”

Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells

“…Although the role of serine proteases in cell death remains largely unexplored, a number of studies have shown that serine protease inhibitors can block apoptosis induced by certain stimuli Adjei et al, 1996, Dong et al, 1997Chow et al, 1995;Weis et al, 1995;Mansat et al, 1997;Shao et al, 1997). For example, TPCK has been shown to inhibit both FAS antigen-and p53-induced apoptosis as well as cell death occurring in response to various chemotherapeutic agents (e.g.…”

“…For example, TPCK has been shown to inhibit both FAS antigen-and p53-induced apoptosis as well as cell death occurring in response to various chemotherapeutic agents (e.g. etoposide, UCN-01, and daunorubicin) in cells of hematopoietic origin Mansat et al, 1997;Shao et al, 1997;Sebestyen et al, 1997;Lotem and Sachs, 1996). TLCK on the other hand, was found to be e ective in inhibiting cell death by camptothecin and glycodeoxycholate in hepatocytes, but TPCK was without e ect (Kwo et al, 1995, Adjei et al, 1996.…”