Shyam S. Biswal

Inhibition of peroxisome-proliferator-activated receptor (PPAR)α by MK886

Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may also indicate that PPAR alpha is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPAR alpha, making it the first compound identified to have such an effect.

Acrolein Causes Inhibitor κB-independent Decreases in Nuclear Factor κB Activation in Human Lung Adenocarcinoma (A549) Cells

Acrolein is a highly electrophilic α,β-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor κB (NF-κB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-κB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-κB. Cells treated for 1 h with 1 mmdiethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-κB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-κB function ∼50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-κB binding, even at doses as low as 0.125 mm that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor κB-α (IκB-α). Furthermore, acrolein decreased NF-κB activation in cells depleted of IκB-α by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-κB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-κB in the cytosol prior to chemical dissociation from IκB with detergent. Together, these data support the conclusion that the inhibition of NF-κB activation by acrolein and DEM is IκB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-κB subunits.

Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis.

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 30% lower in bcl-x(L) cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, as determined by Western blot analyses, were similar in control and bcl-x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.

A Relationship between 5-Lipoxygenase-activating Protein and bcl-xL Expression in Murine Pro-B Lymphocytic FL5.12 Cells

Inhibitors of 5-lipoxygenase-activating protein (FLAP) have been found to induce apoptosis. The current study examined the expression of FLAP and bcl family proteins and the induction of apoptosis in interleukin-3-dependent control andbcl-x L -overexpressing FL5.12 cell lines after treatment with MK886, a specific FLAP inhibitor. FL5.12 cells contained a substantial amount of FLAP protein and mRNA but surprisingly had no measurable 5-lipoxygenase protein or 5-, 12-, or 15-lipoxygenase activity. The basal level of FLAP protein in cells overexpressingbcl-x L was 70% less than in controls. FLAP disappeared 4 h after withdrawal of interleukin-3 inbcl-x L cells but not in control cells, which underwent apoptosis. A dose- and time-response study revealed that 5 nmol of MK886/106 cells was sufficient to induce apoptosis both in control and bcl-x L cells, respectively, but to different degrees. bcl-xL and bcl-2 proteins, but not bax or FLAP, were decreased by 4 h after 5 nmol of MK886/106 cells in both cell lines, although the higher levels of bcl-xL in overexpressors took longer to disappear. This early loss of bcl-xL and bcl-2 was not attributable to generalized proteolysis, as shown by Coomassie Blue staining and by the maintenance of bax. Caspase-3 was activated 2 h after MK886 treatment in control cells but not inbcl-x L cells. Inhibition of caspase-3 decreased MK886-induced apoptosis by 50% in control cells. Inhibition of this caspase after MK886 treatment was unable to prevent the loss of bcl-xL in control cells but did provide partial protection for the loss of the transfected form, but not the endogenous form, in overexpressing cells. These data indicate that MK886 induces extensive apoptosis that is partially caspase-3 dependent and may be related to a rapid loss of bcl-xL. Although caspase-3 inhibitors had no effect on the loss of bcl-xL, other caspases or protease systems may still be involved. The absence of 5-lipoxygenase in cells containing FLAP, the lower level of FLAP in bcl-xL cells, the apoptosis-inducing activity of MK886, and the rapid loss of bcl-xL and bcl-2 proteins after treatment with MK886 strongly indicate that FLAP has activities unrelated to lipoxygenase and suggest a possible functional or regulatory link between these proteins, which share similar subcellular localizations.

Changes in ceramide and sphingomyelin following fludarabine treatment of human chronic B-cell leukemia cells

Fludarabine is used to treat chronic lymphocytic leukemia. Both in vitro and in vivo studies have indicated that apoptosis is an important mode of fludarabine-induced cell death. However, the apoptotic pathways activated are not known. The effects of apoptotic doses of fludarabine on sphingomyelin, ceramide and the production of reactive oxygen species were investigated in the chronic B-cell leukemia lines WSU and JVM-2. Apoptosis, as assessed by an increase in phosphatidylserine externalization, internucleosomal DNA fragmentation and caspase-3-like activity, was evident by 18 h after fludarabine in both cell lines. The general caspase inhibitor t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (OMe, methyl ester) significantly inhibited apoptosis supporting a role for caspases in fludarabine-induced cell death. A 2.5- to threefold elevation in ceramide levels was observed 6 h after fludarabine treatment. Concomitantly, a decrease in sphingomyelin levels was observed. Fumonisin B1 (an inhibitor of ceramide synthase) pretreatment significantly prevented fludarabine-induced ceramide generation and apoptosis. Conversely, C6-ceramide induced apoptosis in both cell lines. No effect of fludarabine on indices of oxidative stress (dichlorofluorescin oxidation and glutathione disulfide formation) were detected, although partial protection from apoptosis, and prevention of ceramide generation and caspase-3 activation, were achieved with N-acetylcysteine. These findings are consistent with the involvement of caspases and ceramide in fludarabine-induced apoptosis in WSU and JVM-2 cells. Oxidative stress does not appear to be induced by fludarabine, although the protective effects of N-acetylcysteine suggest that thiol redox balance may play a role in the apoptotic pathway.