Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation.

BAY 43-9006 is a kinase inhibitor that induces apoptosis in a variety of tumor cells. Here we report that treatment with BAY 43-9006 results in marked cytochrome c and AIF release into the cytosol, caspase-9, -8, -7, and -3 activation, and apoptosis in human leukemia cells (U937, Jurkat, and K562). Pronounced apoptosis was also observed in blasts from patients with acute myeloid leukemia. These events were accompanied by ERK1/2 inactivation and caspase-independent down-regulation of Mcl-1. Inducible expression of a constitutively active MEK1 construct did not prevent Mcl-1 down-regulation, suggesting that this event is not related to MEK/ERK pathway inactivation. Furthermore, BAY 43-9006 did not induce major changes in Mcl-1 mRNA levels monitored by real-time PCR or Mcl-1 promoter activity demonstrated by luciferase reporter assays, but it did enhance Mcl-1 down-regulation in actinomycin D-treated cells. Inhibition of protein synthesis by cycloheximide or proteasome function with MG132 and pulse-chase studies with [35S]methionine demonstrated that BAY 43-9006 did not diminish Mcl-1 protein stability, nor did it enhance Mcl-1 ubiquitination, but instead markedly attenuated Mcl-1 translation in association with the rapid and potent dephosphorylation of the eIF4E translation initiation factor. Finally, ectopic expression of Mcl-1 in leukemic cells markedly inhibited BAY 43-9006-mediated cytochrome c cytosolic release, caspase-9, -7, and -3 activation, as well as cell death, indicating that Mcl-1 operates upstream of cytochrome c release and caspase activation. Together, these findings demonstrate that BAY 43-9006 mediates cell death in human leukemia cells, at least in part, through down-regulation of Mcl-1 via inhibition of translation.

Coadministration of histone deacetylase inhibitors and perifosine synergistically induces apoptosis in human leukemia cells through Akt and ERK1/2 inactivation and the generation of ceramide and reactive oxygen species.

Interactions between histone deacetylase inhibitors (HDACIs) and the alkyl-lysophospholipid perifosine were examined in human leukemia cells. Coadministration of sodium butyrate, suberoylanilide hydroxamic acid (SAHA), or trichostatin with perifosine synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and a marked decrease in cell growth in U937 as well as HL-60 and Jurkat leukemia cells. These events were associated with inactivation of extracellular signal-regulated kinase (ERK) 1/2 and Akt, p46 c-jun-NH2-kinase (JNK) activation, and a pronounced increase in generation of ceramide and reactive oxygen species (ROS). They were also associated with up-regulation of Bak and a marked conformational change in Bax accompanied by membrane translocation. Ectopic expression of Bcl-2 delayed but was ultimately ineffective in preventing perifosine/HDACI-mediated apoptosis. Enforced expression of constitutively active mitogen-activated protein kinase kinase (MEK) 1 or myristoylated Akt blocked HDACI/perifosine-mediated ceramide production and cell death, suggesting that MEK/ERK and Akt inactivation play a primary role in these phenomena. However, inhibition of JNK activation (e.g., by the JNK inhibitor SP600125) did not attenuate sodium butyrate/perifosine-induced apoptosis. In addition, the free radical scavenger N-acetyl-L-cysteine attenuated ROS generation and apoptosis mediated by combined treatment. Finally, the acidic sphingomyelinase inhibitor desipramine attenuated HDACI/perifosine-mediated ceramide and ROS production as well as cell death. Together, these findings indicate that coadministration of HDACIs with perifosine in human leukemia cells leads to Akt and MEK/ERK disruption, a marked increase in ceramide and ROS production, and a striking increase in mitochondrial injury and apoptosis. They also raise the possibility that combining these agents may represent a novel antileukemic strategy.

Cotreatment with suberanoylanilide hydroxamic acid and 17-allylamino 17-demethoxygeldanamycin synergistically induces apoptosis in Bcr-Abl+ Cells sensitive and resistant to STI571 (imatinib mesylate) in association with down-regulation of Bcr-Abl, abrogation of signal transducer and activator of transcription 5 activity, and Bax conformational change.

Interactions between the histone deacetylase (HDAC) inhibitors suberanoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein (Hsp) 90 antagonist 17-allylamino 17-demethoxygeldanamycin (17-AAG) have been examined in Bcr-Abl(+) human leukemia cells (K562 and LAMA84), including those sensitive and resistant to STI571 (imatinib mesylate). Cotreatment with 17-AAG and SAHA or SB synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and growth inhibition. Similar effects were observed in LAMA84 cells and K562 cells resistant to STI571, as well as in CD34(+) cells isolated from the bone marrows of three patients with chronic myelogenous leukemia. These events were associated with increased binding of Bcr-Abl, Raf-1, and Akt to Hsp70, and inactivation of extracellular signal-regulated kinase 1/2 and Akt. In addition, 17-AAG/SAHA abrogated the DNA binding and the transcriptional activities of signal transducer and activator of transcription (STAT) 5 in K562 cells, including those ectopically expressing a constitutively active STAT5A construct. Cotreatment with 17-AAG and SAHA also induced down-regulation of Mcl-1, Bcl-xL, and B-Raf; up-regulation of Bak; cleavage of 14-3-3 proteins; and a profound conformational change in Bax accompanied by translocation to the membrane fraction. Moreover, ectopic expression of Bcl-2 attenuated cell death induced by this regimen, implicating mitochondrial injury in the lethality observed. Together, these findings raise the possibility that combining HDAC inhibitors with the Hsp90 antagonist 17-AAG may represent a novel strategy against Bcr-Abl(+) leukemias, including those resistant to STI571.