The tyrphostin adaphostin interacts synergistically with proteasome inhibitors to induce apoptosis in human leukemia cells through a reactive oxygen species (ROS)-dependent mechanism

Abstract

Interactions between the tyrphostin adaphostin and proteasome inhibitors (eg, MG-132 and bortezomib) were examined in multiple human leukemia cell lines and primary acute myeloid leukemia (AML) specimens. Cotreatment of Jurkat cells with marginally toxic concentrations of adaphostin and proteasome inhibitors synergistically potentiated mitochondrial damage (eg, cytochrome c release), caspase activation, and apoptosis. Similar interactions occurred in other human leukemia cell types (eg, U937, HL-60, Raji). These interactions were associated with a marked increase in oxidative damage (eg, ROS generation), down-regulation of the Raf/MEK/ERK pathway, and JNK activation. Adaphostin/MG-132 lethality as well as mitochondrial damage, down-regulation of Raf/MEK/ERK, and activation of JNK were attenuated by the free-radical scavenger NAC, suggesting that oxidative damage plays a functional role in antileukemic effects. Ectopic expression of Raf-1 or constitutively active MEK/ERK or genetic interruption of the JNK pathway significantly diminished adaphostin/MG-132-mediated lethality. Interestingly, enforced Raf or MEK/ERK activation partially diminished adaphostin/MG-132-mediated ROS generation, suggesting the existence of an amplification loop. Finally, the adaphostin/MG-132 regimen displayed similar toxicity toward 5 primary AML samples but not normal hematopoietic progenitors (eg, bone marrow CD34+ cells). Collectively, these findings suggest that potentiating oxidative damage by combining adaphostin with proteasome inhibitors warrants attention as an antileukemic strategy.

Figure 1.

Cotreatment of adaphostin with MG-132 synergistically induces cell death in human leukemia cells. (A) Jurkat cells were treated alone or in combination with the indicated concentrations of adaphostin and MG-132 (150 or 200 nM) for 24 hours, after which the percentage of apoptotic cells was monitored by annexin V/PI staining as described in “Materials and methods.” (B) Jurkat cells were treated with adaphostin (400 nM) or MG-132 (200 nM) individually as well as in combination, after which induction of apoptosis was monitored at intervals from 0 to 48 hours. (C) Jurkat cells were exposed to a range of adaphostin and MG-132 concentrations alone and in combination at a fixed ratio (eg, 2:1) simultaneously for 24 hours. At the end of this period, the percentage of cells undergoing apoptosis (reflected by annexin V/PI positivity) was determined for each condition. Fractional effect values were determined by comparing results with those of untreated controls, and median dose effect analysis was employed to characterize the nature of the interaction. Combination index values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results. (D) U937 cells were treated with 750 nM adaphostin plus or minus 250 nM MG-132 for 24 hours. (E) HL-60 cells were treated with adaphostin (1.0 μM) plus or minus MG-132 (300 nM) for 48 hours. (F) Raji cells were treated with adaphostin (1.0 μM) plus or minus MG-132 (225 nM) for 36 hours, after which the percentage of apoptotic cells was monitored by annexin V/PI staining and flow cytometry. For panels A, B, D, E, and F, values represent the means plus or minus strandard deviation (SD) for 3 separate experiments performed in triplicate.

Figure 2.

Combined treatment with adaphostin and MG-132 or bortezomib induces apoptosis in leukemia cells through induction of mitochondrial injury, caspase activation, down-regulation of Raf/MEK/ERK, and activation of JNK. Jurkat cells were treated with 400 nM adaphostin plus or minus 200 nM MG-132 or 4.0 nM bortezomib, while U937 cells were treated with 750 nM adaphostin plus or minus 250 nM MG-132, each for 8 hours. (A-B) Cytosolic (S-100) fractions were obtained as described in “Materials and methods,” and expression of cytochrome c, AIF, and Smac/DIABLO was monitored by Western blot. (C-F) At the end of the drug exposure (8 hours), cells were lysed, sonicated, and the proteins denatured and subjected to Western blot analysis using the indicated primary antibodies. For panels A and B, each lane was loaded with 20 μg protein, whereas for panels C-F, 30 μg protein was loaded in each lane. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. Results are representative of 3 separate studies.

Figure 3.

Combined treatment of leukemia cells with adaphostin and MG-132 or bortezomib leads to an increase in ROS generation and lethality, events that are attenuated by the free-radical scavenger NAC. (A) Jurkat cells preteated with or without 6 mM NAC for 3 hours or 1000 U/mL catalase for 1 hour were exposed to 400 nM adaphostin plus or minus 200 nM MG-132 or 4.0 nM bortezomib (Btzmb), and ROS generation was monitored by flow cytometry using DHCF as the dye following 18 hours of drug exposure. (B) Jurkat cells were treated with 400 nM adaphostin plus or minus 200 nM MG-132, after which levels of ROS generation were measured at the indicated interval (eg, 0.5 to 24 hours). (C) Jurkat cells preteated with or without 6 mM NAC for 3 hours or 10 μM Boc-fmk for 30 minutes or 1000 U/mL catalase for 1 hour were exposed to 400 nM adaphostin plus or minus 200 nM MG-132 for 24 hours, after which apoptotic cells were monitored by annexin V/PI staining and flow cytometry as described previously. (D) Jurkat cells were treated with 400 nM adaphostin plus or minus 200 nM MG-132 for 16 hours or with 1 mM BSO for 24 hours. Then cells were harvested and homogenized. The samples were then deproteinated, and GSH level was determined as described in “Materials and methods.” Jurkat cells were (E) pretreated with or without 6 mM NAC for 3 hours or (F) pretreated with or without 10 μM Boc-fmk for 30 minutes followed by exposure to 400 nM adaphostin plus or minus 200 nM MG-132 for 8 hours. Following drug treatment, cytosolic (S-100) fractions and whole-cell lysates were obtained as described in “Materials and methods.” Protein samples were subjected to Western blot analysis using the indicated primary antibodies. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. ^*Significantly less than values for cells exposed to drugs in the absence of NAC or Boc-fmk; P < .005.

Figure 4.

Enforced activation of MEK/ERK attenuates adaphostin/MG-132 lethality. (A) Jurkat cells inducibly expressing a constitutively active MEK1 construct were incubated in medium in the presence or absence of 1 μM doxycycline for 30 hours, followed by exposure to 500 nM adaphostin and 250 nM MG-132 for 24 hours. At the end of 24 hours of drug exposure, apoptosis was monitored by annexin V/PI staining and flow cytometry. (B) Cells treated as in panel A were monitored for ROS generation after 16 hours of drug exposure. (C) Following 8 hours of drug exposure, Western blot analysis was employed to monitor the effect of drugs on expression of the indicated proteins. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. (D) U937 cells expressing a constitutively active MEK1 construct and stably transfected empty-vector cells were treated with 750 nM adaphostin plus or minus 250 nM MG-132. At the end of 24 hours, apoptotic cells were monitored. (E) U937 cells treated identically were assayed for ROS generation after 2 hours of drug treatment. (F) Following 8 hours of exposure of U937 cells to adaphostin and MG-132 as in panel D, Western blot analysis was employed to monitor effects on protein expression. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. ^*Significantly less than values for cells exposed to drugs in the absence of doxycycline (A,B) or control pMM cells (D,E); P < .05.

Figure 5.

Ectopic expression of constitutively active Raf-1 or a c-Jun dominant-negative mutant significantly protects cells from adaphostin/MG-132 induced lethality. (A) Jurkat cells inducibly expressing a constitutively active Raf-1 construct were incubated in medium in the presence or absence of 1 μM doxycycline for 30 hours, followed by exposure to 500 nM adaphostin plus 250 nM MG-132. After 24 hours of drug exposure, apoptotic cells were monitored by annexin V/PI staining and flow cytometry. (B) Alternatively, levels of ROS generation were determined after 16 hours of drug treatment. (C) Following 8 hours of drug exposure as in panel A, Western blot analysis was employed to monitor protein expression of Raf, phospho-ERK, and phospho-JNK. (D) U937 cells ectopically expressing a c-Jun dominant-negative construct (TAM67) or stably transfected empty-vector controls (pMM) were treated with 750 nM adaphostin plus 250 nM MG-132. At the end of 24 hours of drug exposure, apoptotic cells were monitored by annexin V/PI staining followed by flow cytometric analysis. (E) Following 8 hours of drug exposure, Western blot analysis was employed to monitor protein expression of phospho-c-Jun and phospho-ERK. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein (20 μg each lane). ^*Significantly less than values for control cells; P < .05.

Figure 6.

Combined treatment with adaphostin and MG-132 markedly induces death in primary AML cells while exhibiting little toxicity toward normal hematopoietic cells. (A) Primary human AML blasts (FAB M2 subtype in 4 samples; M5 in 1 sample) were suspended in medium containing 10% FCS at a cell density of 0.8 × 10⁶/mL in the presence of 750 nM adaphostin plus or minus 300 nM MG-132 for 24 hours. At the end of drug exposure, apoptotic cells were monitored by annexin V/PI staining. Cell death for control blasts was generally less than 15% to 25%. (B) Primary AML blasts (sample 2) were exposed to drugs alone and in combination as in panel A for 18 hours, after which cell lysates were obtained and Western blot analysis performed to monitor PARP and MEK1 cleavage and levels of phospho-JNK and Raf-1. Each lane was loaded with 30 μg protein, and blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. (C) CD34⁺ cells obtained from the bone marrow of a patient undergoing a routine diagnostic procedure for a nonmyeloid hematologic disorder (eg, thrombocytopenia) were isolated by an immunomagnetic bead separation technique as described in “Materials and methods” and exposed to adaphostin (1.0 μM) ± MG-132 (300 nM) for 24 hours. At the end of this period, the percentage of apoptotic cells was determined by annexin V/PI staining and flow cytometry. A parallel experiment was also performed with normal peripheral-blood mononuclear cells (NPBMNC) from a healthy donor. Values represent the means plus or minus SD for triplicate determinations.