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. 2008 Sep;74(3):872-83.
doi: 10.1124/mol.107.040808. Epub 2008 Jun 12.

Combinatorial antileukemic disruption of oxidative homeostasis and mitochondrial stability by the redox reactive thalidomide 2-(2,4-difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) and flavopiridol

Yun Ge  1 , Jung S Byun, Paola De Luca, Geraldine Gueron, Idalia M Yabe, Sara G Sadiq-Ali, William D Figg, Jesse Quintero, Cynthia M Haggerty, Quentin Q Li, Adriana De Siervi, Kevin Gardner
Affiliations

Combinatorial antileukemic disruption of oxidative homeostasis and mitochondrial stability by the redox reactive thalidomide 2-(2,4-difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) and flavopiridol

Yun Ge et al. Mol Pharmacol. 2008 Sep.

Abstract

2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a recently identified class of redox-reactive thalidomide analogs that show selective killing of leukemic cells by increasing intracellular reactive oxygen species (ROS) and targeting multiple transcriptional pathways. Flavopiridol is a semisynthetic flavonoid that inhibits cyclin-dependent kinases and also shows selective lethality against leukemic cells. The purpose of this study is to explore the efficacy and mechanism of action of the combinatorial use of the redox-reactive thalidomide CPS49 and the cyclin-dependent kinase inhibitor flavopiridol as a selective antileukemic therapeutic strategy. In combination, CPS49 and flavopiridol were found to induce selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. Highest synergy was observed at the level of ROS generation with a strong correlation between cell-specific cytotoxicity and reciprocal coupling of drug-induced ROS elevation with glutathione depletion. Examination of the transcriptional targeting of CPS49 and flavopiridol combinations reveals that the drugs act in concert to initiate a cell specific transcriptional program that manipulates nuclear factor-kappaB (NF-kappaB), E2F-1, and p73 activity to promote enhanced mitochondrial instability by simultaneously elevating the expression of the proapoptotic factors BAX, BAD, p73, and PUMA while depressing expression of the antiapoptotic genes MCL1, XIAP, BCL-xL, SURVIVIN, and MDM2. The coadministration of CPS49 and flavopiridol acts through coordinate targeting of transcriptional pathways that enforce selective mitochondrial dysfunction and ROS elevation and is therefore a promising new therapeutic combination that warrants further preclinical exploration.

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Figures

Fig. 1
Fig. 1
Combinatorial treatment with CPS49 and flavopiridol shows selective cytotoxic synergy against transformed leukemic cells. (A) PBMCs, Jurkat, K562, HeLa, and 8226 cells were incubated with increasing concentrations of CPS49 alone (left panel) or Flavo alone (right panel). Cell viability was measured by MTT assay at 16 h following the drug treatments. * Asterisk indicates that 20 times higher concentrations of CPS49 were used in 8226 cells (left panel). (B) Jurkat cells (left panel) were incubated with increasing concentrations (0−5 μM) of CPS49 alone, Flavo (flavopiridol) alone, or CPS49 plus flavopiridol with a fixed ratio of 1:1. Cell viability was determined after 16 h incubation time with the drugs. CI values (right panel) for each fractional effect were calculated using a commercially available software (Calcusyn; Biosoft). (C) Following 8 h pretreatment with 0.2 μM Flavo (flavopiridol), Jurkat cells (left panel) were incubated with increasing concentrations (0−5 μM) of CPS49 alone, Flavo alone, or CPS49 plus flavopiridol (a fixed ratio of 1:1) for an additional 16 h, and cell viability was assessed at the termination of incubation. The viability of the Jurkat cells after 16 h exposure to flavopiridol at 0.2 μM was around 90%. The data presented are the means of three independent determinations after normalization to those of the untreated cells. CI values (right panel) for each fractional effect were calculated using the software, and a CI value < 1.0 indicates synergism, CI = 1.0 indicates an additive effect, and CI > 1.0 indicates antagonism. (D) FACS measurement of acute increase in annexin positive staining of Jurkat cells 3 hours following addition of increasing doses (0−10 μM) of flavopiridol and CPS49 either alone or in combination as indicated.
Fig. 2
Fig. 2
CPS49 and flavopiridol demonstrate selective disruption of homeostasis of intracellular glutathione (GSH) and reactive oxygen species (ROS) in transformed leukemia cells. (A) The profile of ROS generation in Jurkat cells after 1 h drug treatment and the profile of cellular GSH depletion in Jurkat cells after 16 h exposure to CPS49 alone (top panel), Flavo alone (flavopiridol; middle panel), or CPS49 plus flavopiridol (bottom panel) at the indicated concentrations. (B) The profile of ROS generation in PBMCs, K562, HeLa, and 8226 cells after 1 h drug treatment and the profile of intracellular GSH depletion in these cells after 16 h drug exposure to CPS49 alone (top panels), Flavo alone (flavopiridol; middle panels), or CPS49 plus flavopiridol (bottom panels) at the concentrations indicated. GSH and ROS were measured as described in the Materials and Methods. The data shown are the means of three independent experiments after normalized to those of the untreated cells.
Fig. 3
Fig. 3
CPS49 and flavopiridol combinations synergistically reduce the mitochondrial membrane potential and NF-κB activation but differentially influence E2F-1 and caspase activation in transformed leukemic cells. (A) Flow cytometric measurement of CPS49-and/or flavopiridol-mediated loss of mitochondrial membrane potential in Jurkat cells following exposure to CPS49, flavopiridol, and CPS49 plus flavopiridol, respectively, for 1 h (left panel) or 3 h (right panel), as outlined above. (B) Jurkat cells were preincubated for 15 min in the presence or absence of 10 μM CPS49 and/or flavopiridol prior to stimulation with PHA (1 μg/ml) and PMA (50 ng/ml) for an additional 15 min. Whole cellular lysates were then prepared for immunoblot analysis of phospho-IkB-α (Ser32) and IkB-α (total) levels in the whole cell lysates from the Jurkat cells as described in the Materials and Methods. (C) Immunoblot analysis of phospho-relA (Ser536) and relA (p65) levels in the whole cell lysates from the Jurkat cells treated with the drugs as outlined above. (D) immunoblot analysis of E2F-1 and β−actin (ActB) protein levels. (E) Immunoblot analysis of caspase 3 in whole cell lysates from Jurkat cells pretreated 8 hr with 0.2 μM flavopiridol followed by the addition of 10 μM CPS49 and Flavo alone or in combination for an additional 8 hours.
Fig. 4
Fig. 4
CPS49 and flavopiridol preferentially up-regulate the expression of pro-apoptotic genes, while simultaneously down-regulate the expression of anti-apoptotic genes. (A) Jurkat cells were stimulated with 1 μg/ml PHA and 50 ng/ml PMA in the presence or absence of 10 μM CPS49 and/or flavopiridol for 3 h. Total cellular RNA was then isolated from the cell lysates and cDNA was synthesized. mRNA levels of BAX, BAD, p73, and PUMA gene expression were analyzed by qRT-PCR, as described in the Materials and Methods. (B) mRNA levels of MCL1, XIAP, BCL-2, BCL-XL, Survivin, and MDM2 gene expression were assessed by qRT-PCR analysis in the Jurkat cells treated with the drugs, as outlined above. The data presented are the means of three separate determinations after normalized to those of the house-keeping gene β-actin. * P < 0.05 and ** P < 0.01 vs. the untreated controls. (C) The reversal of CPS49- and/or Flavo (flavopiridol)-induced cell killing by 10 mM of the antioxidant N-acetylcysteine (NAC) in Jurkat cells, as determined by MTT assay. The data shown are the means of three separate experiments after normalized to those of the untreated cells. (D) Jurkat cells were stimulated for 3 h with PHA (1 μg/ml) and PMA (50 ng/ml) in the presence or absence of 10 μM CPS49 and/or flavopiridol, as well as 15 mM of the antioxidant N-acetyl-cysteine (NAC). Total cellular RNA was then isolated from the cell lysates and cDNA was synthesized. The reversal of CPS49- and flavopiridol-mediated mRNA expression of p73 (left panel) and PUMA (right panel) genes by NAC was assessed by qRT-PCR analysis, as described in the Materials and Methods. * P < 0.05 and ** P < 0.01 for the cells with NAC treatment vs. the cells without NAC treatment.
Fig. 5
Fig. 5
The combinatorial synergy by CPS49 and flavopiridol following low dose flavopiridol pretreatment is highly cell type specific . (A) HeLa, LNCaP, OPM2, 8226, and HH cell lines were incubated with increasing concentrations (0−10μM) of CPS49 alone, Flavo (flavopiridol) alone, or CPS49 plus flavopiridol at a fixed ratio of 1:1. Cell viability was determined after 16 h incubation time with the drugs. (B) CI values were determined for HH, OPM2 and 8226 and plotted against fractional effect to construct isobolograms. CI value < 1.0 indicates synergism, CI = 1.0 indicates an additive effect, and CI > 1.0 indicates antagonism. No isobologram for HeLa or LNCaP could be derived.
Fig 6
Fig 6
Flavopiridol down-regulates genes important for mitochondrial stability and survival but CPS49 shows lower efficacy in 8226 myeloma cells. (A) 8226 myeloma cells were stimulated with 1 μg/ml PHA and 50 ng/ml PMA in the presence or absence of 10 μM CPS49 and/or flavopiridol for 3 h. Total cellular RNA was then isolated from the cell lysates and cDNA was synthesized. mRNA levels of BAX, BAD, p73, and PUMA gene expression were analyzed by qRT-PCR. (B) mRNA levels of MCL1, XIAP, BCL-2, BCL-XL, Survivin, and MDM2 gene expression were assessed by qRT-PCR analysis in the 8226 cells treated with the drugs, as outlined above. The data presented are the means of three separate determinations after normalized to those of the house-keeping gene β-actin. * P < 0.05 and ** P < 0.01 vs. the untreated controls. (C) Jurkat cells (107) were treated with and without 8 h pretreatment with 0.2 μM flavopiridol. Whole cell lysates were prepared and analyzed by immunoblot for total and serine 2 C-terminal domain phosphorylated pol II and for total and serine 807/811 phosphorylated Rb. Shown at the right is a densitometer scan showing relative amount of serine 2 phosphorylated pol II compared to serine 808/811 phosphorylated RB normalized to the total levels of each respectively. (D) This schematic diagram shows that CPS49 and flavopiridol increase p73 directly or indirectly through sustaining the transcription factor E2F-1 and down-regulating MDM2. p73 mediates mitochondrial dysfunction via either the transcription of the pro-apoptotic genes PUMA, BAD, and BAX or mitochondrial localization of these proteins. On the other hand, CPS49 and flavopiridol induce cell death by inhibiting NF-κB and down-regulating NF-κB targeting anti-apoptotic genes, such as Survivin, XIAP, Bcl-2, Bcl-XL, and MCL1. Since both CPS49 and flavopiridol cause mitochondrial damage through closely related but distinct mechanisms, they may act collaboratively or synergistically to promote apoptotic death of leukemia cells through p73 and NF-κB and their downstream molecular events. Such a mechanism may serve to integrate the roles of p73 and NF-κB in culmination in mitochondrial damage and cell death, which may underlie the mechanism of CPS49 and flavopiridol mediated mitochondrial dysfunction and cell killing in transformed leukemic cells.
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