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. 2014 Oct 2;9(2):409–421. doi: 10.1016/j.molonc.2014.09.008

Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage

Chengzhi Xie 1,2,3,†, Holly Edwards 1,2,†, J Timothy Caldwell 4,5, Guan Wang 3,7, Jeffrey W Taub 2,6,7, Yubin Ge 1,2,3,✉
PMCID: PMC4305021  NIHMSID: NIHMS632275  PMID: 25308513

Abstract

Resistance to cytarabine and anthracycline‐based chemotherapy is a major cause of treatment failure for acute myeloid leukemia (AML) patients. Overexpression of Bcl‐2, Bcl‐xL, and/or Mcl‐1 has been associated with chemoresistance in AML cell lines and with poor clinical outcome of AML patients. Thus, inhibitors of anti‐apoptotic Bcl‐2 family proteins could be novel therapeutic agents. In this study, we investigated how clinically achievable concentrations of obatoclax, a pan‐Bcl‐2 inhibitor, potentiate the antileukemic activity of cytarabine in AML cells. MTT assays in AML cell lines and diagnostic blasts, as well as flow cytometry analyses in AML cell lines revealed synergistic antileukemic activity between cytarabine and obatoclax. Bax activation was detected in the combined, but not the individual, drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential. Most importantly, in AML cells treated with the combination, enhanced early induction of DNA double‐strand breaks (DSBs) preceded a decrease of Mcl‐1 levels, nuclear translocation of Bcl‐2, Bcl‐xL, and Mcl‐1, and apoptosis. These results indicate that obatoclax enhances cytarabine‐induced apoptosis by enhancing DNA DSBs. This novel mechanism provides compelling evidence for the clinical use of BH3 mimetics in combination with DNA‐damaging agents in AML and possibly a broader range of malignancies.

Keywords: Acute myeloid leukemia, Obatoclax, Cytarabine, DNA damage, Drug combination

Highlights

  • Obatoclax enhances cytarabine‐induced DNA damage prior to induction of apoptosis.

  • Combined obatoclax and cytarabine treatment decreases Mcl‐1 levels.

  • Cytarabine plus obatoclax induces nuclear localization of Bcl‐2, Bcl‐xL & Mcl‐1.

  • Bcl‐2, Bcl‐xL, and Mcl‐1 modulate cytarabine‐induced DNA damage.

1. Introduction

Despite advances in treatment, acute myeloid leukemia (AML) remains a difficult disease to treat. Overall survival in children and adults remain guarded at 64–71% and 25.5%, respectively (seer.cancer.gov) (Rubnitz et al., 2010; Siegel et al., 2013). One major cause of treatment failure in this disease is resistance to cytarabine (ara‐C) and anthracycline [e.g. daunorubicin (DNR)]‐based chemotherapy (Kaspers and Zwaan, 2007). Clearly, AML remains a challenging disease to treat and urgently requires more effective therapies.

Evasion of cell death is a hallmark of human cancer and a major cause of treatment failure (Fulda et al., 2010; Hanahan and Weinberg, 2000; Reed, 2003; Vogler et al., 2009). Defects in the apoptosis pathways are a major mechanism of evasion of cell death and an essential element of tumor pathogenesis (Reed, 2003). One common cancer‐causing defect arises from the overexpression of the anti‐apoptotic Bcl‐2 family members, including Bcl‐2, Bcl‐xL, and Mcl‐1, which inactivate the apoptotic pathway in many types of cancer cells (Hwang et al., 2010). Overexpression of Bcl‐2, Bcl‐xL, and/or Mcl‐1 has been associated with chemoresistance in leukemia cell lines and with a poor clinical outcome for adult patients with leukemia, including AML (Fennell et al., 2001; Keith et al., 1995; Lauria et al., 1997; Saxena et al., 2004; Schimmer et al., 2008). Therefore, small molecules that inhibit the anti‐apoptotic Bcl‐2 family proteins could be useful for the treatment of leukemia.

Obatoclax (GX15‐070) is a hydrophobic small molecule that binds to the BH3‐binding site of Bcl‐2, Bcl‐xL, and Mcl‐1 (Fulda et al., 2010; Goard and Schimmer, 2013; Leber et al., 2010). This pan‐Bcl‐2 inhibitor has been reported to directly induce apoptosis in cultured AML cell lines and primary patient samples by releasing Bak from Mcl‐1, liberating Bim from Bcl‐2 and Mcl‐1, and forming an active Bak/Bax complex (Konopleva et al., 2008; Rahmani et al., 2012). However, our understanding of the biological mechanisms underlying its spectrum of anticancer activities at clinically achievable concentrations is still evolving. Further studies are needed to comprehensively characterize the anticancer mechanisms of clinically achievable doses of obatoclax (Goard and Schimmer, 2013).

Emerging evidence suggests that Bcl‐2 can regulate DNA double‐strand break (DSB) repair, independent of its pro‐survival functions (Kumar et al., 2010; Laulier et al., 2011; Saintigny et al., 2001; Wang et al., 2008). Further, recent studies suggest that Bcl‐xL and Mcl‐1 translocate to the nucleus to facilitate G2/M cell cycle checkpoint activation in response to DNA damage induced by chemotherapeutic drugs or radiation (Jamil et al., 2008, 2010, 2010, 2007). Based on these findings, we hypothesized that obatoclax enhances cytarabine‐induced DNA damage through targeting the non‐apoptotic functions of the anti‐apoptotic Bcl‐2 family proteins.

In this study, we combined clinically relevant concentrations of obatoclax (O'Brien et al., 2009) with cytarabine in AML cell lines and diagnostic AML blast samples. Our results demonstrate that at clinically achievable concentrations obatoclax potentiates cytarabine‐induced DNA DSBs which are followed by a decrease of Mcl‐1 levels, nuclear accumulation of Bcl‐2, Bcl‐xL, and Mcl‐1, and apoptosis. Our new findings strongly support the clinical use of BH3 mimetics in combination with DNA‐damaging agents for treating AML and possibly a broader range of malignancies.

2. Materials and methods

2.1. Clinical samples

Diagnostic bone marrow samples (n = 6) from children with de novo AML were obtained from the Children's Hospital of Michigan leukemia cell bank. Mononuclear cells were purified by standard Ficoll‐Hypaque density centrifugation. Written consent was provided by the parent or legal guardian according to the Declaration of Helsinki. The study was approved by the Human Investigation Committee of the Wayne State University School of Medicine.

2.2. Drugs

Cytarabine and daunorubicin were purchased from Sigma–Aldrich (St Louis, MO, USA). Obatoclax, TW‐37, ABT‐737, and ABT‐199 were purchased from Selleck Chemicals (Houston, TX, USA).

2.3. Cell Culture

The THP‐1, MV4‐11, and U937 AML cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The OCI‐AML3 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The CMS AML cell line was a gift from Dr. A Fuse from the National Institute of Infectious Diseases, Tokyo, Japan. The cell lines were cultured as previously described (Niu et al., 2014; Xie et al., 2013). Diagnostic AML blasts were cultured in RPMI 1640 with 20% fetal bovine serum supplemented with ITS solution (Sigma–Aldrich) and 20% supernatant of the 5637 bladder cancer cell line (as a source of granulocyte‐macrophage colony‐stimulating factor (Taub et al., 1996). All cells were cultured in a 37 °C humidified atmosphere containing 5% CO2/95% air.

2.4. Western blot analysis

Proteins were subjected to SDS‐polyacrylamide gel electrophoresis, and then electrophoretically transferred to polyvinylidene difluoride membranes (Thermo Fisher Inc., Rockford, IL, USA) and immunoblotted with anti‐Bcl‐2 (2876), ‐Bcl‐xL (2764), ‐Mcl‐1 (4572), ‐cleaved‐caspase 3 (9661), ‐caspase 9 (9505), ‐PARP‐1 (9542), ‐γH2AX (2577), ‐MEK1/2 (9122), ‐Bax (2774, Cell Signaling Technology, Danvers, MA, USA), ‐Histone H4 (07‐108, Upstate Biotechnology, Lake Placid, NY, USA) or ‐β‐actin antibody (A2228, Sigma–Aldrich) as described previously (Ge et al., 2006, 2005). Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li‐Cor, Lincoln, NE, USA), as described by the manufacturer. Densitometry measurements were made using Odyssey V3.0 (Li‐Cor), normalized to β‐actin, and graphed as the fold change compared to the no drug treatment control.

2.5. In vitro cytotoxicity assays

In vitro cytotoxicites were measured using MTT (3‐[4,5‐dimethyl‐thiazol‐2‐yl]‐2,5‐diphenyltetrazoliumbromide, Sigma–Aldrich) assays, as previously described (Ge et al., 2006; Xie et al., 2010; Xu et al., 2011). The extent and direction of antileukemic interaction was determined by calculating the combination index (CI) values using CompuSyn software (Combosyn Inc., Paramus, NJ, USA). CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively (Chou, 2006; Tallarida, 2001; Xie et al., 2010).

2.6. Assessment of apoptosis

AML cells were treated with the indicated drug concentrations for 4 or 48 h, washed with cold PBS, resuspended in annexin V Binding Buffer (BD Pharmingen, San Diego, CA, USA), incubated with annexin V‐APC/7‐Amino‐Actinomycin D (7‐AAD, BD Pharmingen) for 15 min at room temperature, and then analyzed on a FACScanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Apoptotic events are expressed as the percent of annexin V+ cells. THP‐1 cells treated with DNR or TW‐37 were assayed with annexin V‐FITC and propidium iodide (PI) as previously described (Caldwell et al., 2013; Xie et al., 2010). CI values were calculated using CompuSyn.

2.7. Assessment of mitochondrial membrane potential (MMP)

Following the indicated drug treatment, 5 × 105 cells were resuspended in fresh growth media containing 1 μM JC‐1 (Sigma–Aldrich) and incubated for 15 min at 37 °C. The samples were washed, resuspended in PBS, and then pipetted into a black‐walled 96‐well plate. JC‐1 monomer fluorescence was measured on a microplate reader with excitation 485 nm and emission 535 nm. JC‐1 aggregates were not measured due to the high degree of spectral overlap with GX15‐070 fluorescence.

2.8. Immunoprecipitation (IP) of Bax

AML cells were treated with cytarabine and/or obatoclax for 6 h or 48 h. Immunoprecipitation of active Bax was performed as previously described using 2 μg of anti‐Bax monoclonal antibody clone 6A7 (556467, BD Pharmingen), and Protein G Dynabeads (Life Technologies, Carlsbad, CA, USA) (Xie et al., 2012). Eluted proteins were analyzed by Western blotting.

2.9. Subcellular fractionation

Nuclear and cytoplasmic fractions were isolated following the REAP protocol (Suzuki et al., 2010). Briefly, cells were lysed in ice‐cold PBS with 0.1% NP40 and centrifuged. The supernatant was kept as the “cytoplasmic” fraction. The nuclear pellet was washed in ice‐cold PBS with 0.1% NP40, centrifuged, resuspended in 200 μl loading buffer and sonicated. 10 μl of cytoplasmic fraction diluted with 10 mM Tris (pH 6.8) or 30 μl of nuclear fraction was subjected to Western blotting.

2.10. Immunofluorescence analysis

Following drug treatment, AML cells were fixed in ice cold 3.3% paraformaldehyde for 30 min, washed with cold PBS, resuspended in deionized water and 1 × 105 cells were applied to gelatin‐coated adhesive microscope slides (LabScientific, Livingston, NJ, USA). The slides were dried at 50 °C for 5 min. A hydrophobic barrier was drawn around the cell smear using an ImmEdge Hydrophobic Barrier Pen (Vector Laboratories, Burlingame, CA, USA). The slides were washed with PBS, incubated with PBS containing 0.1% Triton X‐100 for 5 min at room temperature, and then blocked with 1% BSA in PBS for 1 h. 1:25 anti‐Bcl‐2 (sc‐7382, Santa Cruz Biotechnology, Santa Cruz, CA), 1:50 anti‐Mcl‐1 (ab114026, AbCam, Cambridge, MA, USA), or 1:25 Bcl‐xL (sc‐8392, Santa Cruz), and 1:50 anti‐γH2AX (Ser139, 9718, Cell Signaling Technology) were diluted in 1% BSA in PBS and incubated on the slide overnight at 4 °C. Alexa Fluor 488 anti‐mouse secondary and Alexa Fluor 555 anti‐rabbit secondary antibodies were diluted 1:400 (Cell Signaling Technology) in 1% BSA in PBS and applied to the slides. Slides were incubated for 1 h at room temperature in the dark and then extensively washed with 1% BSA in PBS. Nuclei were counterstained with 3 μM 4′,6‐diamidino‐2‐phenylindole (DAPI)‐dihydrochloride (Sigma–Aldrich) for 5 min. The slides were visualized using a Leica TCS SP5 laser confocal fluorescent microscope (The Microscopy, Imaging and Cytometry Resources Core at Wayne State University School of Medicine) using a 63×/1.25 oil objective lens and analyzed using the Leica Application Suite Advanced Fluorescence Lite Software (1.8.2 build 1465, Leica Microsystems Inc., Buffalo Grove, IL, USA).

2.11. Alkaline comet assay

OCI‐AML3 cells were treated for 24 h with cytarabine and/or obatoclax and subjected to alkaline comet assay as previously described (Xie et al., 2013). Slides were stained with SYBR Gold (Life Technologies), and then imaged on an Olympus BX‐40 microscope equipped with a DP72 microscope camera and Olympus cellSens Dimension software (Olympus America Inc., Center Valley, PA). 50 comets per gel were scored using CometScore (TriTek Corp, Sumerduck, VA, USA). The median percent DNA in the tail from three independent experiments was calculated and graphed.

2.12. Overexpression of Bcl‐xL or Mcl‐1

The transfection was carried out by using Lipofectamine and Plus reagents (Life Technologies) according to the manufacturer's instructions and as previously described (Edwards et al., 2009, 2013, 2010). Briefly, Precision LentiORF Bcl‐xL, Mcl‐1 or RFP (red fluorescent protein) lentivirus vector (Thermo Fisher Scientific Biosciences, Lafayette, CO, USA), pMD‐VSV‐G and delta 8.2 (gifts from Dr. Dong at Tulane University) were cotransfected into TLA‐HEK293T cells and the culture medium was harvested 48 h posttransfection. THP‐1 cells were transduced by adding virus containing supernatant and 4 μg/mL of polybrene and incubated overnight. The cells were washed three times and allowed to proliferate for 48 h before Blasticidin selection.

2.13. Statistical analysis

Differences were compared using the pair‐wise two‐sample t‐test. Statistical analyses were performed with GraphPad Prism 5.0. Error bars represent the standard errors of the means.

3. Results

3.1. Obatoclax enhances cytarabine sensitivity in AML cells

To begin, we determined the expression levels of Bcl‐2, Bcl‐xL, and Mcl‐1 in five AML cell lines and six diagnostic blast samples by Western blotting. The majority of samples expressed all three proteins, though at variable levels (Figure 1A). Then we assessed cytarabine and obatoclax sensitivities using MTT assays (Figure 1B and C). Obatoclax and cytarabine IC50s varied, ranging from 74.2 nM (CMS) to 2.19 μM (A30314) and 220.1 nM (MV4‐11) to 6387.7 nM (A30314) respectively (Table S1). Cytarabine IC50s were reduced by 3.4‐ (A30316) to 26.6‐fold (U937) when combined with the highest clinically achievable obatoclax concentration (175 nM, Table S1) (O'Brien et al., 2009). The combined effects were additive to synergistic, as determined by standard isobologram analysis (Figure 1D and E) and by calculating CI values, for all three clinically achievable obatoclax concentrations tested (Table S1). Our results demonstrate that at clinically achievable concentrations obatoclax enhances the antileukemic effect of cytarabine in AML cells.

Figure 1.

Figure 1

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Bcl‐2 family protein expression and cytarabine and obatoclax drug sensitivities in AML cell lines and diagnostic blasts. Whole cell lysates from AML cell lines and diagnostic AML blast samples were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1 or ‐β‐actin antibody (Panel A). AML cell lines and diagnostic AML blast samples were cultured in 96‐well plates at 37 °C for 72 h, in complete medium with variable concentrations of cytarabine or obatoclax, and viable cells were determined using MTT reagent. The cytarabine and obatoclax IC50 values were calculated as the concentrations of drug necessary to inhibit 50% proliferation compared to control cells cultured in the absence of drug (Panels B&C). THP‐1 cells or patient A30314 diagnostic AML blasts were treated with cytarabine (Cyta) in the presence or absence of obatoclax (Obat) and viable cells were determined using MTT reagent. Standard isobologram analysis was performed to determine the extent and direction of the antileukemic interaction between cytarabine and obatoclax. The IC50 values of each drug are plotted on the axes; the solid line represents the additive effect, while the points represent the concentrations of each drug resulting in 50% inhibition of proliferation. Points falling below the line indicate synergism whereas those above the line indicate antagonism (Panels D&E). The data for the AML cell lines are presented as mean ± standard error from at least 3 independent experiments. The data for the diagnostic AML blast samples are means of duplicates from one experiment. *Sample not used for this study due to extensive protein degradation.

3.2. Obatoclax enhances cytarabine‐induced apoptosis in AML cell lines

To determine if obatoclax enhanced cytarabine‐induced apoptosis, THP‐1 and OCI‐AML3 cell lines (two relatively cytarabine‐resistant lines) were treated with drug for 48 h. Individual drug treatments had minimal effect on apoptosis, as measured by annexin V/7‐AAD staining and flow cytometry analysis. However, combined treatment caused significant induction of apoptosis (Figure 2A and C), which was accompanied by cleavage of caspase‐3, caspase‐9 and PARP (Figure 2B and D). CI values ranged from 0.23 to 0.55 for the combined drug treatments in both cell lines (Figure 2E and F). Then we measured MMP (mitochondrial membrane potential) using the JC‐1 reagent. We found that obatoclax significantly enhanced the induction of JC‐1 monomers by cytarabine, indicating a loss of MMP after the combined treatment (Figure S1A&B). Together, these results demonstrate that at clinically achievable concentrations, obatoclax synergistically enhances cytarabine‐induced apoptosis in AML cells.

Figure 2.

Figure 2

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Synergistic induction of apoptosis by cytarabine and obatoclax in THP‐1 and OCI‐AML3 cells. THP‐1 and OCI‐AML3 cells were treated with cytarabine and/or obatoclax for 48 h. Apoptotic events in the cells post drug treatment were determined by annexin V/7‐AAD staining and flow cytometry analyses (Panels A&C). Whole cell lysates were subjected to Western blotting, and probed with anti‐cleaved caspase‐3, ‐cleaved caspase‐9, ‐PARP, or ‐β‐actin antibody (CF indicates cleaved form, Panels B&D). CI vs. Fa plots (combination index vs. fraction affected) for the apoptosis data are presented in panels E&F. THP‐1 and OCI‐AML3 cells were treated with cytarabine and obatoclax, alone or in combination, for 48 h. 1 mg of protein lysate was used for immunoprecipitation with anti‐Bax 6A7 (activated BAX) antibody. Immunoprecipitated proteins were subjected to Western blotting and probed with anti‐Bax antibody (Panel G). The data are presented as mean ± standard error from one representative experiment which was repeated at least 3 independent times. ** indicates p < 0.005.

In view of the central role Bax activation plays in the intrinsic apoptosis pathway, we examined Bax activation after drug treatment. THP‐1 and OCI‐AML3 cells were treated with obatoclax and/or cytarabine for 48 h and the active form of Bax was immunoprecipitated with the Bax 6A7 antibody from whole cell lysates. Bax activation was only detected in the combined drug treatments and could not be detected in the individual treatments (Figure 2G). This is in contrast to previous reports which showed that obatoclax treatment caused Bax/Bak activation (Konopleva et al., 2008; Rahmani et al., 2012), though the concentrations used were much higher than were clinically achievable. Further testing with 5 μM and 10 μM obatoclax caused activation of Bax (Figure S1C), confirming results from previous studies (Konopleva et al., 2008). Although a low level of apoptosis was detected for cytarabine treatment alone, we did not detect Bax activation, potentially due to the detection limits of the assay.

3.3. Co‐treatment with obatoclax and cytarabine induces a decrease of Mcl‐1 levels and nuclear accumulation of Bcl‐2, Bcl‐xL, and Mcl‐1 in AML cells

Bax activation and loss of MMP in AML cells treated with the drug combination may be attributed to decreased expression levels and/or altered subcellular localization of Bcl‐2, Bcl‐xL, and/or Mcl‐1. To test these possibilities, we examined the expression levels of these proteins after drug treatment. In OCI‐AML3 cells the combined treatment caused a significant decrease in Mcl‐1 at 48 h, while Bcl‐2 and Bcl‐xL levels remained unchanged (Figure 3A). Prior to drug treatment, only a small fraction of Bcl‐2, Bcl‐xL, and Mcl‐1 could be detected in the nuclear fraction, as assessed by western blot (Figure 3B). Co‐treatment with the two agents for 48 h resulted in nuclear accumulation of all three proteins. In contrast, neither Bcl‐2 nor Bcl‐xL nuclear accumulation could be detected after obatoclax treatment and minimal Bcl‐xL nuclear accumulation could be detected in cells treated with cytarabine alone. Nuclear localization of Bcl‐2, Bcl‐xL or Mcl‐1 was confirmed by immunofluorescence staining at 48 h following drug treatments (Figure 3C). Beginning at 24 h, increased Bcl‐2 and Bcl‐xL levels were detected in the nuclear fraction of cells treated with the drug combination, while nuclear levels of Mcl‐1 increased only at 48 h (Figure 3D). Increased nuclear levels of Bcl‐2 or Bcl‐xL could only be detected at 48 h in cells treated with cytarabine alone (Figure 3D). Similar results were obtained in THP‐1 cells (Figure S2). Although we could detect more Bcl‐2 and Bcl‐xL in the nuclear fraction, we could not detect concurrent decrease of the protein levels in the cytoplasmic fractions. However, as shown in the immunofluorescence experiments, cells with more nuclear localized Bcl‐2 and Bcl‐xL had less localized in the cytoplasm. These results suggest that obatoclax cooperates with cytarabine to induce a decrease of Mcl‐1 levels and an increase of nuclear accumulation of Bcl‐2, Bcl‐xL, and Mcl‐1, potentially leading to apoptosis.

Figure 3.

Figure 3

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The effects of cytarabine and obatoclax treatments on Bcl‐2, Bcl‐xL, and Mcl‐1 expression and subcellular localization in OCI‐AML3 cells. OCI‐AML3 cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting, and then probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, or –β‐actin antibody (Panel A, upper panel). Densitometry for Mcl‐1 expression was measured and graphed as fold change compared to the no drug control (Panel A, lower panel). Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. The membranes were probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, or ‐Histone H4 antibody (Panel B). OCI‐AML3 cells were treated with vehicle control or 2 μM cytarabine plus 175 nM obatoclax for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panel C). OCI‐AML3 cells were treated with 2 μM cytarabine with or without 175 nM obatoclax for up to 48 h. Nuclear proteins were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1 or ‐Histone H4 antibody (Panel D). The data are presented as mean ± standard error from at least 3 independent experiments. * indicates p < 0.05.

To address the functional role decreased Mcl‐1 levels have on the combined cytarabine and obatoclax drug treatment, we ectopically overexpressed Mcl‐1 in the THP‐1 cells (THP‐1/Mcl‐1). The red fluorescent protein (THP‐1/RFP) control cells were significantly more resistant to cytarabine treatment than the parental cells, as measured by MTT assays (data not shown), thus a higher dose was used to see a similar effect to the treatment in Figure 2A. Consistent with the results in the parental THP‐1 cells, co‐treatment of the THP‐1/RFP cells resulted in a decrease of Mcl‐1 (Figure 4A). Although co‐treatment of the THP‐1/Mcl‐1 cells also resulted in a decrease of Mcl‐1, its level remained substantially higher than that in the co‐treated THP‐1/RFP cells (Figure 4A). This was accompanied by partially attenuated drug‐induced apoptosis and cleavage of PARP and caspase‐3 compared to the THP‐1/RFP cells (Figure 4B). These results provide evidence that the decrease in Mcl‐1 plays a functional role in apoptosis induced by the combined drug treatment.

Figure 4.

Figure 4

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Ectopic overexpression of Mcl‐1 or Bcl‐xL in THP‐1 cells blocks apoptosis induced by cytarabine or cytarabine plus obatoclax. THP‐1 cells were infected with Precision LentiORF Mcl‐1, Bcl‐xL, (THP‐1/Mcl‐1 or THP‐1/Bcl‐xL respectively) or red fluorescent protein control (THP‐1/RFP) lentivirus overnight, washed and then incubated for 48 h before selection drug (Blasticidin) was added to the culture medium. The cells were treated with cytarabine and/or obatoclax for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti‐Bcl‐xL, ‐Mcl‐1, ‐PARP, ‐cleaved caspase‐3, or ‐β‐actin antibody (Panels A&E). Apoptotic events in the cells post drug treatment were determined by annexin V/7‐AAD staining and flow cytometry analyses (Panels B&F). THP‐1/RFP and THP‐1/Bcl‐xL cells were treated with cytarabine and/or obatoclax for 48 h. Nuclear and cytoplasmic fractions were extracted and subjected to Western blotting. Due to the extent of overexpression of Bcl‐xL 1/10th of the total protein loaded for the THP‐1/RFP was loaded for the THP‐1/Bcl‐xL samples in order to visualize nuclear Bcl‐xL level changes on the same blot. The membranes were probed with anti‐Bcl‐xL, ‐MEK1/2, or ‐Histone H4 antibody (Panels C&D). Experiments were repeated at least 3 independent times with one representative shown. The data are presented as mean ± standard error. *** indicates p < 0.0005.

To address the functional role of the nuclear translocation of Bcl‐xL/Bcl‐2, we overexpressed Bcl‐xL in THP‐1 cells (designated THP‐1/Bcl‐xL). Higher levels of Bcl‐xL were detected in the cytoplasmic and nuclear fractions prior to and post‐drug treatment in the THP‐1/Bcl‐xL cells compared to the THP‐1/RFP control cells (Figure 4C and D). Similar to the parental cell line, there was a higher level of Bcl‐xL in the nuclear fraction after combined drug treatment compared to the individual drug treatments for both THP‐1/RFP and THP‐1/Bcl‐xL. Overexpression of Bcl‐xL attenuated drug‐induced apoptosis and the accompanying cleavage of PARP and caspase‐3 (Figure 4E and F). These results demonstrate that the nuclear translocation of Bcl‐xL after the combined drug treatment still occurs in the overexpression model, though an overabundance in the cytoplasm is possibly maintaining the balance of pro‐ and anti‐apoptotic proteins towards survival.

3.4. Obatoclax enhances cytarabine‐induced DNA DSBs

Emerging evidence indicates that the anti‐apoptotic Bcl‐2 family proteins are involved in the DNA damage response (Jamil et al., 2008, 2010, 2010, 2011, 2010, 2001, 2007, 2008). It is conceivable that obatoclax may enhance cytarabine‐induced apoptosis by enhancing cytarabine‐induced DNA damage. To test this possibility, OCI‐AML3 cells were treated with obatoclax and/or cytarabine for up to 48 h. The combined drug treatment showed a significant increase of phosphorylated H2AX (γH2AX), indicating increased DNA DSBs, compared to the individual drug treatments, as early as 4 h (Figure 5A and B). In contrast, significant increase of apoptosis was not detected when measured by annexin V/7‐AAD staining (Figure 5C), suggesting that DNA damage occurs prior to induction of apoptosis. Similar results were observed in THP‐1 cells (Figure S3A–D). To further confirm that the combined drug treatment caused an increase in DNA damage, OCI‐AML3 cells were treated for 24 h and subjected to the alkaline comet assay. Obatoclax treatment alone did not show a significant increase in DNA strand breaks, as measured by percent DNA in the tail. Cytarabine treatment, as expected, had a small but significant increase in DNA strand breaks compared to the vehicle control treated cells and the combined treatment had a significant increase in DNA strand breaks compared to cytarabine, or obatoclax treatment (Figure 5D).

Figure 5.

Figure 5

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Obatoclax cooperates with cytarabine to induce DNA DSBs in OCI‐AML3 cells. OCI‐AML3 cells were treated with cytarabine and obatoclax, alone or in combination for up to 48 h. Whole cell lysates were extracted and subjected to Western blotting, and probed with anti‐γH2AX or ‐β‐actin antibody (Panel A). Densitometry for γH2AX expression was measured at the 4 h time point and graphed as fold change compared to the no drug control. Data are presented as mean value ± standard error from 4 independent experiments (Panel B). OCI‐AML3 cells treated with cytarabine and/or obatoclax for 4 h or with 20 μM cytarabine for 48 h (positive control) were subjected to annexin V/7‐AAD staining and flow cytometry analyses to determine apoptotic events (Panel C). OCI‐AML3 cells were treated with cytarabine and/or obatoclax for 24 h, and then subjected to alkaline comet assay analyses (Panel D). OCI‐AML3 cells were treated with vehicle control or cytarabine plus obatoclax for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green), or anti‐γH2AX (red) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue). For each treatment group, the squared region in the low magnification image on the left is shown to the right at higher magnification (Panel E). OCI‐AML3 cells were treated with cytarabine plus obatoclax for up to 48 h, whole cell lysates were extracted, subjected to western blotting and probed with anti‐Mcl‐1, ‐γH2AX, ‐cleaved caspase 3, ‐PARP or ‐β‐actin antibody (Panel F). The data are presented as mean ± standard error from one representative experiment, which was repeated at least 3 independent times. * indicates p < 0.05 and ** indicates p < 0.005.

Next, we examined intracellular localization of Bcl‐2, Bcl‐xL, and Mcl‐1 and induction of γH2AX by immunofluorescence in OCI‐AML3 cells treated with the combination of cytarabine and obatoclax for 48 h. Cells treated with vehicle control showed diffuse staining for Bcl‐2, Bcl‐xL and Mcl‐1 and no detectable staining for γH2AX (Figure 5E). Co‐treated cells showed an increase in staining for γH2AX (almost 90% of the cells having foci), which occurred at the same time as nuclear accumulation of Bcl‐2, Bcl‐xL and Mcl‐1. Combined cytarabine and obatoclax time‐course experiments revealed a time‐dependent increase in γH2AX which showed a positive correlation with caspase‐3 and PARP cleavage and an inverse correlation with Mcl‐1 levels (Figure 5F). Mcl‐1 levels initially increased, and then steadily decreased over time. Similar results were also obtained in THP‐1 cells (Figure S3E&F).

To rule out the possibility of off‐target effects for obatoclax, we treated THP‐1 cells with a structurally different pan‐Bcl‐2 inhibitor, TW‐37 (Mohammad et al., 2007). Similar to obatoclax, TW‐37 significantly enhanced cytarabine‐induced apoptosis (Figure S4A). Increased γH2AX, cleaved PARP, and nuclear accumulation of Bcl‐2, Bcl‐xL and Mcl‐1 were all detected after 48 h combined drug treatment (Figure S4B&C). In addition, we also tested ABT‐737, which has been shown to be a true BH3 mimetic (Vogler et al., 2009), in MV4‐11 cells, which are relatively sensitive to ABT‐737 (Figure S4D). The combined ABT‐737 and cytarabine treatment caused a significant increase in apoptotic cells, as well as increased γH2AX and cleaved PARP, which were similar to combined GX15‐070 and cytarabine treatment (Figure S4E&F). These results provide further evidence that the enhancement of cytarabine‐induced DNA DSBs by obatoclax is unlikely from off‐target effects.

To determine if a decrease of Mcl‐1 levels and nuclear accumulation of Bcl‐2, Bcl‐xL and Mcl‐1 represents a generalized mechanism underlying apoptosis induced by DNA‐damaging agents, we treated THP‐1 cells with another DNA‐damaging agent, daunorubicin (DNR), for 48 h. There was a dose‐dependent increase in apoptosis accompanied by increased γH2AX and decreased Mcl‐1 expression (Figure S5A&B). Western blots of nuclear fractions and immunofluorescence showed increased levels of Bcl‐2 and Bcl‐xL in the nucleus post DNR treatment, which coincided with accumulation of γH2AX (Figure S5C&D). These data provide evidence that a decrease of Mcl‐1 levels and nuclear accumulation of Bcl‐2 and Bcl‐xL may represent a generalized mechanism responsible for apoptosis induced by DNA‐damaging agents.

Finally, to determine if Bcl‐xL and Mcl‐1 play a role in cytarabine‐induced γH2AX we treated the THP‐1/RFP, THP‐1/Bcl‐xL, and THP‐1/Mcl‐1 cells with cytarabine, obatoclax or both for 8 h and assessed γH2AX protein levels by Western blotting. Densitometry measurements indicated that Bcl‐xL and Mcl‐1 overexpressing cells had significantly lower levels of γH2AX after cytarabine or cytarabine plus obatoclax treatment (Figure 6A). To determine if Bcl‐2 plays a role, we used the Bcl‐2 selective BH3 mimetic ABT‐199 to treat THP‐1 cells, alone or in combination with cytarabine for 8 h. Western blotting and densitometry measurements indicate that ABT‐199 significantly increased cytarabine‐induced γH2AX levels (Figure 6B). These results suggest that Bcl‐2, Bcl‐xL, and Mcl‐1 play a critical role in cytarabine‐induced DNA damage.

Figure 6.

Figure 6

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Bcl‐xL, Mcl‐1, and Bcl‐2 are involved in cytarabine‐induced DNA damage. THP‐1/RFP, THP‐1/Bcl‐xL, and THP‐1/Mcl‐1 cells were treated with cytarabine and/or obatoclax for 8 h. Whole cell lysates were extracted and subjected to Western blotting, and probed with anti‐γH2AX or ‐β‐actin antibody. Densitometry for γH2AX expression was measured, normalized to β‐actin, and graphed as fold change compared to the no drug control (Panel A). THP‐1 cells were treated with cytarabine alone and in combination with ABT‐199 for 8 h. Whole cell lysates were extracted and subjected to Western blotting, and probed with anti‐γH2AX or ‐β‐actin antibody. Densitometry for γH2AX expression was measured, normalized to β‐actin, and graphed as fold change compared to the no drug control (Panel B). The data are presented as mean ± standard error from at least 3 independent Western blots. * indicates p < 0.05.

4. Discussion

In addition to the traditional roles in apoptosis, emerging evidence suggests that Bcl‐2, Bcl‐xL, and Mcl‐1 also possess nuclear functions and are involved in the DNA damage response including DNA repair and regulation of cell cycle checkpoints (Jamil et al., 2008, 2010, 2010, 2011, 2010, 2001, 2007, 2008). In line with these studies, we have found that clinically achievable concentrations of obatoclax enhanced cytarabine‐induced DNA DSBs which was followed by a decrease of Mcl‐1 levels, nuclear accumulation of Bcl‐2, Bcl‐xL, and Mcl‐1, and apoptosis.

In the present study, we have demonstrated additive to synergistic antileukemic activities for simultaneous treatment of AML cell lines and diagnostic blasts with cytarabine and clinically achievable concentrations of obatoclax. These results are in agreement with earlier studies which demonstrated synergy between obatoclax and standard chemotherapy drugs or other molecularly targeted agents in multiple cancer types including leukemia (Konopleva et al., 2008; Li et al., 2008; Rahmani et al., 2012). Similar to these studies, we found that combined cytarabine and obatoclax treatment caused BAX activation, loss of MMP, and apoptosis in AML cells. However, these were not detected after treatment with clinically achievable obatoclax concentrations, suggesting that obatoclax alone does not disrupt the pro‐survival functions of Bcl‐2, Bcl‐xL, or Mcl‐1. Rather they potentiate the cytotoxic effect of cytarabine in AML cells.

Our combined cytarabine and obatoclax treatment resulted in significantly lower levels of Mcl‐1 at 48 h compared to vehicle control, cytarabine, or obatoclax treatment. This decrease was accompanied by increased apoptosis, in agreement with other studies which demonstrated that the elimination of Mcl‐1 is required for Bak/Bax activation and subsequent apoptosis (Bose and Grant, 2013; Cuconati et al., 2003; Nijhawan et al., 2003). Cytarabine‐ and cytarabine plus obatoclax‐induced apoptosis was partially attenuated by ectopic overexpression of Mcl‐1 and almost completely attenuated by ectopic overexpression of Bcl‐xL.

Our time course experiments revealed that combined cytarabine and obatoclax treatment resulted in an early increase of γH2AX, which was later followed by increased nuclear localization of Bcl‐2, Bcl‐xL, and Mcl‐1, as well as increased cleavage of caspase‐3 and PARP. We also detected an initial increase in Mcl‐1 levels followed by a time‐dependent decrease resulting in significantly lower levels of Mcl‐1 at 48 h compared to vehicle control, cytarabine, or obatoclax treatment. It has been reported that Mcl‐1 levels increase in response to low levels of DNA damage (Jamil et al., 2008; Zhan et al., 1997), which could explain the early increase in Mcl‐1 levels, as it happens to coincide with the appearance of increased γH2AX. Based on all of these findings, it is likely that obatoclax enhances cytarabine‐induced DNA damage, a novel mechanism for the additive‐to‐synergistic antileukemic activity of the combination of cytarabine and obatoclax which has not been reported until now. Accumulation of the DNA damage to a certain level then induces a generic response to DNA damage involving nuclear localization of Bcl‐2, Bcl‐xL, and Mcl‐1, and decrease of Mcl‐1 levels. We speculate that these events could potentially facilitate Bax (and/or Bak) activation and induce apoptosis. It is important to note that these molecular events involving Bcl‐2, Bcl‐xL, and Mcl‐1 were also observed in AML cells treated with DNR (a known DNA DSB inducer), suggesting that they may represent a generalized molecular mechanism underlying apoptosis induced by DNA damage.

In addition to nuclear translocation of Bcl‐2, Bcl‐xL, and Mcl‐1 and decreased levels of Mcl‐1, the proteins likely have nuclear functions. Bcl‐2 has been shown to be involved in calcium homeostasis, DNA end joining, and PARP1 function (Bonneau et al., 2013; Dutta et al., 2012; Kumar et al., 2010; Saintigny et al., 2001). Additionally, Choi et al. have demonstrated that nuclear localized Bcl‐2 has pro‐apoptotic functions (Choi et al., 2013). Also, they demonstrated that aspirin treatment induced nuclear translocation and phosphorylation of Bcl‐2, triggering apoptosis in breast cancer cells. Furthermore, it has been demonstrated that Bcl‐xL interacts with cdk1 and stabilizes G2/M cell cycle arrest after DNA damage (Schmitt et al., 2007; Wang et al., 2012). Also, Mcl‐1 has exhibited nuclear localization and interaction with cdk1 (Jamil et al., 2005, 2010). Our Bcl‐xL and Mcl‐1 overexpression data and ABT‐199 data suggest that these proteins modulate cytarabine‐induced DNA damage. Based on our results and others' published data, Bcl‐2, Bcl‐xL, and/or Mcl‐1 are likely involved in DNA damage repair and/or cell cycle progression, thus obatoclax treatment may result in inhibition of the nuclear functions of these proteins, leading to enhancement of cytarabine sensitivity in AML cells. Studies involving DNA damage repair and cell cycle progression are ongoing to further determine the mechanism of action of the combination.

In this study we used multiple BH3 mimetics, which have different binding specificities, to demonstrate that BH3 mimetics enhance cytarabine‐induced γH2AX. GX15‐070 and TW‐37 bind Bcl‐2, Bcl‐xL, and Mcl‐1 whereas ABT‐737 binds Bcl‐2 and Bcl‐xL (Goard and Schimmer, 2013; Mohammad et al., 2007). All three drugs demonstrated the ability to enhance cytarabine‐induced apoptosis, suggesting that inhibition of their common binding targets Bcl‐2 and Bcl‐xL likely play a major role, while inhibition of Mcl‐1 may play a lesser role. Our results with ABT‐199 also indicate that targeting Bcl‐2 itself can enhance cytarabine‐induced γH2AX (Figure 6B) and apoptosis (unpublished data), suggesting that Bcl‐2 may play the major role in enhancing cytarabine‐induced DNA damage and apoptosis.

Bcl‐2 family proteins also play an important role in autophagy. While autophagy was not investigated in this study, we cannot rule out the role it may play in the antileukemic activity of the combination of obatoclax and cytarabine in AML cells. Obatoclax has been shown to induce autophagy in various malignancies, including leukemia (Maiuri et al., 2007a; Malik et al., 2011; Pan et al., 2010; Urtishak et al., 2013; Vogler et al., 2009; Wei et al., 2010), though induction of autophagy has demonstrated both pro‐death and pro‐survival functions in leukemia (Yu and Liu, 2013). As discussed by Yu and Liu, obatoclax can induce autophagy by inhibiting the interaction of Bcl‐2 and/or Bcl‐xL and Beclin‐1, but there is also evidence that it may inhibit the completion of autophagic flux (Yu and Liu, 2013). While clinically achievable concentrations of obatoclax could induce autophagy, Wei et al. demonstrated that AML cells treated with 200 nM obatoclax did not result in formation of autophagosomes, as determined by transmission electron microscopy, suggesting that autophagy is not likely being induced by the single drug treatment (Wei et al., 2010). However, it may play a role in the combination treatment as DNA damage can induce autophagy (Maiuri et al., 2007b; Munoz‐Gamez et al., 2009). It would be interesting to determine the role autophagy plays in the combination of obatoclax and cytarabine as well as the timing in relation to DNA damage and nuclear translocation of the Bcl‐2 family proteins; however it is beyond the scope of this paper. Studies are ongoing to determine the role autophagy may play in antileukemic activity of the combination of obatoclax and cytarabine.

Although it is possible that obatoclax could have non‐selective effects by which it enhances cytarabine‐induced DNA damage and apoptosis, as suggested by a recent study (Vogler et al., 2009), our studies using TW‐37, ABT‐737 and ABT‐199, structurally different BH3 mimetics, produced similar results. Therefore, it is unlikely that the enhancement of cytarabine‐induced DNA DSBs and apoptosis by obatoclax was due to its off‐target effects. Instead, we suspect that these findings are the result of previously unexplored on‐target effects, suggesting that additional functions of the Bcl‐2 family proteins may represent novel druggable targets. Although further studies are needed to elucidate the mechanism by which the DNA damage is enhanced by obatoclax, our findings may broaden the clinical use of BH3 mimetics.

Conflict of interest disclosure

The authors disclose no potential conflicts of interests.

Supporting information

The following are the supplementary data related to this article:

Supplementary data

Click here for additional data file. (19.9KB, docx)

Figure S1. Clinically achievable concentrations of obatoclax enhance cytarabine‐induced apoptosis. THP‐1 (Panel A) and OCI‐AML3 (Panel B) cells were treated with cytarabine or obatoclax, alone or in combination for 48 h 5 × 105 cells were incubated with JC‐1 for 15 min at 37 °C. Cells were washed, resuspended in PBS, and then plated in black‐walled 96‐well plates. JC‐1 monomer fluorescence was measured using a microplate reader with excitation 485 nm and emission 535 nm. THP‐1 and OCI‐AML3 cells were treated with cytarabine and obatoclax for 6 h and then 1 mg of protein lysate was used for immunoprecipitation with anti‐Bax 6A7 (activated Bax) antibody. Immunoprecipitated proteins were subjected to Western blotting and probed with anti‐Bax antibody (Panel C).

Figure S2. The effects of cytarabine and obatoclax treatment on protein expression and localization in THP‐1 cells. THP‐1 cells were treated with vehicle control or 4 μM cytarabine (Cyta) plus 175 nM obatoclax (Obat) for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panels A&B). THP‐1 cells were treated with vehicle control, 4 μM cytarabine (Cyta), 100 nM or 175 nM obatoclax (Obat), alone or in combination for 48 h. Whole cell lysates were subjected to Western blotting, and then probed with anti‐Bcl‐2, Bcl‐xL, Mcl‐1, or –β‐actin antibody (Panel C).

Figure S3. Obatoclax cooperates with cytarabine to induce DNA Damage in THP‐1 cells. THP‐1 cells were treated with vehicle control, cytarabine, obatoclax, or cytarabine plus obatoclax for 48 h, whole cell lysates were extracted, subjected to Western blotting and probed with anti‐γH2AX or ‐β‐actin antibody (Panel A). THP‐1 cells were treated for 4 h with the indicated concentration of each drug. Whole cell lysates were extracted and subjected to Western blotting, and probed with anti‐γH2AX or ‐β‐actin antibody (Panel B). Densitometry for γH2AX expression from 4 independent experiments was measured and graphed as fold change compared to the no drug control (Panel C). THP‐1 cells were treated with cytarabine and/or obatoclax for 4 h or with 20 μM cytarabine for 48 h. Apoptotic events were determined by Annexin V/7‐AAD staining and flow cytometry analyses (Panel D). THP‐1 cells were treated with cytarabine plus obatoclax for up to 48 h, whole cell lysates were extracted, subjected to Western blotting and probed with anti‐Mcl‐1, ‐γH2AX, ‐cleaved caspase 3, ‐PARP or ‐β‐actin antibody (Panel E). THP‐1 cells were treated with vehicle control or cytarabine plus obatoclax for 48 h. The cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green), or anti‐γH2AX (red) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panel F). * indicates p < 0.05 and ** indicates p < 0.005.

Figure S4. TW‐37 or ABT‐737 plus cytarabine cooperate to induce DNA damage in THP‐1 or MV4‐11 cells. THP‐1 cells were treated with vehicle control, cytarabine, TW‐37, or cytarabine plus TW‐37 for 48 h. Apoptotic events were determined by annexin V/PI staining and flow cytometry analysis (Panel A). γH2AX, PARP, and β‐actin protein levels were determined by Western blotting (Panel B). Cytoplasmic and nuclear fractions were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, and ‐Histone H4 (Panel C). AML cell lines were cultured in 96‐well plates at 37 °C for 72 h, in complete medium with variable concentrations of ABT‐737, and viable cell numbers were determined using MTT reagent and a microplate reader. The IC50 values were calculated as the concentrations of drug necessary to inhibit 50% proliferation compared to control cells cultured in the absence of drug (Panel D). MV4‐11 cells were treated with vehicle control, cytarabine, obatoclax, ABT‐737 (ABT) or a combination of cytarabine plus obatoclax or ABT‐737 for 48 h. Apoptotic events were determined by annexin V/7‐AAD staining and flow cytometry analysis (Panel E). Whole cell lysates were extracted, subjected to Western blotting, and then probed with anti‐γH2AX, – PARP, and β‐actin antibody (Panel F). The data are presented as mean ± standard error from one representative experiment, which was repeated at least 3 independent times. *** indicates p < 0.0005.

Figure S5. DNR induces DNA DSBs accompanied by downregulation of Mcl‐1 and nuclear accumulation of Bcl‐2 and Bcl‐xL. THP‐1 cells were treated with 0 nM, 25 nM, or 50 nM daunorubicin (DNR) for 48 h. Apoptotic events were determined by annexin V/PI staining and flow cytometry analysis (Panel A). γH2AX, Bcl‐2, Bcl‐xL, Mcl‐1, PARP, cleaved caspase 3 and β‐actin protein levels were determined by Western blotting (Panel B). THP‐1 cells treated with 50 nM DNR for 48 h were subjected to cellular fractionation. Cytoplasmic and nuclear fractions were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, or ‐Histone H4 antibody (Panel C). THP‐1 cells were treated with 50 nM DNR and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green), and anti‐γH2AX (red). Nuclei were stained with DAPI (blue, Panel D). The data are presented as mean ± standard error from one representative experiment, which was repeated at least three times.

Click here for additional data file. (3.4MB, pptx)

Acknowledgments

This study was supported by a Start‐up Fund from the Barbara Ann Karmanos Cancer Institute, grants from the St. Baldrick's Foundation, American Cancer Society IRG#11‐053‐01‐IRG, the Herrick Foundation, National Cancer Institute CA120772, Leukemia Research Life, Elana Fund, Justin's Gift Charity, the Buric Family, Sehn Family Foundation, the Ring Screw Textron Endowed Chair for Pediatric Cancer Research, and the National Natural Science Foundation of China (NSFC 31271477 and 81200363). The Microscopy, Imaging and Cytometry Resources Core is supported, in part, by NIH Clinical Center grant P30CA22453 to the Karmanos Cancer Institute, Wayne State University and the Perinatology Research Branch of the National Institutes of Child Health and Development, Wayne State University. Mr. JTC is a predoctoral trainee supported by T32 CA009531 from the National Cancer Institute.

Supplementary data 1.

1.1.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2014.09.008.

Xie Chengzhi, Edwards Holly, Caldwell J. Timothy, Wang Guan, Taub Jeffrey W., Ge Yubin, (2015), Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage, Molecular Oncology, 9, doi: 10.1016/j.molonc.2014.09.008.

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Figure S1. Clinically achievable concentrations of obatoclax enhance cytarabine‐induced apoptosis. THP‐1 (Panel A) and OCI‐AML3 (Panel B) cells were treated with cytarabine or obatoclax, alone or in combination for 48 h 5 × 105 cells were incubated with JC‐1 for 15 min at 37 °C. Cells were washed, resuspended in PBS, and then plated in black‐walled 96‐well plates. JC‐1 monomer fluorescence was measured using a microplate reader with excitation 485 nm and emission 535 nm. THP‐1 and OCI‐AML3 cells were treated with cytarabine and obatoclax for 6 h and then 1 mg of protein lysate was used for immunoprecipitation with anti‐Bax 6A7 (activated Bax) antibody. Immunoprecipitated proteins were subjected to Western blotting and probed with anti‐Bax antibody (Panel C).

Figure S2. The effects of cytarabine and obatoclax treatment on protein expression and localization in THP‐1 cells. THP‐1 cells were treated with vehicle control or 4 μM cytarabine (Cyta) plus 175 nM obatoclax (Obat) for 48 h. Cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panels A&B). THP‐1 cells were treated with vehicle control, 4 μM cytarabine (Cyta), 100 nM or 175 nM obatoclax (Obat), alone or in combination for 48 h. Whole cell lysates were subjected to Western blotting, and then probed with anti‐Bcl‐2, Bcl‐xL, Mcl‐1, or –β‐actin antibody (Panel C).

Figure S3. Obatoclax cooperates with cytarabine to induce DNA Damage in THP‐1 cells. THP‐1 cells were treated with vehicle control, cytarabine, obatoclax, or cytarabine plus obatoclax for 48 h, whole cell lysates were extracted, subjected to Western blotting and probed with anti‐γH2AX or ‐β‐actin antibody (Panel A). THP‐1 cells were treated for 4 h with the indicated concentration of each drug. Whole cell lysates were extracted and subjected to Western blotting, and probed with anti‐γH2AX or ‐β‐actin antibody (Panel B). Densitometry for γH2AX expression from 4 independent experiments was measured and graphed as fold change compared to the no drug control (Panel C). THP‐1 cells were treated with cytarabine and/or obatoclax for 4 h or with 20 μM cytarabine for 48 h. Apoptotic events were determined by Annexin V/7‐AAD staining and flow cytometry analyses (Panel D). THP‐1 cells were treated with cytarabine plus obatoclax for up to 48 h, whole cell lysates were extracted, subjected to Western blotting and probed with anti‐Mcl‐1, ‐γH2AX, ‐cleaved caspase 3, ‐PARP or ‐β‐actin antibody (Panel E). THP‐1 cells were treated with vehicle control or cytarabine plus obatoclax for 48 h. The cells were fixed and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green), or anti‐γH2AX (red) and visualized by confocal microscopy. Nuclei were stained with DAPI (blue, Panel F). * indicates p < 0.05 and ** indicates p < 0.005.

Figure S4. TW‐37 or ABT‐737 plus cytarabine cooperate to induce DNA damage in THP‐1 or MV4‐11 cells. THP‐1 cells were treated with vehicle control, cytarabine, TW‐37, or cytarabine plus TW‐37 for 48 h. Apoptotic events were determined by annexin V/PI staining and flow cytometry analysis (Panel A). γH2AX, PARP, and β‐actin protein levels were determined by Western blotting (Panel B). Cytoplasmic and nuclear fractions were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, and ‐Histone H4 (Panel C). AML cell lines were cultured in 96‐well plates at 37 °C for 72 h, in complete medium with variable concentrations of ABT‐737, and viable cell numbers were determined using MTT reagent and a microplate reader. The IC50 values were calculated as the concentrations of drug necessary to inhibit 50% proliferation compared to control cells cultured in the absence of drug (Panel D). MV4‐11 cells were treated with vehicle control, cytarabine, obatoclax, ABT‐737 (ABT) or a combination of cytarabine plus obatoclax or ABT‐737 for 48 h. Apoptotic events were determined by annexin V/7‐AAD staining and flow cytometry analysis (Panel E). Whole cell lysates were extracted, subjected to Western blotting, and then probed with anti‐γH2AX, – PARP, and β‐actin antibody (Panel F). The data are presented as mean ± standard error from one representative experiment, which was repeated at least 3 independent times. *** indicates p < 0.0005.

Figure S5. DNR induces DNA DSBs accompanied by downregulation of Mcl‐1 and nuclear accumulation of Bcl‐2 and Bcl‐xL. THP‐1 cells were treated with 0 nM, 25 nM, or 50 nM daunorubicin (DNR) for 48 h. Apoptotic events were determined by annexin V/PI staining and flow cytometry analysis (Panel A). γH2AX, Bcl‐2, Bcl‐xL, Mcl‐1, PARP, cleaved caspase 3 and β‐actin protein levels were determined by Western blotting (Panel B). THP‐1 cells treated with 50 nM DNR for 48 h were subjected to cellular fractionation. Cytoplasmic and nuclear fractions were subjected to Western blotting and probed with anti‐Bcl‐2, ‐Bcl‐xL, ‐Mcl‐1, ‐MEK1/2, or ‐Histone H4 antibody (Panel C). THP‐1 cells were treated with 50 nM DNR and stained with anti‐Bcl‐2, ‐Bcl‐xL or ‐Mcl‐1 (green), and anti‐γH2AX (red). Nuclei were stained with DAPI (blue, Panel D). The data are presented as mean ± standard error from one representative experiment, which was repeated at least three times.

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