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Correspondence: Levi J. Beverly, PhD, Department of Medicine, James Graham Brown Cancer Center, University of Louisville, 505 S. Hancock Street, CTRB RM 204, Louisville, KY 40202.
Affiliations
Department of Medicine, James Graham Brown Cancer Center, University of Louisville, Louisville, KentuckyDepartment of Physiology, James Graham Brown Cancer Center, University of Louisville, Louisville, KentuckyDepartment of Pharmacology and Toxicology, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky
Successful treatment of leukemia requires new medications to combat drug resistance, but the development of novel therapies is an arduous and risky endeavor. Repurposing currently approved drugs or those already in clinical development to treat other indications is a more practical approach. Moreover, combinatorial therapeutics are often more efficacious than single agent therapeutics because the former can simultaneously target multiple pathways that mitigate tumor aggressiveness and induce cancer cell death.
Material and Methods
In this study, we combined the class III antiarrhythmic agent amiodarone and the BH3 mimetic ABT-263 based on data from a prior drug screen to assess the degree of apoptotic induction in 2 human leukemia cell lines.
Results
The combination yielded statistically significant increases in apoptosis in both cell lines by downregulating AKT activity and increasing cleaved caspase-3.
Conclusions
Overall, our findings suggest that combining K+ channel blockers with prosurvival Bcl-2 family inhibitors is a promising therapeutic approach in treating leukemia.
The treatment of leukemia has seen many advances in therapy since the 1950s with the development of folate inhibitors that induce partial remission of acute lymphocytic leukemia in children.
However, long-term survival rates for acute myeloid leukemia (AML) and chronic lymphocytic leukemia still lag and have remained steady at around 30% for the past 2 decades.
According to the National Institutes of Health Surveillance, Epidemiology, and End Results (SEER) statistics from 2010-2014, the number of new men and women diagnosed with cancer is 13.7 per 100,000 with 6.8 per 100,000 deaths. There is estimated to be greater than 60,000 new cases of leukemia in 2017. Current therapies have improved 5-year survival rates, but there still is a need for enhancement. Leukemia cells can resist target therapies via activation of alternative signaling pathways by secondary mutation.
Therefore, it is imperative that novel treatment options and combinations be explored to improve remission, combat drug resistant relapsed disease and ultimately increase overall survival rates.
Advances in understanding the basic biological mechanisms that contribute to leukemogenesis has been instrumental in increasing favorable outcomes in patients. One such advancement has been a greater understanding of the role of plasma
in cancer. Membrane channels facilitate the movement of substances across the lipid bilayer, thereby carrying out vital functions in many physiological cellular processes. However, ATP-binding cassette (ABC) transporters can confer resistance to leukemia cells by causing the export of therapeutic medications.
Ion channels are essential for cell survival and seem like an obvious target given their importance to cellular function, accessibility and the availability of pharmacologic inhibitors. Indeed, Na+, K+ and Ca2+ channels have been targeted for possible therapeutic benefit with in vivo evidence.
Combining therapies that inhibit these channels with others that target alternative pathways could potentially enhance cytotoxicity against leukemia cells.
In addition, much work has been done to understand the role of the antiapoptotic Bcl-2 family members in apoptosis and cancer. Bcl-2 family proteins are some of the most studied regulators of apoptosis and consist of over 20 members. The function of these proteins is to act as antagonists or protagonists of proapoptotic proteins, including Bak and Bax that are responsible for the mitochondrial permeabilization step of the intrinsic pathway. Overexpression of Bcl-2, Bcl-xL and Bcl-w confers a survival advantage in several cancer types by preventing mitochondrial outer membrane permeabilization, a crucial step in the intrinsic apoptotic pathway.
BH3 mimetics such as ABT-737, ABT-263 and ABT-199 were developed to inhibit these prosurvival proteins and have shown effectiveness in vitro as well as in animal models.
Although these compounds have shown promise, there are still limitations to potential therapy, including the upregulation of the antiapoptotic protein Mcl-1, which is not targeted by the compounds.
Combinatorial drug therapies may provide the solution to the shortcomings of BH3 mimetics.
Based on a prior drug screen, we chose the channel blockers amitriptyline, amiloride and amiodarone as drugs that could potentially be repurposed by combining them with ABT-263 to kill leukemia cells.
Repurposing experimental or currently approved medications is advantageous because the compositions, modes of action and toxicities of these therapies are already known. Amiodarone was selected based on dose-response data herein and additional data showing the importance of K+ channels in leukemia.
We hypothesize that amiodarone will sensitize human AML cells to ABT-263 as demonstrated by a greater degree of apoptosis when the 2 drugs are used in combination. We then propose a mechanism by which this therapeutic combination enhances apoptosis in these cells.
Materials & Methods
Cell Lines
U937 and MV4-11 cell lines were purchased from ATCC and grown in RPMI-1640 (GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum, 5% l-glutamine and 5% penicillin-streptomycin. Both cell lines underwent short tandem repeat analysis for identity confirmation as well as periodic mycoplasma testing using the MycoSensor PCR assay kit (Agilent).
Drugs
ABT-263 (ChemieTek), amitriptyline (Cayman Chemical Company), amiloride (Cayman Chemical Company and amiodarone (Cayman Chemical Company) were obtained in powder form and dissolved in DMSO (Fisher Scientific).
Alamar Blue
Human leukemia cells were seeded on 96 well dishes (8,000 for U937 and 10,000 for MV4-11) and immediately treated with ABT-263, amitriptyline, amiloride and amiodarone. The drugs were diluted in RPMI-1640 at a 1:2 or 1:3 dilution with the volume of each well totaling 100 µL. Each treatment was done in triplicate. After the cells were incubated for 48 hours, 10 µL of alamar blue (100 µg/µL of resorufin sodium salt [Sigma] in PBS) was added to each well. Plates were incubated for 2 hours and the fluorescence was determined using a SPECTRAmax Gemini plate reader, with readings occurring every hour until all wells had approximately equal readings. Wells with only alamar blue and media were used to subtract background. All treatments were normalized to vehicle wells that contained only DMSO. Each graph represents at least 3 biological replicates. Graphs were designed using GraphPad Prism.
Flow Cytometry
Cell lines were treated with the indicated concentrations of amiodarone and ABT-263. After 24 hours at 37°C, samples were clarified by centrifugation. Cells were suspended in annexin V binding buffer (1 mM HEPES, 15 mM NaCl and 0.25 mM CaCl2) and stained with anti-annexin V antibody (BD Biosciences) and propidium iodide (PI) (Sigma-Aldrich). After 20 minutes at 4°C, samples were analyzed using a FACScan Fluorescence Activated Cell Analyzer (Becton Dickinson) and FlowJo software.
Western Blotting
Cell lines were treated with the indicated concentrations of amiodarone and ABT-263. After 12 hours at 37°C, whole cell lysates were harvested in lysis buffer (1% CHAPS, 150 mM NaCl, 50 mM Tris and 5 mM EDTA). Protein concentration was quantified using a Pierce BCA assay kit (Thermo Fisher Scientific). Samples were heated at 95°C for 5 minutes and 30 µg (U937) or 50 µg (MV4-11) protein was loaded onto Bolt 4-12% Bis-Tris Plus gels (Thermo Fisher Scientific) in MES SDS running buffer (Life Technologies). Proteins were transferred to 0.45-µm PVDF membranes (Millipore), which were subsequently blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% nonfat dry milk for 1 hour at room temperature. Membranes were incubated in either anti-pan-AKT, anti-phospho-AKT or anti-cCASP3 monoclonal antibodies (Cell Signaling Technology) overnight at 4°C. After washing with TBST plus 5% milk, membranes were incubated in HRP-linked secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. Signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and Amersham ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences) and visualized by exposure to X-ray film (Phenix).
Statistical Analysis
The percentage of annexin V-positive cells within each treatment condition was determined by adding quadrants 2 and 3 for each replicate and averaging this value among triplicates. Mean values for each condition were plotted using GraphPad Prism, and 1-way analysis of variance with Tukey’s test was used to determine statistical significance.
Results
Several Channel Inhibitors Reduce Cell Viability in a Dose-Dependent Manner
Previously, we performed a small-molecule drug screen to identify compounds that synergize with the first-generation Bcl-2 inhibitor ABT-737.
However, ABT-263 was selected for this investigation because it has an identical mode of action but possesses superior oral bioavailability and therefore greater clinical relevance.
From the screening data we set forth the following criteria for candidates to further pursue. First, they had to have a synergy score greater than −40 (the more negative the score, the more calculated synergy). Second, they needed to be drugs or clinical candidates that have been used previously in humans. Lastly, the candidates needed to not have been used for cancer indication in human trials. Thus, we assessed the cytotoxic effects of amitriptyline, amiloride and amiodarone in U937 and MV4-11 cell lines using alamar blue (Figure 1). These cell lines were chosen based on their known expression of the Bcl-2 family members and their differential sensitivity to ABT-263 (Figure 1).
Acute myeloid leukemia cells harboring MLL fusion genes or with the acute promyelocytic leukemia phenotype are sensitive to the Bcl-2-selective inhibitor ABT-199.
All channel inhibitors reduced cell viability in a dose-dependent manner. We selected amiodarone for the combination studies since it reduced cell viability to a greater degree compared with the other compounds.
FIGURE 1Assessment of cell viability by alamar blue in U937 and MV4-11. (A) Table showing drugs tested, known mechanisms of action and synergy scores based on prior drug screen data. (B) Dose-response graphs of ABT-263, amiloride, amiodarone and amitriptyline at 1:2 or 1:3 dilution factors.
Individual Doses of Amiodarone and ABT-263 Increase Apoptosis in a Dose-Dependent Manner
We established the dose-response behavior of each drug in U937 to determine the optimal concentrations for the combination assays. Cells were treated with increasing concentrations of amiodarone and ABT-263, initial concentrations were based on the alamar blue dose-response data. After 24 hours, staining levels of the early and late apoptotic markers annexin V and PI, respectively, were assessed using flow cytometry. In U937, only ABT-263 at 1.0 µM showed a statistically significant difference (P < 0.001) in staining compared to vehicle and was selected for the combination study in this cell line (Figure 2A). In the same cell line, amiodarone showed a clear dose response with the 12.5 µM (P < 0.01) and 20 µM (P < 0.0001) arms both showing statistical differences in staining (Figure 2B). The 12.5 µM concentration was chosen for the combination study in U937 since it induced less apoptosis compared to 20 µM.
FIGURE 2Dose-response behaviors of ABT-263 and amiodarone in U937. (A) Cells were treated with increasing doses of (A) ABT-263 or (B) amiodarone for 24 hours and stained with anti-annexin V antibody and propidium iodide (PI) to determine the percentage of apoptotic cells.
To further validate this observation, we tested a second AML cell line using the same procedure. In MV4-11, we used lower ABT-263 concentrations because previous alamar blue data (Figure 1B) indicated that MV4-11 cells were more sensitive. ABT-263 at 25 nM and 250 nM yielded staining increases with statistical significance (P < 0.0001) (Figure 3A), but 25 nM was selected for the combination study because 250 nM resulted in excessive cell death. Similarly, amiodarone at 20 µM produced excessive staining, so 12.5 µM was chosen despite identical statistical significance compared to vehicle (P < 0.0001) (Figure 3B).
FIGURE 3Dose-response behaviors of ABT-263 and amiodarone in MV4-11. (A) Cells were treated with increasing doses of (A) ABT-263 or (B) amiodarone for 24 hours and stained with anti-annexin V antibody and propidium iodide (PI) to determine the percentage of apoptotic cells.
Amiodarone and ABT-263 in Combination Enhance Apoptosis Relative to Either Drug Alone
We next tested 1.0 µM ABT-263 in conjunction with 12.5 µM amiodarone in U937 to determine if this combination would result in an elevated level of staining compared to the individual drugs (Figure 4). The combination did indeed show a marked statistically significant increase in staining relative to vehicle (P < 0.0001) and each drug in isolation (P < 0.0001). Next, the combination of 25 nM ABT-263 and 12.5 µM amiodarone in MV4-11 was assessed. Similar to U937, the combination in MV4-11 showed a considerable statistically significant increase in staining compared to vehicle (P < 0.0001) and the individual drugs (P < 0.0001) (Figure 5).
FIGURE 4Amiodarone and ABT-263 in combination enhance apoptosis in U937. Cells were treated with either amiodarone (12.5 µM), ABT-263 (1.0 µM) or both drugs in combination for 24 hours and stained with anti-annexin V antibody and propidium iodide (PI) to determine the percentage of apoptotic cells.
FIGURE 5Amiodarone and ABT-263 in combination enhance apoptosis in MV4-11. Cells were treated with either amiodarone (12.5 µM), ABT-263 (25 nM) or both drugs in combination for 24 hours and stained with anti-annexin V antibody and propidium iodide (PI) to determine the percentage of apoptotic cells.
Amiodarone Reduces AKT Phosphorylation and Increases cCASP3 Cleavage When Combined With ABT-263
To establish a mechanism by which the amiodarone/ABT-263 combination yielded greater apoptotic staining, we treated U937 and MV4-11 cells with each drug individually or in combination for 12 hours. This time period was chosen because we expected 12 hours to be sufficient to observe intracellular changes. The expression of pan-AKT, phospho-AKT (p-AKT) and cleaved caspase 3 (cCASP3) was assayed by western blot (Figure 6A and 6B). In both cell lines, pan-AKT remained constant across all arms while the combination of ABT and amiodarone resulted in a dramatic decrease in p-AKT and an increase in cCASP3 compared to the individual drugs. To begin to understand the possible mechanism by which combining amiodarone with ABT-263 could potentiate apoptosis we determined the expression of the canonical BCL-2 family member, BCL-2. Our previous worked examined the expression of all 6 antiapoptotic BCL-2 proteins and demonstrated that U937 cells do not express appreciable amounts of BCL-2.
To our surprise, treatment of U937 cells with amiodarone for 6 hours caused an increase in the levels of BCL-2 proteins (Figure 6C). This is likely a compensatory increase the cells are using to attempt to block apoptosis. As before, we see a dramatic reduction in p-AKT following amiodarone treatment. To determine if the mechanisms of action of other potentially synergistic candidates are the same, we performed the same experiment with amitryptyline. Treatment with this drug alone did not cause a decrease in p-AKT, but interestingly did cause a modest increase in BCL-2. Blocking the compensatory antiapoptotic BCL-2 increase with ABT-263 may be the explanation for increased apoptosis when these drugs are used in combination.
FIGURE 6ABT-263 and amiodarone decrease p-AKT and increase cCASP3. (A) U937 cells were treated with either ABT-263 (1.0 µM), amiodarone (12.5 µM) or both drugs in combination for 12 hours. (B) MV4-11 cells were treated with either ABT-263 (25 nM), amiodarone (12.5 µM) or drugs in combination for 12 hours. Subsequently, western blots for pan-AKT, p-AKT and cleaved caspase 3 (cCASP3) were performed. (C) U937 cells were treated with ABT-263 (1.0 µM), amiodarone (12.5 µM), amitriptyline (12.5 µM) or both drugs in combination for 6 hours.
AML is a heterogeneous hematologic cancer in which abnormally differentiated hematopoietic cells undergo excessive proliferation, impairing their normal function and causing a host of other complications.
Clearly, additional therapies need further exploration. However, the process of developing novel therapeutics is lengthy, expensive and fraught with regulatory hurdles. A more feasible approach is the repurposing of approved drugs or those currently undergoing clinical trials.
Amiodarone, a class III antiarrhythmic agent approved for the treatment of arrhythmias, lengthens the cardiac action potential duration and refractory period by inhibiting K+ channels.
VEGFR-1 (FLT-1), beta1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome.
HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors.
demonstrated that human ether-a-gó-gó-related gene (HERG) K+ channels are downregulated in normal peripheral blood mononuclear cells but are constitutively expressed in primary human AML cells. Furthermore, amiodarone has been shown to inhibit heterologously expressed HERG channels.
The Bcl-2 family proteins have been shown to be important in leukemia, with overexpression of Bcl-2 leading to chemoresistance of AML cells and poor long-term prognoses.
Bcl-2, Bcl-xL and Bcl-w inhibit apoptosis and promote cell survival by preventing the multimerization of the proapoptotic proteins Bak and Bax in the outer mitochondrial membrane.
ABT-263, a BH3 mimetic currently in clinical development, binds and inhibits Bcl-2, Bcl-xL and Bcl-w, allowing Bak and Bax to oligomerize and initiate mitochondrial outer membrane permeabilization, which culminates in cell apoptosis.
In this study, we sought to explore the role of the potassium channel inhibitors when combined with ABT-263 in leukemia cells. We hypothesized that the combination treatment would increase cell apoptosis. To test this hypothesis, we first established that individual treatments of amiodarone and ABT-263 resulted in dose-dependent increases in apoptosis in the human AML cell lines by both alamar blue and flow cytometry. We next treated these cells with a combination of the 2 drugs and observed a significant increase in the percentage of apoptotic cells compared to either drug alone. Inhibition of potassium channels has been shown by others to interfere with the PI3K/AKT pathway, a major signaling regulator in cell proliferation.
K+ Channel inhibition differentially regulates migration of intestinal epithelial cells in inflamed vs. non-inflamed conditions in a PI3K/Akt-mediated manner.
In several cancer models, the potassium channel hERG1 has been shown to recruit and activate the PI3K/AKT pathway and inhibition of hERG1 reduced AKT phosphorylation.
Based on this information, the status of AKT was assessed as a possible mechanism for the ability of amiodarone to increase cellular apoptosis when combined with ABT-263. Our data showed that p-AKT levels were reduced when amiodarone was present. This indicates that the increase in apoptosis seen in the combinatorial treatment is likely through a reduction in AKT promotion of cell survival, growth and proliferation,
allowing the cells to be more sensitive to the apoptotic mechanisms of ABT-263. The increase in apoptosis was confirmed with the evaluation of cCASP3 levels, which were markedly elevated in the combination arm. CASP3 in its cleaved form signals that an irreversible step in the apoptotic pathway has been reached and that cell death is imminent.
Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer’s disease. Evidence for apoptotic cell death.
have shown that potassium channels play a role in programmed cell death and the Kv1.3 channel on mitochondrial membranes has been implicated in the regulation of Bax induced apoptosis in lymphocytes. As a lipophilic drug, amiodarone can penetrate cellular membranes and could conceivably access the mitochondrial membrane. Others have demonstrated that intracellular accumulation of amiodarone does occur using chasing experiments.
Cellular accumulation of amiodarone and desethylamiodarone in cultured human cells. Consequences of drug accumulation on cellular lipid metabolism and plasma membrane properties of chronically exposed cells.
Therefore, it is possible that enhancement of apoptosis by amiodarone may be aided by inhibition of the Kv1.3 channel, inducing release of reactive oxygen species. The value of combining these compounds is further explained by the interaction of amiodarone with the P-glycoprotein (MDR1), an ATP-binding cassette (ABC) transporter. ABC transporters have been implicated in chemotherapeutic resistance in certain cancers.
studied the kinetics of P-glycoprotein by using amiodarone as a reverser of this transporter and showed an increase in intracellular cytotoxin and cell death. Also, ABT-263 has been revealed to be a substrate of P-glycoprotein.
Treatment of leukemia cells with amiodarone may allow greater intracellular accumulation of ABT-263, thereby inducing greater apoptotic signals. Figure 7 summarizes the proposed modes of action of the 2 drugs and shows how they are interconnected in a manner that could explain enhancement of cell death observed in this study.
FIGURE 7Mechanism for the actions of ABT-263 and amiodarone. Summary of proposed mechanism that allow amiodarone to increase leukemia cell sensitivity to ABT-263. The ( – ) symbol indicates negative or down regulation and the ( + ) symbol indicates upregulation. Inhibition of antiapoptotic BCL-2 family proteins by ABT-263 is shown, leading to greater cytochrome C release and more apoptosis. Amiodarone reduces p-AKT by interfering with PI3K phosphorylation of AKT, likely through reduction in recruitment to potassium channels near the plasma membrane, such as with hERG1. The inhibition of Kv1.3 by amiodarone allows greater release of ROS, which leads to potentiation of cytochrome C release. Furthermore, P-glycoprotein efflux of ABT-263 is reduced by amiodarone, allowing more apoptotic signaling to occur by increasing intracellular levels of ABT-263. ROS, reactive oxygen species.
Although the data support efficacy of the in vitro combination of amiodarone with ABT-263, in vivo experiments may show some limitations. One obvious constraint regarding the use of amiodarone in cancer in vivo is its effects on cardiac rhythmicity. However, further experiments would need to be conducted to assess the effective concentration of amiodarone in vivo. When combining drugs, it would be expected that the effective doses of each drug would be lower than if they were used individually. This may limit the cardiac impact and side effects of amiodarone. In addition, targeting the drug to cancer cells and avoiding systemic effects altogether would be ideal. The lipophilicity of amiodarone allows it to be packaged into liposomes, and this technique was utilized in a human prostate carcinoma model. Liposomes containing the combination of doxorubicin and amiodarone incubated with the prostate carcinoma cells showed a decrease in cell survival.
If a similar technique were utilized to target amiodarone and ABT-263 specifically to leukemia cells in an in vivo model, it is likely to improve efficacy and reduce side effects. Future studies will attempt to further elucidate the mechanism by which amiodarone downregulates AKT signaling and understand other possible effects related to apoptosis. In addition, establishment of more clinically relevant data by exploring this therapeutic combination in vivo and in other cancer types will improve our understanding of the therapeutic potential of amiodarone.
CONCLUSIONS
In conclusion, amiodarone reduces AKT phosphorylation in leukemia cells and may improve ABT-263 effectiveness by increasing reactive oxygen species production and inhibiting P-glycoproteins. The enhanced cytotoxicity of combinatorial amiodarone and ABT-263 observed in this study suggests that using antiapoptotic Bcl-2 family inhibitors in conjunction with K+ channel blockers may be a unique and effective therapeutic approach in treating leukemia.
Acknowledgments
We are grateful for the input and help we received from colleagues in the Beverly-Siskind laboratory. CJK developed the hypothesis, designed and performed experiments and wrote the manuscript.
Authors Contributions
CJK developed the hypothesis, designed and performed experiments and wrote the manuscript. CK performed experiments and wrote the manuscript. AB helped with experimental design and reviewed manuscript. LJB supported work and provided feedback and guidance on experiments and result interpretation.
References
Farber S.
Diamond L.K.
Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-aminopteroyl-glutamic acid.
Acute myeloid leukemia cells harboring MLL fusion genes or with the acute promyelocytic leukemia phenotype are sensitive to the Bcl-2-selective inhibitor ABT-199.
VEGFR-1 (FLT-1), beta1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome.
HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors.
K+ Channel inhibition differentially regulates migration of intestinal epithelial cells in inflamed vs. non-inflamed conditions in a PI3K/Akt-mediated manner.
Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer’s disease. Evidence for apoptotic cell death.
Cellular accumulation of amiodarone and desethylamiodarone in cultured human cells. Consequences of drug accumulation on cellular lipid metabolism and plasma membrane properties of chronically exposed cells.
☆This work was supported by National Cancer Institute, United States Grant R25-CA134283 to Dr. David Hein for the Cancer Education Program summer research at the University of Louisville; Kosair pediatric cancer research program to LJB; James Graham Brown Cancer Center to LJB; IPIBS to CK.
☆☆The authors have no conflicts of interest to disclose.
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