Diosmetin Induces Apoptosis of Acute Myeloid Leukemia Cells

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Diosmetin Induces Apoptosis of Acute Myeloid Leukemia Cells Alessia Roma,† Sarah G. Rota,† and Paul A. Spagnuolo*,†,‡ †

Department of Food Science, University of Guelph, 50 Stone Road E, Guelph, Ontario, Canada, N1G 2W1 School of Pharmacy, University of Waterloo, 10A Victoria Street S, Kitchener, Ontario, Canada, N2G 1C5

‡

S Supporting Information *

ABSTRACT: Acute myeloid leukemia is an aggressive disease with limited and nonselective therapeutic options. This study explored the bioactivity and cell death inducing mechanism of diosmetin, a novel compound identified in a nutraceutical screen to impart selective anti-AML activity. Diosmetin, a citrus flavone, induced apoptosis characterized by increases in caspases 8 and 3/7 and the death inducing cytokine TNFα. In fact, through protein and mRNA expression analysis, activity was shown to be dependent on expression of estrogen receptor (ER) β. Treatment with diosmetin also delayed tumor growth in AML mouse xenografts. In summary, these studies highlight diosmetin as a novel therapeutic that induces apoptosis through estrogen receptor β. KEYWORDS: apoptosis, cancer, diosmetin, leukemia, estrogen receptor

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including bergamot, mandarin, and orange,12−14 to target estrogen receptor (ER) β in AML.15 Estrogen signaling is mediated through two ERs, denoted ERα and ERβ, which participate in a variety of biological processes and diseases including cancer.16 While structurally similar, the ERs demonstrate differential tissue expression and biological activity.17 ERα is predominantly expressed in vaginal, uterine, and pituitary tissues, where it exerts a proliferative effect.18,19 In contrast, ERβ elicits an antiproliferative response and is preferentially expressed in lung, colon, prostate, mammary gland, and bone marrow stem cells.20−24 Modulation of estrogen signaling has been studied as a therapeutic strategy in a variety of cancers. ERα is involved in the onset of breast cancer where tamoxifen, an ERα antagonist, is clinically used to combat proliferative estrogenic effects.25 Synthetic ERβ agonists have been shown to induce apoptosis of prostate and hepatic cancer tissue.26,27 In hematological malignancies, ERβ was found to be the predominant ER expressed in lymphoma and targeting ERβ strongly inhibited lymphoma cell growth.28 Additionally, knockout of ERβ in mice led to the development of a myeloproliferative disease resembling chronic myeloid leukemia (CML), indicating a role of ERβ in regulating hematopoietic differentiation.24 Our previous work showed that diosmetin was an ERβ agonist that selectively targeted LSCs in vitro and in vivo.15 Indeed, preclinical studies clearly demonstrated the ability of diosmetin to reduce engraftment of patient derived AML cells in primary and secondary mouse models. In addition, we showed that patient-derived AML cells with high ERβ expression were

INTRODUCTION Acute myeloid leukemia (AML) is a hematological malignancy characterized by the improper differentiation of myeloid cells that accumulate in the blood and bone marrow.1 It is the most common acute leukemia in adults with over 21 000 new diagnoses annually in the United States.2 Cytarabine and anthracyclines3 are the backbone of AML therapy; however, their use is associated with intolerable side effects particularly for patients over the age of 60, which comprise the majority of the AML patient population.4 These factors contribute to a 5-year survival rate of 40% in patients under the age of 60, which significantly decreases to only 5−15% in those over 60 years of age.5 It is evident that AML is a disease that requires investigation for novel therapeutic targets to improve patient outcomes. The suboptimal nature of induction therapy (i.e., low rates of survival) is, in part, attributed to the inability of drugs to target and eliminate leukemia stem cells (LSCs), a rare subpopulation of the bulk AML cells responsible for disease onset and patient relapse.6 As such, previous research has identified various metabolic and molecular targets with potential to preferentially eradicate this rare population. The antimicrobial agent tigecycline was shown to inhibit mitochondrial translation of LSCs and AML blasts to induce cytotoxicity.7,8 Avocatin B, a fatty alcohol derived from avocados, selectively killed AML stem and leukemia progenitor cells by targeting mitochondria and inhibiting fatty acid oxidation.9,10 Venetoclax targets the overexpression of BCL-2 in LSCs but not hematopoietic stem cells.11 Although these compounds have shown selective targeting toward AML blasts and leukemia progenitor cells, they are all still under preclinical or early clinical evaluation. We recently explored the ability of diosmetin, a flavone aglycon found in parsley, rosemary, and a variety of citrus fruits © XXXX American Chemical Society

Received: December 27, 2017 Accepted: January 16, 2018

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DOI: 10.1021/acs.molpharmaceut.7b01151 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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measured by the MTS assay. Compounds which exhibited the greatest reduction in cell growth were then further tested in the TEX and K562 cell lines at increasing concentrations with the MTS assay. Immunoblotting. Whole cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Sigma Chemical) with the addition of a protease inhibitor cocktail (Sigma Chemical). Samples were then reduced in SDS at 95 °C for 5 min and loaded into an SDS−PAGE gel, where proteins were separated at 150 V for 75 min. Proteins were then transferred to a PVDF membrane and blocked in a 5% bovine serum albumin (BSA) solution in tris-buffered saline Tween (TBS-T) for 1 h. Membranes were then incubated overnight with ERα or ERβ primary antibody (1:750, Santa Cruz Biotechnologies, Dallas, TX) at 4 °C. For U2OS-ERβ cells, anti-FLAG antibody was used (1:1000, Thermo Fisher). GAPDH (1:15000) was used as the loading control. Following incubation, membranes were washed in TBS-T and incubated with the appropriate secondary antibody (1:8000, Santa Cruz Biotechnologies) for 1 h. Proteins were visualized using enhanced chemiluminescence (ECL) solution (Biorad Laboratories, Hercules, CA) and imaged with the Kodak Image Station 4000MM Pro. Kodak Molecular Imaging Software Version 5.0.1.27 was used to perform densitometric analysis. Quantitative PCR. To detect mRNA, quantitative PCR (qPCR) was performed, as described previously.31 Briefly, RNA was isolated using a mammalian RNA isolation kit (Qiagen, Hilden, Germany) and then used to make cDNA with a commercially available kit (Thermo Fisher, Waltham, MA). Next, 5 ng of RNA equivalent cDNA, SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA), and 300 nM of ER-specific primers (forward ERβ 5′-TGCTCAATTCCAGTATGTACC-3′, reverse ERβ 5′-ATGAGGTGAGTGTTTGAGAG-3′; forward ERα 5′-CATTATGGAGTCTGGTCCTG-3′, reverse ERα 5′TTCGTATCCCACCTTTCATC-3′) were added together, and PCR products were measured on the ABI 7900 Sequence Detection System (Applied Biosystems). ERα or ERβ were normalized to their respective GADPH values using the ΔΔCT method, as previously described31 to obtain relative mRNA expression. Primer efficiency was calculated using LinRegPCR analysis software.32,33 SubG1 Peak Analysis. To assess DNA fragmentation, a hallmark of apoptosis, subG1 peak analysis was performed on TEX cells treated with diosmetin (10 μM).34 The microtubule depolymerizing agent nocodazole (1 μM) was used as a positive control.35 Following treatment, cells were collected and suspended in PBS and cold absolute ethanol. Next, 100 ng/mL of DNasefree RNase A (Invitrogen; Carlsbad, CA) was added, and cells were incubated for 30 min at 37 °C. Cells were washed, and PI was added and incubated for 15 min. DNA content was measured by flow cytometry and analyzed with the Guava Cell Cycle software (Millipore). ROS Detection. To study the oxidative effects of diosmetin, TEX cells were treated with diosmetin (10 μM) or 50 μM antimycin A, as a positive control.36 Following treatment, intracellular ROS was measured with the fluorescent dye 2′,7′-dicholorofluorescein diacetate (DCFH/DA; Sigma Chemical) as previously described.9 Caspase Activity. TEX cells were treated with diosmetin and then lysed at various time points. Caspase 3/7 and 8 activation was measured using the Apo-ONE Homogeneous Caspase-3/7 (Promega) kit, as previously described.37 Caspase 8 (Ac-LETDAFC; Enzo Life Sciences, Farmingdale, NY) and caspase 9 substrates (Ac-LEHD-AMC; Enzo Life Sciences) were used according to the manufacturer’s protocol. Equal volumes of cell lysate and

increasingly sensitive to diosmetin and that ERβ was functionally important to diosmetin’s cytotoxicity using genetic knock-down and knock-in studies.15 In the present study, we extend these findings and report that diosmetin induces apoptosis in AML cells that is characterized by increases in TNFα and activation of caspase 8. Furthermore, TNFα activation was dependent on ERβ expression, which represents a potential relationship between ERβ and TNFα-induced AML cell apoptosis. Together, these studies demonstrate the ERβ-modulating effects of diosmetin and the potential benefit of ERβ targeting for the treatment of AML.

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EXPERIMENTAL SECTION Cell Culture. The AML cell line OCI-AML2 and the CML line K562 were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies; Grand Island, NY). The surrogate leukemia stem cell line TEX29 was grown in IMDM medium supplemented with 15% (v/v) fetal bovine serum (FBS; Seradigm;VWR; Mississauga, ON) and 20 ng/mL stem cell factor, 2 ng/mL IL3 (Peprotech; Hamburg, Germany), and 2 mM L-glutamine (Sigma Chemical; St. Louis, MO). The prostate cancer cell line, DU145, and human myeloma cell line, LP1, were cultured in RPMI medium (Life Technologies). U2OS human osteosarcoma cells with epitope (FLAG)-tagged ERβ under the doxycycline promoter (U2OS-ERβ) were created by Monroe et al.30 and maintained in RPMI medium. Unless otherwise stated, all media were supplemented with 10% FBS and an antibiotic solution consisting of 200 μg of streptomycin and 200 units of penicillin per milliliter of medium. All cells were maintained in an incubator at 37 °C with 5% CO2 and 95% humidity. Cell Growth and Viability. To measure cell proliferation and viability, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) reduction assay (Promega, Madison, WI), ANN V and propidium iodide (ANN/PI) staining, or 7-aminoactinomycin D (7-AAD; Caymen Chemicals, Ann Arbor, MI) were used. For the MTS assay, cells were incubated with 20 μL of MTS for 2 h at 37 °C and 5% CO2 and then absorbance at 490 nm was read using a SpectraMax M5 spectrophotometer (Molecular Devices; Sunnyvale, CA). For ANN/PI or 7-AAD staining, cells were suspended in 250 μL of staining solution consisting of annexin V binding buffer (Biovision; Milpitas, CA) supplemented with 2% (v/v) FBS and 1 μL each of annexin V-FITC (150 μg/mL) (Biovision; Milpitas, CA) and propidium iodide (100 μg/mL) (PI; Biovision) or 7-AAD (1 μg/mL) and incubated at room temperature for 15 min. Fluorescence was measured with the Guava EasyCyte 8HT Benchtop Flow Cytometer (Merck Millipore; Dermstadt, Germany). The pan-caspase inhibitor z-VAD-FMK (Z-VAD; R&D Systems, Minneapolis, MN) was also used to study the role of caspase enzymes in diosmetin’s cytotoxicity. Here, TEX cells were treated with diosmetin in the presence or absence of 50 μM Z-VAD. Following a 72 h incubation period, cell viability was measured using the PI exclusion assay and flow cytometry. Nutraceutical Screen. A unique nutraceutical library was created and screened as described previously.7 Nutraceuticals were obtained from Chengdu Biopurify Phytochemicals Ltd. (n = 288, Sichuan, China). All compounds, including the lead compound, diosmetin (Sigma Chemical; St. Louis, MO), were reconstituted in dimethyl sulfoxide (DMSO) and diluted in phosphate buffered saline (PBS) to ensure that in vitro concentrations of DMSO did not exceed 0.05%. TEX and K562 cells were seeded in 96-well clear bottom tissue culture plates at a concentration of 1.5 × 105 cells/mL. Cells were then treated with 10 μM of each library compound for 72 h. Following treatment, cell viability was B


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Molecular Pharmaceutics reaction buffer (1 M DTT, 100 mM EDTA, and 20 mM pH 7.4 Tris buffer) were mixed in a white 96-well plate and incubated for 60 min at 37 °C. Fluorescent measurements were taken from the wells using the appropriate emission and excitation wavelengths. z-VAD-FMK, a pan-caspase inhibitor, was used as a control. Detection of Intracellular TNFα. TEX and K562 cells were treated with diosmetin (20 μM) for 12 h or lipopolysaccharide (LPS, 5 mg/mL), as a positive control, for 4 h.38 Cells were then collected, fixed, and permeabilized using 3% paraformaldehyde (Sigma Chemical) in BD Perm/Wash buffer (BD Biosciences; San Jose, CA). Next, cells were stained with APC mouse antihuman TNFα antibody (BD Biosciences), according to the manufacturer’s protocol, and fluorescence was measured via flow cytometry. In addition, U2OS-ERβ cells were incubated with 100 ng/mL of doxycycline for 24 h, as previously described30 to induce expression of ERβ prior to measurement of intracellular TNFα. To further study the importance of TNFα activation, TEX cells were pretreated with a TNFα neutralizing antibody (Biolegend;San Diego, CA) for 2 h and then coincubated with diosmetin. After 72 h, cell viability was assessed using the PI exclusion assay and flow cytometry. In Vivo Studies. Four- to six-week-old nondiabetic severe combined immunodeficient mice (NOD/SCID) were obtained from Jackson Laboratory (Bar Harbor, ME) and acclimatized for 1 week. Mice were inoculated subcutaneously in the left flank with TEX leukemia cells (2.5 × 106). After the formation of a palpable tumor (day 7), mice were then randomly separated into two groups and given either diosmetin (75 mg/kg/every other day; in 0.9% NaCl and 0.01% Tween-80) or vehicle control (0.9% NaCl and 0.01% Tween-80) via an intraperitoneal injection three times weekly. After 17 days, mice were euthanized, and tumors were excised and weighed. All animal studies were carried out according to the regulations of the Canadian Council on Animal Care and with the approval of the University of Waterloo, Animal Care Committee. Statistical Analysis. All statistical analysis was conducted using the GraphPad 5.0 Prism software. Results are expressed as the mean ± standard deviation unless otherwise stated. Inhibitory concentrations were calculated using the nonlinear regression 4-parameter logistic model. Where appropriate, significance between values was determined by a paired, two-tailed t test or a one-way ANOVA paired with a Tukey’s post hoc test or a twoway ANOVA with a Bonferroni post hoc test. Mann−Whitney t tests were used for animal experiments. p < 0.05 was considered to be statistically significant.

Figure 1. Diosmetin induces selective toxicity toward AML cells. (A, B) (left) A screen of a nutraceutical library identified diosmetin (arrow), as the agent which imparted the greatest reduction in the growth of TEX cells, a surrogate LSC cell line, (right) without affecting the growth of K562 cells, a CML cell line lacking stem cell activity. Cell growth was assessed using the MTS assay, and compounds were ranked based on their ability to reduce growth of TEX and K562 cells. The cytotoxicity of diosmetin (structure: C) was measured in a dose-dependent manner by (D) the ANN/PI assay following a 72 h incubation period. Data presented as an average of 3 independent experiments; ***p < 0.001.

Cell Line Sensitivity Is Linked to ERβ. The activity of diosmetin was tested in a panel of cell lines. TEX and LP1 cells displayed the highest level of sensitivity while K562 and DU145 cells were insensitive to diosmetin (Figure 2A). Since a previous study completed by our lab linked diosmetin’s activity to estrogen receptors,15 we examined whether diosmetin’s sensitivity in these cell lines was related to ER levels by quantifying mRNA using qPCR. Diosmetin-sensitive TEX cells displayed high levels of ERβ but undetectable amounts of ERα while the opposite trend was observed in the insensitive K562 cells (Figure 2B). In support of this, immunoblotting of ER proteins revealed increased levels of ERβ in TEX and LP1 cells (i.e., diosmetin-sensitive) but not in K562 and DU145 cells (i.e., diosmetin-insensitive, Figure 2D). No such pattern was observed with ERα protein (Figure 2C). Together, this indicates that cell line sensitivity to diosmetin is related to expression of ERβ (Figure 2E). Diosmetin Induces Extrinsic Apoptosis. The mechanism of diosmetin-induced death was initially explored using the ANN/PI assay. TEX cells treated with diosmetin displayed a timedependent increase in the percent of apoptotic cells (i.e., ANN+/ PI−; Figure 3A). Diosmetin also increased the percentage of cells in the subG1 phase of the cell cycle, indicative of DNA fragmentation, a hallmark of apoptosis (Figure 3B).34 DNA damage and apoptosis are often preceded by oxidative stress; thus, to measure levels of reactive oxygen species (ROS) in diosmetin-treated TEX cells, DCFH-DA was used.34 Diosmetin increased ROS levels in TEX cells at times preceding the onset of apoptosis, suggesting ROS involvement in diosmetin’s cytotoxicity (Figure 3C).

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RESULTS Diosmetin Is Selectively Toxic toward TEX over K562 Cells. A unique library consisting of 288 nutraceuticals was created to identify novel compounds with selective toxicity toward AML cells. Each compound was screened against TEX cells, an AML cell line with LSC characteristics, as well as K562 cells, a CML line lacking any LSC properties (Supplementary Figure 1). Following treatment with library compounds for 72 h, cell growth was measured using the MTS assay (Figure 1A). The top 12 compounds exerting effects toward TEX but not K562 cells were then assessed using the MTS assay (Figure 1B). Through this analysis, diosmetin (Figure 1C, compound 73) was confirmed as the compound imparting the greatest activity toward TEX cells without affecting K562 cell viability. Selective toxicity was further confirmed in dose response studies using the ANN/PI assay (Figure 1D; EC50 6.8 ± 1.7 μM). C


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The effects of diosmetin were measured on caspases 8 and 9, which are upstream enzymes indicative of extrinsic and intrinsic apoptosis, respectively, and caspases 3/7, which are terminal caspases that execute apoptotic signaling.34 Treatment with diosmetin resulted in a time-dependent increase in caspases 3/7 that was attenuated by the pan-caspase inhibitor Z-VAD (Figure 3D). Diosmetin also activated caspase 8 (Figure 3E) but not caspase 9 (Figure 3F), suggesting that diosmetin’s cytotoxicity occurs through the extrinsic pathway of apoptosis. However, we do note the limitation that mRNA and/or protein levels of caspase enzymes were not measured. Finally, cotreatment of TEX cells with ZVAD-FMK attenuated diosmetin’s cytotoxicity, further highlighting the role of caspase activity in diosmetin-induced death (Figure 3G). TNFα Increases in Diosmetin Sensitive Cells. Since diosmetin induced the extrinsic or death receptor pathway of apoptosis, we next wanted to establish whether it increased TNFα, a ligand that plays a central role in extrinsic apoptosis.39 To test our hypothesis that TNFα was involved in diosmetin’s activity, we first measured levels of TNFα in TEX and K562 cells, which differ based on their diosmetin sensitivity and levels of ERβ expression. Cells were treated with diosmetin for 12 h and levels of intracellular TNFα were measured by flow cytometry. Diosmetin increased intracellular TNFα in the diosmetin and ERβ expressing TEX cells but not in the insensitive and low ERβ expressing K562 cells (Figure 4A). To confirm the relationship between diosmetin activity and ERβ expression, a doxycycline inducible flag-tagged ERβ cell line (U2OS-ERβ) was used.30 In this system, the addition of doxycycline increased ERβ expression 4-fold (Figure 4B). Interestingly, diosmetin increased TNFα expression only in the presence of doxycycline, and this increased expression was absent without doxycycline addition (i.e., in the absence of ERβ; Figure 4C,D). Additionally, cotreatment of TEX cells with an antibody capable of neutralizing TNFα (Figure 4E) blocked diosmetin-induced cytotoxicity (Figure 4F). Given our previous report showing that diosmetin also binds to ERα, we hypothesized that blocking ERα

Figure 2. Diosmetin targets estrogen receptor β. (A) A panel of cell lines were tested for sensitivity to diosmetin using the MTS assay following a 72 h treatment period. (B) TEX and K562 cells were analyzed for ERα and ERβ mRNA expression by qPCR. Data represents the relative mRNA expression normalized to the GAPDH gene. (C, D) Protein levels of ERα or ERβ in the cell line panel were assessed by immunoblotting. (E) High ERβ to ERα ratios positively correlate with sensitivity to diosmetin. Data represented as the relative densitometry of each cell line normalized to their respective GAPDH loading control. Values were normalized to TEX cells. Data presented as an average of 3 independent experiments and representative blots are shown; ***p < 0.001.

Figure 3. Diosmetin activates the extrinsic pathway of apoptosis. (A) TEX cells were treated with 10 μM diosmetin, and apoptosis was measured by the ANN/PI assay. Data was normalized to the vehicle control and is presented as a mean percentage of apoptotic cells ± SD from 3 independent experiments. (B) Following treatment with diosmetin, DNA fragmentation of TEX cells was measured by quantifying the sub G1 phase of the cell cycle. Data represent the percent of cells in the sub G1 phase, relative to the vehicle control. Nocodazole, at 48 h of incubation, was used a positive control. (C) Reactive oxygen species (ROS) were measured in TEX cells treated with 10 μM diosmetin using DCFH-DA and flow cytometry. Antimycin A at 12 h of incubation was used as a positive control. Data are presented as fold increase compared to vehicle control ± SD. (D−F) TEX cells were treated with diosmetin (10 μM), and activation of caspases 3/7, 8, and 9 were measured by a commercially available activation assay. The pan-caspase inhibitor ZVAD-FMK was used as a negative control for caspase activation. (G) TEX cells were cotreated with increasing concentrations of diosmetin and ZVAD-FMK (50 μM) for 72 h. Cell viability was measured by PI staining and flow cytometry. Data was normalized to the vehicle control; **p < 0.01, ***p < 0.001. D


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vehicle control via intraperitoneal injection, three times weekly for 17 days (Figure 5A). Tumor volumes were monitored every 2−3 days. Tumor growth was delayed in mice treated with diosmetin compared to control treated mice (Figure 5B). At end point, mice were euthanized, and tumors were excised and weighed. Tumors from diosmetin treated mice weighed significantly less than those from the vehicle control group, indicating that diosmetin is able to delay tumor growth in vivo (Figure 5C).

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DISCUSSION Through this investigation, the citrus flavone diosmetin was shown to induce apoptosis characterized by increases in TNFα. The activity of diosmetin was further linked to the expression of ERβ, and its bioactivity was demonstrated in vitro and in vivo. In our previous study, there was a direct positive correlation between ERβ expression and AML cell sensitivity to diosmetin.15 Additionally, diosmetin preferentially bound (>3-fold) to ERβ, and when ERβ was genetically silenced, these AML knockdown cells became diosmetin insensitive.15 In this study, we extend these observations to demonstrate that cell lines expressing ERβ are diosmetinsensitive whereas cell lines without ERβ are diosmetin-insensitive. Moreover, we linked ERβ to diosmetin-induced apoptosis characterized by elevated caspases 3/7 and 8 and increases in intracellular TNFα. Diosmetin belongs to a class of secondary plant metabolites known as phytoestrogens, which are reported to exhibit estrogenic activity.12 Phytoestrogens, defined as plant-derived compounds that are similar in structure and/or function to mammalian estrogen, have been widely shown to possess anticancer activity. Other phytoestrogens (i.e., quercetin and genistein) have been shown to reduce cancer cell proliferation and induce ER-dependent apoptosis in breast, ovarian, and cervical cancer cell lines.40−43 The antiproliferative effects of phytoestrogens are also observed in non sex hormone dependent tissues. In T-cell leukemia cells, genistein induced cell cycle arrest and apoptosis through inhibition of the antiapoptotic NF-κB pathway;44 resveratrol and quercetin performed similarly in chronic lymphocytic leukemia cells.45 Quercetin has also been shown to inhibit tumor growth in vitro and in vivo in AML cells through activation of caspases and mitochondrial membrane depolarization.46 Collectively, phytoestrogens possess anticancer effects and, we demonstrate here, the anti-AML effects of the phytoestrogen diosmetin. Diosmetin increased intracellular levels of TNFα only in cells expressing ERβ. Moreover, cotreatment with a TNFα neutralizing antibody blocked the activity of diosmetin. TNFα plays a role in several biological processes, and in this study, intracellular rather than extracellular TNFα was examined. This is primarily because of our initial observation that diosmetin activated caspase 8 and not caspase 9, suggesting initiation of the extrinsic or deathreceptor mediated pathway of apoptosis. Intracellular TNFα is capable of inducing extrinsic apoptosis resulting in activation of caspase 8 or regulation of cell death through NF-κB.34 NF-κB is a transcription factor that is constitutively active in leukemia but not normal hematopoietic stem cells; it has been explored as an anti-AML therapeutic target.47,48 In prostate cancer, ERβ was found to negatively regulate NF-κB to overcome apoptotic evasion, and loss of ERβ contributed to prostate cancer development.49 Similarly, in hepatocellular carcinoma, ERβ overexpression increased expression and activity of TNFα and caspase 8 signaling.27 TNFα and ERβ are also responsible for regulation of monocyte differentiation and proliferation to regulate immunity. It has been postulated that TNFα-induced activation of the extrinsic apoptotic pathway occurs only in monocytic cells

Figure 4. Diosmetin activation of TNFα is ERβ expression dependent. (A) TEX cells were treated with 20 μM diosmetin for 12 h or 5 ng/mL LPS (as a positive control) for 4 h and were then fixed and permeabilized. Levels of intracellular TNFα were measured by flow cytometry. (B) Flagtagged U2OS-ERβ doxycycline inducible cells were treated with doxycycline for 24 h to induce expression of ERβ, which was measured with an antiflag antibody by immunoblotting. Data represents the relative protein expression normalized to GAPDH. (C) U2OS-ERβ cells were treated with diosmetin (20 μM) for 12 h with or without doxycycline. Data was normalized to the untreated control. (D) Schematic outline of ERβ associated activation of TNFα in doxycycline inducible cells. (E) TEX cells were treated with 50 ng/mL of TNFα neutralizing antibody for 72 h, and intracellular TNFα was measured by flow cytometry. (F) TEX cells were pretreated with 50 ng/mL of TNFα neutralizing antibody for 2 h and then treated with diosmetin. Following 72 h, cell viability was analyzed using the 7-AAD stain and flow cytometry. (G, H) TEX or OCI-AML2 cells were treated with diosmetin or a combination of diosmetin and the ERα antagonist, tamoxifen (10 μM). Following a 72 h incubation, cell viability was measured by 7-AAD staining and flow cytometry. Data was normalized to the untreated control. Data presented as a summary of 3 independent experiments.

binding sites would increase diosmetin’s activity. To test this hypothesis, we assessed diosmetin’s activity in the presence of the well-known ERα antagonist, tamoxifen. Interestingly, tamoxifen does not impart toxicity in AML cells (Supplementary Figure 2), but significantly increases diosmetin’s cytotoxicity (Figure 4G,H). Together, these results confirm that diosmetin’s activity is dependent on ERβ expression and mediated by TNFα. Diosmetin Inhibits Growth of AML Xenograft Tumors in Vivo. To study the effects of diosmetin in vivo, 4−6-week-old NOD/SCIDγ (NSG) mice were subcutaneously injected with TEX cells in the left flank. Following the formation of a palpable tumor (7 days), mice were treated with 75 mg/kg diosmetin or a E


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Figure 5. Diosmetin delays tumor growth in vivo. (A) NOD/SCID mice were subcutaneously injected with TEX cells. Following the formation of palpable tumors (1 week; see arrow in panel B), mice were treated with 75 mg/kg diosmetin or vehicle control three times a week for 17 days. (B) Tumor volumes were measured throughout the study, and volume was calculated as L × W × 0.5236. (C) Following euthanasia, tumors were excised and measured. Mean tumor weights between groups were compared using the Mann−Whitney method; **p < 0.01.

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expressing ERβ and is lost in more mature macrophages, which only express ERα.50 In myeloid leukemia, TNFα elicits an antiproliferative response, which was demonstrated through introduction of exogenous TNFα in various cell lines;51 patients with lower TNFα apoptosis inducing ligand (TRAIL) had a predicted shorter overall survival.52 Our findings that diosmetin increased TNFα levels that were linked to ERβ expression complement these studies showing a link between ERβ, TNFα, and cancer cell apoptosis. Future studies are needed to further elucidate the interplay between these factors and NF-κB in diosmetin-induced apoptosis. Phytoestrogens are also linked with anti-inflammatory effects and decreased production of TNFα through ERβ. When ERβ was stably transfected into osteosarcoma or U937 cells that did not previously express ERs, TNFα levels were significantly lower following estradiol exposure compared to similar cells stably transfected with ERα.53−55 The differential response of TNFα to estrogen signaling may be a result of tissue-specific expression and activity of ERs as well as expression of TNF receptors.18 Indeed, physiological factors including age, genotype, and health status as well as molecular factors that include tissue specificity and accumulation of ligand are all factors that contribute to the cellular response to estrogen, phytoestrogens, and the selective estrogen receptor modifiers.56,57 Although this study focused on the link between TNFα, disometin, and ERβ, future studies should also explore the role of ERα as well as other more potent ERβββ agonists to further define the anti-AML role of ERβ signaling. In summary, diosmetin induced apoptosis that was mediated by ERβ and TNFα. These findings complement our previous report that showed in vitro and in vivo activity against leukemia and leukemia stem cells, which together highlight diosmetin as a novel anti-AML agent.

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AUTHOR INFORMATION

Corresponding Author

*Department of Food Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. Phone: (519) 824-4120 x53732. E-mail: [email protected]. ORCID

Paul A. Spagnuolo: 0000-0002-2431-4368 Author Contributions

A.R. and S.G.R performed experiments, analyzed data, and cowrote the manuscript. P.A.S. designed experiments, analyzed data, and cowrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge Drs. John E. Dick and David G. Monroe for their generous gift of TEX cells and doxycyclineinducible ERβ cells, respectively. This work was supported by grants to P.A.S. by the Stem Cell Network and the University of Waterloo, Ontario Research Fund, and the Canadian Foundation of Innovation.

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ABBREVIATIONS USED AML, acute myeloid leukemia; CML, chronic myeloid leukemia; DCFH/DA, 2′,7′-dicholorofluorescein diacetate; Dios, diosmetin; DMSO, dimethyl sulfoxide; ER, estrogen receptor; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; LPS, lipopolysaccharide; LSC, leukemia stem cell; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; PI, propidium iodide; ROS, reactive oxygen species; TNF, tumor necrosis factor

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ASSOCIATED CONTENT

REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b01151. Supplementary Figures 1 and 2 (PDF) F


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Diosmetin Induces Apoptosis of Acute Myeloid Leukemia Cells

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