Curcumin and Dimethoxycurcumin Induced Epigenetic Changes in Leukemia Cells
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Curcumin is an ideal chemopreventive and antitumor agent characterized by poor bioavailability and low stability. The development of synthetic structural analogues like dimethoxycurcumin (DMC) could overcome these drawbacks. In this study we compared the cytotoxicity, metabolism and the epigenetic changes induced by both drugs in leukemia cells.
Apoptosis and cell cycle analysis were analyzed by flow cytometry. Real-time PCR was used for gene expression analysis. DNA methylation was analyzed by DNA pyrosequencing. The metabolic stability was determined using human pooled liver microsomes. Chromatin Immunoprecipitation was used to quantify histone methylation.
Clinically relevant concentration of curcumin and DMC were not cytotoxic to leukemia cells and induced G2/M cell cycle arrest. DMC was more metabolically stable than curcumin. Curcumin and DMC were devoid of DNA hypomethylating activity. DMC induced the expression of promoter methylated genes without reversing DNA methylation and increased H3K36me3 mark near the promoter region of hypermethylated genes.
DMC is a more stable analogue of curcumin that can induce epigenetic changes not induced by curcumin. DMC induced the expression of promoter methylated genes. The combination of DMC with DNA methyltransferase inhibitors could harness their combined induced epigenetic changes for optimal re-expression of epigenetically silenced genes.
KEY WORDScurcumin dimethoxycurcumin DNA methylation DNA pyrosequencing histone methylation
- 5 AC
- Annexin V-PE
Ten Eleven Translocation
Uridine 5′-diphosphoglucuronic acid
Curcumin, a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa (turmeric) has a wide spectrum of pharmacological activities (1). Curcumin is the active ingredient of the Indian spice turmeric in addition to the other two ingredients, demethoxycurcumin and bisdemethoxycurcumin. Curcumin demonstrated anti-inflammatory (2), antimicrobial (3), antiviral (4), antioxidant (5) and anti-tumorigenic activity (6, 7, 8) in several studies. Curcumin is considered an ideal chemopreventive and antitumor agent because of its multiple targets. Curcumin is safe when administered at high doses; however, its low bioavailability due to poor absorption and rapid metabolism is a major drawback. Different formulation based approaches were adopted to overcome its low bioavailability like liposomal curcumin and curcumin nanoparticles. Additionally, several structural analogues like pyrimidine-substituted curcumin analogues (9), carbonyl moiety modified analogues (10), dimethoxycurcumin (DMC) (11), T63 (12), EF31 (13), UBS109 (13) and C086 (14) were also synthesized to improve the solubility and bioavailability of curcumin.
Curcumin and its analogues were shown to induce epigenetic changes in tumor cells. Curcumin modulated histone acetylation by inhibiting histone deacetylase (HDAC) and histone acetyltransferase (HAT) enzymes in tumor cells (15). Curcumin modulated microRNAs (miRNAs) expression in tumor cells (16). Moreover, curcumin demonstrated a DNA hypomethylating effect and induced the expression of silenced promoter-methylated genes in several studies (13, 17, 18, 19, 20, 21, 22). However, other reports demonstrated that curcumin lacks a DNA hypomethylating effect or only modify DNA methylation in partially methylated loci (23, 24). The controversy regarding the DNA hypomethylating effect of curcumin and its analogues remains to be further elucidated.
Curcumin was also shown to induce apoptosis and cell cycle arrest in tumor cells (1, 8). Unfortunately, most of the previous reports that studied the epigenetic changes or the cytotoxic effect of curcumin used very high concentrations of curcumin (5–30 μM) that are considered clinically irrelevant. Curcumin achievable concentration in plasma was reported as 1.77 ± 1.87 μmol/L.
In this study, we are reporting the DNA and histone methylation changes induced by clinically relevant concentrations of pure curcumin and its synthetic analogue DMC in leukemia cells. Clinically relevant concentrations of curcumin and DMC were not cytotoxic to leukemia cells but induced G2/M cell cycle arrest. DMC but not curcumin induced the expression of promoter-methylated genes like p15 and CDH-1, indicating a possible DNA hypomethylating effect of DMC. Surprisingly, both drugs lacked any significant gene-specific and global DNA hypomethylating activity. Analysis of histone methylation in the CpG island near the promoter region of the p15 and CDH-1 genes showed an increase in H3K36me3 mark after DMC treatment, a mark associated with actively transcribed genes. Our results demonstrate that although both compounds lack a DNA hypomethylating effect, DMC induced the expression of promoter-methylated genes by a mechanism that does not involve DNA methylation reversal.
Materials and Methods
Cell Culture and Chemicals
CEM, BV-173 and Kasumi-1 leukemia cells were grown in RPMI medium supplemented with 10% fetal bovine serum (life Technologies, CA) in a humidified atmosphere containing 5% CO2 at 37°C. Curcumin analytical standard grade (Sigma, WI) was dissolved in DMSO as 10 mM stock. DMC (Cayman, MI) was dissolved in DMSO as a 10 mM stock. 5-azacytidine (5 AC, Sigma, WI) was dissolved in PBS as 10 mM stock. PCR primers were purchased from Integrated DNA Technologies (Coralville, IA).
Apoptosis quantitation was performed by double staining and fluorescence detection using flow cytometry as described previously (25). Briefly, 1 × 106 cells were stained by Guava Nexin Reagent (EMD Millipore, MA) and incubated at room temperature in the dark for 20 min. Guava Nexin Reagent is a mixture of Annexin V-phosphatidylethanolamine (PE) and the cell impermeant dye, 7-aminoactinomycin D (7-AAD). Samples were acquired on a Guava easyCyte 5 system.
Cell Cycle Analysis
Analysis of cell cycle populations was performed by propidium iodide (PI) staining as described previously (26). Sample acquisition was performed on a Guava easyCyte 5 system.
Metabolism of Curcumin and DMC by Human Liver Microsomal Enzymes
The time course metabolism of curcumin and DMC was evaluated using human pooled liver microsomes followed by HPLC analysis. A validated HPLC method with fluorescence detector was used to quantify both curcumin and DMC. The HPLC system consisted of a quaternary pump and a Waters H – class autosampler (Milford, MA). A Waters Symmetry C – 18 analytical column (4.6 × 150 mm, 5 μm) was used. The mobile phase was run on an isocratic condition and consisted of acetonitrile and 10 mM potassium phosphate containing 0.1% TEA (pH = 4.5) (70:30 (v/v)) at a flow rate of 1.0 ml/min. Fluorescence detection wavelengths were 420 nm for excitation and 549 nm for emission. The lower limit of quantification was 0.0025 μg/ml and 0.005 μg/ml, with a linearity range of 0.0025 to 15 μg/ml and 0.005 to 10 μg/ml for curcumin and DMC, respectively. Accuracy and precision were determined by replicate injection of quality control samples. Both precision and accuracy were of satisfactory results below 17% of CV.
Where CLint indicates intrinsic clearance, Vmax indicates the maximum rate achieved at maximum substrate concentration, Km indicates the substrate concentration when the reaction rate is half of Vmax, V0 indicates the initial metabolic rate and C0 is the substrate concentration at time 0. The intrinsic clearance values were calculated separately from each of the replicates. CLint values are presented as mean ± SD from three replicates performed for each reaction.
Quantitative Real-Time PCR Analysis of Gene Expression
RNA was extracted using RNeasy kit according to the manufacturer’s instructions (Qiagen, CA). RNA was treated with DNase enzyme to remove any DNA contamination associated with the process of RNA extraction using the Turbo DNA-free kit (Ambion-Life Technologies, NY) according to the manufacturer’s instructions. cDNA was generated using the Verso cDNA synthesis kit (ThermoScientific, MA). Real-time PCR was performed on a RealPlex II thermal cycler (Eppendorf, NY) using Kapa SYBR Fast qPCR kit (KapaBiosystems, MA) and a two-step cycling protocol with annealing/extension temperature of 60°C. Supplementary Table I shows the primers sequences used for p15, CDH-1, DNA methyltransferase (DNMT) and Ten Eleven Translocation (TET) isotypes.
Pyrosequencing is a sequencing by synthesis method used for quantitation of DNA methylation (27). Biotin-labelled, single-stranded PCR products generated from bisulfite-treated DNA are used as a template with an internal primer to perform the pyrosequencing reaction. DNA was extracted using the Quick-gDNA microprep kit (Zymo Research, CA). Bisulfite treatment of DNA (500 ng) was performed using the EZ DNA Methylation-Gold kit (Zymo Research). Amplification of template DNA for pyrosequencing was performed using a PyroMark PCR kit (Qiagen). Primers for CDH-1 and p15 genes were designed using the PyroMark Assay Design Software (Qiagen) and the reverse primer was biotin labelled. The amplicon length was 170 and 207 bp for the p15 and the CDH-1, respectively. The amplification and the size of the amplicon were verified by agarose gel electrophoresis (supplementary Fig. 1). The sequence to analyze for the p15 gene was CGGGCCGCTGCGCGTCTGGGGGCTGCGGAATGCGCGA and included seven CpG sites (underlined). The sequence to analyze for the CDH-1 gene was CGGCAGCGCGCCCTCACCTCTGCCCAGGACGCGGC and included five CpG sites (underlined). Pyrosequencing was performed on a PyroMark Q24 instrument (Qiagen).
Analysis of global DNA methylation was performed using the Long Interspersed Nuclear Element-1 (LINE-1) assay (28). The CpG sites in LINE-1 sequences are normally heavily methylated and can be used as a surrogate marker for global DNA methylation. PyroMark Q24 CpG LINE-1 kit (Qiagen) was used to quantify the methylation level of three CpG sites in positions 318 to 331 of LINE-1 sequence (GenBank accession number X58075). Briefly, bisulfite treated DNA was used as a template to amplify a 146 bp fragment by PCR using a biotin-labelled reverse primer. The sequence to analyze after bisulfite conversion was TTYGTGGTGYGTYGTTT (Y indicates C or T) and included three CpG sites (underlined).
Chromatin Immunoprecipitation (ChIP)
Data are represented as the average of the number of replicates ± the standard deviation (SD). Statistical difference between the control and drug-treated samples was calculated using Student’s t-test or ANOVA followed by Bonferroni post-hoc test where appropriate. p < 0.05 was considered statistically different.
Clinically Relevant Concentrations of Curcumin Does not Induce Apoptosis in Tumor Cells
Curcumin was reported to induce cell cycle arrest in different tumors (30). Although curcumin (2 μM) and DMC (1 μM) did not induce significant apoptosis in CEM and Kasumi-1 cells, their effect on cell cycle arrest is unknown. Figure 1c shows that both drugs increased the M4 marker population significantly (p < 0.05) after 72 h from 22.8 ± 0.3% to 26.1 ± 0.2% for curcumin and to 26.9 ± 0.2% for DMC, indicating G2/M phase cell cycle arrest. The same effect was also observed with Kasumi-1 cells (data not shown). These results show that although low concentrations of curcumin and DMC are non-toxic to leukemia cells, they induce G2/M arrest which may contribute to their antitumor effect.
Curcumin is More Rapidly Cleared Than DMC by the Human Liver Microsomal Enzymes
Intrinsic Clearance Values for Curcumin and DMC in Human Pooled Liver Microsomes
Intrinsic clearance (microliters per minute per milligram of protein)
Absence of UDPGA
Presence of UDPGA
Absence of UDPGA
Presence of UDPGA
140.18 ± 2.32
196.37 ± 19.13a
177.48 ± 6.52b
173.80 ± 4.88c
DMC Induces the Expression of Promoter-Methylated Genes in Leukemia Cells
Curcumin and DMC Lack DNA Hypomethylating Activity
To further confirm the lack of hypomethylating effect of curcumin and DMC in other genes, we investigated their effect on CDH-1 gene methylation reversal in both CEM and Kasumi-1 cells. CEM cells were treated with curcumin (1 and 2 μM) and DMC (0.5 and 1 μM) for 72, 120 and 168 h and the methylation changes in five CpG sites within exon 1-associated CpG island of CDH-1 gene were monitored. The first four CpG sites were highly methylated (above 85%) and the average methylation of the fifth CpG site was 61 ± 5.2% in the CEM control cells (Fig. 3b). Similar to the p15 gene, no significant DNA methylation reversal was observed in any of the CpG sites after 72 h (Fig. 3b), 120 h and 168 h (data not shown). On the other hand, 5 AC reversed methylation in all the five CpG sites. Similar results were also observed in Kasumi-1 cells after 72 h (supplementary Fig. 5), 120 h and 168 h (data not shown). Collectively, these data confirm that clinically relevant concentration of curcumin and DMC lack significant gene-specific DNA hypomethylating activity.
It is possible that we did not observe any hypomethylating activity of curcumin because of the low concentrations used. To address this concern, CEM cells were treated with high concentrations of curcumin (5 and 10 μM) for 72 and 120 h and methylation reversal in both p15 and CDH-1 genes was monitored. It is worth mentioning here that similar high concentrations of DMC are highly toxic to leukemia cells and cannot be used. Figure 3c shows that even high concentrations of curcumin did not reverse p15 or CDH-1DNA methylation in CEM cells after 72 h and after 120 h (data not shown). Kasumi-1 cells showed similar results as CEM cells after 72 h (Supplementary Fig. 6).
Curcumin and DMC do not Reverse Global DNA Methylation
Curcumin and DMC Upregulate the Expression of TET Enzymes
Effect of Curcumin, DMC and 5 AC on the Expression of the Enzymes Controlling DNA Methylation
1.21 ± 0.2
1.39 ± 0.3
1.1 ± 0.1
1.28 ± 0.2
1.47 ± 0.3
1.51 ± 0.3
1.15 ± 0.1
1.29 ± 0.1
1.49 ± 0.3
2.2 ± 0.1*
2.3 ± 0.4*
2.4 ± 0.2*
1.9 ± 0.1*
1.8 ± 0.2*
2 ± 0.2*
1.4 ± 0.1*
1.64 ± 0.3*
1.65 ± 0.2*
DMC increases H3K36me3 mark in the putative CpG island of p15 and CDH-1 genes
An ideal chemopreventive and chemotherapeutic agent should affect multiple molecular targets in cancer cells with minimal toxicity in normal healthy cells. Chemotherapeutic agents lack the multiple target effect and are highly non selective. On the other hand, the polyphenolic phytochemical curcumin meets both criteria. Curcumin was shown to inhibit multiple vital pathways in cancer cells and was found to be cytoprotective for normal cells because of its antioxidant effect. However, the poor bioavailability of curcumin is a major hurdle in its successful use as an antitumor agent and the development of modified synthetic analogues provides a feasible strategy to improve its pharmacokinetic properties. In this study, we are comparing the cytotoxicity, metabolism and the epigenetic changes induced by clinically relevant concentrations of curcumin and its synthetic analogue, DMC. DMC was more toxic to leukemia cells than curcumin; concordant with being more metabolically stable. Unlike curcumin, DMC induced the expression of promoter-methylated genes like p15 and CDH-1; similar to the potent DNA hypomethylating agent 5 AC. Both curcumin and DMC lacked any significant gene-specific or global DNA hypomethylating activity in leukemia cells. DMC increased the H3K36me3 mark near the promoter region of the p15 and CDH-1 genes, while curcumin did not show any significant changes in H3K36me3 or H3k436me3 level. These data elucidate significant differences between curcumin and DMC and demonstrate that although both compounds lack DNA hypomethylating activity, DMC can induce the expression of promoter methylated genes.
Previous reports investigated the epigenetic changes and cytotoxicity induced by curcumin using high concentrations (5–50 μM) that are considered clinically irrelevant (15, 18, 21). In this study, we used a lower concentration of curcumin (2 μM) that is close to the achievable plasma concentration of curcumin (29). A serious concern for using such low concentration would be the lack of any pharmacological action associated with curcumin treatment. However, our data provide four evidences that refute this concern. First, low concentrations of curcumin induced G2/M cell cycle arrest that was comparable to the more stable DMC (Fig. 1c). Second, treatment of BV-173 cells with 2 μM curcumin for 168 h induced massive apoptosis comparable to DMC. This also demonstrates the differential sensitivity of leukemia cells to curcumin and that resistance to curcumin-induced apoptosis in kasumi-1 and CEM leukemia cells could be attributed to factors other than curcumin poor bioavailability. Third, low concentration of curcumin induced the expression of the three TET isoforms similar to DMC and the potent DNA hypomethylating agent 5 AC. Finally, when low curcumin concentrations failed to reverse DNA methylation and induce the expression of promoter-methylated genes, we used higher concentrations of curcumin (5 and 10 μM) and could not detect any methylation reversal or induction of gene expression, indicating that the lack of DNA hypomethylating activity is not attributed to the low concentration of curcumin used. Collectively, low concentration of curcumin was generally non-toxic to leukemia cells, induced cell cycle arrest and gene expression changes, which are all considered desirable effects for an epigenetic modifier.
DMC chemical structure differs from curcumin in that the two phenolic hydroxyl groups in curcumin are replaced by methoxy groups in DMC (supplemental Fig. 2). Since compounds with hydroxyl groups can be readily subjected to phase II metabolism (e.g., glucuronidation); it was expected that under physiological conditions and at equimolar concentrations, that DMC would be more resistant to phase II metabolism as opposed to curcumin. Indeed, our metabolism studies demonstrated that DMC has significantly lower intrinsic clearance values (P < 0.05), and hence is more metabolically stable than curcumin in presence of UDPGA, which accounts for phase II metabolism (p < 0.05) (Table I). These results are in line with our data reported herein which shows DMC as a more potent compound than curcumin in inducing apoptosis in leukemia cells. Nevertheless, other factors like the impact of both drugs on the expression of pro-apoptotic and anti-apoptotic genes could also contribute to that. Also, It is worth mentioning that the intrinsic clearance values of DMC in presence and absence of UDPGA were comparable (P > 0.05), demonstrating the minimal effect of UDPGA on the metabolic stability of DMC; which was not the case with curcumin (Table I). Consistently, previous in vivo reports that employed higher concentrations (10–20 μM) of DMC and curcumin indicated that DMC is more metabolically stable and has higher bioavailability than curcumin (37, 38).
DNA hypomethylating agents induce methylation reversal and re-expression of epigenetically silenced genes. Molecular docking studies suggested a possible covalent interaction between curcumin and the catalytic pocket of DNMT1 (22). Global DNA methylation analysis demonstrated a comparable methylation reversal to the potent DNA hypomethylating agent decitabine after treatment of leukemia cells with 3 and 30 μM of a commercial curcumin mixture (consists of curcumin 80.3%, demethoxycurcumin 20.2% and bisdemethoxycurcumin 10.8%). Another study also used the curcumin mixture in prostate cancer cells but in different proportions (curcumin 70% and 30% for dimethoxycurcumin and bisdemethoxycurcumin) and demonstrated significant reversal of Nrf2 gene promoter methylation after treatment with the curcumin mixture (21). In our study, we used pure analytical grade curcumin and not a commercial mixture of curcumin and this may contribute to the contradicting results. It is possible that demethoxycurcumin and/or bisdemethoxycurcumin are the active DNA hypomethylating agents in the mixture.
A different study used pure analytical grade curcumin to monitor DNA methylation reversal at the promoter region of the Neurog1 gene in LNCaP prostate cancer cells (20). Curcumin induced CpG demethylation and induced the expression of Neurog1 in LNCaP cells contrary to our findings in leukemia cells. We believe that the contribution of using cells from different tissues to the conflicting results is minimal if any, based on the documented activity of other DNA hypomethylating agents (decitabine and 5-azacytidine) in both leukemia cells and solid tumors. A possible explanation could be provided by the findings of another group that showed that curcumin selectively demethylate partially-methylated loci and not fully-methylated CpG sites (23). Indeed, the CpG sites studied in this report were almost fully methylated compared to 26% average CpG methylation in 47 CpG sites at the Neurog1 promoter region (20). On the other hand and in support of this study, curcumin was shown to have no significant global DNA demethylating activity in both leukemia and colorectal cancer cells after 6 days of treatment (23, 24). Taken together, factors like using curcumin mixture versus pure curcumin and the methylation density of the CpG sites to be analyzed could contribute to the controversy of the activity of curcumin as a DNA hypomethylating agent. Nonetheless, we are presenting solid evidence that DMC and pure curcumin, used at either low or high concentrations, did not reverse DNA methylation of highly methylated (70–95%) CpG sites, which is consistent with previous findings (23).
The analysis method used to quantitate DNA methylation reversal could also contribute to the conflicting results. In this study, we are using DNA pyrosequencing, which is considered the gold standard in quantitative analysis of both gene-specific and global DNA methylation (33). Other studies used techniques like bisulfite genomic sequencing (21), which is considered semi-quantitative and LC-MS/MS (39), which measures only global methylation changes without any information about gene-specific methylation. It is worth mentioning that DNA pyrosequencing was also used by Link et al. (23) and their results are concordant with our findings.
The combination of curcumin with other epigenetic modifiers like DNMT inhibitors (decitabine and 5 AC) could harness the effect of curcumin on histone acetylation and the effect of DNMT inhibitors on DNA methylation to induce optimum re-expression of epigenetically silenced tumor suppressor genes; similar to the sequential administration of DNMT inhibitors and HDAC inhibitors (32). Consequently, it is important to study the effect of curcumin on the expression of genes that control the DNA methylation machinery as this could augment or antagonize the action of DNMT inhibitors. DNMT isotypes (DNMT1, DNMT3a and DNMT3b) catalyze the transfer of methyl group from the universal methylation donor S-adenosyl-L-methionine (SAM) to cytosine. On the other hand, the TET isotypes (TET1, TET2 and TET3) catalyze the conversion of methylcytosine to 5-hydroxymethylcytosine with consequent active DNA demethylation (40). Curcumin and DMC did not induce the expression of any of the DNMT isotypes in leukemia cells suggesting the absence of an antagonistic effect between curcumin and DNMT inhibitors. On the other hand, the expression of the TET enzymes was induced by both curcumin and DMC suggesting a possible potentiation of the action of DNMT inhibitors on DNA methylation reversal when combined with curcumin, which needs to be validated.
The impact of curcumin and DMC on histone methylation is largely unknown. The position of the histone amino acid residue and its degree of methylation (mono, di or trimethylation) affects chromatin configuration. H3K4me3 and H3k36me3 are associated with transcriptionally active chromatin; while H3K9me3 and H3K27me3 mark closed heterochromatin domains (36). Curcumin induced a decrease of the H3K27me3 mark near the promoter region of Neurog1 and globally in prostate cancer cells (20). The decrease in H3K27me3 was associated with induction of Neurog1 expression. In this study, both DMC and 5 AC increased the H3K36me3 but not the H3K4me3 mark near the methylated promoter region of p15 and CDH-1 genes in leukemia cells and that was associated with induction of their expression. In contrast to 5 AC, DMC did not reverse promoter DNA methylation in both genes, indicating that induction of expression by DMC is independent on DNA methylation reversal. Previous reports demonstrated induction of gene expression in presence of promoter methylation. The expression of the estrogen receptor alpha (ER-alpha) gene is silenced by DNA methylation in breast cancer. ER-alpha expression was induced by treatment with the HDAC inhibitor trichostatin A (TSA) without reversing DNA methylation (41). Moreover, the class III HDAC SIRT1 was shown to localize to promoter methylated silenced tumor suppressor genes. Inhibition of SIRT1 re-expressed the silenced genes despite retention of promoter methylation (42). Furthermore, treatment of colorectal cancer cells with different HDAC inhibitors was able to induce the expression of promoter methylated genes without reversing DNA methylation, indicating that DNA methylation could not prevent gene reactivation by drug-induced resetting of the chromatin state (43). Other DMC-induced epigenetic changes like histone acetylation cannot be ruled out and may also contribute to the observed induction of gene expression. The mechanism of H3K36me3 increase by DMC and 5 AC is not clear. A direct effect on the expression or activity of histone methyltransferases or histone demethylases is possible and needs to be investigated.
In conclusion, we highlighted major differences between curcumin and its synthetic analogue, DMC. The fact that DMC can induce the expression of promoter methylated genes without reversing DNA methylation suggests a possible synergistic induction mechanism of gene expression upon combining DMC with DNMT inhibitors; similar to the previously reported combination of HDAC inhibitors and DNMT inhibitors.
Acknowledgments & Disclosures
This work was supported by the Scholarship of Discovery Intramural Research Grant Program (SDIRGP) from Albany College of Pharmacy to TEF and University of Maryland intramural research grant to HH. The work was also supported by NIH Grant Numbers NIGMS R15GM104865 to KCG, 5 P30 RR032135 from the COBRE Program of the National Center for Research Resources and 8 P30 GM 103498 from the National Institute of General Medical Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.
Conflicts of Interest
The authors have no financial disclosures or conflicts of interest to declare.
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