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Melatonin overcomes resistance to clofarabine in two leukemic cell lines by increased expression of deoxycytidine kinase
Experimental Hematology 2014;-:-–-
Melatonin overcomes resistance to clofarabine in two leukemic cell lines by increased expression of deoxycytidine kinase Miho Yamanishi, Hidehiko Narazaki, and Takeshi Asano Department of Pediatrics, Nippon Medical School Chiba Hokusoh Hospital, Inzai, Japan (Received 2 July 2014; revised 16 October 2014; accepted 3 November 2014)
Drug resistance remains a serious problem in leukemia therapy. Among newly developed nucleoside antimetabolites, clofarabine has broad cytotoxic activity showing therapeutic promise and is currently approved for relapsed acute lymphoblastic leukemia. To investigate the mechanisms responsible for clofarabine resistance, we established two clofarabine-resistant lymphoblastic leukemia cell lines from parental lines. To elucidate the mechanisms against clofarabine resistance in two newly established clofarabine-resistant cell lines, we measured the expression of export pumps multidrug resistance protein 1, multidrug resistance–associated protein 1, and ATP-binding cassette subfamily G member 2. There were no differences in the expression between clofarabine-sensitive and -resistant cell lines. Next, we determined expression of deoxycytidine kinase (dCK), which phosphorylates clofarabine to exert cytotoxicity, in clofarabinesensitive and -resistant cells. Clofarabine-resistant cells showed significantly decreased expression of dCK RNA when compared with sensitive cells. To elucidate the mechanisms of decreased dCK expression in clofarabine-resistant cells, we analyzed the methylation status of CpG islands of the dCK promoter and found no differences in methylation status between clofarabine-sensitive and -resistant cells. Next, we measured the acetylation status of histone and found that total histone acetylation, and histone H3 and H4 acetylation on chromatin immunoprecipitation assay were significantly decreased in resistant cells. Melatonin is an indolamine that functions in the regulation of chronobiological rhythms to exert cytotoxic effects. We examined the effects of melatonin in clofarabine-resistant cells and found that melatonin treatment led to significantly increased cytotoxicity with clofarabine in resistant cells via increased acetylation. Melatonin may be a useful candidate for overcoming clofarabine resistance in two newly established clofarabine resistant leukemia cell lines. Copyright Ó 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.
Drug resistance remains a serious problem in leukemia therapy, and children with leukemia cells exhibiting in vitro resistance to antileukemic agents have substantially worse prognosis than children whose leukemia cells are drug-sensitive [1]. Nucleoside antimetabolites are the most widely used and effective types of anticancer drugs [2]. Recently, several improved purine nucleoside analogues derived from adenine have been developed; one such compound, clofarabine (2-chloro-2-arabino-fluro-2deoxyadenosine), has broad cytotoxic activity, showing therapeutic promise, and is currently approved for relapsed acute lymphoblastic leukemia [3]. Clofarabine is readily phosphorylated by deoxycytidine kinase (dCK) and exerts Offprint requests to: Dr. Takeshi Asano, Department of Pediatrics, Nippon Medical School Chiba Hokusoh Hospital, 1715 Kamagari, Inzai, Chiba Prefecture 270-1694, Japan; E-mail: [email protected]
cytotoxicity against both proliferating and nonproliferating cells [4–6]. Resistance to nucleoside derivatives reportedly involves either impaired cellular uptake or reduced conversion to nucleoside monophosphates. By reducing the concentration of nucleoside monophosphate by either increased export pump function or decreased phosphorylation function, a reduction in higher phosphorylated nucleosides leads to reduced cytotoxicity. The increased export pump function of ATP-binding cassette subfamily G member 2 (ABCG2) has been reported as a novel mechanism of clofarabine resistance [7]. On the other hand, resistance to these nucleoside antimetabolites has been linked to deficiency or decreased expression of dCK [8–10]. Aberrant methylation has been shown to play a potent role in tumorigenesis, where genome-wide hypomethylation and regional hypermethylation of tumor suppressor
0301-472X/Copyright Ó 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.exphem.2014.11.001 EXPHEM3200_proof ■ 4-2-2015 4-8-24
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gene promoters are characteristic hallmarks in many cancers [11]. DNA methylation occurs in eukaryote DNA at CpG sites, usually enriched in the promoters of genes. Increasing evidence has shown that epigenetic changes can be a crucial driving force behind the acquisition of drug resistance, with changes in gene expression occurring after chemotherapy without gene mutations [12]. Histones also control gene expression by modulating the structure of chromatin, and the accessibility of regulatory DNA sequences to transcriptional activators and repressors [13,14]. Acetylation of histone increases gene expression by relaxing chromatin structure, allowing access of transcription factors to DNA. Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine that functions in the regulation of chronobiological rhythms and endocrine function [15]. In addition, melatonin has antiproliferative and cytotoxic effects on brain tumors [16]. Melatonin also increases the methylation of the ABCG2 promoter and decreases ABCG2 expression and function [16], and it has been reported to significantly increase histone H3 and histone H4 acetylation in the hippocampus [17]. Here, we found that melatonin reduces clofarabine resistance by epigenetic mechanisms related to dCK regulation in two newly established clofarabine-resistant leukemia cell lines.
Materials and methods Drugs and chemicals Clofarabine, melatonin, and dimethyl sulfoxide were obtained from Wako Pure Chemical Industries (Osaka, Japan). Phosphatebuffered saline without metal salt solution was from Nissui (Tokyo, Japan). Roswell Park Memorial Institute medium 1640, Hanks’ Balanced Salt Solution without Ca2þ or Mg2þ, fetal calf serum and gentamicin were purchased from Life Technologies (Gaithersburg, MD). Vorinostat (N-Hydroxy-N’-phenyloctanediamide) was purchased from TOKYO Chemical Industry Company (Tokyo, Japan). Cell lines NALM6/P, a parental cell line of human B-cell lymphoblastic leukemia, and SKW3/P, a parental cell line of human T-cell leukemia, were purchased from RIKEN (Tsukuba, Japan). Clofarabine-resistant cells (NALM6/Clo, SKW3/Clo) were selected by stepwise and continuous exposure to clofarabine using the limiting dilution method [12]. Before use in each experiment, clofarabine-resistant cells were cultured without clofarabine for 2 weeks. All cell lines were free from mycoplasma organisms, as confirmed by the MycoAlert mycoplasma detection kit (Lonza Japan, Tokyo, Japan). Cytotoxicity assay Cytotoxicity was assessed by trypan blue dye exclusion assay, as described previously [12]. Briefly, 1 105 cells/mL were incubated with various concentrations of anticancer drugs, including clofarabine, and melatonin for 48 hours. Viable cells were then
counted after trypan blue staining. The synergistic effect of melatonin with clofarabine was analyzed by combination index method [18]. Genomic DNA isolation and quality assessment DNA extractions from fresh cells were performed using the QIAamp DNA Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. Genomic DNA quality was assessed by low agarose gel (0.5%) electrophoresis under low voltage. Thresholds for genomic DNA quality check were: (a) showing a high molecular band (!40 kbp) in 0.6% agarose gel low-voltage electrophoresis (3 hours) and no strong bands of low molecular weight (!2.0 kbp) and (b) OD260/280 within a range of 1.8–2.0. Bisulfite conversion Bisulfite conversion of genomic DNA was performed with the methylSEQr bisulfite conversion kit (Applied Biosystems, Tokyo, Japan), in accordance with the manufacturer’s instructions. Bisulfite-converted samples were immediately subject to methylation-specific polymerase chain reaction (MSP) analysis, as described below. RNA extraction Total RNA from each sample was isolated individually using the QIAGEN RNA Mini kit (QIAGEN) according to the manufacturer’s protocol, and RNA integrity was confirmed using 1% agarose gel electrophoresis. Flow cytometric analysis Flow cytometric analysis was performed using MACSQuant Analyzer (Miltenyli Biotech, Tokyo, Japan) with FlowJo (Treestar, Tokyo, Japan), in accordance with the manufacturer’s instructions. Antihuman multidrug resistance protein 1 (MDR1), multidrug resistance–associated protein 1 (MRP1) and ABCG2 antibodies were purchased from R&D (Minneapolis, MN). Quantitative real-time polymerase chain reaction To evaluate mRNA levels of dCK, quantitative real-time polymerase chain reaction (qPCR) was performed using the ABI Prism 7500 sequence detection system (Applied Biosystems). All primers for dCK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were purchased from Applied Biosystems. We normalized dCK expression levels against GAPDH expression levels. Methylation-specific polymerase chain reaction analysis For MSP analysis, genomic DNA was obtained and 300 ng of DNA per sample was treated as described above. Primer pairs for MSP of dCK promoter were designed based on methylated and unmethylated DNA sequences in the promoter region, as follows [19]: dCK1 methylation-specific primers (dCK1M); dCK promoter 233–243 (50 -TTTATTTTTTTTTTTTATTCG ATTTCG-30 , 50 -ATCACGTAAAAACCGAAACG-30 ); dCK1 unmethylation-specific primers (dCK1U); dCK promoter 233– 243 (50 -GGTTTATTTTTTTTTTTTATTTGATTTTG-30 , 50 -ACA AATCACATAAAAACCAAAACA-30 ); dCK2M; dCK promoter 292–299 (50 -GTTGGAGGCGGGCG-30 , 50 -AATACGCACACT AAAACTCGC-30 ); dCK2U; dCK promoter 292–299 (50 AATTTGTGTTGGAGGTGGGTG-30 , 50 -ACCACAAATACACA CACTAAAACTCACA-30 ); dCK3M; dCK promoter 333-345
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Figure 1. IC50 against clofarabine (mmol/L) in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with various doses of clofarabine for 48 hours with (þ) or without () melatonin, and cytotoxicity was evaluated by trypan blue dye exclusion. Data are means 6 standard deviation from three independent experiments. *p ! 0.02, NALM/P vs. NALM/ Clo; **p ! 0.02, NALM/Clo without melatonin treatment vs. NALM/ Clo with melatonin treatment; #p ! 0.03, SKW3/P vs. SKW3/Clo; ## p ! 0.03, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
(50 -GAGGAGGGCGGGGTC-30 , 50 -GCACACTAAAACTCGCG ACG-30 ), dCK3U; dCK promoter 333-345 (50 -TTTGAGG AGGGTGGGGTT-30 , 50 -CACAAATACACACACTAAAACTCA CAACA-30 ); dCK4M; dCK promoter 388-396 (50 -GGTAG TTAGGGAGCGCG-30 , 50 -CTAATAAACTCACCGACCCG-30 ); dCK4U; and dCK promoter 388-396 (50 -GAGTGTGTAGTGG GAATTTGTG-30 , 50 -CAACTAATAAACTCACCAACCCA-30 ). Polymerase chain reaction was performed using the EpiScope MSP kit (Takara Biotechnology Company, Ohtsu, Japan) and the ABI Prism 7500 sequence detection system (Applied Biosystems). Amplification was performed with an initial denaturation at 95 C for 30 sec, 45 cycles of denaturation at 98 C for 5 sec, annealing at 55 C for 30 sec and extension at 72 C for 60 sec. Subsequently, melting curve analysis was performed on PCR products. Determination of total histone acetylation To determine total histone acetylation, a commercial colorimetric kit (EpiQuik Total Histone H3 or H4 Acetylation Detection Fast Kit; Epigentek Group, Farmingdale, NY) was used, in accordance with the manufacturer’s instructions. Chromatin immunoprecipitation analysis Chromatin immunoprecipitation (ChIP) analysis was measured using EpiQuik acetyl-histone H3 ChIP Kit and EpiQuik acetylhistone H4 ChIP Kit (Epigentek), in accordance with the manufacturer’s instructions, and the ABI Prism 7500 sequence detection
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Figure 2. dCK mRNA expression in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and dCK RNA expression was evaluated by qPCR. After normalization against GAPDH expression, relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from nine independent experiments. *p 5 0.05, NALM/P vs. NALM/Clo; **p 5 0.035, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; # p ! 0.0001, SKW3/P vs. SKW3/Clo; ##p 5 0.03, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
system (Applied Biosystems). Briefly, leukemic cells were disaggregated and incubated in 1% formaldehyde (crosslink). Chromatin was sheared by sonication. Samples were incubated with primary antibody (anti–acetyl H3 and anti–acetyl H4) or with an equivalent amount of normal immunoglobulin G (antimouse). A portion of sonicated DNA was left untreated to serve as an input control. Immunoprecipitated DNA was analyzed by qPCR with specific primers for dCK (50 -CCTCCCCACCCGACTCCG GAACC-30 , 50 -CAGCTGAGGACACTGGCGGGCCTG-30 ) [20] for immunoprecipitated DNA and b-actin (50 -GTGGGGCG CCCCAGGCACA-30 , 50 -CTCCTTAATGTCACGCACGATTTC30 ) [21], for input DNA using the SYBR Premix Ex TaqÔ II kit (Takara Biotechnology Company). We normalized Ct values for dCK promoter against Ct values for b-actin from input DNA. Amplification was performed with an initial denaturation at 95 C for 30 sec, 40 cycles of denaturation at 95 C for 5 sec, annealing at 59 C for 34 sec, and extension at 95 C for 5 sec, 59 C for 60 sec, and 95 C for 15 sec. We then subjected PCR products to melting curve analysis. Statistical analysis Statistical analysis was performed using the Kruskall-Wallis H test. Mann-Whitney U test was used to determine the significance of differences between each group.
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Figure 3. (A) ABCG2, MDR, and MRP1 expression analysis by flow cytometric analysis in NALM6 cells with or without melatonin treatment. Flow cytometric analysis was performed using MACSQuant Analyzer with FlowJo. Red curve represents negative control, and blue curve represents expression with ABCG2, MDR, and MPR1, respectively. (B) ABCG2, MDR, and MRP1 expression analysis by flow cytometric analysis in SKW3 cells with or without melatonin treatment. Flow cytometric analysis was performed using MACSQuant Analyzer with FlowJo. Red curve represents negative control, and blue curve represents expression with ABCG2, MDR, and MPR1, respectively.
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Results Cytotoxicity of clofarabine was partially restored with melatonin treatment in clofarabine-resistant cell lines As shown in Figure 1, clofarabine-resistant cell lines (NALM6/Clo, SKW3/Clo) were strongly resistant to clofarabine when compared with the parent cell lines (NALM6/P, Skw3/P). Next, we determined the optimal concentration of melatonin in NALM6 (IC50 of NALM/P, 3.3 6 0.2 mmol/ L; NALM/Clo, 3.3 6 0.18 mmol/L) and SKW3 cells ((IC50 of SKW3/P, 1.20 6 0.18 mmol/L; SKW3/Clo, 1.25 6 0.20 mmol/L) and we used a 100 mmol/L melatonin treatment for 48 hours in subsequent experiments [22]. Melatonin treatment significantly increased the cytotoxicity of clofarabine in NALM6/Clo cells and SKW3/Clo cells (Fig. 1). Combination index with clofarabine and melatonin showed 1.16 in NALM/P, 0.06 in NALM6/Clo, 1.58 in SKW3/P, and 0.1 in SKW3/Clo. These results strongly indicated that melatonin and clofarabine synergistically acted in clofarabine resistant cells. Deoxycytidine kinase RNA expression decreased in clofarabine-resistant cell lines and increased with melatonin treatment We next examined mRNA expression in clofarabineresistant lines NALM6/Clo and SKW3/Clo, as compared with the parental lines NALM6/P and SKW3/P cells, with or without melatonin treatment (Fig. 2). Clofarabineresistant cells showed decreased expression in dCK mRNA when compared with parental cells. Melatonin treatment increased dCK mRNA in NALM6/Clo and SKW3/Clo cells, but had marginal effects in NALM/P and SKW/P cells (Fig. 2). By contrast, expression of ABCG2, MDR1, and MRP1 by flow cytometric analysis was not altered in NALM/P and NALM/Clo or SKW3/P and SKW3/Clo leukemic cells with or without melatonin treatment (Fig. 3A and B). CpG islands in deoxycytidine kinase promoter did not contribute to changes in expression after melatonin treatment in clofarabine-resistant leukemia cell lines We next measured the methylation status in the dCK promoter by MSP analysis in NALM6/P, NALM6/Clo, SKW3/P, and SKW3/Clo cells with or without melatonin treatment. All NALM6/P, NALM6/Clo, SKW3/P, and SKW3/Clo cells showed all methylated CpG islands in all dCK promoters examined (Fig. 4). Melatonin treatment did not alter the methylation status of dCK promoters (Fig. 4). Decreased acetylation in clofarabine-resistant cells, as compared with parental cells and melatonin treatment, induced increases in acetylation in clofarabine-resistant cells Because a decrease in histone acetylase activity has been shown to induce gene silencing, we next examined histone
Figure 4. Methylation status in dCK promoter in NALM6, SKW3 parent cells and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and methylated and unmethylated dCK promoters were evaluated by methylation-specific PCR using qPCR. Relative values were calculated based on expression of dCK in NALM6 parent cells without melatonin treatment (set as 1). PCR amplification with unmethylated primers did not detect PCR products in all sets of experiments. Data are averages from two independent experiments.
H3 and H4 acetylation activity at the dCK promoter. First, as shown in Figure 5A and B, decreased acetylation of total histone H3 and H4 was observed in clofarabine-resistant cell lines (NALM6/Clo, SKW3/Clo) when compared with parental cell lines (NALM6/P, SKW3/P). Melatonin treatment increased total histone H3 and H4 acetylation in clofarabine-resistant cells (Fig. 5A and B). However, melatonin showed marginal effects in parental cells (Fig. 5A and B). Decreased acetylation of histone H3 at the dCK promoter in clofarabine-resistant NALM6 cells was observed when compared with parental cells on ChIP assay, but not in SKW3/Clo cells (Fig. 6A). Decreased acetylation of histone H4 at the dCK promoter in clofarabine-resistant cells was observed when compared with parental cells on ChIP assay (Fig. 6B). Furthermore, melatonin treatment increased acetylation of both histone H3 and H4 at the dCK promoter in NALM6/Clo cells (Fig. 6A and B). In SKW3/Clo cells, increased acetylation of histone H4 at
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Figure 5. (A) Total histone H3 acetylation in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and total histone H3 expression was evaluated. Relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; # p ! 0.001, SKW3/P vs. SKW3/Clo; ##p ! 0.01, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment. (B) Total histone H4 acetylation in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and total histone H4 expression was evaluated. Relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; #p ! 0.01, SKW3/P vs. SKW3/Clo; ## p 5 0.003, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
the dCK promoter was observed, but no increased acetylation of histone H3 was noted (Fig. 6A and B). Vorinostat restored the sensitivity against clofarabine in acute lymphoid leukemia cell lines We showed that melatonin restored clofarabine sensitivity in clofarabine-resistant cells via increased acetylation.
These results prompted us to investigate whether other Histone Diacetylase (HDAC) inhibitors might increase sensitivity against clofarabine in clofarabine resistant cells. We used vorinostat (N-Hydroxy-N’-phenyloctanediamid). We firstly determined the optimal concentration of vorinostat in NALM6 (IC50 of NALM/P, 15.2 6 3.5 mmol/L; NALM/Clo, 10.1 6 1.1 mmol/L) and SKW3 cells (IC50
Figure 6. (A) Total histone H3 acetylation at dCK promoter as evaluated by ChIP assay in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and total histone H3 acetylation was evaluated. After normalization by input DNA, relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.001, NALM/P vs. NALM/Clo; **p ! 0.02, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; #p ! 0.01, SKW3/P vs. SKW3/Clo; ##p ! 0.01, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment. (B) Total histone H4 acetylation at dCK promoter as evaluated by ChIP assay in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours and total histone H3 acetylation was evaluated. After normalization by input DNA, relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; #p ! 0.001, SKW3/P vs. SKW3/Clo; ## p ! 0.04, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
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Figure 7. IC50 against clofarabine (mmol/L) in NALM6, SKW3 parent cells and clofarabine-resistant cells with or without vorinostat treatment. Leukemia cells were incubated with various doses of clofarabine for 48 hours with (þ) or without () vorinostat and cytotoxicity was evaluated by trypan blue dye exclusion. Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without vorinostat treatment vs. NALM/Clo with vorinostat treatment; #p ! 0.01, SKW3/P vs. SKW3/Clo; ## p ! 0.01, SKW3/Clo without vorinostat treatment vs. SKW3/Clo with vorinostat treatment.
of SKW3/P, 12.3 6 1.2 mmol/L; SKW3/Clo, 11.0 6 1.0 mmol/L) and we used a 0.1 mmol/L vorinostat treatment for 72 hours in subsequent experiments [23]. Vorinostat treatment also significantly increased the cytotoxicity of clofarabine in NALM6/Clo cells and SKW3/Clo cells (Fig. 7). Combination index with clofarabine and vorinostat showed 4.00 in NALM/P, 0.04 in NALM6/Clo, 1.67 in SKW3/P, and 0.04 in SKW3/Clo. These results strongly indicated that vorinostat, another HDAC inhibitor, and clofarabine synergistically acted in clofarabine resistant cells (Fig. 7).
Discussion The results of this study showed that the development of clofarabine resistance in two newly established clofarabine-resistant acute lymphoblastic leukemia cell lines was accompanied by downregulation of a key gene in clofarabine metabolism, dCK. This downregulation was not due to gene promoter hypermethylation, as demonstrated by MSP, but was due to gene promoter hypoacetylation, as confirmed by ChIP assay. The mechanisms of clofarabine resistance are reported to be ABCG2 overexpression [24] and decreased expres-
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sion of dCK gene with methylated promoter regions [19]. Our results showed no changes in ABCG2 expression in addition to other export pumps, such as MDR1 and MRP1, nor any changes in methylation status in clofarabine-resistant cells, as compared with sensitive cells. We assessed the histone deacetylation status of dCK promoter in clofarabine resistant cells. Acetylation of histone increases gene expression and is known to be an epigenetic mechanism. To our knowledge, this is the first report to find that histone deacetylation of the dCK promoter is responsible for the decreased expression of dCK in clofarabine-resistant leukemic cells. We also found that histone deacetylation status differs between NALM6 cells, which are derived from B-cell leukemia, and SKW3 cells, which are derived from T-cell leukemia. These findings are important to gain further knowledge of the mechanisms of chemotherapy drug resistance. Gene inactivation by DNA methylation is the epigenetic mechanism shown to be directly involved in the acquisition of chemotherapy resistance in vitro [12]. However, the involvement of other epigenetic mechanisms, such as histone deacetylation, in the inactivation process of genes involved in chemotherapy sensitivity and resistance has been reported [25–28]. Our results strongly suggest a novel model for clofarabine resistance in these newly established acute lymphoblastic leukemia cell lines. In addition, we found that histone acetylation patterns varied between NALM6/Clo and SKW3/Clo. The precise mechanisms were unknown. In some transforming conditions, such as thyroid tumor and endometriosis, different acetylation of histone H3 and H4 occurred separately during transformation [29,30]. We considered that T-cell leukemia and B-cell leukemia might show different acetylation of histones during development of drug resistance. Melatonin plays a role in the regulation of chronobiological rhythms and has been used to ameliorate jet lag. Recently, melatonin was reported to have other important functions, such as increasing promoter methylation [24] and increasing histone H3 and histone H4 acetylation [17]. Increased methylation typically induces decreased gene expression, and increased histone acetylation typically induces increased gene expression. Thus, melatonin exerts both actions on gene expression (increases and decreases expression). In our experiments, melatonin induced increased dCK expression in two clofarabine-resistant leukemia cell lines, which we established. Acquired resistance to clofarabine may be responsible for treatment failure; hence, melatonin may reverse the resistance to and potentially increase the efficacy of clofarabine. In addition, the finding that histone acetylation may lead to chemotherapy resistance deserves testing in other experimental models. In conclusion, to our knowledge, this is the first study to find that melatonin can influence the mechanisms of clofarabine resistance in leukemia cells. Although the mechanism of drug resistance was multifunctional, we consider that, at
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least, we found additional mechanisms in clofarabineresistant leukemia cell lines. Incubation with melatonin resulted in a marked increase in cytotoxicity against clofarabine in resistant cells. These data indicate that melatonin alters DNA accessibility via histone acetylation and relaxation of the chromatin structure, which could allow clofarabine to more efficiently target the DNA. Conflict of interest disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.
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14. Berger SL. Histone modifications in transcriptional regulation. Curr Opin Genet Dev. 2002;12:142–148. 15. Lewy AJ. Melatonin and human chronobiology. Cold Spring Harb Symp Quant Biol. 2007;72:623–636. 16. Martın V, Sanchez-Sanchez AM, Herrera F, et al. Melatonin-induced methylation of the ABCG2/BCRP promoter as a novel mechanism to overcome multidrug resistance in brain tumour stem cells. Br J Cancer. 2013;108:2005–2012. 17. Niles LP, Pan Y, Kang S, Lacoul A. Melatonin induces histone hyperacetylation in the rat brain. Neurosci Lett. 2013;541:49–53. 18. Zhao L, Wientjes MG, Au JL. Evaluation of combination chemotherapy: integration of nonlinear regression, curve shift, isobologram, and combination index analyses. Clin Cancer Res. 2004;10: 7994–8004. 19. Peters GJ, Hodzic J, Ortega B, et al. Methylation specific PCR to characterize methylation of the promoter of deoxycytidine kinase. Nucleosides Nucleotides Nucleic Acids. 2010;29:408–413. 20. Candelaria M, de la Cruz-Hernandez E, Taja-Chayeb L, et al. DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells. PLoS One. 2012;7:e29181. 21. Asano T, Tsutsuda-Asano A, Fukunaga Y. Indomethacin overcomes doxorubicin resistance by decreasing intracellular content of glutathione and its conjugates with decreasing expression of gamma-glutamylcysteine synthetase via promoter activity in doxorubicin-resistant leukemia cells. Cancer Chemother Pharmacol. 2009;64:715–721. 22. Wang J, Xiao X, Zhang Y, et al. Simultaneous modulation of COX2, p300, Akt, and Apaf-1 signaling by melatonin to inhibit proliferation and induce apoptosis in breast cancer cells. J Pineal Res. 2012;53:77–90. 23. Yu CC, Pan SL, Chao SW, et al. A novel small molecule hybrid of vorinostat and DACA displays anticancer activity against human hormone-refractory metastatic prostate cancer through dual inhibition of histone deacetylase and topoisomerase I. Biochem Pharmacol. 2014;90:320–330. 24. de Wolf C, Jansen R, Yamaguchi H, et al. Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides. Mol Cancer Ther. 2008;7: 3092–3102. 25. Huang Y, Greene E, Murray Stewart T, et al. Inhibition of lysinespecific demethylase 1 by polyamine analogues results inreexpression of aberrantly silenced genes. Proc Natl Acad Sci U S A. 2007;104: 8023–8028. 26. Ougolkov AV, Bilim VN, Billadeau DD. Regulation of pancreatic tumor cell proliferation and chemoresistance by the histone methyltransferase enhancer of zeste homologue 2. Clin Cancer Res. 2008;14: 6790–6796. 27. Huo H, Magro PG, Pietsch EC, Patel BB, Scotto KW. Histone methyltransferase MLL1 regulates MDR1 transcription and chemoresistance. Cancer Res. 2010;70:8726–8735. 28. Ma J, Dong C, Ji C. MicroRNA and drug resistance. Cancer Gene Ther. 2010;17:523–531. 29. Puppin C, Passon N, Lavarone E, et al. Levels of histone acetylation in thyroid tumors. Biochem Biophys Res Commun. 2011;411: 679–683. 30. Xiaomeng X, Ming Z, Jiezhi M, Xiaoling F. Aberrant histone acetylation and methylation levels in woman with endometriosis. Arch Gynecol Obstet. 2013;287:487–494.
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Melatonin overcomes resistance to clofarabine in two leukemic cell lines by increased expression of deoxycytidine kinase Miho Yamanishi, Hidehiko Narazaki, and Takeshi Asano Department of Pediatrics, Nippon Medical School Chiba Hokusoh Hospital, Inzai, Japan (Received 2 July 2014; revised 16 October 2014; accepted 3 November 2014)
Drug resistance remains a serious problem in leukemia therapy. Among newly developed nucleoside antimetabolites, clofarabine has broad cytotoxic activity showing therapeutic promise and is currently approved for relapsed acute lymphoblastic leukemia. To investigate the mechanisms responsible for clofarabine resistance, we established two clofarabine-resistant lymphoblastic leukemia cell lines from parental lines. To elucidate the mechanisms against clofarabine resistance in two newly established clofarabine-resistant cell lines, we measured the expression of export pumps multidrug resistance protein 1, multidrug resistance–associated protein 1, and ATP-binding cassette subfamily G member 2. There were no differences in the expression between clofarabine-sensitive and -resistant cell lines. Next, we determined expression of deoxycytidine kinase (dCK), which phosphorylates clofarabine to exert cytotoxicity, in clofarabinesensitive and -resistant cells. Clofarabine-resistant cells showed significantly decreased expression of dCK RNA when compared with sensitive cells. To elucidate the mechanisms of decreased dCK expression in clofarabine-resistant cells, we analyzed the methylation status of CpG islands of the dCK promoter and found no differences in methylation status between clofarabine-sensitive and -resistant cells. Next, we measured the acetylation status of histone and found that total histone acetylation, and histone H3 and H4 acetylation on chromatin immunoprecipitation assay were significantly decreased in resistant cells. Melatonin is an indolamine that functions in the regulation of chronobiological rhythms to exert cytotoxic effects. We examined the effects of melatonin in clofarabine-resistant cells and found that melatonin treatment led to significantly increased cytotoxicity with clofarabine in resistant cells via increased acetylation. Melatonin may be a useful candidate for overcoming clofarabine resistance in two newly established clofarabine resistant leukemia cell lines. Copyright Ó 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.
Drug resistance remains a serious problem in leukemia therapy, and children with leukemia cells exhibiting in vitro resistance to antileukemic agents have substantially worse prognosis than children whose leukemia cells are drug-sensitive [1]. Nucleoside antimetabolites are the most widely used and effective types of anticancer drugs [2]. Recently, several improved purine nucleoside analogues derived from adenine have been developed; one such compound, clofarabine (2-chloro-2-arabino-fluro-2deoxyadenosine), has broad cytotoxic activity, showing therapeutic promise, and is currently approved for relapsed acute lymphoblastic leukemia [3]. Clofarabine is readily phosphorylated by deoxycytidine kinase (dCK) and exerts Offprint requests to: Dr. Takeshi Asano, Department of Pediatrics, Nippon Medical School Chiba Hokusoh Hospital, 1715 Kamagari, Inzai, Chiba Prefecture 270-1694, Japan; E-mail: [email protected]
cytotoxicity against both proliferating and nonproliferating cells [4–6]. Resistance to nucleoside derivatives reportedly involves either impaired cellular uptake or reduced conversion to nucleoside monophosphates. By reducing the concentration of nucleoside monophosphate by either increased export pump function or decreased phosphorylation function, a reduction in higher phosphorylated nucleosides leads to reduced cytotoxicity. The increased export pump function of ATP-binding cassette subfamily G member 2 (ABCG2) has been reported as a novel mechanism of clofarabine resistance [7]. On the other hand, resistance to these nucleoside antimetabolites has been linked to deficiency or decreased expression of dCK [8–10]. Aberrant methylation has been shown to play a potent role in tumorigenesis, where genome-wide hypomethylation and regional hypermethylation of tumor suppressor
0301-472X/Copyright Ó 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.exphem.2014.11.001 EXPHEM3200_proof ■ 4-2-2015 4-8-24
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gene promoters are characteristic hallmarks in many cancers [11]. DNA methylation occurs in eukaryote DNA at CpG sites, usually enriched in the promoters of genes. Increasing evidence has shown that epigenetic changes can be a crucial driving force behind the acquisition of drug resistance, with changes in gene expression occurring after chemotherapy without gene mutations [12]. Histones also control gene expression by modulating the structure of chromatin, and the accessibility of regulatory DNA sequences to transcriptional activators and repressors [13,14]. Acetylation of histone increases gene expression by relaxing chromatin structure, allowing access of transcription factors to DNA. Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine that functions in the regulation of chronobiological rhythms and endocrine function [15]. In addition, melatonin has antiproliferative and cytotoxic effects on brain tumors [16]. Melatonin also increases the methylation of the ABCG2 promoter and decreases ABCG2 expression and function [16], and it has been reported to significantly increase histone H3 and histone H4 acetylation in the hippocampus [17]. Here, we found that melatonin reduces clofarabine resistance by epigenetic mechanisms related to dCK regulation in two newly established clofarabine-resistant leukemia cell lines.
Materials and methods Drugs and chemicals Clofarabine, melatonin, and dimethyl sulfoxide were obtained from Wako Pure Chemical Industries (Osaka, Japan). Phosphatebuffered saline without metal salt solution was from Nissui (Tokyo, Japan). Roswell Park Memorial Institute medium 1640, Hanks’ Balanced Salt Solution without Ca2þ or Mg2þ, fetal calf serum and gentamicin were purchased from Life Technologies (Gaithersburg, MD). Vorinostat (N-Hydroxy-N’-phenyloctanediamide) was purchased from TOKYO Chemical Industry Company (Tokyo, Japan). Cell lines NALM6/P, a parental cell line of human B-cell lymphoblastic leukemia, and SKW3/P, a parental cell line of human T-cell leukemia, were purchased from RIKEN (Tsukuba, Japan). Clofarabine-resistant cells (NALM6/Clo, SKW3/Clo) were selected by stepwise and continuous exposure to clofarabine using the limiting dilution method [12]. Before use in each experiment, clofarabine-resistant cells were cultured without clofarabine for 2 weeks. All cell lines were free from mycoplasma organisms, as confirmed by the MycoAlert mycoplasma detection kit (Lonza Japan, Tokyo, Japan). Cytotoxicity assay Cytotoxicity was assessed by trypan blue dye exclusion assay, as described previously [12]. Briefly, 1 105 cells/mL were incubated with various concentrations of anticancer drugs, including clofarabine, and melatonin for 48 hours. Viable cells were then
counted after trypan blue staining. The synergistic effect of melatonin with clofarabine was analyzed by combination index method [18]. Genomic DNA isolation and quality assessment DNA extractions from fresh cells were performed using the QIAamp DNA Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. Genomic DNA quality was assessed by low agarose gel (0.5%) electrophoresis under low voltage. Thresholds for genomic DNA quality check were: (a) showing a high molecular band (!40 kbp) in 0.6% agarose gel low-voltage electrophoresis (3 hours) and no strong bands of low molecular weight (!2.0 kbp) and (b) OD260/280 within a range of 1.8–2.0. Bisulfite conversion Bisulfite conversion of genomic DNA was performed with the methylSEQr bisulfite conversion kit (Applied Biosystems, Tokyo, Japan), in accordance with the manufacturer’s instructions. Bisulfite-converted samples were immediately subject to methylation-specific polymerase chain reaction (MSP) analysis, as described below. RNA extraction Total RNA from each sample was isolated individually using the QIAGEN RNA Mini kit (QIAGEN) according to the manufacturer’s protocol, and RNA integrity was confirmed using 1% agarose gel electrophoresis. Flow cytometric analysis Flow cytometric analysis was performed using MACSQuant Analyzer (Miltenyli Biotech, Tokyo, Japan) with FlowJo (Treestar, Tokyo, Japan), in accordance with the manufacturer’s instructions. Antihuman multidrug resistance protein 1 (MDR1), multidrug resistance–associated protein 1 (MRP1) and ABCG2 antibodies were purchased from R&D (Minneapolis, MN). Quantitative real-time polymerase chain reaction To evaluate mRNA levels of dCK, quantitative real-time polymerase chain reaction (qPCR) was performed using the ABI Prism 7500 sequence detection system (Applied Biosystems). All primers for dCK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were purchased from Applied Biosystems. We normalized dCK expression levels against GAPDH expression levels. Methylation-specific polymerase chain reaction analysis For MSP analysis, genomic DNA was obtained and 300 ng of DNA per sample was treated as described above. Primer pairs for MSP of dCK promoter were designed based on methylated and unmethylated DNA sequences in the promoter region, as follows [19]: dCK1 methylation-specific primers (dCK1M); dCK promoter 233–243 (50 -TTTATTTTTTTTTTTTATTCG ATTTCG-30 , 50 -ATCACGTAAAAACCGAAACG-30 ); dCK1 unmethylation-specific primers (dCK1U); dCK promoter 233– 243 (50 -GGTTTATTTTTTTTTTTTATTTGATTTTG-30 , 50 -ACA AATCACATAAAAACCAAAACA-30 ); dCK2M; dCK promoter 292–299 (50 -GTTGGAGGCGGGCG-30 , 50 -AATACGCACACT AAAACTCGC-30 ); dCK2U; dCK promoter 292–299 (50 AATTTGTGTTGGAGGTGGGTG-30 , 50 -ACCACAAATACACA CACTAAAACTCACA-30 ); dCK3M; dCK promoter 333-345
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Figure 1. IC50 against clofarabine (mmol/L) in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with various doses of clofarabine for 48 hours with (þ) or without () melatonin, and cytotoxicity was evaluated by trypan blue dye exclusion. Data are means 6 standard deviation from three independent experiments. *p ! 0.02, NALM/P vs. NALM/ Clo; **p ! 0.02, NALM/Clo without melatonin treatment vs. NALM/ Clo with melatonin treatment; #p ! 0.03, SKW3/P vs. SKW3/Clo; ## p ! 0.03, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
(50 -GAGGAGGGCGGGGTC-30 , 50 -GCACACTAAAACTCGCG ACG-30 ), dCK3U; dCK promoter 333-345 (50 -TTTGAGG AGGGTGGGGTT-30 , 50 -CACAAATACACACACTAAAACTCA CAACA-30 ); dCK4M; dCK promoter 388-396 (50 -GGTAG TTAGGGAGCGCG-30 , 50 -CTAATAAACTCACCGACCCG-30 ); dCK4U; and dCK promoter 388-396 (50 -GAGTGTGTAGTGG GAATTTGTG-30 , 50 -CAACTAATAAACTCACCAACCCA-30 ). Polymerase chain reaction was performed using the EpiScope MSP kit (Takara Biotechnology Company, Ohtsu, Japan) and the ABI Prism 7500 sequence detection system (Applied Biosystems). Amplification was performed with an initial denaturation at 95 C for 30 sec, 45 cycles of denaturation at 98 C for 5 sec, annealing at 55 C for 30 sec and extension at 72 C for 60 sec. Subsequently, melting curve analysis was performed on PCR products. Determination of total histone acetylation To determine total histone acetylation, a commercial colorimetric kit (EpiQuik Total Histone H3 or H4 Acetylation Detection Fast Kit; Epigentek Group, Farmingdale, NY) was used, in accordance with the manufacturer’s instructions. Chromatin immunoprecipitation analysis Chromatin immunoprecipitation (ChIP) analysis was measured using EpiQuik acetyl-histone H3 ChIP Kit and EpiQuik acetylhistone H4 ChIP Kit (Epigentek), in accordance with the manufacturer’s instructions, and the ABI Prism 7500 sequence detection
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Figure 2. dCK mRNA expression in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and dCK RNA expression was evaluated by qPCR. After normalization against GAPDH expression, relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from nine independent experiments. *p 5 0.05, NALM/P vs. NALM/Clo; **p 5 0.035, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; # p ! 0.0001, SKW3/P vs. SKW3/Clo; ##p 5 0.03, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
system (Applied Biosystems). Briefly, leukemic cells were disaggregated and incubated in 1% formaldehyde (crosslink). Chromatin was sheared by sonication. Samples were incubated with primary antibody (anti–acetyl H3 and anti–acetyl H4) or with an equivalent amount of normal immunoglobulin G (antimouse). A portion of sonicated DNA was left untreated to serve as an input control. Immunoprecipitated DNA was analyzed by qPCR with specific primers for dCK (50 -CCTCCCCACCCGACTCCG GAACC-30 , 50 -CAGCTGAGGACACTGGCGGGCCTG-30 ) [20] for immunoprecipitated DNA and b-actin (50 -GTGGGGCG CCCCAGGCACA-30 , 50 -CTCCTTAATGTCACGCACGATTTC30 ) [21], for input DNA using the SYBR Premix Ex TaqÔ II kit (Takara Biotechnology Company). We normalized Ct values for dCK promoter against Ct values for b-actin from input DNA. Amplification was performed with an initial denaturation at 95 C for 30 sec, 40 cycles of denaturation at 95 C for 5 sec, annealing at 59 C for 34 sec, and extension at 95 C for 5 sec, 59 C for 60 sec, and 95 C for 15 sec. We then subjected PCR products to melting curve analysis. Statistical analysis Statistical analysis was performed using the Kruskall-Wallis H test. Mann-Whitney U test was used to determine the significance of differences between each group.
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Figure 3. (A) ABCG2, MDR, and MRP1 expression analysis by flow cytometric analysis in NALM6 cells with or without melatonin treatment. Flow cytometric analysis was performed using MACSQuant Analyzer with FlowJo. Red curve represents negative control, and blue curve represents expression with ABCG2, MDR, and MPR1, respectively. (B) ABCG2, MDR, and MRP1 expression analysis by flow cytometric analysis in SKW3 cells with or without melatonin treatment. Flow cytometric analysis was performed using MACSQuant Analyzer with FlowJo. Red curve represents negative control, and blue curve represents expression with ABCG2, MDR, and MPR1, respectively.
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Results Cytotoxicity of clofarabine was partially restored with melatonin treatment in clofarabine-resistant cell lines As shown in Figure 1, clofarabine-resistant cell lines (NALM6/Clo, SKW3/Clo) were strongly resistant to clofarabine when compared with the parent cell lines (NALM6/P, Skw3/P). Next, we determined the optimal concentration of melatonin in NALM6 (IC50 of NALM/P, 3.3 6 0.2 mmol/ L; NALM/Clo, 3.3 6 0.18 mmol/L) and SKW3 cells ((IC50 of SKW3/P, 1.20 6 0.18 mmol/L; SKW3/Clo, 1.25 6 0.20 mmol/L) and we used a 100 mmol/L melatonin treatment for 48 hours in subsequent experiments [22]. Melatonin treatment significantly increased the cytotoxicity of clofarabine in NALM6/Clo cells and SKW3/Clo cells (Fig. 1). Combination index with clofarabine and melatonin showed 1.16 in NALM/P, 0.06 in NALM6/Clo, 1.58 in SKW3/P, and 0.1 in SKW3/Clo. These results strongly indicated that melatonin and clofarabine synergistically acted in clofarabine resistant cells. Deoxycytidine kinase RNA expression decreased in clofarabine-resistant cell lines and increased with melatonin treatment We next examined mRNA expression in clofarabineresistant lines NALM6/Clo and SKW3/Clo, as compared with the parental lines NALM6/P and SKW3/P cells, with or without melatonin treatment (Fig. 2). Clofarabineresistant cells showed decreased expression in dCK mRNA when compared with parental cells. Melatonin treatment increased dCK mRNA in NALM6/Clo and SKW3/Clo cells, but had marginal effects in NALM/P and SKW/P cells (Fig. 2). By contrast, expression of ABCG2, MDR1, and MRP1 by flow cytometric analysis was not altered in NALM/P and NALM/Clo or SKW3/P and SKW3/Clo leukemic cells with or without melatonin treatment (Fig. 3A and B). CpG islands in deoxycytidine kinase promoter did not contribute to changes in expression after melatonin treatment in clofarabine-resistant leukemia cell lines We next measured the methylation status in the dCK promoter by MSP analysis in NALM6/P, NALM6/Clo, SKW3/P, and SKW3/Clo cells with or without melatonin treatment. All NALM6/P, NALM6/Clo, SKW3/P, and SKW3/Clo cells showed all methylated CpG islands in all dCK promoters examined (Fig. 4). Melatonin treatment did not alter the methylation status of dCK promoters (Fig. 4). Decreased acetylation in clofarabine-resistant cells, as compared with parental cells and melatonin treatment, induced increases in acetylation in clofarabine-resistant cells Because a decrease in histone acetylase activity has been shown to induce gene silencing, we next examined histone
Figure 4. Methylation status in dCK promoter in NALM6, SKW3 parent cells and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and methylated and unmethylated dCK promoters were evaluated by methylation-specific PCR using qPCR. Relative values were calculated based on expression of dCK in NALM6 parent cells without melatonin treatment (set as 1). PCR amplification with unmethylated primers did not detect PCR products in all sets of experiments. Data are averages from two independent experiments.
H3 and H4 acetylation activity at the dCK promoter. First, as shown in Figure 5A and B, decreased acetylation of total histone H3 and H4 was observed in clofarabine-resistant cell lines (NALM6/Clo, SKW3/Clo) when compared with parental cell lines (NALM6/P, SKW3/P). Melatonin treatment increased total histone H3 and H4 acetylation in clofarabine-resistant cells (Fig. 5A and B). However, melatonin showed marginal effects in parental cells (Fig. 5A and B). Decreased acetylation of histone H3 at the dCK promoter in clofarabine-resistant NALM6 cells was observed when compared with parental cells on ChIP assay, but not in SKW3/Clo cells (Fig. 6A). Decreased acetylation of histone H4 at the dCK promoter in clofarabine-resistant cells was observed when compared with parental cells on ChIP assay (Fig. 6B). Furthermore, melatonin treatment increased acetylation of both histone H3 and H4 at the dCK promoter in NALM6/Clo cells (Fig. 6A and B). In SKW3/Clo cells, increased acetylation of histone H4 at
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Figure 5. (A) Total histone H3 acetylation in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and total histone H3 expression was evaluated. Relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; # p ! 0.001, SKW3/P vs. SKW3/Clo; ##p ! 0.01, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment. (B) Total histone H4 acetylation in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and total histone H4 expression was evaluated. Relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; #p ! 0.01, SKW3/P vs. SKW3/Clo; ## p 5 0.003, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
the dCK promoter was observed, but no increased acetylation of histone H3 was noted (Fig. 6A and B). Vorinostat restored the sensitivity against clofarabine in acute lymphoid leukemia cell lines We showed that melatonin restored clofarabine sensitivity in clofarabine-resistant cells via increased acetylation.
These results prompted us to investigate whether other Histone Diacetylase (HDAC) inhibitors might increase sensitivity against clofarabine in clofarabine resistant cells. We used vorinostat (N-Hydroxy-N’-phenyloctanediamid). We firstly determined the optimal concentration of vorinostat in NALM6 (IC50 of NALM/P, 15.2 6 3.5 mmol/L; NALM/Clo, 10.1 6 1.1 mmol/L) and SKW3 cells (IC50
Figure 6. (A) Total histone H3 acetylation at dCK promoter as evaluated by ChIP assay in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours, and total histone H3 acetylation was evaluated. After normalization by input DNA, relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.001, NALM/P vs. NALM/Clo; **p ! 0.02, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; #p ! 0.01, SKW3/P vs. SKW3/Clo; ##p ! 0.01, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment. (B) Total histone H4 acetylation at dCK promoter as evaluated by ChIP assay in NALM6, SKW3 parent cells, and clofarabine-resistant cells with or without melatonin treatment. Leukemia cells were incubated with (þ) or without () melatonin (100 mmol/L) for 48 hours and total histone H3 acetylation was evaluated. After normalization by input DNA, relative values were calculated based on expression of dCK in parent cells without melatonin treatment (set as 1). Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without melatonin treatment vs. NALM/Clo with melatonin treatment; #p ! 0.001, SKW3/P vs. SKW3/Clo; ## p ! 0.04, SKW3/Clo without melatonin treatment vs. SKW3/Clo with melatonin treatment.
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Figure 7. IC50 against clofarabine (mmol/L) in NALM6, SKW3 parent cells and clofarabine-resistant cells with or without vorinostat treatment. Leukemia cells were incubated with various doses of clofarabine for 48 hours with (þ) or without () vorinostat and cytotoxicity was evaluated by trypan blue dye exclusion. Data are means 6 standard deviation from three independent experiments. *p ! 0.01, NALM/P vs. NALM/Clo; **p ! 0.01, NALM/Clo without vorinostat treatment vs. NALM/Clo with vorinostat treatment; #p ! 0.01, SKW3/P vs. SKW3/Clo; ## p ! 0.01, SKW3/Clo without vorinostat treatment vs. SKW3/Clo with vorinostat treatment.
of SKW3/P, 12.3 6 1.2 mmol/L; SKW3/Clo, 11.0 6 1.0 mmol/L) and we used a 0.1 mmol/L vorinostat treatment for 72 hours in subsequent experiments [23]. Vorinostat treatment also significantly increased the cytotoxicity of clofarabine in NALM6/Clo cells and SKW3/Clo cells (Fig. 7). Combination index with clofarabine and vorinostat showed 4.00 in NALM/P, 0.04 in NALM6/Clo, 1.67 in SKW3/P, and 0.04 in SKW3/Clo. These results strongly indicated that vorinostat, another HDAC inhibitor, and clofarabine synergistically acted in clofarabine resistant cells (Fig. 7).
Discussion The results of this study showed that the development of clofarabine resistance in two newly established clofarabine-resistant acute lymphoblastic leukemia cell lines was accompanied by downregulation of a key gene in clofarabine metabolism, dCK. This downregulation was not due to gene promoter hypermethylation, as demonstrated by MSP, but was due to gene promoter hypoacetylation, as confirmed by ChIP assay. The mechanisms of clofarabine resistance are reported to be ABCG2 overexpression [24] and decreased expres-
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sion of dCK gene with methylated promoter regions [19]. Our results showed no changes in ABCG2 expression in addition to other export pumps, such as MDR1 and MRP1, nor any changes in methylation status in clofarabine-resistant cells, as compared with sensitive cells. We assessed the histone deacetylation status of dCK promoter in clofarabine resistant cells. Acetylation of histone increases gene expression and is known to be an epigenetic mechanism. To our knowledge, this is the first report to find that histone deacetylation of the dCK promoter is responsible for the decreased expression of dCK in clofarabine-resistant leukemic cells. We also found that histone deacetylation status differs between NALM6 cells, which are derived from B-cell leukemia, and SKW3 cells, which are derived from T-cell leukemia. These findings are important to gain further knowledge of the mechanisms of chemotherapy drug resistance. Gene inactivation by DNA methylation is the epigenetic mechanism shown to be directly involved in the acquisition of chemotherapy resistance in vitro [12]. However, the involvement of other epigenetic mechanisms, such as histone deacetylation, in the inactivation process of genes involved in chemotherapy sensitivity and resistance has been reported [25–28]. Our results strongly suggest a novel model for clofarabine resistance in these newly established acute lymphoblastic leukemia cell lines. In addition, we found that histone acetylation patterns varied between NALM6/Clo and SKW3/Clo. The precise mechanisms were unknown. In some transforming conditions, such as thyroid tumor and endometriosis, different acetylation of histone H3 and H4 occurred separately during transformation [29,30]. We considered that T-cell leukemia and B-cell leukemia might show different acetylation of histones during development of drug resistance. Melatonin plays a role in the regulation of chronobiological rhythms and has been used to ameliorate jet lag. Recently, melatonin was reported to have other important functions, such as increasing promoter methylation [24] and increasing histone H3 and histone H4 acetylation [17]. Increased methylation typically induces decreased gene expression, and increased histone acetylation typically induces increased gene expression. Thus, melatonin exerts both actions on gene expression (increases and decreases expression). In our experiments, melatonin induced increased dCK expression in two clofarabine-resistant leukemia cell lines, which we established. Acquired resistance to clofarabine may be responsible for treatment failure; hence, melatonin may reverse the resistance to and potentially increase the efficacy of clofarabine. In addition, the finding that histone acetylation may lead to chemotherapy resistance deserves testing in other experimental models. In conclusion, to our knowledge, this is the first study to find that melatonin can influence the mechanisms of clofarabine resistance in leukemia cells. Although the mechanism of drug resistance was multifunctional, we consider that, at
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M. Yamanishi et al./ Experimental Hematology 2014;-:-–-
least, we found additional mechanisms in clofarabineresistant leukemia cell lines. Incubation with melatonin resulted in a marked increase in cytotoxicity against clofarabine in resistant cells. These data indicate that melatonin alters DNA accessibility via histone acetylation and relaxation of the chromatin structure, which could allow clofarabine to more efficiently target the DNA. Conflict of interest disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.
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