Epigenetic therapy with chidamide alone or combined with 5‑azacitidine exerts antitumour effects on acute myeloid leukaemia cells in vitro
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- Published online on: January 31, 2022 https://doi.org/10.3892/or.2022.8277
- Article Number: 66
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Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Numerous studies have indicated that cancer may be caused both by mutations in DNA and by two specific types of ubiquitous epigenetic modifications: DNA methylation and histone acetylation (1,2). These two epigenetic modifications may regulate gene expression, remodel chromatin, alter cell phenotypes and promote cancer development (3,4). In addition, methylation and acetylation modifications may regulate cellular pathways that are involved in cell differentiation, proliferation and apoptosis, which are attributed to the genesis of leukaemia (5).
DNA methylation has been widely studied in DNA epigenetic modification. In acute myeloid leukaemia (AML), abnormal DNA methylation may silence the expression of tumour suppressor genes by binding to the upstream promoter region (6). Changes in DNA methylation are prevalent in myelodysplastic syndrome (MDS) and secondary AML and they appear to have an important role in the transformation of MDS to AML (7). Hence, hypomethylating agents (HMAs) are important in the treatment of higher-risk patients with MDS and AML, particularly in those patients who are ineligible for haematopoietic stem cell transplantation (8,9). 5-azacitidine (5-AZA), which is a cytidine analogue prodrug, is an HMA for the treatment of higher-risk MDS and/or AML.
Histone deacetylases (HDACs) are able to alter gene expression and chromatin modification by inducing the deacetylation of histones and other processes (10). In addition, the conversion of DNA-histone complexes from an open state to a compact configuration is closely related to gene transcription silencing (11). In addition to histone deacetylation-induced gene silencing, DNA methylation is also engaged in histone methylation, which is another important epigenetic modification and may regulate proliferation, ageing, tumourigenesis and other biological processes (12). Chidamide is an original new drug in China and a novel selective HDAC inhibitor (HDACI). It is able to selectively inhibit HDAC-1, −2, −3 and −10, particularly −2 and −3. Chidamide has been researched in numerous clinical trials in the USA and China and has been ratified to treat cutaneous T-cell lymphoma and peripheral T-cell lymphoma in China (13). Chidamide is highly toxic to leukaemia cells in a concentration-dependent manner, and less toxic, more tolerated and more stable than other drugs (14). An increasing number of studies have confirmed its antileukaemia effect. Both DNA methylation and histone acetylation are reversible to a certain extent (2), which is worth considering for the design of therapeutic strategies. This mechanism suggests that the combination of HDACI and HMA may be effective against leukaemia. Therefore, HDACI and HMA have become novel strategies for leukaemic and epigenetic treatments in AML (15).
In the present study, the antileukaemia effects of chidamide alone and in combination with 5-AZA on AML cells were assessed. The results suggested that chidamide and its combination with 5-AZA had a strong antileukaemic effect by inhibiting cell proliferation and by inducing apoptosis of AML cells. These results revealed that chidamide is a potential drug for leukaemia treatment, particularly in combination with 5-AZA.
Materials and methods
Cell lines, primary AML samples and reagents
The AML cell lines KG1a, Kasumi-1, NB4, OCI-AML3, U937, MV4-11 and SKM1 were obtained from the Cell Biology Research Institute (Chinese Academy of Sciences) (Table SI). U937 was validated by short tandem repeat analysis. The cell lines were cultured in RPMI 1640 containing 10% foetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin in an incubator at 37°C in a humidified atmosphere consisting of 95% air and 5% CO2.
Primary AML cells were isolated from peripheral blood (PB) or bone marrow (BM) containing >50% blasts. These specimens were cultured in IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS, 50 ng/ml recombinant human (rh) stem cell factor, 100 ng/ml fms-related receptor tyrosine kinase 3 (FLT3) ligand, 25 ng/ml rhIL-3 and 10 ng/ml rhIL-6 (PeproTech, Inc.). The present study was approved by the Research Ethics Committee of The First Affiliated Hospital of Soochow University (Suzhou, China) and written informed consent was provided by all subjects.
Chidamide (Chipscreen Biosciences Ltd.) was dissolved in DMSO (Thermo Fisher Scientific, Inc.) to a concentration of 20 mM to prepare a stock solution. The stock solution was stored at −80°C. 5-AZA (MilliporeSigma) and Venetoclax (MCE MedChemExpress) was respectively dissolved in DMSO to a 100 mM concentration and kept at −80°C. The stock solutions were diluted to working concentrations in subsequent experiments with growth media.
Mononuclear cell separation
BM or PB samples were collected from 5 patients (P1: Male, 57 years; P2: Male, 81 years; P3: Female, 55 years; P4: Female, 75 years; P5: Male, 56 years) who had been diagnosed with AML (non-acute promyelocytic leukaemia) at the First Affiliated Hospital of Soochow University between January 2020 to May 2021 (Table SII). Mononuclear cells were separated by using Ficoll solution (TBD Science) according to the manufacturer's protocol (16).
Cell viability assay
The cytotoxic effects of chidamide and 5-AZA alone (or the combination of the two drugs) on the AML cell lines were determined via the use of the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Inc.). Cell lines were seeded at a density of 1–3×104/ml in 96-well flat-bottomed microtiter plates at 100 µl/well and exposed to chidamide (0–20 µM) or 5-AZA (0–40 µM) at varying concentrations (either alone or in combination) for 72 h, after which they were subjected to a standard CCK-8 assay. The plate was read at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Inc.).
Cell cycle analysis
The cells were treated with chidamide (1 µM) for 48 h. Subsequently, the cells were collected, washed with PBS and then fixed overnight in 75% ice-cold ethanol at 4°C. The fixed cells were then harvested, stained with propidium iodide (PI)/RNase A (BD Pharmingen; BD Biosciences) and incubated in the dark at room temperature for 30 min after being washed with PBS. The DNA content was analyzed by flow cytometry (FCM) with an LSR2 instrument (BD Biosciences). FlowJo 7.6 software (Tree Star, Inc.) was used for data analysis.
Total RNA isolation and reverse transcription-quantitative (RT-q)PCR
The cells were treated with chidamide (1 µM) for 72 h and total cellular RNA was extracted from the cells using Direct-zol™ RNA MircoPrep (ZYMO Research Corp.) according to the manufacturer's protocols. RNA was eluted with RNase-free water, quantified at an absorbance at 260/280 nm and subjected to RT. Total mRNA was reverse transcribed into cDNA using a SuperScript™IV One-Step RT-PCR System (cat. no. 12594025; Thermo Fisher Scientific, Inc.). Real-time qPCR was performed using TaqMan master mix (Thermo Fisher Scientific, Inc.) with 0.2 mM TaqMan probe (Thermo Fisher Scientific, Inc.). The real-time qPCR conditions were as follows: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 20 sec and 40 cycles at 95°C for 1 sec and 60°C for 20 sec. Signals were detected with a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher Scientific, Inc.). Relative expression levels were determined by normalizing to GAPDH levels. GAPDH-qPCR Taqman probe (cat. no. Hs02786624_g14453320; Thermo Fisher Scientific, Inc.) and early growth response 1 (EGR1)-qPCR Taqman probe (cat. no. Hs00152928_m14331182; Thermo Fisher Scientific, Inc.) were used. The results were analyzed using the 2−ΔΔCq method, in which ∆Cq=Cq (target gene)-Cq (internal reference) and ∆∆Cq=∆Cq (sample)-∆Cq (control) (17). Each sample was measured in triplicate.
Cell apoptosis assay
The effects of chidamide alone or combined with 5-AZA on apoptosis of AML cell lines were analysed by using FCM analysis. In brief, cells were harvested, washed with cold PBS and resuspended in 400 µl Annexin Binding Buffer. Subsequently, 1 µl allophycocyanin (APC)-conjugated Annexin V and 5 µl PI (BD Pharmingen; BD Biosciences) were added to each sample. Stained samples were analysed by using FCM with an LSR2 instrument (BD Biosciences).
AML cell differentiation analysis
AML cell lines and AML patient samples were treated with chidamide for three days prior to analysis. Phycoerythrin/cyanin 7-conjugated mouse anti-human CD11b monoclonal antibody (clone ICRF44; cat. no. 301322; BioLegend, Inc.) and APC-conjugated mouse anti-human CD86 monoclonal antibody (clone IT2.2; cat. no. 305412; BioLegend, Inc.) were used for staining at a 1:200 dilution (30 min at 4°C). Stained samples were analysed by using FCM with an LSR2 instrument (BD Biosciences). The results were then analysed with the use of FlowJo 7.6 software (TreeStar, Inc.).
Western blot analysis
Cultured cells were harvested, washed with PBS and then lysed in RIPA buffer. The protein lysates were clarified via centrifugation at 12,000 × g for 30 min at 4°C and the supernatant was collected. The protein concentration was measured by using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Equal amounts (30 µg per lane) of protein were separated by using 12% SDS/PAGE, after which they were electrotransferred onto a PVDF membrane (MilliporeSigma). The membranes were blocked with 5% skimmed milk (BD Biosciences) and incubated with primary antibodies at 4°C overnight. The primary antibodies were as follows: BCL-2 (cat. no. sc-7382; dilution, 1:500; Santa Cruz Biotechnology, Inc.), BAX (cat. no. sc-7480; dilution, 1:500; Santa Cruz Biotechnology, Inc.), BCL-XL (cat. no. sc-8392; dilution, 1:500; Santa Cruz Biotechnology, Inc.) and MCL-1 (cat. no. sc-12756; dilution, 1:500; Santa Cruz Biotechnology, Inc.). GAPDH (cat. no. 97166; dilution, 1:1,000; Cell Signaling Technology, Inc.) was used as a loading control. The membrane was then incubated with secondary antibodies (cat. no. A0216; dilution, 1:1,000; Beyotime Institute of Biotechnology) for 1 h at room temperature, and visualized with the use of an enhanced chemiluminescent western blotting detection reagent (cat. no. RPN2209; Cytiva) via an Gel Doc™ EZ imaging system (Bio-Rad Laboratories, Inc.).
Statistical analysis
All of the experiments were performed in triplicate wells as three independent replicates. All of the data are presented as the mean ± standard deviation. One-way ANOVA was used to compare multiple independent groups by GraphPad Prism 5 (GraphPad Software, Inc.). Further statistical comparisons were performed by using Bonferroni's and Dunnett's test and the corresponding bar charts or linear graphs were drawn by using GraphPad Prism 5 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference. The half-maximal inhibitory concentration (IC50) values were calculated with GraphPad. The combination index (CI) value was calculated from the fraction-affected value of each combination (according to the Chou-Talalay method) by using CompuSyn software 1.0.1 (ComboSyn, Inc.).
Results
Chidamide and 5-AZA synergistically inhibit the proliferation of AML cell lines and primary cultured AML cells
To determine the effect of chidamide and 5-AZA on proliferation, the IC50 values for chidamide and 5-AZA alone were first determined at different concentrations when applied to the AML cell lines M1-M5 for 72 h. The M1-M5 cell lines represent different AML subtypes in accordance with the French-American-British (FAB) classification (Table SI). Chidamide and 5-AZA caused growth inhibition of the M1-M5 cell lines in a dose-dependent manner (Fig. 1A and B). Chidamide markedly inhibited AML cell proliferation at low concentrations (except for KG1a). By contrast, KG1a and Kasumi-1 cells were insensitive to 5-AZA compared with the other cell lines.
Next, the effect of the chidamide and 5-AZA combination on cell viability was evaluated. Cell lines were treated with different concentrations, in accordance with the IC50 of each drug. Chidamide plus 5-AZA inhibited AML cell proliferation in a dose-dependent manner and the combination had a stronger inhibitory effect than either drug alone (Fig. 2A-F). In addition, chidamide and 5-AZA (alone or in combination) had marked cytotoxic effects on the proliferation of primary cultured AML cells from patients and the inhibitory effect was dose-dependent (Fig. 2G and H). The calculation of the CI value suggested that chidamide combined with 5-AZA also had a clear synergistic effect at their suitable concentrations (Fig. 2I). Furthermore, the results of the cell cycle analysis indicated that chidamide was able to cause G1 phase arrest in the MV4-11 cell line (Fig. S1).
Apoptosis is significantly induced by chidamide plus 5-AZA in AML cell lines
An FCM analysis was performed to determine the ability of the drugs to induce apoptosis in AML cells. It was observed that the percentage of Annexin V-positive cells significantly increased in the M1-M5 cell line at 72 h after the use of chidamide or 5-AZA treatment as well as with the combination treatment (Fig. 3A and B). Of note, treatment with chidamide plus 5-AZA induced significantly higher rates of apoptosis than single treatments with either drug (Fig. 3C). The results also suggested that chidamide plus 5-AZA not only increased the early apoptotic population (Annexin V+/PI−) in KG1a, Kasumi-1, U937 and MV4-11 cells [as well as the late apoptotic population (Annexin V+/PI+) in NB4 cells], but also caused increases in both early and late apoptotic populations in OCI-AML3 cells.
Chidamide induces the differentiation of AML cell lines and primary AML samples
To investigate the effect of chidamide on AML cell differentiation, the ability of the drug to upregulate the differentiation markers CD11b (integrin subunit αM) and CD86 was assessed in AML cells. CD11b is a myeloid differentiation marker and CD86 is a monocytic/dendritic differentiation marker. Analysis of The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/) and TAGERT (https://ocg.cancer.gov/programs/target) databases indicated that from the M1 to M5 subtypes of AML, the expression levels of CD11b and CD86 gradually increased at different stages of cell differentiation and maturation (Fig. 4A). Chidamide induced the expression of CD11b in Kasumi-1, NB4, OCI-AML3, U937 and MV4-11 cells (Fig. 4B). Chidamide also upregulated the expression of CD86 (4-fold increase) in primary AML cells but the effect on CD11b (2-fold increase) expression was only slight (Fig. 4C). To investigate the mechanism of differentiation, additional experiments were performed, which indicated that chidamide upregulates the expression of EGR-1 in U937 cells (Fig. S2).
Chidamide and 5-AZA downregulate the antiapoptotic proteins BCL-2 and MCL-1 in AML cell lines
BCL-2 family proteins are critical regulators of apoptotic pathways. To understand the molecular mechanism underlying chidamide- and 5-AZA-induced apoptosis, western blot assays were performed, which indicated that AML cell lines exhibited decreased levels of antiapoptotic proteins (BCL-2 and MCL-1) in response to the single drug treatments after 72 h (Fig. 5A). Chidamide also downregulated the expression of BCL-XL and upregulated BAX in MV4-11 and Kasumi-1 (Fig. S3).
Chidamide downregulates BCL-2 and induces the differentiation of the venetoclax-resistant U937 cell line
Venetoclax markedly inhibited the proliferation of different AML cell lines (M1-M5) at low concentrations (except for U937; IC50=6 µM) (Fig. 5B); thus, the U937 cell line was selected for further analysis. Next, a venetoclax-resistant U937 cell line (R-VEN) was established by gradually increasing the concentration of venetoclax to 12 µM in vitro. Western blot analysis suggested that the expression of MCL-1 protein increased in R-VEN; however, exposure to chidamide led to a modest reduction in MCL-1 expression in R-VEN cells (Fig. 5C). Furthermore, chidamide upregulated the expression of CD11b in the R-VEN cell line (Fig. 5D).
Discussion
From the 1970s until 2017, the progress in the frontline treatment of AML was limited. In other words, since the establishment of ‘7+3′ chemotherapy (cytarabine and anthracycline) in the mid-1970s, the development of agents for patients with AML has proven to be a daunting challenge (18). With improvements in supportive care, long-term survival has improved in younger patients with AML, with a 5-year overall survival (OS) in the range of 40–50%. However, among patients above the age of 60 years who make up the bulk of AML cases, the long-term outlook is dismal, with a 5-year OS of 10–20% (19). Therefore, there remains to be a clear requirement for newer therapies and a more individualized approach for the treatment of AML.
Genes that may be silenced by abnormal methylation or acetylation modifications are promising targets for cancer therapies, particularly in AML (20). In clinical practice, using HDACIs and HMAs in combination or with other antitumour drugs may be an effective antileukaemia strategy. To explore this, clinical trials of HMA combined with HDACI or other drugs for AML have been performed (21–23). HDACI plus HMA has already demonstrated promising effects in the clinical treatment of AML (11,24,25).
In the present study, different types of AML cell lines were used as models, and chidamide and 5-AZA alone or in combination inhibited the proliferation of the M1-M5 AML cell lines in a dose-dependent manner. Different cell lines represent different subtypes of the AML FAB classification. MV4-11 represent patients harbouring the FLT3-internal tandem duplication mutation, which has the highest mutation frequency in AML. When compared with other AML cell lines, both drugs had a weak effect on the growth inhibition of KG1A cells and the IC50 values of chidamide and 5-AZA were 5.57 and 11.11 µM, respectively. The reason may be the slow growth rate of the cell itself; therefore, it is not sensitive to chemotherapy drugs. However, it remains elusive why Kasumi-1 was insensitive to 5-AZA compared with other cell lines; this may possibly be due to special cytogenetics (chromosome and gene mutations). When the two drugs were combined, the inhibitory effect on proliferation was more potent than that of a single drug and the significant synergistic effect was confirmed by the CI value at certain suitable concentrations. It was also observed that chidamide and 5-AZA alone or in combination markedly inhibited the proliferation of AML cells from patient samples; however, the time-point of the test was important, as it was observed that 72 h was too toxic to cells and the synergistic effect was not obvious, and the incubation time was thus decreased to 24 h, at which the synergistic effect was more obvious. In the subsequent experiment, the minimum value of the CI was not selected as the combination concentration, but the intermediate concentration combination was chosen for apoptosis in order to obtain a significant difference in the results. When measuring differentiation, a concentration close to the IC50 was selected, as changes in the expression rate of CD11b were most obvious with this concentration. Meanwhile, the chidamide-induced cell death did not affect the results of the differentiation assay.
Numerous studies have confirmed that HDACI induces cell cycle arrest in G0/G1 phase in vitro (13,26,27). The experimental results of the present study suggested that chidamide is able to cause G1 phase arrest. HDACI and HMA are able to overcome drug resistance by reactivating silenced tumour suppressor genes and inducing cell cycle arrest (28). When chidamide was combined with 5-AZA, chidamide potently and synergistically enhanced the cytotoxicity in AML cell lines. This synergistic cytotoxicity was obvious, as the two agents were able to cooperatively induce cell apoptosis. The present study indicated that the single drug-induced inhibition was incomplete; however, when chidamide plus 5-AZA was added, the inhibition was significantly improved. Different cell lines had different apoptosis types; specifically, certain cell lines exhibited early apoptosis, while others exhibited late apoptosis, and certain cells exhibited both types of apoptosis.
The FAB classification system is based on morphology for defining specific immunotypes (29). From M1 to M5, the maturity of cells increased and the expression levels of CD11b and CD86 also tended to increase, which was also confirmed by the results from the TCGA and TARGET databases. CD11b is considered a myeloid differentiation marker of leukaemia cells (30). For instance, the expression of CD11b may be significantly upregulated when AML-M3 cells are treated with ATRA and a similar change may also be observed in U937 cells treated with Brequinar (31). CD86 is considered a monocytic/dendritic differentiation marker (32). The present results indicated that the expression of CD11b on Kasumi-1, NB4, OCI-AML3, U937 and MV4-11 cells was upregulated (except for KG1a cells) after treatment with chidamide. Perhaps KG1a has different biological characteristics from other AML cell lines, thus making them more primitive and more difficult to differentiate, based on drug induction. Further research on the combined effect of chidamide and 5-AZA on the differentiation of AML cells requires to be performed. EGR-1 is a differentiation-related transcription factor. The results of the present study indicated that chidamide was able to upregulate the expression of EGR-1 in U937. However, when primary cultured cells from patients with AML were treated with chidamide in vitro, it was observed that both CD11b and CD86 were upregulated, thus indicating that the cells further differentiated into monocytic/dendritic cells. As most patients are diagnosed with M4 or M5, they are more likely to exhibit monocytic/dendritic differentiation. It was speculated that this may be related to the subtypes of AML patients, which requires further exploration.
BCL-2 family proteins are important for cell fate, as they may regulate cells in two ways: Anti- and pro-apoptosis (33,34). Our previous research also found that chidamide is able to induce apoptosis in AML cell lines via antiapoptotic BCL-2 family proteins (BCL-2 and MCL-1) (35). However, in the present study, no significant synergistic effect of chidamide combined with 5-AZA to downregulate the BCL-2 and MCL-1 was observed (data not shown). In addition, chidamide was also able to downregulate the expression of BCL-XL and upregulate BAX in MV4-11 and Kasumi-1. Various HDACIs (in combination with decitabine) shortened tumour cell survival at the mRNA and protein levels (36). The results of the present study demonstrated that chidamide and 5-AZA alone were able to degrade the antiapoptotic proteins BCL-2 and MCL-1 in several AML cell lines. Of note, it was observed that the expression of MCL-1 was compensatorily increased in the venetoclax-resistant U937 cell line (35). Chidamide degraded MCL-1 in venetoclax-resistant U937 cells and upregulated the expression of CD11b, and venetoclax slightly promoted CD11b expression in venetoclax-resistant U937 cells as compared with U937 (data not shown). There is reason to believe that the downregulation of MCL-1 may be a promising strategy for the treatment of venetoclax-resistant leukaemia. Further investigations will be performed in in vivo models (e.g. PDX animal models or an increased number of patient samples).
Other studies have reported that chidamide is able to degrade the AML1-ETO fusion gene and also inhibit leukaemia cells with mixed lineage leukemia (MLL) rearrangements (37,38). Chidamide may become a novel option in the treatment of certain subtypes of AML, such as t(8;21) and MLL rearrangement, in the future.
It has been demonstrated that antitumour drugs have multiple functions and targets. HDACI combined with 5-AZA may have multiple pathways for inducing cell apoptosis, differentiation and proliferation inhibition. In conclusion, chidamide in combination with 5-AZA has a synergistic antileukaemia effect in vitro and may be considered a therapeutic strategy for AML.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This work was supported by grants from the Social Development-Clinical Frontier Project of Jiangsu Province (grant no. BE2018652), the National Natural Science Foundation of China (grant no. 81970138), Translational Research Grant of the National Clinical Research Center for Hematology (grant no. 2020ZKMB05) and the Project of State Key Laboratory of Radiation Medicine and Protection (grant no. GZK1202002).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
HYQ contributed conceptualization, funding acquisition, supervision, resources, study design and confirmed the authenticity of all the raw data. SLX contributed to conceptualization, funding acquisition, supervision, resources, study design and confirmed the authenticity of all the raw data. ZL designed and performed the experiments. JZ, MZ and JLL performed the experiments. QCQ and JHF analyzed data. ZL wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by The First Affiliated Hospital of Soochow University (Suzhou, China) and all patients provided written informed consent.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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