Chidamide acts on the histone deacetylase‐mediated miR ‐34a/Bcl‐2 axis to regulate NB4 cell line proliferation and apoptosis

Abstract

Acute promyelocytic leukemia (APL), a biologically and clinically distinct variant of acute myelogenous leukemia, is characterized by the fusion of the N‐terminus of promyelocytic leukemia protein to the C terminus of retinoic acid receptor alpha, mostly due to chromosomal translocation t(15;17). Chidamide, a synthetic analogue of MS‐275 identified from a group of benzamide‐type compounds, has been found to have efficient anticancer activity in basic and clinical research studies. However, the concrete role and underlying mechanism of Chidamide in the treatment of APL has not been well characterized. Our data demonstrate that Chidamide inhibited the expression of histone deacetylase (HDAC) to induce apoptosis and suppress proliferation in NB4 cells. Mechanistically, Chidamide increases the expression of miR‐34a by suppressing HDAC. Furthermore, B‐cell lymphoma‐2 (Bcl‐2) is a direct target of miR‐34a, the expression of which is regulated by miR‐34a. Functionally, Chidamide inhibits cell proliferation and promotes apoptosis through miR‐34a/Bcl‐2. Chidamide exerts its anticancer effect via the HDAC‐mediated miR‐34a/Bcl‐2 axis, providing potential targets for APL therapy.

Abbreviations
• AML
• acute myeloid leukemia
• APL
• acute promyelocytic leukemia
• Bcl‐2
• B‐cell lymphoma‐2
• Bax
• Bcl‐2 associated X protein
• Caspase‐3
• cysteine aspartic acid specific protease 3
• FITC
• fluorescein isothiocyanate
• GAPDH
• glyceraldehyde‐3‐phosphate dehydrogenase
• Caspase‐3
• cysteine aspartic acid specific protease 3
• HDACs
• histone deacetylases
• HDACIs
• histone deacetylase inhibitors
• miRNAs
• microRNAs
• p21
• cyclindependent kinase inhibitor 1
• PBS
• phosphate‐buffered saline
• qRT‐PCR
• quantitative realtime polymerase chain reaction

INTRODUCTION

Acute promyelocytic leukemia (APL), characterized by the fusion of the N‐terminus of promyelocytic leukemia protein (PML) to the C terminus of retinoic acid receptor alpha (RARα), mostly due to chromosomal translocation t(15;17), is a biologically and clinically distinct variant of acute myelogenous leukemia (AML).¹ In the clinic, chemotherapy with anthracycline, such as daunorubicin and idarubicin, and cytarabine arabinoside is the preferred treatment for APL. However, the results are discouraging because only 35% to 45% of APL patients are cured by standard chemotherapy alone.² With better understanding of the pathogenesis of APL, the importance of PML‐RARα degradation in therapy against APL has gained attention, accompanied by the introduction of new drugs, such as all‐trans retinoic acid and arsenic trioxide.³ New drugs or strategies are still needed, and combination therapy is also vital for patients.

Histone deacetylases (HDACs) are a class of enzymes that deacetylate the acetyl group of the ε‐N‐acetyl lysine amino acid of histones. HDACs are the main pathogenic factors in leukemia, such as chronic myeloid leukemia, chronic lymphocytic leukemia, pediatric acute myeloid leukemia, and APL.⁴ The pathogenic effect of HDACs has also been found in renal cancer, colorectal cancer, gastric cancer, and breast cancer.⁵ Histone deacetylase inhibitors (HDACIs) are considered promising drugs to treat cancer, and their positive effects on cell differentiation, cell cycle arrest, and apoptosis in leukemic cells have been reported.⁶ To date, panobinostat, belinostat, romidepsin, and vorinostat, the only HDACIs, have been approved by the US Food and Drug Administration (FDA) in several cancer treatments.⁷

Chidamide (HBI‐8000 or CS055) is a synthetic analogue of MS‐275 identified from a set of benzamide‐type compounds, followed by further rational design by using molecular docking and an HDAC‐like protein. Chidamide is an innovative drug independently developed by Chipscreen Biosciences in China.⁸ Chidamide selectively suppresses the activity of class I HDACs (HDAC 1, 2, 3) and class IIb HDAC (HDAC 10) by targeting the catalytic pocket.⁹ The efficient anticancer activity of Chidamide has been revealed in basic and clinical research.¹⁰ In non‐small cell lung cancer cells, Chidamide synergistically increases the DNA damage and apoptosis induced by platinum.¹¹ Additionally, Chidamide induces apoptosis in human colon cancer cells¹² and exhibits antitumor activity in hepatocellular carcinoma cell lines.¹³ In pancreatic cancer cells, Chidamide inhibits aerobic metabolism to induce growth arrest mediated by Mcl‐1 degradation.¹⁴ Additionally, Chidamide was found to induce G0/G1 arrest and apoptosis in myelodysplastic syndromes.¹⁵ In human leukemia cells, Chidamide participates in reactive oxygen species (ROS)‐dependent apoptosis and differentiation.¹⁶ When Chidamide and decitabine were used together, apoptosis was enhanced through DNA damage in adult acute lymphoblast leukemia, especially in patients with p16 deletion.¹⁷ In NK/T lymphoma cells, Chidamide induces growth inhibition and apoptosis.¹⁸ However, further detailed research on Chidamide in the treatment of APL is necessary. The concrete role of Chidamide in APL therapy has not been well characterized.

In our study, we focused on microRNAs (miRNAs). miRNAs are a family of small, noncoding, endogenous RNAs (19‐24 nucleotides in length). miRNAs bind to the 3′‐untranslated region (3′‐UTR) of the mRNA sequence and lead to translational degradation or repression to ultimately inhibit gene expression. miRNAs are thought to play crucial roles in cell proliferation, apoptosis, migration, and invasion.¹⁹ Dysregulation of miRNAs that act as tumor suppressors or oncogenes can lead to tumorigenesis. In acute leukemia, the association of miRNAs expression with lineage and survival has been found.²⁰ In addition, HDACI treatment can also lead to the suppression of miRNAs, which are an important class of regulatory genes.²¹ Furthermore, miR‐34a, located on chromosome 1p36.23, was originally identified as a target gene of p53, the upregulation of which induces apoptosis and cell cycle arrest.²² miR‐34a is downregulated and functions as a tumor suppressor in many human cancers.²³ For example, in human colon cancer cells, miR‐34a induces senescence‐like growth arrest mediated by the regulation of the E2F pathway.²⁴ miR‐34a downregulates the expression of c‐Met and inhibits the migration and invasion of human hepatocellular carcinoma cells.²⁵ Research has found that miR‐34a expression inhibits leukemia stem cells and their metastasis.²⁶ In addition, Zenz et al found that miR‐34a is part of the chemotherapy resistance network in chronic lymphocytic leukemia.²⁷ Mraz et al also found that miR‐34a is downregulated in chronic lymphocytic leukemia patients with TP53 abnormalities.²⁸ This indicates that aberrant miR‐34a expression is a marker of chemotherapy resistance in a variety of cancers.²⁹

Chidamide is an innovative antitumor drug, and its mechanism requires further investigation in APL. In the present study, the function of Chidamide in the apoptosis and viability of NB4 cells as well as its potential regulatory mechanism on miR‐34a and HDAC were explored.

MATERIALS AND METHODS

Cell culture and cell transfection

The human APL cell line NB4 was obtained from the Shanghai Institute of Cell Biology (Chinese Academy of Science, Shanghai, China). Cells were cultured with RPMI 1640 containing 10% fetal bovine serum (Gibco, Life Technologies, NY), 100 U/mL penicillin, and 100 μg/mL streptomycin (1 × P/S) in a CO2 incubator at 37°C. The NB4 cell line was treated with serial dilutions of Chidamide (0, 5, 25, 50 μM) for 24 hours. According to the manufacturer's instructions, NB4 cells were transfected with pcDNA‐HDAC, pcDNA‐B‐cell lymphoma‐2 (Bcl‐2), miR‐34a inhibitor, or miR‐34a mimic using Lipofectamine 2000 (Invitrogen). Then, the cells (transfection efficiency >90%) were further stimulated with Chidamide for 24 hours.

RNA isolation and quantitative real‐time polymerase chain reaction (qRT‐PCR)

Total RNA was extracted from treated cells using the RNeasy Micro Kit (Qiagen). Then, we performed reverse transcription using an Omniscript RT kit (Qiagen). Real‐time quantitative PCR was run on an iCycler iQ (Bio‐Rad) with the Quantitect SYBR Green PCR kit (Qiagen). Primers for qRT‐PCR were designed using the Primer Express 5.0 software (Applied Biosystems, Foster City, CA). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as the housekeeping gene for transcript normalization, and the mean values were used to calculate relative transcript levels with the ΔΔCT method as per the instructions from Qiagen. All assays were performed in duplicate, and assay products were validated using melting curves to confirm the presence of single PCR products.

Western blot analysis

Western blot analysis was performed using standard procedures. Briefly, cells were lysed on ice in radio Immunoprecipitation assay (RIPA) buffer. The protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to polyvinylidene membranes (Millipore, Billerica, MA), and probed with antibodies. Primary antibodies against cleaved cysteine aspartic acid specific protease 3 (caspase‐3) (1:1000), Bcl‐2 associated X protein (Bax) (1:1000), Bcl‐2 (1:1000), and GAPDH (1:1000) were obtained from Cell Signaling Technology (CST, MA). Following three Tris‐buffered saline Tween 20 washes, the blots were incubated with horseradishperoxidase (HRP)‐conjugated secondary antibody for 1 hour. Blotted proteins were visualized using an enhanced chemiluminescence kit.

Flow cytometric analysis

Apoptosis was investigated using an Annexin V‐fluorescein isothiocyanate (FITC) apoptosis kit according to the manufacturer's protocol (BD Biosciences, San Jose, CA). The cells were harvested and washed. The number of cells was adjusted to 1 × 10⁶ cells/mL in binding buffer. FITC‐labeled Annexin V (5 μL) and propidium iodide (PI, 5 μL) were added to the cells and incubated for 15 minutes at room temperature. Finally, we used a flow cytometer (BD Biosciences) to detect the stained cells. The storing and processing of data was analyzed with the FlowJo software v10.0.7 (Tree Star, Inc., Ashland, OR).

Colony forming assay

NB4 cells were stimulated with Chidamide for 24 hours. After washing with phosphate‐buffered saline, NB4 cells were plated in methylcellulose medium in triplicate. Clonogenicity was detected after 7 days. NB4 cells were transfected with miR‐34a inhibitor, pcDNA‐Bcl‐2, or pcDNA vector for 24 hours. Then, the transfected cells were stimulated with Chidamide for an additional 24 hours before being assayed for their colony forming ability after 7 days.

Luciferase assay

We used TargetScan (http://www.targetscan.org/) and miRDB (http://mirdb.org/) to predict the targets and the potential binding between miR‐34a and the 3′‐UTR of Bcl‐2.

NB4 cells (3 × 10⁴/well) were seeded into 24‐well plates and then transfected with the Renilla luciferase pRL‐TK plasmid plus the recombinant firefly luciferase pGL3 reporter containing the 3′‐UTR of human Bcl‐2 (GenePharma Technology Co., Ltd, Shanghai, China) in combination with the miR‐34a mimic or NC mimic by using Lipofectamine 2000. After transfection for 24 hours, we collected the cells and used a Dual‐Luciferase Reporter Assay Kit (Promega, WI) to measure firefly and Renilla luciferase activity.

Cell viability assay

Cell viability was determined using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) proliferation assay kit (Biosharp Life Sciences, Anhui, China). NB4 cells were seeded in 96‐well plates (1 × 10⁴ cells/well, 180 μL) and cultured for 24 hours. Cells were treated with 25 μM Chidamide for 24 hours. Cells were subsequently incubated with 10 μL of MTT reagent for 4 hours. The spectrophotometric absorbance (490 nm) was measured by a SPECTRAmax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).

Statistical analysis

Statistical evaluation was performed using the SPSS 20.0 software (SPSS, Inc., Chicago, IL). All data are expressed as the means ± SD of at least three experiments. Student t test was performed to analyze comparisons between two groups. One‐way analysis of variance (ANOVA) followed by Tukey's test was conducted to analyze comparisons among three or more groups. P < .05 was considered statistically significant.

RESULTS

Chidamide inhibits the expression of HDAC in leukemic cells

To investigate the action of Chidamide on HDAC, we detected the mRNA and protein expression of HDAC by quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blot analysis, respectively, in leukemic cells. When we stimulated NB4 cells with different concentrations of Chidamide, we found reduced mRNA expression of HDAC in a dose‐dependent manner (Figure 1A), as well as reduced protein expression (Figure 1B). These results showed that Chidamide reduced the expression of HDAC in leukemic cells.

1 FIGURE. Chidamide inhibits HDAC mRNA and protein expression in leukemic cells. A, qRT‐PCR analysis of HDAC mRNA levels after treatment with serial dilutions of Chidamide. NB4 cells were treated with Chidamide for 24 hours at the indicated concentrations. B, Western blot analysis of HDAC protein expression in NB4 cells incubated with Chidamide. GAPDH was used as a loading control. The data are the means ± SD of three independent sets of analyses. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HDAC, histone deacetylase; qRT‐PCR, quantitative real‐time polymerase chain reaction. *P < .05; **P < .01; ***P < .001

Chidamide regulates the proliferation and apoptosis of leukemic cells by inhibiting HDAC

To further verify the effect of Chidamide on HDAC, we constructed an HDAC overexpression plasmid (pcDNA‐HDAC). As shown in Figure 2A, the decreased HDAC mRNA level in HDAC‐transfected cells was restored compared with that in vector‐transfected cells after Chidamide stimulation. Subsequently, the MTT proliferation assay indicated that Chidamide reduced cell viability, which was reversed by overexpression of HDAC (Figure 2B). Moreover, apoptosis analysis showed that HDAC overexpression decreased the apoptosis induced by Chidamide (Figure 2C‐D). Western blot analysis indicated enhanced protein expression of cleaved caspase‐3 and Bax and decreased expression of Bcl‐2 in Chidamide‐treated NB4 cells, while these effects were counteracted in pcDNA‐HDAC‐transfected cells (Figure 2E). These findings suggest that Chidamide inhibits cell proliferation and induces apoptosis by inhibiting HDAC in NB4 cells.

2 FIGURE. Chidamide inhibits proliferation and promotes apoptosis by inhibiting HDAC. A, qRT‐PCR analysis of HDAC mRNA expression in pcDNA‐HDAC‐ or vector‐transfected cells after 25 μM Chidamide stimulation. B, Overexpression of HDAC recovered cell viability after 25 μM Chidamide stimulation, as demonstrated by the MTT proliferation assay. C‐D, HDAC overexpression decreased the apoptosis induced by Chidamide. Apoptosis was determined by flow cytometric analysis after NB4 cells were treated with 25 μM Chidamide for 24 hours. E, The expression of Bcl‐2, Bax, and cleaved caspase‐3 was assayed by a standard Western blot. The data are the means ± SD of three independent sets of analyses. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HDAC, histone deacetylase; qRT‐PCR, quantitative real‐time polymerase chain reaction. *P < .05; **P < .01; ***P < .001

Chidamide upregulates the expression of miR‐34a mediated by HDAC

We further explored the underlying molecular mechanism of Chidamide in the progression of anti‐proliferation and pro‐apoptosis. We found that Chidamide treatment increased the miR‐34a level (Figure 3A). HDAC overexpression inhibited the enhanced expression of miR‐34a, as did the miR‐34a inhibitor (Figure 3A). MTT proliferation assays showed that miR‐34a inhibitor‐transfected cells recovered the viability induced by Chidamide (Figure 3B). As expected, upregulation of miR‐34a induced by Chidamide promoted cell apoptosis, while inhibiting miR‐34a alleviated Chidamide‐induced apoptosis (Figure 3C‐D). Compared with the inhibitor NC group, the miR‐34a inhibitor group presented reverted protein expression levels of caspase‐3, Bax, and Bcl‐2 (Figure 3E). Overall, the anti‐proliferative and pro‐apoptotic characteristics of Chidamide are mediated by the upregulation of miR‐34a in APL.

3 FIGURE. Chidamide upregulates the expression of miR‐34a mediated by HDAC. A, qRT‐PCR showed the expression of miR‐34a in NB4 cells transfected with pcDNA‐HDAC, inhibitor NC, or miR‐34a inhibitor following 25 μM Chidamide stimulation. HDAC overexpression inhibited the enhanced expression of miR‐34a. B, MTT proliferation assay showed recovered cell viability in miR‐34a inhibitor‐transfected cells following Chidamide treatment. C‐D, The indicated four groups of cells were exposed to Chidamide, and apoptosis was determined by flow cytometric analysis. The miR‐34a inhibitor decreased the apoptosis induced by Chidamide. E, Western blotting was used to detect the protein expression of caspase‐3, Bcl‐2, and Bax in the four groups of cells. The data are the means ± SD of three independent sets of analyses. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HDAC, histone deacetylase; qRT‐PCR, quantitative real‐time polymerase chain reaction. *P < .05; **P < .01; ***P < .001

Bcl‐2 is a direct target of miR‐34a in NB4 cells

Using the TargetScan and miRDB databases, we found that Bcl‐2 might be a target gene of miR‐34a (Figure 4A). Then, we characterized the binding site of miR‐34a in the 3′‐UTR of Bcl‐2 mRNA and observed that miR‐34a specifically decreased the luciferase activity of the WT reporter (Figure 4B). When cells were cotransfected with miR‐34a, the relative luciferase activity of the mutant reporter construct did not decrease significantly. Furthermore, overexpression of miR‐34a reduced the mRNA level of Bcl‐2 in NB4 cells, while inhibition of miR‐34a increased it (Figure 4C). Western blot analysis further confirmed this result (Figure 4D). In conclusion, Bcl‐2 is a target gene of miR‐34a in the progression of Chidamide regulating the proliferation and apoptosis of leukemic cells.

4 FIGURE. miR‐34 targets Bcl‐2 in AML. A, The sequences of miR‐34a binding sites in the Bcl‐2 3′‐UTR. B, The relative luciferase activities of Bcl‐2 WT and Bcl‐2 MUT were measured using a Dual‐Luciferase Reporter Assay Kit following transfection with miR‐34a mimic (50 pmoL/mL). Bcl‐2 WT is the reporter constructs containing the entire 3′‐UTR sequence of Bcl‐2. Bcl‐2 MUT is the reporter constructs containing mutated nucleotides. C, qRT‐PCR was performed to detect the expression of Bcl‐2 mRNA in cells transfected with mimic NC, inhibitor NC, miR‐34a mimic, or miR‐34a inhibitor. D, Western blotting was performed to determine the expression of Bcl‐2 protein in the indicated groups of cells. The data are the means ± SD of three independent sets of analyses. WT, wild type; MUT, mutant type; AML, acute myelogenous leukemia; qRT‐PCR, quantitative real‐time polymerase chain reaction; UTR, untranslated region. *P < .05; **P < .01; ***P < .001

Chidamide inhibits cell proliferation and promotes apoptosis through miR‐34a/Bcl‐2

To further confirm that Chidamide inhibits cell proliferation and promotes apoptosis through miR‐34a/Bcl‐2, we cotransfected miR‐34 mimics and pcDNA‐Bcl‐2 in NB4 cells followed by Chidamide stimulation. As shown, the mRNA level of Bcl‐2 was reduced in Chidamide‐treated cells and was restored in Bcl‐2 overexpressing cells. When miR‐34a mimics were cotransfected with pcDNA‐Bcl‐2, the high mRNA level of Bcl‐2 was inhibited (Figure 5A). In addition, we also found suppressed proliferation in cells cotransfected with miR‐34a mimics and pcDNA‐Bcl‐2 as well as in normal cells stimulated by Chidamide, while overexpression of Bcl‐2 enhanced cell viability (Figure 5B). Colony forming assays further showed similar reduced cell proliferation in cells cotransfected with miR‐34a mimics and pcDNA‐Bcl‐2 compared to Bcl‐2 overexpressing cells (Figure 5C). Moreover, flow cytometric analysis showed that miR‐34a overexpression could enhance the cell apoptosis rate, which was inhibited in Bcl‐2‐overexpressing cells following Chidamide treatment (Figure 5D). The recovered apoptotic protein expression also further confirmed these results (Figure 5E). Here, the results showed that Bcl‐2 overexpression could counteract the increased apoptosis and suppress viability induced by Chidamide. However, when cells were cotransfected with miR‐34a mimics and pcDNA‐Bcl‐2, the variation in the apoptosis or proliferation of cells overexpressing pcDNA‐Bcl‐2 was abolished. Combined, these results further confirm that Chidamide acts on leukemic cell proliferation and apoptosis through the HDAC‐mediated miR‐34a/Bcl‐2 axis.

5 FIGURE. Chidamide regulates cell proliferation and apoptosis through miR‐34a/Bcl‐2. A, qRT‐PCR was performed to measure the expression of Bcl‐2 mRNA in cells transfected with the plasmids indicated below following Chidamide treatment. B, MTT proliferation assay was used to detect cell viability. C, A colony formation assay was used to further investigate proliferation in different groups of cells following transfection with miR‐34a inhibitor, pcDNA‐Bcl‐2, and pcDNA vector. Cell viability was determined by MTT assay. D, Apoptosis was determined by flow cytometric analysis after NB4 cells were transfected as indicated and treated with Chidamide. E, Western blotting was performed to detect the protein expression of caspase‐3, Bax, and Bcl‐2. The data are the means ± SD of three independent sets of analyses. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; qRT‐PCR, quantitative real‐time polymerase chain reaction. *P < .05; **P < .01; ***P < .001

DISCUSSION

Although significant progress has been achieved in the treatment of APL in recent years, poor long‐term clinical outcomes after conventional chemotherapy have been a major problem for patients. Regardless of the poor long‐term clinical outcomes, conventional chemotherapeutic drugs remain the most preferred and efficacious standard agents in the clinic for the therapy of APL. However, these drugs cannot completely eliminate tumor cells and result in a high recurrence rate. Recently, HDACIs have emerged as a promising clinical treatment strategy for leukemia by inducing cell differentiation and apoptosis.³⁰ However, unlike other hematologic malignancies, such as cutaneous T‐cell lymphoma, some studies have shown that HDACIs have limited clinical activity as a single agent and that combination therapy with hypomethylated drugs does not always result in a significantly improved response.³¹ The possible reason for this may be the different pharmacologic potency, rapid metabolism, and/or off‐target activity (eg, acetylation of nonhistone substrates) specific to HDACIs.³² In view of this, the underlying mechanisms responsible for the effect of Chidamide on APL require further exploration. These studies will provide the basis for the clinical improvement of the therapeutic effect of Chidamide in the treatment of APL.

As a novel HDACI with a similar chemical structure to entinostat (MS‐275), Chidamide is less toxic, better tolerated, and more stable.³³ In multiple myeloma, Chidamide was reported to induce a marked anti‐myeloma effect by promoting G0/G1 arrest through a decrease in the expression of cyclin D1 and c‐myc and an increase in the expression of phosphorylated cell tumor antigen p53 and cyclin‐dependent kinase inhibitor 1 (p21).⁷ In human leukemia cells, Chidamide induces apoptosis and differentiation in a manner that is ROS‐dependent.¹⁶ In combination therapy for leukemia, Chidamide was found to have a synergistic antitumor effect in combination with cytarabine in FLT3‐ITD‐positive acute myeloid leukemia.³⁴ Similarly, we found that Chidamide inhibited proliferation and promoted apoptosis by inhibiting HDAC. Overexpression of HDAC recovered the cell viability and decreased the apoptosis induced by Chidamide. Chidamide upregulated Bax (Bcl‐2 family proteins) and downregulated Bcl‐2 (anti‐apoptotic Bcl‐2 family proteins) to further induce the intrinsic apoptotic pathways. Here, we also detected increased cleaved caspase‐3 protein expression in Chidamide‐treated cells. Chidamide was reported to suppress proliferation by promoting mitochondrial pathway‐dependent cell apoptosis through upregulation of the Bax/Bcl‐2 ratio in pancreatic cancer.³⁵ Whether Chidamide acts on mitochondrial function and metabolism remains a topic of our future studies. In our study, we found reduced mRNA expression of HDAC by Chidamide in a dose‐dependent manner. Chidamide was reported to exhibit antitumor activity similar to its effects in colon cancer, hepatocellular carcinoma, pancreatic cancer, and myelodysplastic syndromes.^12–15 Chidamide selectively suppresses the activity of class I HDACs (HDAC 1, 2, 3) and class IIb HDAC (HDAC 10) by targeting the catalytic pocket.⁹ HDACs are frequently overexpressed in many cancers, and inhibition of HDACs arrests cell cycling and often drives cancer cells into apoptosis.³⁶ Consistent with previous reports, we further observed that HDAC overexpression reversed Chidamide‐induced apoptosis, indicating that HDAC plays an antiapoptotic role in APL cells.

To further explore the mechanism of Chidamide on HDAC‐mediated proliferation and apoptosis regulation, we focused on miRNAs. HDAC was verified to exhibit a strong effect on miRNAs and induce ~40% changed expression of miRNAs.³⁷ Here, we observed enhanced miR‐34a levels induced by Chidamide to promote apoptosis and suppress proliferation. Recently, the induction of apoptosis has become a promising clinical therapeutic strategy for the exploitation of novel anticancer agents. The identification of additional miR‐34a targets regulating apoptosis may help to find further points of intervention in the clinic. Interestingly, miR‐34a is rarely hypermethylated in chronic lymphocytic leukemia, while miR‐34b/c is frequently methylated in patient samples of chronic lymphocytic leukemia.³⁸ No significant association was found between miR‐34b/c methylation status and clinical parameters (age, gender, diagnostic Hb, lymphocyte or platelet count, Rai stage, or survival).³⁸ In APL, whether the reported increased miR‐34a expression after Chidamide treatment is associated with clinical rehabilitation index and the function of the miR‐34 family in AML remain to be further studied. In addition, we also found that miR‐34a‐targeted Bcl‐2 to induce apoptosis in Chidamide‐treated NB4 cells. miR‐34a was originally identified as a target gene of p53.²² Studies have shown that PML can activate Chk2 and that phosphorylation is important for the induction of p53‐independent apoptosis in APL.³⁹ Here, we wondered whether, in addition to the miR‐34a/Bcl‐2 axis, miR‐34a could be a regulator mediated by p53. In APL cells with PML/RAR, apoptosis is inhibited, at least in part, because of the dominant negative action of PML/RAR over PML.⁴⁰ Here, we found that Chidamide induced apoptosis in NB4 cells. Interestingly, Chidamide was also reported to regulate p53 in multiple myeloma,⁷ which also helps to determine whether Chidamide could affect some players that are also important regulators associated with PML/RARα.

In summary, we showed that Chidamide regulates miR‐34a/Bcl‐2 by inhibiting HDAC and acts on cell proliferation and apoptosis in APL. However, the correlation between Chidamide and miR‐34a/Bcl‐2 and the exact underlying mechanism in regulating proliferation and apoptosis remain to be further investigated.

CONFLICT OF INTERESTS

The authors declare no potential conflicts of interest.