Contributed equally
Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignant tumor type, and epigenetic modifications such as acetylation or deacetylation serve vital roles in its development. Chidamide, a novel histone deacetylase inhibitor, exerts an anticancer effect against various types of cancer. The present study aimed to evaluate the cellular effect of chidamide on a number of DLBCL cell lines and to investigate its underlying mechanism. The results demonstrated that chidamide induced the death of these cells in a concentration-(0–30 µmol/l) and time-dependent (24–72 h) manner, as determined using the Cell Counting Kit-8 cell viability assay. Moreover, chidamide promoted cellular apoptosis, which was identified via flow cytometry and western blot analysis, with an increase in cleaved caspase-3 expression and a decrease in Bcl-2 expression. Chidamide treatment also decreased the expression level of STAT3 and its phosphorylation, which was accompanied by the downregulation of a class-I histone deacetylase (HDAC) inhibitor, chidamide. Collectively, these data suggested that chidamide can be a potent therapeutic agent to treat DLBCL by inducing the apoptotic death of DLBCL cells by inhibiting the HDACs/STAT3/Bcl-2 pathway.
Diffuse large B-cell lymphoma (DLBCL) is a genetically and clinically heterogeneous lymphoid malignancy type, which represents the most common category and disease entity of non-Hodgkin lymphoma in adults and accounts for ~30-40% of all cases in different geographic regions, such as Europe and the USA (
STAT3 is a transcription activation factor, and participates in the malignant progression of multiple tumors, including DLBCL (
Chidamide is a benzamide histone deacetylase (HDAC) inhibitor, and it has been marketed in China for the treatment of peripheral T-cell lymphoma (PTCL) since 2015 (
To provide evidence for the clinical application of chidamide in DLBCL, the present study examined the cytotoxic effect of chidamide on DLBCL cells and its possible underlying molecular mechanism.
Human DLBCL cell lines SU-DHL2 and MZ were purchased from Jennio-Bio, Co., Ltd., and the OCI-LY3 cell line was from Beina Chuanglian Biological Research Institute (Beijing, China). SU-DHL2 and OCI-LY3 cells of the ABC-subtype, and the MZ cells of the GCB-subtype of DLBCL cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured in a 37°C incubator with 5% CO2. All three cell lines were tested for mycoplasma contamination and authenticated by STR profiling.
Chidamide was purchased from Absin Bioscience, Inc., and dissolved in DMSO (Sigma-Aldrich; Merck KGaA) to prepare the stock solution (30 mM), which was stored at −20°C in the dark. A Cell Counting Kit-8 (CCK-8) was obtained from Beijing Solarbio Biotechnology Co., Ltd. The Annexin V-FITC/PI kit, which was used for apoptotic cell death determination via flow cytometry, was purchased from Beyotime Institute of Biotechnology. Antibodies against cleaved caspase-3 (cat. no. 9664S; 1:1,000), Bcl-2 (cat. no. 15071S; 1:1,000), HDAC1 (cat. no. 34589S; 1:1,000), HDAC2 (cat. no. 57156S; 1:1,000), HDAC3 (cat. no. 85057S; 1:1,000), STAT3 (cat. no. 9139S; 1:1,000), phosphorylated (P)-STAT3-705 (cat. no. 9145S; 1:1,000), P-STAT3-727 (cat. no. 49081S; 1:1,000) and β-actin (cat. no. 3700S; 1:5,000) were purchased from Cell Signaling Technology, Inc. An antibody against HDAC8 (cat. no. JJ0845; 1:1,000) was purchased from Novus Biologicals, LLC.
SU-DHL2, OCI-LY3 and MZ DLBCL cells (2×105 cells/ml) were seeded into 96-well plates at a volume of 100 µl/well and then treated with 5 µl/well chidamide at different final concentrations (0, 0.1, 0.3, 1, 3, 10 and 30 µM) for 24, 48 and 72 h in a 37°C incubator with 5% CO2, respectively, to examine the concentration- and time-dependence of cellular chidamide effects. Equal molar concentrations of DMSO were used in the control group. At the indicated time of the reaction, 10 µl CCK-8 reagent was added to each well and incubated at 37°C for 2 h, according to the manufacturer's instructions. The final absorbance [optical density (OD) value] of the cultured cells was measured at a wavelength of 450 nm with a SynergyHTX microplate reader (BioTek Instruments, Inc.). Cell viability was calculated and expressed as the ratio of the OD450 values for chidamide-treated cells over those of the DMSO-treated control cells. The drug concentration and cell survival rate were entered into GraphPad Prism software 5.0 (GraphPad Software, Inc.) to obtain a concentration-survival rate curve. The half maximal inhibitory concentration (IC50), which represents the concentration of an inhibitor (drug) that is required for 50% inhibition of cells (
Cells were seeded in 6-well flat-bottomed plates at a density of 3×105 cells/well and treated with chidamide at different concentrations (0, 1, 2.5, 5 and 10 µM) in a 37°C incubator with 5% CO2 for 48 h. The cells were then collected, and their density was adjusted to 5×105 cells/ml. Then, 100 µl cell suspension was incubated with 5 µl Annexin V-FITC at room temperature for 10 min in the dark, followed by centrifugation at 1,000 × g for 5 min at room temperature. Subsequently, 10 µl PI staining solution was added to the Annexin V-FITC stained cells for 15 min at room temperature in dark, which were then analyzed via flow cytometry (FACSCanto II; BD Biosciences) and FACSCanto Clinical Software (v3.0; BD Biosciences) to determine the percentage of Annexin V+/PI− (early apoptotic) and Annexin V+/PI+ (late apoptotic) cells. The Annexin V-FITC apoptotic cell detection kit (Beijing TransGen Biotech Co., Ltd.) used included PI to label the cellular DNA of dead cells with a compromised total membrane. Thus, the kit allows the differentiation among early apoptotic cells (Annexin V+, PI−), late apoptotic cells (Annexin V+, PI+) and viable cells (Annexin V−, PI−) in chidamide-treated DLBCL cells. Early and late apoptosis were analyzed, and the apoptotic rate was calculated as the percentage of early and late apoptotic cells.
SU-DHL2, OCI-LY3 and MZ DLBCL cells were treated for 48 h with chidamide (0, 2.5, 5 and 10 µM) in a 37°C incubator with 5% CO2, and total RNAs were extracted from the cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. RT-qPCR was performed with a UltraSYBR One Step RT-qPCR kit (cat. no. CW0659S; CWBio) using the ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in duplicate for each reaction. The reverse transcription of total RNA into cDNA was performed at 45°C for 10 min. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 5 min; followed by 30 cycles at 95°C for 10 sec and 60°C for 45 sec. Averages of the obtained Cq values were used for further calculations. Gene expression levels were normalized to the expression of the endogenous control gene, β-actin. The gene expression levels were calculated using the 2−ΔΔCq method (
After SU-DHL2, OCI-LY3 and MZ DLBCL cells were treated for 48 h with chidamide (at 0, 2.5, 5 and 10 µM) in a 37°C incubator with 5% CO2, proteins were extracted from these cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using the BCA protein assay reagent (CWBio). SDS-PAGE with 10% gels was used to isolate proteins (30 µg per lane). The proteins were transferred to PVDF membranes, which were then blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. After incubation with the primary antibodies overnight at 4°C, the membranes were washed three times with Tween-20 (1:1,000 dilution)-PBS for 10 min each. Finally, the membranes were treated with the appropriate HRP-conjugated secondary antibodies (cat. nos. CW0103S and CW0102S; 1:5,000; CWBio) for 1 h at room temperature, and protein bands were visualized with ECL reagents (Corning, Inc.). Images were captured and analyzed with a Universal Hood II Chemiluminescence Imaging system (Bio-Rad Laboratories, Inc.). Densitometric analysis was performed on the instrument.
Data are presented as the mean ± SD for the number of indicated replicates. Statistical analyses were conducted using GraphPad Prism version 5.0 (GraphPad Software, Inc.). A Student's t-test was used to compare the difference between two groups. Homogeneity of variance was assessed using Brown-Forsythe test. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.
To investigate the cellular effect of chidamide on DLBCL cells, three cell lines of SU-DHL2, OCI-LY3 and MZ were selected for this study. These cells were treated with chidamide at different final concentrations (0, 0.1, 0.3, 1, 3, 10 and 30 µM) for 24, 48 and 72 h. As presented in
As the malignancy of DLBCL cells has been associated with the inhibition of apoptosis (
Western blot analysis of total proteins extracted from the cells exposed to chidamide revealed an enhanced expression level of cleaved caspase-3 and a decreased expression level of Bcl-2 (
Chidamide is a selective and potent inhibitor of the activities of class I HDAC enzymes, including HDAC1, HDAC2, HDAC3 and HDAC8 (
Activation or phosphorylation of STAT3 has been reported to serve a critical role in the malignant progression of multiple tumors, including DLBCL (
It has been well documented that histone acetylation, which is regulated by the opposing activities of histone acetyltransferases and HDACs, serves a critical role in the development and progression of cancer by modulating gene transcription, chromatin remodeling and nuclear architecture (
A high level of HDAC expression has been well established in DLBCL cell lines and tissue sections, especially for class I HDACs that are closely associated with the survival of patients with DLBCL (
To evaluate the clinical potential of chidamide in the treatment of DLBCL, the current study determined whether chidamide affects the viability of three different DLBCL cell lines; SU-DHL2 and OCI-LY3 cells of the ABC-subtype and MZ cells of the GCB-subtype. The present study found that exposure of these DLBCL cells to chidamide for 72 h evoked both concentration- and time-dependent inhibition of cell viability, displaying IC50 values of 2.722 µM for SU-DHL2 cells, 1.353 µM for OCI-LY3 cells and 4.183 µM for MZ cells. While these data do suggest that chidamide is a potent inhibitor of cell viability in DLBCL cells, they cannot provide conclusive information on which subtype of DLBCL cells is more sensitive to chidamide as only three cell lines were analyzed here.
To further examine the mechanism of chidamide-evoked inhibition of cell viability in DLBCL cells, flow cytometry and western blot analyses were performed to determine whether chidamide induces apoptosis in these DLBCL cells. There is evidence that apoptosis is involved in the initiation, progress and chemotherapy resistance of DLBCL (
As a transcription factor, STAT3 is constitutively activated in various cancer cell lines and tumor tissues, where it promotes tumor cell proliferation, invasion and migration (
The present study demonstrated that the total mRNA and protein expression levels of STAT3 were progressively decreased by the administration of increasing concentrations of chidamide in these DLBCL cells. A concentration dependent suppression of STAT3 phosphorylation (Tyr705 and Ser727) was also induced by chidamide treatment in DLBCL cells. Interestingly, recent evidence has indicated that valproic acid, another HDAC inhibitor, can inhibit STAT3 Tyr705 phosphorylation, but had no effect on the total protein expression level of STAT3 in natural killer cells (
In conclusion, the present study demonstrated that chidamide can have a tumor-suppressive effect on DLBCL cells by inducing apoptotic cell death, which is regulated by the HDACs/STAT3/Bcl-2 pathway. As well as inhibiting the activity of HDAC, chidamide may also inhibit the activity of STAT3 to function as an apoptosis inducer in these DLBCL cells. Therefore, the present results suggested that chidamide has great potential as a therapeutic agent for the management of DLBCL. While these results are encouraging, the current research is limited to determine which of the ABC- and GCB-subtype cells or both are more sensitive to chidamide as only three cell lines (two ABC subtypes and one GCB subtype) were used. Additionally, the current data were only obtained from these cell lines, which possess their inherent limitations as any other
Not applicable.
This work was supported by Nature Science Project of Shanxi, China (grant nos. 201701D121165 and 201901D111190), the Research Project Supported by Shanxi Scholarship Council of China (grant no. 2020-194), Key R & D Projects in Shanxi, China (grant no. 201703D421023), the Open Fund from Key Laboratory of Cellular Physiology (Shanxi Medical University), Ministry of Education, China (grant no. KLMEC/SXMU-202011) and the Shanxi ‘1331 Project’ Key Subjects Construction, China (grant no. 1331KSC).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
BYu and LS designed the project. HZ, FC, KQ, XM and LW performed the experiments. HZ, BYa, YW, MB and ZL analyzed the data. HZ and BYa wrote the manuscript. All authors read and approved the final manuscript. BYu and LS confirm the authenticity of all the raw data.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
diffuse large B-cell lymphoma
Cell Counting Kit-8
histone deacetylase
Chidamide inhibits diffuse large B-cell lymphoma cell viability. (A) SU-DHL2, (B) OCI-LY3 and (C) MZ cells were treated with chidamide at the indicated concentrations for 24, 48, and 72 h. Cell viability was determined and the IC50 was calculated accordingly. The experiments were repeated three times. Data are presented as the mean ± SD from three independent experiments performed in triplicate.
Flow cytometry analysis of chidamide-induced apoptosis in DLBCL cells. (A) SU-DHL2, (B) OCI-LY3 and (C) MZ cells were treated with chidamide at the indicated concentrations for 48 h, double stained with Annexin V-FITC and PI, and analyzed via quantitative flow cytometry assay. Quantification of apoptosis results for (D) SU-DHL2, (E) OCI-LY3 and (F) MZ cells. The experiments were repeated three times in triplicates for each. **P<0.01 and ***P<0.001 vs. control group without chidamide.
Western blot analysis of chidamide-induced apoptosis in DLBCL cells. (A and B) SU-DHL2, (C and D) OCI-LY3 and (E and F) MZ cells were treated with chidamide at the indicated concentrations for 48 h, and western blotting was used to determine the expression levels of the apoptosis-related cleaved-caspase-3 and Bcl-2 proteins. β-actin served as the internal control. Data are present as the mean ± SD from three sets of independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control group without chidamide.
Chidamide decreases the expression of HDACs in DLBCL cells. (A and B) SU-DHL2, (C and D) OCI-LY3 and (E and F) MZ cells were treated with chidamide at concentrations from 0–10 µM for 48 h, and the expression levels of HDAC1, HDAC2, HDAC3 and HDAC8 were determined using western blotting. β-actin served as the internal control in all experiments. Data are presented as the mean ± SD of three sets of experimental data. *P<0.05, **P<0.01 and ***P<0.001 vs. control group without chidamide. HDAC, histone deacetylase.
Chidamide represses the mRNA expression of STAT3 in DLBCL cells. SU-DHL2, OCI-LY3 and MZ cells were treated with chidamide at 0, 1, 2.5, 5 and 10 µM for 48 h. The mRNA expression level of STAT3 was assessed via reverse transcription-quantitative PCR, and results are expressed as ratios of 2−ΔΔCq values. All data are presented as the mean ± SD of three sets of experimental data. *P<0.05, **P<0.01 and ***P<0.001 vs. control group without chidamide.
Chidamide inhibits the activation of STAT3 in DLBCL cells. (A and B) SU-DHL2, (C and D) OCI-LY3 and (E and F) MZ cells were treated with chidamide at 0, 1, 2.5, 5 or 10 µM for 48 h. The protein expression levels of STAT3 and its phosphorylation were measured via western blotting. β-actin served as the internal control in all experiments. Data are presented as the mean ± SD of three sets of experimental data. *P<0.05, **P<0.01 and ***P<0.001 vs. control group without chidamide. P, phosphorylated.