Chidamide, a histone deacetylase inhibitor, induces growth arrest and apoptosis in multiple myeloma cells in a caspase-dependent manner

Abstract

Chidamide, a novel histone deacetylase (HDAC) inhibitor, induces antitumor effects in various types of cancer. The present study aimed to evaluate the cytotoxic effect of chidamide on multiple myeloma and the underlying mechanisms involved. Viability of multiple myeloma cells upon chidamide treatment was determined by the Cell Counting Kit-8 assay. Apoptosis induction and cell cycle alteration were detected by flow cytometry. Specific apoptosis-associated proteins and cell cycle proteins were evaluated by western blot analysis. Chidamide suppressed cell viability in a time- and dose-dependent manner. Chidamide treatment markedly suppressed the expression of type I HDACs and further induced the acetylation of histones H3 and H4. In addition, it promoted G₀/G₁ arrest by decreasing cyclin D1 and c-myc expression, and increasing phosphorylated-cellular tumor antigen p53 and cyclin-dependent kinase inhibitor 1 (p21) expression in a dose-dependent manner. Treatment with chidamide induced cell apoptosis by upregulating the apoptosis regulator Bax/B-cell lymphoma 2 ratio in a caspase-dependent manner. In addition, the combination of chidamide with bortezomib, a proteasome inhibitor widely used as a therapeutic agent for multiple myeloma, resulted in enhanced inhibition of cell viability. In conclusion, chidamide induces a marked antimyeloma effect by inducing G₀/G₁ arrest and apoptosis via a caspase-dependent pathway. The present study provides evidence for the clinical application of chidamide in multiple myeloma.

Keywords: G0/G1 arrest; apoptosis; cell cycle; chidamide; histone deacetylase inhibitors; multiple myeloma.

Figure 1.

Chidamide inhibits the viability of myeloma cells and sensitizes myeloma cells to bortezomib. (A) The structural formula of chidamide (molecular weight, 747.25). (B) RPMI8226 cells and (C) U266 cells were treated with 0.25–8 µmol/l chidamide for 24, 48 and 72 h, and cell viability was measured using the CCK-8 assay. (D) Primary myeloma cells from 4 patients were treated with 0.25–8 µmol/l chidamide for 48 h and cell viability was measured using the CCK-8 assay. CCK-8, Cell Counting Kit-8.

Figure 2.

Chidamide induces G₀/G₁ phase arrest in myeloma cells. (A) RPMI8226 cells were treated with (0, 0.5, 1, 2 µmol/l) chidamide for 48 h and (B) U266 cells were treated with (0, 2, 4, 8 µmol/l) chidamide for 48 h, and subsequently cell cycle phases were assessed using flow cytometry (typical results from one experiment are presented). The G₁ phase fraction of (C) RPMI8226 cells and (D) U266 cells following 48-h chidamide treatment. The quantitative data were pooled from three independent experiments. *P<0.05 vs. no treatment.

Figure 3.

Chidamide induces apoptosis in myeloma cells. (A) RPMI8226 cells were treated with (0, 0.5, 1, 2 µmol/l) chidamide for 48 h and (B) U266 cells were treated with (0, 2, 4, 8 µmol/l) chidamide for 48 h, then the percentages of apoptotic cells were assessed by Annexin V-FITC/PI flow cytometry (typical results from one experiment are presented). The total apoptosis rate of (C) RPMI8226 cells and (D) U266 cells following chidamide treatment for 48 h. The quantitative data were pooled from three independent experiments. *P<0.05 vs. no treatment. Giemsa-Wright staining of (E) RPMI8226 and (F) U266 cells was performed following treatment in the presence or absence of chidamide (2 µmol/l for RPMI8226 cells and 8 µmol/l for U266 cells) for 48 h (×400 magnification). The arrows indicate typical apoptotic cells including nuclear fragmentation, chromatin condensation and apoptotic bodies. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 4.

Molecular mechanisms of chidamide-mediated cell cycle arrest and apoptosis induction in myeloma cell lines. (A) RPMI8226 cells were treated with 0, 0.5, 1 or 2 µmol/l chidamide for 48 h and (B) U266 cells were treated with 0, 2, 4 or 8 µmol/l chidamide for 48 h, and HDACs expression was assessed using western blot analysis. The expression of cyclin D1, p53, p-p53, c-myc and p21 in (C) RPMI8226 and (D) U266 cells, whereas Bax, Bcl-2 and mcl-1 in (E) RPMI8226 and (F) U266 cells following chidamide treatment were assessed using western blot analysis. GAPDH served as the internal control in all experiments. HDAC, histone deacetylase; Bcl-2, B-cell lymphoma 2; Bax, apoptosis regulator Bax; mcl-1, myeloid cell leukemia-1; p-, phosphorylated; t-, total; p21, cyclin-dependent kinase inhibitor 1; p53, cellular tumor antigen p53.

Figure 5.

Chidamide induces apoptosis in a caspase-dependent manner in myeloma cells. (A) RPMI8226 cells were treated with (0, 0.5, 1, 2 µmol/l) chidamide for 48 h and (B) U266 cells were treated with (0, 2, 4, 8 µmol/l) chidamide for 48 h, and then the expression of the cleaved and full-length caspase-8, caspase-9, caspase-3 and PARP was determined using western blot analysis. GAPDH served as the internal control. PARP, poly ADP ribose polymerase.

Figure 6.

CM sensitizes myeloma cells to BTZ. (A) RPMI8226 and (B) U266 cell lines were exposed to (2.5 g/ml and 5 ng/ml) BTZ alone and in combination with CM (0.5 µmol/l for RPMI8226 cells and 2 µmol/l for U266 cells) for 24 h, and subsequently cell viability was evaluated using the Cell Counting Kit-8. *P<0.05. BTZ, bortezomib; CM, chidamide.