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J. He et al.
Chidamide has been shown to induce cell apoptosis and
cell cycle arrest in a variety of cancers, such as lung cancer, pancreatic cancer, leukemia and lymphoma.4-7 Moreover, HDAC3 was recently shown to be overexpressed in MM and was a key factor that propelled myeloma cell prolifer- ation.8,9 Additionally, HDAC1 and HDAC2 are important positive regulators of osteoclastogenesis.10,11 Based on these data, we hypothesize that chidamide may exert dual anti-myeloma and bone-protective effects.
In the work herein, we investigated the dual anti- myeloma and bone-protective effects on myeloma cells and preclinical models. To the best of our knowledge, this compound is the first HDACi that was independently developed in China and shows both anti-myeloma and anti-osteolytic bone disease activity in vitro and in vivo, supporting the future clinical application of this drug as a treatment for MM.
Methods
Drugs
Chidamide was a generous gift from the Chipscreen Company, Shenzhen, China. Additional information regarding drugs and reagents is provided in the Online Supplementary Materials.
Human myeloma cell lines, primary myeloma samples, bone marrow stromal cells (BMSCs), healthy peripheral blood mononuclear cells (PBMCs) and bone marrow-derived mononuclear cells (BMMCs)
The human myeloma cell lines RPMI-8226, MM.1s, and U266B1 were purchased from the cell bank of the Chinese Academy of Science; H929, CAG, ARP-1, LP-1, and OPM2 cells were generous gifts from Dr. Qing Yi (Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA). ANBL-6 cells were a generous gift from Professor Zhiqiang Liu, Tianjin Medical University, China. The luciferase- expressing myeloma cell line RPMI-8226-luc was constructed by infecting RPMI-8226 cells with a lentivirus encoding the luciferase gene, and infected cells were selected with puromycin. Primary myeloma samples and BMSCs were collected from newly diag- nosed patients, and PBMCs and BMMCs were collected from healthy donors. Primary CD138(+) cells were sorted using CD138 microbeads (Miltenyi Biotech, CA, USA).
Cell culture
ing 50ng/ml RANKL and 25ng/ml monocyte colony stimulating factor (M-CSF) supplemented with 10% fetal bovine serum and 1% L-glutamine was used as osteoclastogenic medium for cells cultured in the presence or absence of chidamide. PBMCs from healthy donors were cultured as pre-OCs for 14 days and as mature OCs for 21 days. The F-actin ring formation assay, assess- ment of the resorption ability of OCs and tartrate-resistant acid phosphatase (TRAP)+ staining were performed as described in a previous report.12
Drug treatments in different mouse models
Two tumor-bearing murine models and a non-tumor-bearing model were employed to investigate the anti-tumor and bone-pro- tective effects of chidamide.4,13-15 Micro computed tomography (CT) was used to evaluate MM bone disease. Further details are provided in the Online Supplementary Materials.16,17
Statistical analysis
All data are expressed as means ± SEM and are representative of at least three experiments with similar results performed in tripli- cate, unless indicated otherwise. A two-tailed Student’s t-test was used to determine the significance of differences between two groups, and one-way analysis of variance was used to estimate the differences between three or more groups. GraphPad Prism 5.0 software (GraphPad Software, CA, USA) was used for the analy- sis.
Ethical approval
Primary myeloma samples, BMSCs, PBMCs, and BMMCs were collected after obtaining informed consent from the patients and approval from the Ethics Committee of the First Affiliated Hospital of Zhejiang University. The animal experiments were carried out after obtaining approval from the Ethics Committee of the First Affiliated Hospital of Zhejiang University.
Results
Chidamide exhibits HDAC inhibitory activity in MM cell lines
We first investigated the basal expression levels of HDAC1, 2, 3, and 10, which are chidamide targets, in MM cell lines. As shown in Figure 1A, HDAC1, HDAC2 and HDAC3 were all expressed in the nine myeloma cell lines (H929, LP-1, ARP-1, U266B1, RPMI-8226, ANBL-6, OPM2, CAG, and MM.1s), and both HDAC2 and HDAC3 were expressed at slightly higher levels than HDAC1. HDAC10 was barely detectable in human myeloma cell lines. Next, we measured HDAC activity and the acetyla- tion of lysine residues on histones H3 and H4 to deter- mine the inhibitory effect of chidamide on HDAC. As shown in Figure 1B,C, chidamide inhibited HDAC activi- ty and significantly increased the acetylation of H3K8, H3K18 and H4K8. However, the expression of HDAC1, HDAC2, HDAC3 and HDAC10 was not affected (Figure 1D).
Chidamide not only exerts anti-myeloma effects but also overcomes the resistance conferred by the BM microenvironment
Subsequently, we evaluated the anti-myeloma effect of chidamide. Cell lines were treated with increasing concen- trations of chidamide (0.5-8μM) for different times (24, 48, or 72h). CCK-8 assays revealed a dose-dependent and time-dependent pattern of chidamide cytotoxicity (Online Supplementary Figure S1A). Flow cytometry (Annexin V
Myeloma cell lines were cultured in RPMI 1640 medium sup- plemented with 10% fetal bovine serum and 1% L-glutamine at 37°C in a 5% CO atmosphere. BMSCs were cultured in mini-
2
mum essential medium a (MEM-a) supplemented with 10% fetal
bovine serum and 1% L-glutamine. BMSCs were cultured as pre- mature osteoblasts for seven days and as mature osteoblasts for 21 days in the human mesenchymal stem cell (MSC) osteogenic dif- ferentiation basal medium.
Cell proliferation, cell cycle, and cell apoptosis assays
Cell counting kit-8 (CCK-8) assays were used to detect MM cell viability. Flow cytometry was used to assess the cell cycle and apoptosis. A detailed description of each procedure is provided in the Online Supplementary Materials.
In vitro OC differentiation, pit formation and F-actin ring formation
Osteoclasts were differentiated from PBMCs in osteoclasto- genic medium as previously described.12 Briefly, MEM-a contain-
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