ONCOLOGY LETTERS 18: 411-419, 2019 Chidamide, a histone deacetylase inhibitor, induces growth arrest and apoptosis in multiple myeloma cells in a caspase‑dependent manner XIANG-GUI YUAN*, YU-RONG HUANG*, TENG YU, HUA-WEI JIANG, YANG XU and XIAO-YING ZHAO Department of Hematology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, P.R. China Received August 1, 2018; Accepted March 29, 2019 DOI: 10.3892/ol.2019.10301 Abstract. Chidamide, a novel histone deacetylase (HDAC) Introduction inhibitor, induces antitumor effects in various types of cancer. The present study aimed to evaluate the cytotoxic effect of Multiple myeloma (MM) is the second most frequent hemato- chidamide on multiple myeloma and the underlying mecha- logical neoplasm in the USA in 2018 (1) and is characterized nisms involved. Viability of multiple myeloma cells upon by the infiltration of clonal plasma cells in the bone marrow, chidamide treatment was determined by the Cell Counting secretion of monoclonal immunoglobulins and end organ Kit-8 assay. Apoptosis induction and cell cycle alteration were damage (2). Over the last decade, the introduction of protea- detected by flow cytometry. Specific apoptosis-associated some inhibitors [bortezomib (BTZ) and carfilzomib] and proteins and cell cycle proteins were evaluated by western immunomodulatory drugs (thalidomide and lenalidomide), blot analysis. Chidamide suppressed cell viability in a time- combined with autologous stem cell transplantation, have and dose-dependent manner. Chidamide treatment markedly significantly improved the prognosis of patients with MM. suppressed the expression of type I HDACs and further The 5-year overall survival (OS) rate of patients diagnosed induced the acetylation of histones H3 and H4. In addition, between 2005 and 2011 was nearly double the rate of patients it promoted G0/G1 arrest by decreasing cyclin D1 and c-myc diagnosed between 1975 and 1977 (49 and 25%, respec- expression, and increasing phosphorylated-cellular tumor tively) (3). However, MM remains an incurable disease, and antigen p53 and cyclin-dependent kinase inhibitor 1 (p21) the majority of patients will eventually relapse and become expression in a dose-dependent manner. Treatment with chemorefractory to currently available drugs. Therefore, chidamide induced cell apoptosis by upregulating the apoptosis further development of novel therapies is required. regulator Bax/B-cell lymphoma 2 ratio in a caspase-dependent Histone deacetylase (HDAC) inhibitors (HDACIs), are manner. In addition, the combination of chidamide with bort- believed to be promising therapeutic drugs in the treatment ezomib, a proteasome inhibitor widely used as a therapeutic of cancer. To date, only 4 HDACIs (panobinostat, belinostat, agent for multiple myeloma, resulted in enhanced inhibition romidepsin and vorinostat) have been approved by the US of cell viability. In conclusion, chidamide induces a marked Food and Drug Administration (FDA) for the treatment of antimyeloma effect by inducing G0/G1 arrest and apoptosis several types of cancer. Deregulation of histone acetylation via a caspase-dependent pathway. The present study provides has been recognized to serve a critical role in the pathogen- evidence for the clinical application of chidamide in multiple esis of MM (4). HDACs are overexpressed in plasma cells myeloma. derived from patients with MM, compared with those from healthy controls. High expression levels of HDAC1 protein are associated with decreased overall survival time (OS) in patients with MM (4). In addition, HDACI treatment induces the upregulation of 21 genes, which were reported to be associ- Correspondence to: Professor Xiao-Ying Zhao, Department of ated with improved OS in MM (5). Therefore, HDACs have Hematology, The Second Affiliated Hospital, Zhejiang University been considered promising targets for MM therapies. Several School of Medicine, 88 Jiefang Road, Hangzhou, Zhejiang 310009, HDACI-based regimens are in clinical trials for MM, with P. R. Ch i na E-mail: [email protected] overall response rates of 42-61% (6-8). The phase 3 study PANORAMA-1 demonstrated that the chemotherapy regimen *Contributed equally including panobinostat (a pan-HDACI) resulted in a longer progression-free survival, when compared with that in the Key words: chidamide, histone deacetylase inhibitors, cell cycle, placebo group (7). As a result, panobinostat was approved by G0/G1 arrest, apoptosis, multiple myeloma the FDA for the treatment of relapsed/refractory MM in 2015. However, a number of adverse events have been observed with pan-HDACIs, including diarrhea, anorexia, nausea, fatigue, 412 YUAN et al: CHIDAMIDE TREATMENT FOR MULTIPLE MYELOMA thrombus and thrombocytopenia (7). To reduce the adverse from Xian Janssen Pharmaceutical Ltd. (Beijing, China). events of pan-HDACIs, selective HDACIs targeting class I Anti-c-myc (cat. no. 9402), anti-caspase-3 (cat. no. 9662), isoforms with improved efficacy and lower toxicity may be anti-caspase-8 (cat. no. 4790), anti-cleaved-caspase-8 (cat. promising drugs for the treatment of MM (9). no. 9496), anti-caspase-9 (cat. no. 9502), anti-Bcl-2 (cat. Chidamide (CM), a novel benzamide type HDACI, selec- no. 15071), anti-myeloid cell leukemia 1 (mcl-1; cat. no. 94296), tively suppresses the activity of class I HDACs, including anti-t-p53 (cat. no. 2524), anti-p-p53 (cat. no. 2521), anti-Bax HDAC1, HDAC2, HDAC3 and HDAC10 (10,11). Previous (cat. no. 5023), anti-p21 (cat. no. 2947), anti-cyclin D1 (cat. studies have revealed that CM induces cell proliferation inhi- no. 2922), anti-HDAC1 (cat. no. 5356), anti-HDAC2 (cat. bition and apoptosis in several hematological malignancies, no. 2540) and anti-HDAC3 (cat. no. 3949) antibodies were including myelodysplastic syndromes (12), leukemia (13-15) obtained from Cell Signaling Technology, Inc. (Danvers, and natural killer (NK)/T-cell lymphoma (16), as well as MA, USA). Anti-GAPDH (cat. no. ab181602), horseradish non-hematological malignancies, including lung cancer (17), peroxidase (HRP)-conjugated goat anti-rabbit secondary hepatocellular carcinoma (18), colon cancer (19) and pancre- antibody (cat. no. ab6721), HRP-conjugated goat anti-mouse atic cancer (20). CM was approved by the China FDA for the secondary antibody (cat. no. ab6789), anti-acetyl-histone treatment of peripheral T-cell lymphoma (PTCL) in 2014 (21). H3 (acetyl K9 + K14 + K18 + K23 + K27; cat. no. ab47915) A study in 2017 revealed that the overall response rate to CM and anti-acetyl-histone H4 (acetyl K5 + K8 + K12 + K16; alone or in combination was 39.06 and 51.18%, respectively, cat. no. ab177790) antibodies were obtained from Abcam for relapsed/refractory PTCL (22). At present, clinical trials (Cambridge, MA, USA). The anti-poly ADP ribose poly- of CM are being conducted in China and the US for the treat- merase (PARP) antibody (cat. no. sc53643) was obtained ment of solid tumors. However, to the best of our knowledge, from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All its biological effects on MM and relevant mechanisms have antibodies from Cell Signaling Technology and Santa Cruz not been investigated. Biotechnology were diluted at 1:1,000 and all antibodies from In the present study, the cytotoxic effect of CM on MM Abcam were diluted at 1:10,000. cells was evaluated and the possible mechanisms involved were investigated. The aim of the present study was to provide Cell viability assay. Cells were seeded in 96-well plates at evidence for the clinical application of chidamide in multiple 2x104 cells/well and incubated in 0.25-8 µmol/l CM for 24, 48 myeloma. or 72 h. Following treatment, cell viability was measured using the Cell Counting Kit-8 (CCK-8) viability assay (Dojindo Materials and methods Molecular Technologies, Inc., Kumamoto, Japan). According to the manufacturer's instruction, 10 µl CCK-8 solution was Cell lines and cell culture. The human MM RPMI8226 and added to each well for 3 h at 37˚C. The optical density (OD) U266 cell lines were obtained from the Hematology Institute was measured at 450 nm using a microplate reader (Biotek, of Zhejiang University (Hangzhou, China). All cells were Winooski, VT, USA). To determine whether CM sensi- cultured in a humidified atmosphere containing 5% CO2 at tized MM cells to BTZ, the cells were cultured with media 37˚C. All cells were maintained in RPMI-1640 medium with containing 0.5 µmol/l CM for RPMI8226 cells or 2 µmol/l CM 10% fetal bovine serum, penicillin (100 U/ml) and strepto- for U266 cells in the absence or presence of BTZ (2.5 and mycin (100 µg/ml) (all Gibco; Thermo Fisher Scientific, Inc., 5 ng/ml) for 24 h, and subsequently cell viability was assessed Waltham, MA, USA). Bone marrow was collected from 4 using the CCK-8 assay. The cell viability was calculated as newly diagnosed patients with MM admitted to the Second [OD (treatment)-OD (control)]/[OD (control)-OD (blank)] Affiliated Hospital, Zhejiang University School of Medicine x100. The viability inhibition rate (%) was calculated as (Hangzhou, China) between January 2018 and March 2018. [100-cell viability rate (%)]. Time- and dose-response curves Patient 1 was a 59-year-old male with IgG/κ subtype, patient 2 were produced using GraphPad Prism 5 (GraphPad Software was a 58-year-old male with IgG/λ subtype, patient 3 was Inc., San Diego, CA, USA). IC50 values were predicted based a 72-year-old male with IgG/λ subtype and patient 4 was a on the available data using the SPSS probit model analysis 77-year-old female with IgG/λ subtype. Once informed consent (SPSS v17.0; SPSS, Inc., Chicago, IL, USA). was obtained, an additional 3 ml of bone marrow was extracted during the diagnostic bone marrow puncture. Subsequently, Western blot analysis. Following exposure to CM (0, 0.5, 1, primary myeloma cells were isolated by Ficoll-Hypaque 2 µmol/l for PRMI8226 cells, and 0, 2, 4, 8 µmol/l for U266 density gradient centrifugation (2,000 x g, 15 min at room cells) for 48 h, the cells were harvested and washed with temperature).