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J. He et al.
tion was inhibited in the presence of chidamide during osteoclastogenesis (Figure 6C). Next, we evaluated func- tional changes in OCs cultured on calcium substrate-coat- ed slides. Chidamide treatment induced a dose-dependent reduction in the area of resorption pits. With increasing drug doses, the resorption area was substantially reduced (Figure 6C, Figure 6E ***P<0.001).
Effect of chidamide on osteoblasts
HDAC1 and HDAC3 are regarded as negative regula- tors of osteoblastogenesis during bone formation, thus we also evaluated the effect of chidamide on osteoblasts. As shown in Online Supplementary Figure S7, primary BMSCs from patients with MM (n=6) were cultured in human MSC osteogenic differentiation basal medium in the pres- ence of different chidamide concentrations. Alkaline phos- phatase (ALP) was measured as a positive marker of osteoblast differentiation, and its expression was slightly increased by chidamide treatment. Additionally, Alizarin red staining (ARS) of calcium deposits showed that chi- damide had no promotion or inhibitory effect on osteoblast differentiation (Online Supplementary Figure S7A,B). Finally, the expression of osteocalcin (OCN), Activin A and semaphorins at the messenger ribonucleic acid (mRNA) level was also examined; OCN (day 21) was increased, while Activin A (day 21) was down-regulated (Online Supplementary Figure S7C).
Chidamide exerts bone-protective effects on non-tumor-bearing mice
Chidamide was administered via oral gavage to non- tumor-bearing C57BL/6 mice at a dose of 25mg/kg for 21 days to establish whether chidamide directly exerts its bone-preserving effect on the bone tissue or exerts an indi- rect effect by decreasing the tumor burden. As shown in Figure 7A, serum CTX-I levels were not significantly dif- ferent between the vehicle (n=5) and chidamide (n=5) groups, whereas the serum PINP level was clearly increased after treatment with chidamide (***P<0.001). Thereafter, in vivo intraperitoneal injections of soluble receptor activator of nuclear factor-κB ligand (sRANKL; three doses) were administered within 50h followed by gavage with chidamide for 21 days to mimic OC stimula-
tion by myeloma-derived sRANKL. Serum CTX-I levels increased in both the vehicle group (n=5) and the chi- damide group (n=5) after sRANKL injections, but the level in the chidamide-treated group was increased to a lesser extent than the level in the vehicle group (*P<0.05). Serum PINP levels were reduced in both groups; however, chi- damide attenuated the reduced PINP level in the chi- damide-treated group (***P<0.001). TRAP+ staining of bone showed a reduced number of OCs (Figure 7B,C) in the chidamide-treated group (***P<0.001). Moreover, the bone morphometric parameters evaluated by micro-CT (Figure 7D,E) indicated that chidamide increased the tra- becular number (***P<0.001) and reduced trabecular sepa- ration compared to the control group (**P<0.01). Although bone volume density over total volume (BV/TV, P>0.05) showed an increasing trend in the chidamide-treated group, the difference between the two groups was not sig- nificant. Based on these results, chidamide increased the bone volume of healthy mice to some extent and directly prevented RANKL-induced OC activation.
Discussion
HDACs are an important family of enzymes with cru- cial roles in carcinogenesis through their repressive effects on tumor suppressor gene transcription and are proposed as therapeutic targets in oncology.18,19 HDAC inhibitors induce cancer cell apoptosis, cell cycle arrest and promote differentiation, particularly in hematological malignancies. However, the anti-cancer effects of HDAC inhibitors dif- fer and their pharmacological effects vary, depending on the cancer cell types, HDAC targets and doses. Chidamide, a novel HDACi which is currently being widely used in China to treat patients with T-cell lym- phoma, shows good efficacy and tolerability.5 In our investigation herein, we evaluated and explored the effica- cy of chidamide in treating myeloma and its associated osteolytic bone disease.
Chidamide reduced myeloma cell viability in both pri- mary MM cells and MM cell lines, even in the presence of BMSCs or chidamide-pretreated OCs. As the microenvi- ronment is crucial for myeloma drug resistance and
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Figure 4. The molecular mechanisms underlying chidamide activity in myeloma cells. (A) Western blot analysis of Mcl-1, Myc, Bcl-xL, Bcl-2, p21, p27, CDK4, CDK6, and Cyclin-D2 levels; a-tubulin was used as the loading control. (B) SOCS-3, p-JAK2, JAK2, p-STAT3-727, p-STAT3-705, and STAT3 levels were analyzed by Western blotting; a-tubulin was used as the loading control.
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