Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo
Abstract
Song XU1, 2, 3, Kim DE VEIRMAN2, 3, Holly EVANS4, Gaia Cecilia SANTINI2, Isabelle VANDE BROEK3, Xavier LELEU5, Ann DE BECKER2, Ben VAN CAMP3, Peter CROUCHER6, Karin VANDERKERKEN3, Ivan VAN RIET2, 3
1Department of Lung Cancer Surgery, Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China; 2Stem Cell Laboratory-Division Clinical Hematology, Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium; 3Department of Hematology and Immunology-Vrije Universiteit Brussel (VUB), Myeloma Center Brussels, Brussels, Belgium; 4Department of Human Metabolism, University of Sheffield Medical School, Sheffield, UK; 5Service d’Hématologie, Centre Hospitalier Universitaire (CHU), Lille, France; 6Garvan Institute for Medical Research, Sydney, Australia
Aim: Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo.
Methods: Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis.
Results: Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 μmol/L). The low concentration of vorinostat (1 μmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 μmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 μmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen.
Conclusion: Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.
Keywords: histone deacetylase (HDAC); vorinostat; mesenchymal stem cell; osteogenesis; alkaline phosphatase (ALP); Runx2; osteocalcin; multiple myeloma
We would like to thank Nicole ARRAS, Wim RENMANS, Angelo WILLEMS, and Carine SEYNAEVE for their expert technical assistance and Prof Qing-hua ZHOU for his administrative support. We thank Merck & Co for providing us with vorinostat. Our research was supported by grants from the FWO-Vlaanderen, “Vlaamse Liga tegen Kanker” (Stichting Emmanuel Van der Schueren), the Vrije Universiteit Brussel (HOA) and the Scientific Foundation Willy Gepts (WFWG) UZ Brussel. Song XU is supported by the CSC-VUB scholarship.
* To whom correspondence should be addressed.
E-mail ivan.vanriet@uzbrussel.be
Received 2012-08-21 Accepted 2012-12-05
Keywords:
1Department of Lung Cancer Surgery, Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China; 2Stem Cell Laboratory-Division Clinical Hematology, Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium; 3Department of Hematology and Immunology-Vrije Universiteit Brussel (VUB), Myeloma Center Brussels, Brussels, Belgium; 4Department of Human Metabolism, University of Sheffield Medical School, Sheffield, UK; 5Service d’Hématologie, Centre Hospitalier Universitaire (CHU), Lille, France; 6Garvan Institute for Medical Research, Sydney, Australia
Aim: Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo.
Methods: Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis.
Results: Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 μmol/L). The low concentration of vorinostat (1 μmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 μmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 μmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen.
Conclusion: Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.
Keywords: histone deacetylase (HDAC); vorinostat; mesenchymal stem cell; osteogenesis; alkaline phosphatase (ALP); Runx2; osteocalcin; multiple myeloma
We would like to thank Nicole ARRAS, Wim RENMANS, Angelo WILLEMS, and Carine SEYNAEVE for their expert technical assistance and Prof Qing-hua ZHOU for his administrative support. We thank Merck & Co for providing us with vorinostat. Our research was supported by grants from the FWO-Vlaanderen, “Vlaamse Liga tegen Kanker” (Stichting Emmanuel Van der Schueren), the Vrije Universiteit Brussel (HOA) and the Scientific Foundation Willy Gepts (WFWG) UZ Brussel. Song XU is supported by the CSC-VUB scholarship.
* To whom correspondence should be addressed.
E-mail ivan.vanriet@uzbrussel.be
Received 2012-08-21 Accepted 2012-12-05