Division of Hematology
Contributions
Type: Publication Only
Background
Osteolytic bone disease is a common manifestation of multiple myeloma (MM) that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases CHIT1 and YKL40 is associated with osteoclast (OCs) differentiation and bone digestion.
Aims
In the current study, we investigated the effect of two Bortezomib’s concentration (BO) (2.5 nM and 5nM) on osteoclastogenesis by analyzing regulation of chitinase expression.
Methods
In order to obtained the OCs, the conditioned medium was supplemented with 25 ng/ml soluble rhRANK ligand (Peproteck, BDA, Italy) and 20 ng/ml rhM-CSF (Peproteck, BDA, Italy), for 21 days w/o Bortezomib (2.5nM or 5nM). The medium was replaced every 3 days. Cells and supernatants were harvested every 3 days for enzymatic assay, qRT-PCR, immunofluorescence and Western blotting. The supernatants were stored at − 20 °C. To confirm that macrophages achieved OCs differentiation, suitable markers were analyzed by qRT-PCR. Finally, in order to evaluate the ability of MM cell lines (U266) to digest bone, dentine discs were added to the wells before cell seeding. U266 cultured with conditioned medium (without BO) for 24 h were used as a control.
Results
OCs exposition to BO was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover,, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disc assay. After 3 days of incubation, we observed that U266 cells were able to form resorption pits on a dentin disc. Silencing the chitinase proteins in U266 cell line with specific siRNAs, resulted in pits number reduction on dentine discs
Summary
In conclusion we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients.
Keyword(s): Bone disease, Bortezomib, Multiple myeloma
Session topic: Publication Only
Type: Publication Only
Background
Osteolytic bone disease is a common manifestation of multiple myeloma (MM) that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases CHIT1 and YKL40 is associated with osteoclast (OCs) differentiation and bone digestion.
Aims
In the current study, we investigated the effect of two Bortezomib’s concentration (BO) (2.5 nM and 5nM) on osteoclastogenesis by analyzing regulation of chitinase expression.
Methods
In order to obtained the OCs, the conditioned medium was supplemented with 25 ng/ml soluble rhRANK ligand (Peproteck, BDA, Italy) and 20 ng/ml rhM-CSF (Peproteck, BDA, Italy), for 21 days w/o Bortezomib (2.5nM or 5nM). The medium was replaced every 3 days. Cells and supernatants were harvested every 3 days for enzymatic assay, qRT-PCR, immunofluorescence and Western blotting. The supernatants were stored at − 20 °C. To confirm that macrophages achieved OCs differentiation, suitable markers were analyzed by qRT-PCR. Finally, in order to evaluate the ability of MM cell lines (U266) to digest bone, dentine discs were added to the wells before cell seeding. U266 cultured with conditioned medium (without BO) for 24 h were used as a control.
Results
OCs exposition to BO was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover,, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disc assay. After 3 days of incubation, we observed that U266 cells were able to form resorption pits on a dentin disc. Silencing the chitinase proteins in U266 cell line with specific siRNAs, resulted in pits number reduction on dentine discs
Summary
In conclusion we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients.
Keyword(s): Bone disease, Bortezomib, Multiple myeloma
Session topic: Publication Only