Bortezomib inhibits the proliferation of bone marrow stromal cells (BMSCs). MS-5 cells (A), BMSCs from 3 healthy individuals (B), and BMSCs from 5 myeloma patients (C) were incubated without or with bortezomib (5-500 nM) in 96-well plates in serum-free X-VIVO medium, and cell proliferation was measured by colorimetric assay. Data are the mean±SD of the relative proliferation indices from 3 independent experiments. (D) MS-5 cells were incubated with or without bortezomib for 24 hr prior to cell cycle analysis. ^a)P<0.05, as compared with the control (no bortezomib). (E) BMSCs from a normal individual, incubated for 24 hr, are shown.

Bortezomib induces delayed apoptosis of bone marrow stromal cells (BMSCs). Cells were incubated in appropriate growth media, without or with bortezomib (5-500 nM), for 24-72 hr. Apoptosis was measured by flow cytometry after staining the cells for annexin V. (A) Representative flow cytometric profiles of bortezomib-treated U266 myeloma cells. (B) Representative flow cytometric profiles of bortezomib-treated MS-5 cells. (C) Apoptosis of MS-5 cells induced by bortezomib. Data are the mean±SD of 3 independent experiments. (D) Apoptosis of bortezomib-exposed BMSCs from 3 healthy individuals. Data are the mean±SD. (E) Apoptosis of bortezomib-exposed BMSCs from 5 myeloma patients. Data are the mean±SD. ^a)P<0.05, as compared with the control (no bortezomib).

Knockdown of chemokine (CXC motif) ligand 12 (CXCL12) inhibits the spontaneous proliferation of bone marrow stromal cells (BMSCs). BMSCs from 3 normal individuals (A) and 3 multiple myeloma patients (B) were transfected with 25 nM CXCL12 siRNA or control siRNA for 24 hr, incubated for up to 72 hr, and subjected to cell proliferation assays. ^a)P<0.05, compared with the control siRNA.

Bortezomib downregulates the expression and production of chemokine (CXC motif) ligand 12 (CXCL12) in MS-5 cells. MS-5 cells were incubated without or with bortezomib (5-500 nM) in X-VIVO medium for 24 hr prior to reverse transcription-PCR (A), real-time quantitative reverse transcription-PCR (B), and western blotting for CXCL12 (C). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (D) Concentrations of CXCL12α in 3-day MS-5 culture supernatants were measured by ELISA. Data are mean±SD of 3 independent experiments. ^a)P<0.05, compared with the control (no bortezomib).

Bortezomib downregulates the expression and production of chemokine (CXC motif) ligand 12 (CXCL12) in bone marrow stromal cells (BMSCs). BMSCs from 3 normal individuals (A, B) and 3 multiple myeloma patients (C, D) were incubated without or with bortezomib (5-500 nM) in serum-free X-VIVO medium. After 24 hr, CXCL12 mRNA levels were measured using quantitative reverse transcription-PCR (A, C). After 72 hr, media concentrations of CXCL12α were measured by ELISA (B, D). ^a)P<0.05, compared with the control (no bortezomib). (E) Serum concentrations of CXCL12α before, and 3 days after, intravenous administration of bortezomib (1.3 mg/m²) to multiple myeloma patients (N=3). ^a)P<0.05. (F) Transmigration of RPMI8226 cells induced by 3-day bortezomib-treated or -non-treated MS-5 cell culture media. ^a)P<0.05, compared with the control (no bortezomib). (G) MS-5 cell monolayers were treated with (right panel) or without (left panel) 5 nM bortezomib for 24 hr. RPMI8226 cells were added to the MS-5 monolayers, and migration of cells beneath the monolayers (the dark phases) was studied by inverted microscopy 24 hr later. Short-term bortezomib treatment inhibited the localization of RPMI8226 cells under monolayers. Representative results are shown.

Bortezomib and dexamethasone exert an additive downregulation of chemokine (CXC motif) ligand 12 (CXCL12) levels in MS-5 cells. (A) MS-5 cells were treated with dexamethasone for 24 hr prior to western blotting for CXCL12. (B) Concentrations of CXCL12α in 3-day MS-5 culture media were measured by ELISA. Data are the mean±SD of 3 independent experiments. ^a)P<0.05, compared with the control (no bortezomib). (C) MS-5 cells were treated with bortezomib (5 nM) and/or dexamethasone (Dexa; 0.1 µM) and CXCL12 mRNA was measured by quantitative reverse transcription-PCR. (D) MS-5 cells were incubated with bortezomib (5 nM) and/or dexamethasone (0.1 µM) in X-VIVO medium for 72 hr. Media concentrations of CXCL12α were measured by ELISA. ^a)P<0.05.