Myeloma cells inhibit osteogenic differentiation of mesenchymal stem cells and kill osteoblasts via TRAIL-induced apoptosis

Abstract

Introduction: Myeloma bone disease (MBD) is the result of the increased activity of osteoclasts (OCs), which is not accompanied by a comparable increase of osteoblast (OB) function, thus leading to enhanced bone resorption. Osteoblasts can also regulate osteoclast activity through expression of cytokines, such as receptor activator of nuclear factor-κB ligand (RANKL), which activates osteoclast differentiation, and osteoprotegerin (OPG), which inhibits RANKL by acting as a decoy receptor.

Material and methods: Based on a series of 21 patients with multiple myeloma (MM) and human osteoblast cell line HFOB1.19, we provide evidence that the bone marrow-derived mesenchymal stem cells (BMMSCs) of patients with MM exhibit normal phenotype, but showed reduced efficiency to differentiate into OBs as compared with normal controls.

Results: In vitro assays showed that MM cells inhibited the potential of osteogenic differentiation of BMMSCs from healthy controls and rendered the OBs sensitive to TRAIL-induced apoptosis. There was no evidence of the formation of tartrate-resistant acid phosphatase positive OCs. The osteogenic differentiation of HFOB1.19 was also inhibited in the presence of RPMI 8266 or XG7 MM cells, as confirmed by von Kossa and ALP staining. Osteoblast s induced from BMMSCs supported survival and proliferation of MM cells, especially when the MM cells were cultured in medium containing rhTRAIL and dexamethasone. Multiple myeloma cells proliferated and grew well in the presence of residual OBs.

Conclusions: Besides OCs, our results demonstrated that OBs and MM cells were dependent upon each other and made a microenvironment suitable for MM cells.

Keywords: multiple myeloma; myeloma bone disease (MBD); osteoblast; recombinant human tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL).

Figure 1

Morphological characterization of BMMNCs. A – BMMNCs cultured for 5 days, B – BMMNCs at day 12 (100×)

Figure 2

von Kossa staining mineralized crystal generated from BMMSCs. A and a – from normal controls and MM patients respectively after 21 days incubation with osteogenic inducing medium (100×). B and b – from normal controls and MM patients respectively after incubation with osteogenic condition medium and RPMI 8266

Figure 3

The change of ALP and mineralized deposition in HFOB1.19 before and after cells were cultured in osteogenic condition medium and RPMI 8266. Before the HFOB1.19 cells were incubated in osteogenic inducing medium with RPMI 8266, mineralized deposition and ALP activity were seen with von Kossa and ALP staining. A – von Kossa staining, a – ALP staining (100×). When the HFOB1.19 cells were incubated in osteogenic inducing medium with RPMI 8266 for 7 days, decreased of mineralized deposition and downregulation of ALP activity were seen as cells was stained with von Kossa and staining (B – von Kossa staining, b – ALP staining (100×), C – there was no TRAP positive found in coculture experiments

Figure 4

The OBs induced from BMMSCs or human osteoblast cell line HFOB1.19 grew well in medium containing rhTRAIL and were not sensitive to rhTRAIL induced apoptosis in vitro as MTT assays or flow cytometry using the annexin V/PI binding assay (A and B). The percentage of apoptosis increased significantly whether the OBs or HFOB1.19 were precultured or co-cultured with XG7/RPMI 8266 MM cells, but the proportion of apoptosis decreased significantly when anti-TRAIL-R2 mab were added to the cultured (C)

Figure 5

RPMI 8266 and XG7 MM cells were sensitive to TRAIL induced apoptosis (A), but the percentage of apoptosis decreased when the cells were co-cultured with osteoblasts (OBs); (B) MM cells and OBs were incubated in medium containg 2 × 10−⁷ M dexa-methasone; (C) MM cells and OBs were incubated in medium containing 50 ng/ml rhTRAIL