Blood Res.  2015 Jun;50(2):87-96. 10.5045/br.2015.50.2.87.

Bortezomib inhibits the survival and proliferation of bone marrow stromal cells

• Kim HY
• Moon JY
• Ryu H
• Choi YS
• Song IC
• Lee HJ
• Yun HJ
• Kim S
• Jo DY

• KMID: 2172772
• DOI: http://doi.org/10.5045/br.2015.50.2.87

Abstract

BACKGROUND
Bortezomib is widely used for the treatment of multiple myeloma. Bone marrow stromal cells (BMSCs) endow myeloma cells with survival and growth advantages. However, the influence of bortezomib on BMSCs is not well elucidated. We examined the effects of bortezomib on the survival and growth of BMSCs in vitro.
METHODS
The effects of bortezomib on the survival and proliferation of the BMSC MS-5 cell line and on BMSCs obtained from healthy individuals (N=4) and newly diagnosed myeloma patients (N=5) were investigated in vitro. Transmembrane cell migration was evaluated using the Transwell system. A short interfering RNA strategy was used to knock down the expression of chemokine (CXC motif) ligand 12 (CXCL12) mRNA. To examine the effects of bortezomib-exposed BMSCs on the migration and localization of myeloma cells, MS-5 monolayers were treated with bortezomib for 24 hr, washed, and then overlaid with human RPMI8226 myeloma cells.
RESULTS
Bortezomib inhibited BMSC proliferation in a concentration-dependent manner, and induced cellular apoptosis. Bortezomib decreased CXCL12 production by BMSCs. Knockdown of CXCL12 mRNA in BMSCs revealed that CXCL12 served as an autocrine growth factor. Short-term bortezomib treatment of BMSC monolayers reduced the tendency of myeloma cells to locate to positions under the monolayers.
CONCLUSION
Bortezomib inhibits the survival and growth of BMSCs via downregulation of CXCL12, which may contribute to the clinical effects of this agent.

Keyword

Bone marrow stromal cells; Bortezomib; CXCL12; Multiple myeloma; Proliferation; Survival

MeSH Terms

Apoptosis
Cell Line
Cell Movement
Down-Regulation
Humans
Mesenchymal Stromal Cells*
Multiple Myeloma
RNA, Messenger
RNA, Small Interfering
Bortezomib
RNA, Messenger
RNA, Small Interfering

Figure

• 

  Fig. 1 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.

• 

  Fig. 2 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).

• 

  Fig. 3 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.

• 

  Fig. 4 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).

• 

  Fig. 5 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.

• 

  Fig. 6 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.

Reference

1. Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 2001; 61:3071–3076. PMID: 11306489.

2. Mitsiades N, Mitsiades CS, Richardson PG, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood. 2003; 101:2377–2380. PMID: 12424198.

3. Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med. 2003; 348:2609–2617. PMID: 12826635.

4. Huang N, Kawano MM, Harada H, et al. Heterogeneous expression of a novel MPC-1 antigen on myeloma cells: possible involvement of MPC-1 antigen in the adhesion of mature myeloma cells to bone marrow stromal cells. Blood. 1993; 82:3721–3729. PMID: 8260709.

5. Okado T, Hawley RG. Adhesion molecules involved in the binding of murine myeloma cells to bone marrow stromal elements. Int J Cancer. 1995; 63:823–830. PMID: 8847141.

6. Uchiyama H, Barut BA, Mohrbacher AF, Chauhan D, Anderson KC. Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion. Blood. 1993; 82:3712–3720. PMID: 8260708.

7. Chauhan D, Uchiyama H, Akbarali Y, et al. Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B. Blood. 1996; 87:1104–1112. PMID: 8562936.

8. Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS. Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood. 1999; 93:1658–1667. PMID: 10029595.

9. Landowski TH, Olashaw NE, Agrawal D, Dalton WS. Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-kappa B (RelB/p50) in myeloma cells. Oncogene. 2003; 22:2417–2421. PMID: 12717418.

10. Wang X, Li C, Ju S, Wang Y, Wang H, Zhong R. Myeloma cell adhesion to bone marrow stromal cells confers drug resistance by microRNA-21 up-regulation. Leuk Lymphoma. 2011; 52:1991–1998. PMID: 21718132.

11. Wallace SR, Oken MM, Lunetta KL, Panoskaltsis-Mortari A, Masellis AM. Abnormalities of bone marrow mesenchymal cells in multiple myeloma patients. Cancer. 2001; 91:1219–1230. PMID: 11283920.

12. Feng Y, Wen J, Mike P, et al. Bone marrow stromal cells from myeloma patients support the growth of myeloma stem cells. Stem Cells Dev. 2010; 19:1289–1296. PMID: 20121456.

13. Mitsiades N, Mitsiades CS, Poulaki V, et al. Apoptotic signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma cells: therapeutic implications. Blood. 2002; 99:4525–4530. PMID: 12036884.

14. Lazarini M, Traina F, Winnischofer SM, Costa FF, Queiroz ML, Saad ST. Effects of thalidomide on long-term bone marrow cultures from patients with myelodysplastic syndromes: induction of IL-10 expression in the stromal layers. Leuk Res. 2011; 35:1102–1107. PMID: 21511336.

15. Wobus M, Benath G, Ferrer RA, et al. Impact of lenalidomide on the functional properties of human mesenchymal stromal cells. Exp Hematol. 2012; 40:867–876. PMID: 22705469.

16. Roccaro AM, Hideshima T, Raje N, et al. Bortezomib mediates antiangiogenesis in multiple myeloma via direct and indirect effects on endothelial cells. Cancer Res. 2006; 66:184–191. PMID: 16397231.

17. Maruyama D, Watanabe T, Heike Y, et al. Stromal cells in bone marrow play important roles in pro-inflammatory cytokine secretion causing fever following bortezomib administration in patients with multiple myeloma. Int J Hematol. 2008; 88:396–402. PMID: 18989635.

18. Jo DY, Rafii S, Hamada T, Moore MA. Chemotaxis of primitive hematopoietic cells in response to stromal cell-derived factor-1. J Clin Invest. 2000; 105:101–111. PMID: 10619866.

19. Barbieri F, Bajetto A, Stumm R, et al. Overexpression of stromal cell-derived factor 1 and its receptor CXCR4 induces autocrine/paracrine cell proliferation in human pituitary adenomas. Clin Cancer Res. 2008; 14:5022–5032. PMID: 18698020.

20. Kojima Y, Acar A, Eaton EN, et al. Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts. Proc Natl Acad Sci U S A. 2010; 107:20009–20014. PMID: 21041659.

21. Lee HJ, Lee K, Lee DG, et al. Chemokine (C-X-C motif) ligand 12 is associated with gallbladder carcinoma progression and is a novel independent poor prognostic factor. Clin Cancer Res. 2012; 18:3270–3280. PMID: 22553346.

22. Kim SW, Hwang JH, Jin SA, et al. Role of pertussis toxin-sensitive G protein-coupled receptor signaling in the proliferation of bone marrow mesenchymal stem cells. Korean J Hematol. 2007; 42:24–32.

23. Kortesidis A, Zannettino A, Isenmann S, Shi S, Lapidot T, Gronthos S. Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood. 2005; 105:3793–3801. PMID: 15677562.

24. Ponomaryov T, Peled A, Petit I, et al. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest. 2000; 106:1331–1339. PMID: 11104786.

25. Psenák O, Sefc L, Sýkora V, Chang KT, Necas E. Cytokine gene expression in regenerating haematopoietic tissues of mice after cyclophosphamide treatment. Acta Haematol. 2003; 109:68–75. PMID: 12624490.

26. Lerman OZ, Greives MR, Singh SP, et al. Low-dose radiation augments vasculogenesis signaling through HIF-1-dependent and -independent SDF-1 induction. Blood. 2010; 116:3669–3676. PMID: 20631377.

27. Kim SW, Kim HY, Lee HJ, Yun HJ, Kim S, Jo DY. Dexamethasone and hypoxia upregulate CXCR4 expression in myeloma cells. Leuk Lymphoma. 2009; 50:1163–1173. PMID: 19391039.

28. Azab AK, Runnels JM, Pitsillides C, et al. CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. Blood. 2009; 113:4341–4351. PMID: 19139079.