The Mechanisms Of Abnormal Expression Of BDNF/TrkB On The Pathogenesis Of Multiple Myeloma

Multiple myeloma (MM) is a B-cell neoplasma characterized by clonal expansion of malignant plasma cells in the bone marrow. Despite recent new insights into the pathogenesis of MM, it remains an incurable disease with the currently available therapeutic modalities. New biologically based therapies are therefore urgently needed. Laboratory and preclinical studies have shown that signals from the bone marrow microenviroment play a critical role in sustaining the growth and survival of MM cells during tumor progression. Recently, it has been reported that BDNF levels are elevated in the plasma of patients with active myeloma compared with normal control and the levels of BDNF correlated with the progression of disease. To explore the significance of abnormal expression of BDNF/TrkB in the development and evolution of MM, we investigated the expression of BDNF and its receptor TrkB and p75NTR in human MM cell lines (HMCLs) and clinical samples; we further investigated the in vitro and in vivo proangiogenic effects of BDNF and the relationship between expression of BDNF in MM cells and MM-induced angiogenesis; following that the biological effects of BDNF on MM cells were examined.These observations suggest a major role for BDNF in the pathophysiology of MM and support targeting BDNF/TrkB in novel MM treatment strategies.PART I Expression of brain-derived neurotrophic factor and its receptors in multiple myeloma Background and objective: brain-derived neurotrophic factor (BDNF) is a member of Neurotrophins (NTs) family, which plays an important and well described role in the survival, development, differentiation and regeneration of neuronal cells in the central and peripheral nervous system. Despite their well-known effects on neurons, recent studies found that NTs activation of Trks has been implicated in the pathogenesis of a number of neuronal and non-neuronal solid tumors. Recently, it has been reported that BDNF and/or TrkB also produced in hematological malignancies including MM, BDNF levels was elevated in the plasma of patients with active myeloma compared with normal control and the levels of BDNF correlated with the progression of disease. To explore whether BDNF/TrkB pathway is a potential therapeutic target in MM, we investigate the expression of BDNF and its receptor TrkB and p75NTR in HMCLs and clinical samples.Methods: Primary myeloma cell was isolated and cultured successfully. Transcript and protein expression of BDNF and its receptors (TrkB and p75NTR) in HMCLs (RPMI 8226, U266, KM3) and primary myeloma cells was determined by RT-PCR and Western blotting, respectively. BDNF levels in culture supernatant were determined by enzyme-linked immunosorbent assay. The expression of BDNF and TrkB by myeloma cells was studied by immunohistochemistry in bone marrow biopsy samples from 16 patients with MM. results from these MM patients were compared with the degree of protein expression in bone marrow biopsy obtained at diagnosis from 9 patients with different diseases but with normal bone marrow morphology.Results: The expression of BDNF and its high affinity receptor TrkB transcripts in marrow myeloma cells from 12 patients and HMCLs was demonstrated by RT-PCR. In contrast, BDNF and TrkB mRNA was not detected in normal peripheral blood B lymphocytes. By Western blot analysis, we confirmed that HMCLs expressed BDNF and TrkB protein. Median BDNF concentrations on different cell culture supernatant were (14.7±2.4) ng/ml, (8.2±1.3) ng/ml, (13.2±1.2) ng/ml, respectively, BDNF secretion in myeloma cell lines were significantly higher than in the normal human plasma (median 2.0±0.6 ng/ml). In bone marrow biopsy specimens from patients with MM, a diffuse cytoplasmic expression of BDNF was demonstrated immunohistochemiacally in 12 of 16 patients, TrkB expression by the plasma cells was also detected in 15 of 16 patients, whereas control bone marrow samples showed weaker BDNF and TrkB protein staining. In contrast, p75NTR, the low affinity receptor for BDNF, was not detected on HMCL or MM plasma cells by either RT-PCR or immunohistochemistry.Conclusion: Not only BDNF but also its high affinity receptor TrkB are expressed by MM cells and that BDNF/TrkB pathway may be a potential therapeutic target in MM.PART II Proangiogenic properties of brain-derived neurotrophic factor on angiogenesisBackground and objective: The importance of angiogenesis or new vessel formation (occurring in the bone marrow) in the pathophysiology of MM is well established. Studies have shown that MM cells are capable of producing various cytokines contributing to new vessel formation in the bone marrow in myeloma. Recently, it has been reported that BDNF and its high affinity receptor TrkB are co-expressed by MM cells and the level of BDNF in the plasma of MM patients correlated with the level of vascular endothelian growth factor (VEGF) significantly. Studies have proved that various endothelial cells including human umbilical vein endothelial cells (HUVEC), human cerebral endothelial cells (HCEC) and cultured vascular smooth muscle cells are capable of secreting BDNF, and BDNF plays an important role in regulating cardiovascular development and maintaining vessel stability in the heart. In the present study, we investigate the in vitro and in vivo proangiogenic effects of BDNF and the relationship between expression of BDNF in MM cells and MM-induced angiogenesis.Methods: HUVEC was isolated and cultured successfully. The effect of BDNF on the in vitro proliferation of HUVEC was examined by MTT assay. The effects of BDNF on HUVEC migration, tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively. Matrigel plug assay and chorioallantoic membrane assay were used to evaluate the effect of BDNF on angiogenesis in vivo. Culture medium of RPMI 8226 and KM3 cells were collected. Proliferation of HUVEC in medium mixed with MM culture medium at different concentrations in the presence or absence of neutralizing antibody to BDNF was examined by MTT assay. The effects of MM culture medium on HUVEC migration, tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively.Results: BDNF stimulated the migration and tube formation of HUVEC in vitro significantly, although it did not induce HUVEC proliferation. BDNF enhanced migration of HUVEC in a dose-dependent fashion, migrating index were 1.24±0.18 at 25ng/ml, 1.56±0.23 at 50ng/ml, and 1.89±0.29 at 100ng/ml compared with nonstimulated controls. The effect of 100ng/ml BDNF was comparable with the optimal concentration of VEGF (25ng/ml), which is the known stimulus of HUVEC migration. Using two-dimension Matrigel, the treatment of 100ng/ml BDNF led to the formation of elongated and robust tube-like structures,which were organized by much larger number of cells compared with the control(P<0.05). BDNF also stimulate angiogenesis both in Matrigel plug of mouse model and in chick chorioallantoic membrane. In mouse Matrigel plug assay, BDNF produced more neovessels than control, and the vascular density and the number of mature vascular structures were significantly increased (P<0.01). In the CAM assay, the micorvessels around the BDNF treated gelatin sponge were obviously increased and demonstrated a radial neovascularization pattern, while negative response were elicited by control subjects only. On the other hand, MM culture medium induced a concentration-dependent increase in proliferation of HUVEC, the number of HUVEC at a concentration of 50% KM3 culture medium and complete KM3 culture medium were significantly higher than that of control(P<0.05). When anti-human BDNF antibody was added to the culture medium, the proliferative activity of HUVEC was reduced. MM culture medium also stimulated the migration and differentiation of HUVEC in vitro, the chemotactic index of HUVEC at a concentration of 50% KM3 culture medium and complete KM3 culture medium were 1.85±0.23 and 2.16±0.29, respectively(P<0.05), complete KM3 culture medium also stimulated capillary-like tube formation in HUVEC (P<0.01), addition of antihuman BDNF antibody neutralized these effects significantly.Conclusion: BDNF can stimulate the angiogenesis markedly in vitro and in vivo, may be developed as a new angiogenic factor. BDNF expressed by MM cells may be involved, at least in part, in MM-induced angiogenesis.PARTⅢBrain-derived neurotrophic factor activates TrkB signaling cascades mediating growth and migration of multiple myeloma cellsBackground and objective: Most of the tumor evolution in MM takes place in the bone marrow, indicating that signals from bone marrow microenviroment play a critical role in sustaining the growth and survival of MM cells during tumor progression. Expression of BDNF and its high affinity receptor TrkB has been demonstrated in MM cells. Recent studies have shown that abnormal expression of BDNF/TrkB plays an important role in growth, survival, migration, and invasion of various human malignancies, including those of neurogenic and nonneurogenic origin. In this study, we examined the biological effects of BDNF on MM cells.Methods: Primary myeloma cells were isolated and cultured successfully. The effects of BDNF on the cell viability of HMCLs (RPMI8226, U266, KM3) and primary myeloma cells were determined by trypan blue dye-exclusion method. Cell growth was determined by [3H] thymidine incorporation assay. The effects of BDNF on MM cells migration and invasion were studied by modified Boyden chamber assay. MTT assay was used to evaluate the effects of BDNF on cytotoxicity of chemotherapeutic agents. The effects of BDNF on the phosphorylation of TrkB in MM cells were determined by Western blotting.Results: (1) BDNF was a potent growth factor for MM. BDNF (5-500 ng/ml) had strong proliferative effects on both MM cell lines and primary MM cells, and DNA synthesis of MM cells was enhanced by BDNF in a concentration-dependent manner. (2) BDNF promoted migration and invasion of MM cells. BDNF (12.5-200 ng/ml) triggered significant increase in migration in RPMI8266 (1.63- to 3.11-fold), KM3 (1.92- to 3.76-fold) and primary myeloma (1.63- to 2.73-fold) cells. The maximal responses were seen at 50 ng/ml for RPMI 8266 (P < 0.05) and 25 ng/mL for KM3 (P < 0.01) and patient MM cells (P < 0.05) compared with nonstimulated controls. In contrast, BDNF only modestly enhances (1.2- to 2.1-fold) migration of U226 cells. (3) BDNF protected KM3 cells from melphalan and vincristine. The viability of KM3 cells in varying concentrations of melphalan with and without 50 ng/ml BDNF showed that BDNF induced almost a 2-fold shift in melphalan toxicity. BDNF induced a similar shift (3-fold) in vicristine toxicity. Similar effects could also be demonstrated in primary myeloma cells. (4) BDNF treatment increased growth of xenografted MM cells in a nude mice model. A significant increase in the size and weight of tumors in mice treated with BDNF was demonstrated when compared to controls. Intratumoral injection of BDNF starting after the development of measurable tumor also significantly reduced survival of mice. (5) TrkB present in MM cells is a functional signaling receptor for BDNF. A fraction of TrkB was tyrosine-phosphorylated under base-line culture conditions, and phosphorylated TrkB levels were increased following the addition of exogenous BDNF. BDNF-triggered increases in migration in RPMI 8226 cells, as well as patient MM cells, were completely abrogated by a relatively selective Trk tyrosine kinase inhibitor K252a.Conclusion: BDNF activates TrkB signaling cascades mediating growth, migration, and chemoprotection of multiple myeloma cells and appears to have a major contribution to the pathophysiology of MM.