Role of brain-derived neurotrophic factor in bone marrow angiogenesis in multiple myeloma / 华中科技大学学报（医学）（英德文版）

This study examined the expression of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM) and its role in bone marrow angiogenesis. The peripheral blood plasma was harvested from 71 MM patients and 63 patients without hematological malignancy. The BDNF level in the blood plasma was determined by ELISA. Human bone marrow endothelial cells (HBMECs) were cultured. The mRNA and protein expression levels of the BDNF receptor TrkB in HBMECs were detected by using RT-PCR and flow cytometry, respectively. The viability of HBMECs treated with recombinant human (rh) BDNF or not was measured by using MTT assay. The migration of HBMECs in the presence of rhBDNF or not was determined by modified Boyden chamber assay. In vitro tube formation assay was used to assess the effect of rhBDNF on HBMECs differentiation. The results of ELISA revealed that the BDNF level was significantly higher in peripheral blood plasma of MM patients than in that of control patients (4.39±0.67 vs. 1.96±0.39 ng/mL, P<0.05). The BDNF receptor TrkB was expressed in HBMECs at mRNA and protein level. MTT assay manifested that rhBDNF could significantly concentration-dependently promote the HBMECs proliferation. The number of HBMECs treated with 160 ng/mL rhBDNF for 48 h was 1.57±0.10 folds higher than that in control group (P<0.05). Moreover, rhBDNF could enhance HBMECs migration in a concentration-dependent manner and the maximal migration was reached in the presence of 100 ng/mL rhBDNF. The migration indexes were 1.40±0.11, 1.64±0.16, 2.06±0.25 and 2.18±0.21 in 25, 50, 100 ng/mL rhBDNF groups and 25 ng/mL rhVEGF group, respectively. In vitro tube formation assay demonstrated that the area of the formed tubular structure was increased with the rhBDNF concentration. In control group, there was no formation of intact tubular structure and the HBMECs on the matrigel were irregularly dispersed. HBMECs treated with 100 ng/mL rhBDNF could form intact tubular structure and the area and the diameter of tubes were significantly greater than those in control group (P<0.05). There was no significant difference in the formed tubular area between 25 ng/mL VEGF group and 100 ng/mL rhBDNF group. It was concluded that BDNF plays an important role in myeloma cell-induced angiogenesis, and it may become a new target of anti-angiogenesis treatment for MM.

Role of brain-derived neurotrophic factor in bone marrow angiogenesis in multiple myeloma / 华中科技大学学报（医学）（英德文版）

This study examined the expression of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM) and its role in bone marrow angiogenesis. The peripheral blood plasma was harvested from 71 MM patients and 63 patients without hematological malignancy. The BDNF level in the blood plasma was determined by ELISA. Human bone marrow endothelial cells (HBMECs) were cultured. The mRNA and protein expression levels of the BDNF receptor TrkB in HBMECs were detected by using RT-PCR and flow cytometry, respectively. The viability of HBMECs treated with recombinant human (rh) BDNF or not was measured by using MTT assay. The migration of HBMECs in the presence of rhBDNF or not was determined by modified Boyden chamber assay. In vitro tube formation assay was used to assess the effect of rhBDNF on HBMECs differentiation. The results of ELISA revealed that the BDNF level was significantly higher in peripheral blood plasma of MM patients than in that of control patients (4.39±0.67 vs. 1.96±0.39 ng/mL, P<0.05). The BDNF receptor TrkB was expressed in HBMECs at mRNA and protein level. MTT assay manifested that rhBDNF could significantly concentration-dependently promote the HBMECs proliferation. The number of HBMECs treated with 160 ng/mL rhBDNF for 48 h was 1.57±0.10 folds higher than that in control group (P<0.05). Moreover, rhBDNF could enhance HBMECs migration in a concentration-dependent manner and the maximal migration was reached in the presence of 100 ng/mL rhBDNF. The migration indexes were 1.40±0.11, 1.64±0.16, 2.06±0.25 and 2.18±0.21 in 25, 50, 100 ng/mL rhBDNF groups and 25 ng/mL rhVEGF group, respectively. In vitro tube formation assay demonstrated that the area of the formed tubular structure was increased with the rhBDNF concentration. In control group, there was no formation of intact tubular structure and the HBMECs on the matrigel were irregularly dispersed. HBMECs treated with 100 ng/mL rhBDNF could form intact tubular structure and the area and the diameter of tubes were significantly greater than those in control group (P<0.05). There was no significant difference in the formed tubular area between 25 ng/mL VEGF group and 100 ng/mL rhBDNF group. It was concluded that BDNF plays an important role in myeloma cell-induced angiogenesis, and it may become a new target of anti-angiogenesis treatment for MM.

Effect of RNA interference targeting brain-derived neurotrophic factor gene on HeLa cell proliferation and apoptosis / 中华病理学杂志

<p><b>OBJECTIVE</b>To screen effective sequences of short hairpin RNA on brain-derived neurotrophic factor (BDNF) gene and the effect of RNA interference on the proliferation and apoptosis of HeLa cells, a cervix carcinoma cell line with high expression of BDNF.</p><p><b>METHODS</b>Two recombinant eukaryotic human-BDNF siRNA expression vectors were designed and constructed. Sequences were confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector pGenesil-1 and two recombinant plasmids, pGenesil-shRNA-BDNF1 and pGenesil-shRNA-BDNF2, were transfected into HeLa cells using Lipofectamine 2000 (groups: P(0), P(1) and P(2), respectively). The mRNA and protein levels of BDNF in HeLa cells were detected by RT-PCR and Western blot, respectively. The cellular proliferation rates were determined by MTT assay and the apoptotic rates were measured by flow cytometry and Hoechest 33258.</p><p><b>RESULTS</b>The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. The expression of mRNA and protein of BDNF in P(1) group were significantly decreased, comparing with non-transfected group, P(0) and P(2) groups (F = 48.19, P < 0.01). P(2) group failed to meet the expected results (P > 0.05). In addition, the proliferation activity was reduced in P(1) group and the peak point of proliferation curve was prolonged. Moreover, the early cell apoptotic rates were statistically increased in P(1)[(53.4 +/- 4.2)%] VS. non-transfected [(0.8 +/- 0.4)%], P(0) [(5.1 +/- 1.8)%] and P(2)[(7.9 +/- 2.4)%] groups (F = 269.77, P < 0.01).</p><p><b>CONCLUSION</b>HeLa cells express a high level of BDNF. BDNF gene silencing by RNA interference increases the apoptosis of HeLa cells and inhibits cell proliferation, offering a possible target for efficient tumor therapy.</p>

Study of brain-derived neurotrophic factor activating TrkB signaling cascades in the pathogenesis of multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the significance of abnormal expression of brain-derived neurotrophic factor (BDNF)/TrkB in the development and evolution of multiple myeloma (MM) and the involved signaling pathways.</p><p><b>METHODS</b>The effect of BDNF on the cell viability of human myeloma cell line (HMCL) (RPMI8226, U266, KM3) was determined by trypan blue dye-exclusion. MTT assay was used to evaluate the cytotoxicity of tested chemotherapeutic agents. The effect of BDNF on the phosphorylation of TrkB was determined by Western blot. A human myeloma xenograft animal model was used to evaluate the effects of BDNF on tumor growth and survival time.</p><p><b>RESULTS</b>BDNF at 50 microg/L triggered significant increase in cell viability of HMCL. BDNF protected KM3 cells from melphalan and vincristine. The viability of KM3 cells exposed to varying concentrations of melphalan with and without 50 microg/L BDNF showed that BDNF induced almost a 2-fold and a 3-fold increase in melphalan and vincristine toxicity respectively. BDNF treatment increased MM cell growth in xenografted MM model (3240.9 mm3 vs 1032.7 mm3 ) (P <0.05). Intratumoral injection of BDNF also significantly reduced survival time (13 d vs 21 d) (P <0.05). The phosphorylated TrkB level was increased significantly after treated by exogenous BDNF. BDNF-triggered migration in RPMI8226 cells was completely abrogated by a Trk tyrosine kinase inhibitor K252a.</p><p><b>CONCLUSION</b>BDNF can activate TrkB signaling cascades resulting in MM cells growth, migration, and chemoprotection and appears to have a major contribution to the pathogenesis of MM.</p>

Brain-derived neurotrophic factor promotes the secretion of MMP-9 in human myeloma cell through modulation of nucleus factor-kappaB / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of brain-derived neurotrophic factor (BDNF) promoting human multiple myeloma (MM) cells secreting matrix metalloproteinase-9 ( MMP-9).</p><p><b>METHODS</b>Gelatin zymography of culture supernatants was performed to visualize the content of MMPs in myeloma RPMI 8226 cells stimulated by BDNF. NF-kappaB activity was determined by chemiluminescent electrophoretic mobility shift assay (EMSA).</p><p><b>RESULTS</b>Treatment with 25, 50, 100 and 200 microg/L BDNF for 24 h significantly (P < 0.01) enhanced the level of MMP-9 (2.03+/-0.48, 2.99+/-0.046, 4.63+/-0.62 and 5.62+/-1.29 microg/L, respectively, vs 1.00 microg/L of the control) secreted by RPMI8226 cells in a dose-dependent manner, while that of MMP-2 was not changed significantly (P > 0.05). The BDNF-induced activation of MMP-9 was inhibited by pretreatment with pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, or K252 alpha, a specific tyrosine inhibitor of TrkB which is the receptor for BDNF. Pretreated with 1 mmol/L PDTC or 500 nmol/L K252 alpha significantly downregulated MMP-9 secreted by the 100 microg/L of BDNF stimulated RPMI 8226 cells (the optical density values were 867.52+/-101.81 and 727.98 +/-92.05, respectively, vs 1,159.01+/-233.15 of the control). The activity of NF-kappaB was enhanced by BDNF in a dose-dependent manner, and pretreatment with K252 alpha could significantly inhibit this activation at 1, 6, 12 and 24 h (P < 0.05) in a time-dependent manner.</p><p><b>CONCLUSION</b>BDNF plays an important role in the angiogenesis of MM to promote the up-regulation of MMP-9, which may be induced by enhanced NF-kappaB activity in MM cells.</p>

Antimyeloma effects of resveratrol through inhibition of angiogenesis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In multiple myeloma (MM), bone marrow angiogenesis parallels tumour progression and correlates with disease activity. Recent studies have proved resveratrol possesses antiangiogenic activity in vitro and in vivo. In this study, we examined the effects of resveratrol on myeloma cell dependent angiogenesis and the effects of resveratrol on some important angiogenic factors of RPMI 8226 cells.</p><p><b>METHODS</b>RPMI 8226 cells were cocultured with human umbilical vein endothelial cells (HUVECs) to evaluate the effects of myeloma cells on angiogenesis. The RPMI 8226 cells were treated with various concentrations of resveratrol (6.25 - 50.00 micromol/L) for different times (12 - 72 hours). Reverse transcriptase polymerase chain reaction (RT-PCR) was used to assay vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), metalloproteinases (MMP)-2 and MMP-9 mRNA. Gelatin zymography was used to analyze MMP-2 and MMP-9 activity. VEGF and bFGF proteins secreted by the cells in the medium were quantified by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Cell proliferation, migration and differentiation of HUVECs markedly increased by coculture with RPMI 8226 cells. Resveratrol inhibited proliferation, migration and tube formation of HUVECs cocultured with myeloma cells in a dose dependent manner. Treatment of RPMI 8226 cells with resveratrol caused a decrease in MMP-2 and MMP-9 activity. Resveratrol inhibited VEGF and bFGF protein expression in a dose and time dependent manner. Furthermore, decreased levels of VEGF, bFGF, MMP-2 and MMP-9 mRNA from cells treated with various concentrations of resveratrol confirmed its antiangiogenic action at the level of gene expression.</p><p><b>CONCLUSIONS</b>Resveratrol inhibits multiple myeloma angiogenesis by regulating expression and secretion of VEGF, bFGF, MMP-2 and MMP-9. Resveratrol may be a potential candidate for the treatment of multiple myeloma.</p>