Zhang-bo Chu

miR-15a and miR-16 affect the angiogenesis of multiple myeloma by targeting VEGF

Deregulated microRNAs (miRNAs) and their roles in cancer development have attracted much attention. Two miRNAs, miR-15a and miR-16, which act as putative tumor suppressor by targeting the oncogene BCL2, have been implicated in cell cycle, apoptosis and proliferation. In this study, we investigated the possible role of miR-15a/16 in the angiogenesis of multiple myeloma (MM). Using a stem-loop quantitative reverse transcription-PCR, we analyzed miR-15a/16 expressions in bone marrow samples from newly diagnosed MM patients and a panel of MM cell lines. miRNA transfection, western blotting analysis and assay of luciferase activity were used to examine whether vascular endothelial growth factor (VEGF) is the target of miR-15a/16. The functional roles of miR-15a/16 on tumorigenesis and angiogenesis were examined by in vitro angiogenesis models and in vivo tumor xenograft model. We showed that miR-15a and miR-16 were significantly underexpressed in primary MM cells as well as in MM cell lines. The aberrant expression of miR-15a/16 was detected especially in advanced stage MM. In human MM cell lines and normal plasma cells, expression of miR-15a/16 inversely correlated with the expression of VEGF-A. Western blotting combined with the luciferase reporter assay demonstrated that VEGF-A was a direct target of miR-15a/16. Ectopic overexpression of miR-15a/16 led to decreased pro-angiogenic activity of MM cells. Finally, infection of lentivirus-miR-15a or lentivirus-miR-16 resulted in significant inhibition of tumor growth and angiogenesis in nude mice. This study suggest that miR-15a/16 could play a role in the tumorigenesis of MM at least in part by modulation of angiogenesis through targeting VEGF-A.

Brain-derived neurotrophic factor induces proliferation, migration, and VEGF secretion in human multiple myeloma cells via activation of MEK-ERK and PI3K/AKT signaling

This study investigated the signaling pathways involved in the different biological effects of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM). The effects of BDNF on proliferation of MM cell lines and primary myeloma cells were examined by [3H]thymidine incorporation assay. The effects of BDNF on MM cells migration were studied by transwell migration assay. Stimulation by BDNF of vascular endothelial growth factor (VEGF) production was analyzed by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The signal-transduction pathways that are activated in response to BDNF were determined by Western blots. VEGF is induced by BDNF in a dose-dependent manner in MM cells. Stimulation of MM cells with BDNF led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the MEK-extracellular signal-regulated protein kinase pathways. Using specific signal-transduction inhibitors, we demonstrated that MEK is required for BDNF-induced proliferation, whereas activation of PI3K is required for BDNF-stimulated migration and VEGF production. BDNF affects different cell signaling pathways mediating growth, migration, and VEGF secretion in MM cells. Our observations provided the framework for novel therapeutic strategies targeting BDNF signaling cascades in MM.

Lentiviral shRNA silencing of BDNF inhibits in vivo multiple myeloma growth and angiogenesis via down-regulated stroma-derived VEGF expression in the bone marrow milieu

Bone marrow (BM) neovascularization and vascular endothelial growth factor (VEGF) expression in multiple myeloma (MM) correlate with disease progression. Brain derived neurotrophic factor (BDNF) is highly expressed by malignant plasma cells isolated from the majority of MM patients. Recently, BDNF was identified as a potential proangiogenic factor for the promotion of endothelial cell survival, induction of neoangiogenesis in ischemic tissues, and increase of VEGF expression in neuroblastoma. Since tropomyosin receptor kinase B (TrkB), the receptor of BDNF, is expressed by stromal cells within the BM milieu, here we sought to evaluate the involvement of BDNF/TrkB in myeloma–marrow stroma interaction and its effects on BM angiogenesis. TrkB was abundantly expressed by bone marrow stromal cells (BMSCs) isolated from healthy donors. Stimulation of BMSCs with BDNF induced a time‐ and dose‐ dependent increase in VEGF secretion, which was completely abolished by K252α, an inhibitor of TrkB. BDNF triggered activation of signal transducer and activator of transcription 3 (STAT3) and activator protein‐1 (AP‐1), whereas STAT3 was involved in mediating VEGF expression. We further delineated the biological significance of BDNF in MM by using lentiviral short‐interfering RNA (shRNA). When myeloma cells were cocultured with BMSCs in a noncontact Transwell system, VEGF levels in supernatants were significantly decreased when BDNF expression was knocked down. Furthermore, silencing of BDNF expression significantly inhibited xenograft tumor growth and angiogenesis, and prolonged survival in mouse model. Our studies demonstrate that BDNF, as a potential stimulator of angiogenesis, contributes to MM tumorgenesis; it mediates stromal–MM cell interactions via selective activation of specific receptor TrkB and downstream signal transducer STAT3, regulating VEGF secretion.

Brain-derived neurotrophic factor is a potential osteoclast stimulating factor in multiple myeloma.

Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain‐derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In our study, we investigated whether BDNF was possibly involved in MM cell‐induced osteolysis. We showed that BDNF was elevated in MM patients and the bone marrow plasma levels of BDNF positively correlated with extent of bone disease. In osteoclast formation assay, bone marrow plasma from patients with MM increased osteoclast formation and the effect was significantly blocked by neutralizing antibody to BDNF, suggesting a critical role for BDNF in osteoclast activation. Furthermore, the direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF, demonstrating that BDNF used TrkB for its effects on osteoclast. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein‐1α and receptor activator of nuclear factor‐κB ligand, two major osteoclast stimulatory factors in MM. These results support an important role for BDNF in the development of myeloma bone disease.

Inhibition of BDNF in Multiple Myeloma Blocks Osteoclastogenesis via Down-Regulated Stroma-Derived RANKL Expression Both In Vitro and In Vivo

Brain-derived neurotrophic factor (BDNF) was recently identified as a factor produced by multiple myeloma (MM) cells, which may contribute to bone resorption and disease progression in MM, though the molecular mechanism of this process is not well understood. The purpose of this study was to test the effect of BDNF on bone disease and growth of MM cells both in vitro and in vivo. Co- and triple-culture systems were implemented. The in vitro results demonstrate that BDNF augmented receptor activator of nuclear factor kappa B ligand (RANKL) expression in human bone marrow stromal cells, thus contributing to osteoclast formation. To further clarify the effect of BDNF on myeloma bone disease in vivo, ARH-77 cells were stably transfected with an antisense construct to BDNF (AS-ARH) or empty vector (EV-ARH) to test their capacity to induce MM bone disease in SCID–rab mice. Mice treated with AS-ARH cells were preserved, exhibited no radiologically identifiable lytic lesions and, unlike the controls treated with EV-ARH cells, lived longer and showed reduced tumor burden. Consistently, bones harboring AS-ARH cells showed marked reductions of RANKL expression and osteoclast density compared to the controls harboring EV-ARH cells. These results provide further support for the potential osteoclastogenic effects of BDNF, which may mediate stromal–MM cell interactions to upregulate RANKL secretion, in myeloma bone diseases.

Gene silencing of the BDNF/TrkB axis in multiple myeloma blocks bone destruction and tumor burden in vitro and in vivo

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF‐κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain‐derived neurotrophic factor (BDNF) was identified as a MM‐derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma–osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM‐derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time‐ and dose‐dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID‐RPMI8226 mice model was constructed using lentiviral short hairpin RNA‐transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma–myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.

The effect of brain-derived neurotrophic factor on angiogenesis.

To investigate the in vitro and in vivo proangiogenic effects of brain-derived neurotrophic factor (BDNF), human umbilical vein endothelial cells (HUVECs) were isolated and cultured in primary culture. The effect of BDNF on the proliferation of HUVECs was examined by MTT assay. The effects of BDNF on HUVEC migration and 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 effects of BDNF on angiogenesis in vivo. Our results showed that BDNF substantially stimulated the migration and tube formation of HUVECs in vitro, although it did not induce HUVEC proliferation. BDNF also induced angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. In conclusion, BDNF can promote angiogenesis both in vitro and in vivo, and may be a proangiogenic factor.SummaryTo investigate the in vitro and in vivo proangiogenic effects of brain-derived neurotrophic factor (BDNF), human umbilical vein endothelial cells (HUVECs) were isolated and cultured in primary culture. The effect of BDNF on the proliferation of HUVECs was examined by MTT assay. The effects of BDNF on HUVEC migration and 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 effects of BDNF on angiogenesis in vivo. Our results showed that BDNF substantially stimulated the migration and tube formation of HUVECs in vitro, although it did not induce HUVEC proliferation. BDNF also induced angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. In conclusion, BDNF can promote angiogenesis both in vitro and in vivo, and may be a proangiogenic factor.

Efficacy and Safety of Bortezomib Maintenance in Patients with Newly Diagnosed Multiple Myeloma: A Meta-Analysis.

Multiple myeloma (MM) is a B-cell neoplasm with a high incidence of relapse. Bortezomib has been extensively studied for the maintenance treatment of MM. Here, we carried out a meta-analysis to determine the efficacy and safety of maintenance therapy with bortezomib. We searched for clinical trials in PubMed (Medline), Embase (OVID), and the Cochrane Library. Two randomized controlled trials (RCTs) enrolling a total of 1338 patients were included. Bortezomib maintenance statistically significantly improved both progression-free survival (PFS) (hazard ratio (HR) 0.67, 95% confidence interval (CI) = 0.51 to 0.87, P=0.003) and overall survival (OS) (HR = 0.75 therapy, 95% CI = 0.63 to 0.89, P=0.001) more than did non-bortezomib maintenance therapy. Our analysis revealed higher incidence of neutropenia (risks ratios (RR) = 1.39; 95% CI = 1.08 to 1.79), peripheral neuropathy (PN) (RR = 2.23; 95% CI = 1.38 to 3.61, P=0.001), and cardiologic events (RR = 1.91; 95% CI = 1.12 to 3.28, P=0.02) in patients with bortezomib maintenance therapy. Our meta-analysis demonstrates OS and PFS benefits of bortezomib maintenance therapy in patients with newly diagnosed MM. However, the therapy is associated with increased risk of adverse events. Additionally, more RCTs are needed for better understanding and determination of optimal bortezomib maintenance therapy in MM.

Role of brain-derived neurotrophic factor in bone marrow angiogenesis in multiple myeloma

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.SummaryThis 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.

Successful treatment of myeloid neoplasms associated with PDGFRA rearrangement with imatinib mesylate.

Hypereosinophilic syndromes (HES) constitute a rare and heterogeneous group of disorders, defined as persistent and marked blood eosinophilia associated with evidence of eosinophil-induced organ damage. Cardiac dysfunction is the most frequent result of end-organ damage and is the major cause of morbidity and mortality among patients with HES. Despite patients with FIP1-like-1-platelet-derived growth factor alpha (FIP1L1-PDGFRA) associated HES (myeloid neoplasms associated with PDGFRA rearrangement) have been shown to respond to low-dose imatinib with a complete and durable hematological and cytogenetic remission, influences of imatinib on clinical manifestations related to hypereosinophilia heart involvement are variable. Here we describe the case of a young male patient with severe heart involvement who had a prompt, clinical and hematological complete remission following administration of imatinib. However, as endomyocardial fibrosis and related loss of function are deteriorated after initiation of imatinib therapy, valvular replacement and tricuspid annuloplasty had to perform to restore his heart function. Our finding concurs with recent reports that severe heart involvement was irreversible with imatinib treatment.