OBJECTIVETo observe the effect of rosiglitazone (RGZ) on the mRNA expression of HIF1α and IGF1 genes in the myeloma cells and explore possible mechanism of angiogenesis inhibition.

METHODSHuman myeloma cell line RPMI8226 and primary myeloma cells from five patients enriched by using CD138 immunomagnetic beads were treated with different concentrations (10, 20, 40 μmol/L) of RGZ. The mRNA expression of HIF1α and IGF1 was analyzed in cells treated with RGZ after 48h by RT-PCR, The levels of phosphorylated AKT and ERK proteins were detected by Western blotting. Bone marrow mononuclear cells from five patients with iron deficiency anemia were regarded as control.

RESULTSHigher mRNA expression of HIF1α and IGF1 genes in RPMI8226 and in primary myeloma cells was showed as compared to those in control. Treated with RGZ of 20 μmol/L after 48 h, the mRNA expression of HIF1α (1.21 ± 0.08 vs 0.75 ± 0.06) and IGF1 (0.62 ± 0.06 vs 0.32 ± 0.04) in RPMI8226 cells was declined as compared to those without RGC treatment. The same declination was also seen in primary myeloma cells (HIF1α: 2.02 ± 0.16 vs 0.53 ± 0.04; IGF1: 1.92 ± 0.13 vs 0.58±0.03). RGZ could inhibit the expression of pAKT and pERK, nor the total AKT and ERK proteins, in RPMI8226 cells in a dose-dependent manner at the concentration of 10 μmol/L, 20 μmol/L, and 40 μmol/L.

CONCLUSIONRGZ could inhibit the mRNA expression of HIF1α and IGF1. Inhibition of angiogenesis by RGZ may be associated with down-regulation of pAKT and pERK expression.

Cell Line, Tumor ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Multiple Myeloma ; blood supply ; metabolism ; Neovascularization, Pathologic ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; genetics ; Thiazolidinediones ; pharmacology

Humans ; Multiple Myeloma ; blood supply ; therapy ; Neovascularization, Pathologic ; therapy ; Prognosis

OBJECTIVETo investigate effects of low-dose cyclophosphamide and prednisone (CP) metronomic chemotherapy on microvessel density of bone marrow, serum vascular endothelial growth factor (VEGF) and platelet derived growth factor BB (PDGF-BB)in multiple myeloma (MM) patients.

METHODS54 refractory or relapsed MM patients were treated with CP metronomic chemotherapy consisted of oral cyclophosphamide (CTX, 50 mg/d) and prednisone (Pred, 15 mg/d). Bone marrow and peripheral blood of each patient were collected before and 2, 4, 6 months after treatment. Among the 37 assessable patients, 30 cases were responsive with the response rate of 81.08%. Another 17 cases were follow-uped less than 6 months or failure to obtain serum samples or lost to follow-up. Microvessel density of bone marrow was measured by immunohistochemistry and serum VEGF/PDGF-BB expression was analyzed by ELISA in the 37 assessable patients.

RESULTS2, 4, 6 months following CP metronomic chemotherapy, microvessel densities of bone marrow in the responders were 33.1 ± 4.8/HP, 24.8 ± 3.7/HP, 19.7 ± 2.1/HP respectively; the expressions of VEGF were (394 ± 57) ng/L, (268 ± 32) ng/L and (217 ± 20) ng/L respectively; the expressions of PDGF-BB were (304 ± 31) ng/L, (274 ± 31) ng/L and (196 ± 22) ng/L respectively. After CP metronomic chemotherapy, there were significantly lower of microvessel density, VEGF and PDGF-BB levels than pretreatment \[MVD 48.5 ± 5.9/HP, VEGF (517 ± 60) ng/L, PDGF-BB (484 ± 60) ng/L\]in the responders (P < 0.01). While in the non-responders, after treated by CP metronomic chemotherapy for 2 months, microvessel density, the expression of VEGF and the expression of PDGF-BB were 32.5 ± 4.7/HP, 512 ± 39 ng/L and (452 ± 39) ng/L respectively. There were no significant changes of MVD, VEGF and PDGF-BB levels compared with pretreatment \[MVD 33.2 ± 5.6/HP,VEGF (498 ± 55) ng/L, PDGF-BB (488 ± 44) ng/L\] (P > 0.05).

CONCLUSIONSOur findings suggested that continuous low-dose CP metronomic chemotherapy could decrease microvessel density of bone marrow in MM patients. Furthermore, it down-regulated expression of serum VEGF and PDGF-BB to exert its anti-angiogenesis in MM.

Aged ; Aged, 80 and over ; Angiogenesis Inhibitors ; administration & dosage ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cyclophosphamide ; administration & dosage ; Female ; Humans ; Male ; Microvessels ; drug effects ; Middle Aged ; Multiple Myeloma ; blood ; blood supply ; drug therapy ; Prednisone ; administration & dosage ; Proto-Oncogene Proteins c-sis ; blood ; Vascular Endothelial Growth Factor A ; blood

Aged ; Diagnosis, Differential ; Drug Therapy, Combination ; Female ; Humans ; Liver ; blood supply ; pathology ; Multiple Myeloma ; diagnostic imaging ; drug therapy ; pathology ; Neovascularization, Pathologic ; diagnosis ; pathology ; Tomography, X-Ray Computed

The aim of this study was to investigate the effect of [(-)-epigallocatechin-3-gallate (EGCG)] on angiogenesis induced by multiple myeloma cell line KM3 and its mechanism. The effects of KM3 cell supernatant after being treated with EGCG in different concentrations on migration and vascular formation ability of endothelial cell line HUVEC were investigated through culture of MM cell line KM3 in vitro. The secretion level of vascular endothelial growth factor (VEGF) in KM3 cell supernatant and the expression level of VEGF mRNA in KM3 were detected by ELISA and RT-PCR respectively. The results indicated that the KM3 cell supernatant significantly induced endothelial cell migration and vascular formation in vitro. EGCG inhibited the effect of endothelial cell migration induced by KM3 cell supernatant, and the numbers of migrated cells were 414 +/- 27, 299 +/- 70, 202 +/- 42 and 116 +/- 13 at 5, 25, 50, 100 micromol/L respectively. The numbers of migrated cells showed negative correlation with the dose of EGCG (r = -0.952, p < 0.05). The areas of the capillary-like structures decreased while the concentrations of EGCG increased, 88343.9 +/- 3231.1 microm(2) at 25 micromol/L, 60897.5 +/- 914.1 microm2 at 50 micromol/L, which were significantly less than that in the control (p < 0.01) and showed negative correlation with the dose of EGCG (r = -0.888, p < 0.05). 48 hours after treatment with EGCG at concentrations of 5, 25, 50 and 100 micromol/L, the levels of VEGF in the culture supernatant were 1399.0 +/- 47.4, 660.1 +/- 5.7, 108.5 +/- 5.8 and 26.2 +/- 18.6 pg/ml respectively. Except 5 micromol/L, all the other groups showed significant changes while compared with the controls (p < 0.01). Furthermore, EGCG depressed the mRNA expression of VEGF in KM3 cells in a dose-dependent manner. It is concluded that the EGCG can significantly inhibit angiogenic ability of multiple myeloma KM3 cells, its pharmacological mechanism may be downregulation of VEGF mRNA expression and reduction of VEGF secretion.
Angiogenesis Inhibitors ; pharmacology ; Catechin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Down-Regulation ; Humans ; Multiple Myeloma ; blood supply ; metabolism ; RNA, Messenger ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

BACKGROUNDIn 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.

METHODSRPMI 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).

RESULTSCell 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.

CONCLUSIONSResveratrol 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.

Angiogenesis Inhibitors ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Fibroblast Growth Factor 2 ; analysis ; genetics ; Humans ; Matrix Metalloproteinase 2 ; analysis ; Matrix Metalloproteinase 9 ; analysis ; Multiple Myeloma ; blood supply ; drug therapy ; pathology ; RNA, Messenger ; analysis ; Stilbenes ; pharmacology ; Vascular Endothelial Growth Factor A ; analysis ; genetics

In order to explore the probability of curcumin treating multiple myeloma (MM) via the inhibition of angiogenesis, the expressions of brain derived neurotrophic factor (BDNF) and its specific receptor in human MM cells and endothelial cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The angiogenic activity was evaluated by endothelial cell migration assay and tubule formation assay in vitro. The results showed that exogenous BDNF significantly induced endothelial cell tubule formation and endothelial cell migration, these two effects were inhibited by curcumin. Furthermore, BDNF was detected in the MM cell and TrkB was detected in the endothelial cell and curcumin depressed the mRNA expression of BDNF and TrkB in the dose- and time-dependent manners. It is concluded that BDNF is a novel angiogenesis protein. Curcumin interrupts the interaction between multiple myeloma cells and endothelial cells by reducing TrkB expression in endothelial cells and inhibiting BDNF production in multiple myeloma cells, eventually, resulting in inhibition of angiogenesis. This is probably one part of the mechanism of the curcumin treating MM via the inhibition of angiogenesis.
Angiogenesis Inhibitors ; pharmacology ; Antineoplastic Agents ; pharmacology ; Brain-Derived Neurotrophic Factor ; antagonists & inhibitors ; biosynthesis ; genetics ; Cell Line, Tumor ; Curcumin ; pharmacology ; Endothelial Cells ; cytology ; Humans ; Multiple Myeloma ; blood supply ; pathology ; Neovascularization, Pathologic ; prevention & control ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the expression of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM) cells and the correlation between BDNF and MM angiogenesis.

METHODSThe expressions of BDNF mRNA transcripts and protein in MM cell lines (RPMI 8226, KM3) were determined by RT-PCR and Western blot, respectively, BDNF levels in culture supernatant by enzyme-linked immunosorbent assay. Proliferation of human umbilical vein endothelial cells (HUVEC) mixed with MM culture medium at different concentrations was examined by MTT assay. The effects of MM culture medium on HUVEC migration and tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively.

RESULTSBDNF was expressed in and secreted by MM cell lines RPMI 8226 and KM3. BDNF concentrations in culture supernatants were within the range of its biological activity. MM culture medium induced a concentration-dependent proliferation of HUVEC. The number of HUVEC at a concentration of 50% KM3 culture medium and at full KM3 culture medium were (1.85 +/- 0.23)-fold and (2.16 +/- 0.29) -fold increase, respectively (P <0.05), compared with that of control. The proliferative activity of HUVEC was reduced on the addition of BDNF antibody to the culture medium. 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 at full KM3 culture medium were 1.85 +/- 0.23 and 2.16 +/- 0.29, respectively (P < 0.05). Full KM3 culture medium also stimulated capillary-like tube formation in HUVEC (P <0.01), and addition of anti-human BDNF antibody neutralized these effects significantly.

CONCLUSIONMM cell lines expressed and secreted biologically active BDNF, which may be involved, at least in part, in MM angiogenesis.

Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Endothelial Cells ; drug effects ; physiology ; Humans ; Multiple Myeloma ; blood supply ; metabolism ; Neovascularization, Pathologic ; metabolism ; RNA, Messenger ; genetics

In order to investigate the expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in multiple myeloma patients and the in vitro and in vivo proangiogenic effects of BDNF, the plasma concentrations of BDNF and VEGF in MM patients and control group were determined by ELISA, the effect of BDNF on the in vitro proliferation of human umbilical vein endothelial cells (HUVEC) 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 effect of BDNF on angiogenesis in vivo. The results demonstrated that the concentration of BDNF was (4.22 +/- 0.64) ng/ml and (2.03 +/- 0.38) ng/ml in MM group and control group, respectively, (P = 0.01). There was also a significant difference between VEGF levels of two groups [(79.35 +/- 13.25) pg/ml vs (34.41 +/- 1.78) pg/ml, P = 0.006]. The levels of BDNF and VEGF correlated significantly (r = 0.430, P = 0.025). BDNF stimulated the migration and tube formation in vitro significantly, although it had no effect on the proliferation of HUVEC. BDNF also stimulated angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. It is concluded that the concentrations of BDNF and VEGF in MM patients' peripheral blood are at high level; BDNF can stimulate the angiogenesis markedly in vitro and in vivo. Therefore, BDNF may act as an important regulator in angiogenesis of MM.
Adult ; Aged ; Aged, 80 and over ; Angiogenesis Inducing Agents ; blood ; pharmacology ; Animals ; Brain-Derived Neurotrophic Factor ; blood ; pharmacology ; Cell Line ; Cell Movement ; drug effects ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; drug effects ; embryology ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Middle Aged ; Multiple Myeloma ; blood ; pathology ; Neovascularization, Physiologic ; drug effects ; Vascular Endothelial Growth Factor A ; blood ; pharmacology

Angiogenesis is a necessary step in tumor progression, and it correlates an unfavorable prognosis. In multiple myeloma, bone marrow microvessel density and angiogenesis grading correlated with plasma cell labeling index and are poor survival predictors, but the study of myeloma's angiogenesis is very rare. This article was to study the effect of multiple myeloma cell line conditioned media on the proliferation, migration and angiogenesis of human bone marrow endothelial cells (HBMEC). The multiple myeloma cell line conditioned media were obtained by using RPMI 1640 media containing 2% fetal bovine serum (FBS) to cultivate myeloma cell lines for 18 hours. Proliferation and migration of HBMEC were detected by using those media to cultivate HBMEC. Capillary tube formation was performed by using microcarriers cytodex-3 covered with HBMEC in three-dimensional fibrin matrices. The results showed that myeloma conditioned media induced HBMEC's proliferation and migration (P < 0.001), and those media induced capillary tube formation (length and width) of HBMEC (P < 0.001). It was concluded that myeloma cell lines induce HBMEC's proliferation, migration, and capillary tube formation by secreting several cytokines.
Bone Marrow Cells ; cytology ; Cell Division ; Cell Movement ; Endothelial Growth Factors ; analysis ; physiology ; Humans ; Intercellular Signaling Peptides and Proteins ; analysis ; physiology ; Lymphokines ; analysis ; physiology ; Multiple Myeloma ; blood supply ; chemistry ; pathology ; Neovascularization, Pathologic ; etiology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors