Value of (11)C-PiB PET/MRI in the evaluation of organ involvement in primary systemic light chain amyloidosis / 中华血液学杂志

Objective: To analyze the value of (11)C-PiB PET/MRI for evaluating organ involvement in patients with primary light chain amyloidosis (pAL) . Methods: The clinical data of 20 patients with pAL and 3 healthy volunteers from January 2019 to October 2021 were retrospectively analyzed. The correlation between the organ involvement evaluated by clinical standards and PET/MRI was compared. The relationship between cardiac-related biological indicators, disease stage, and the maximum standardized uptake value (SUVmax) were analyzed. The relationship between 24-hour urinary protein quantification and kidney SUVmax was analyzed. Results: ①In 20 patients (18 newly diagnosed patients and 2 non-newly diagnosed patients) ,(11)C-PiB positive uptake was observed in the heart (15 patients, 75%) , lung (8 patients, 40%) , bone marrow (10 patients, 50%) , muscle (10 patients, 50%) , tongue muscle (7 patients, 35%) , thyroid (6 patients, 30%) , salivary gland (4 patients, 20%) , spleen (2 patients, 10%) , and stomach wall (1 patient, 5%) . ②Organ involvement on (11)C-PiB PET/MRI showed good correlations with the clinical evaluation criteria for the heart and bone marrow. The positive rate of PET/MRI evaluation in the lung, spleen, gland, muscle, and tongue muscle was significantly higher than the clinical criteria. However, (11)C-PiB PET/MRI has limitations in the evaluation of the nervous system and fat tissue. ③To analyze the relationship between cardiac-related biological indexes and the SUVmax of the heart in 13 newly diagnosed patients. Patients with left ventricular ejection fraction (LVEF) <50% and interventricular septal thickness (ISV) ≥1.2 cm showed a higher SUVmax than patients with LVEF ≥50% and ISV<1.2 cm (P<0.05) .There are significant differences in the SUVmax of the heart between the Mayo2004 stage and the Mayo2012 stage. The later the disease stage, the higher the SUVmax (P<0.05) . The SUVmax of the heart was positively correlated with cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) (P<0.01) .There was no significant correlation between renal SUVmax and 24-hour urine protein (P>0.05) . Conclusion: Whole body (11)C-PiB PET/MRI, as a visualization system of amyloid protein, is used to qualitatively evaluate organ involvement, which can improve the level of early non-invasive diagnosis. Whole body (11)C-PiB PET/MRI can be used to perform quantitative evaluation of organ levels, especially the heart, which is expected to evaluate organ function and predict disease prognosis more accurately.

Establishment of the international pharmacopoeia standard for norethisterone tablets / 中国药学杂志

OBJECTIVE: To establish the International Pharmacopoeia standard for norethisterone tablets. METHODS: In accordance with the requirements of the International Pharmacopoeia standard, the existent quality standards of norethisterone tablets and related literatures were referred, appropriate methods were selected, and verification test was carried out to determine the best method. RESULTS: In the system suitability test of thin layer chromatography identification, the spots of norethisterone and ethinylestradiol were separated well. Nine known impurities and other unknown impurities could be separated well by the HPLC method for the examination of related substances, and the impurity with relative retention time of 1.1 was shown to be ethinylestradiol by LC-MS. In 0.1 mol·L-1 hydrochloric acid solution containing 0.09% SDS, norethisterone tablets achieved a dissolution of more than 90% in 30 min and the dissolution curve reached plateau. Under the liquid chromatographic condition for the assay, the linear range was 0.746-29.840 μg·mL-1 (r=0.999 9), and the resolution between norethisterone and ethinylestradiol was over 4.0. CONCLUSION: The established quality standard of norethisterone tablets is simple and accurate, which fully considers the feasibility of the method and accessibility of reagents, thus can effectively control the quality of products.

Hydroxysafflor yellow A attenuate lipopolysaccharide-induced endothelium inflammatory injury / 中国结合医学杂志

<p><b>OBJECTIVE</b>This study observed attenuating effect of hydroxysafflor yellow A (HSYA), an effective ingredient of aqueous extract of Carthamus tinctorius L, on lipopolysaccharide (LPS)-induced endothelium inflammatory injury.</p><p><b>METHODS</b>Eahy926 human endothelium cell (EC) line was used; thiazolyl blue tetrazolium bromide (MTT) test was assayed to observe the viability of EC; Luciferase reporter gene assay was applied to measure nuclear factor-κB (NF-κB) p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase (MAPKs) and NF-κB activation. Reverse transcription polymerase chain reaction (RT-PCR) method was applied to observe intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin mRNA level; EC surface ICAM-1 expression was measured with flow cytometry and leukocyte adhesion to EC was assayed with Rose Bengal spectrophotometry technology.</p><p><b>RESULTS</b>HSYA protected EC viability against LPS-induced injury (P <0.05). LPS-induced NF-κB p65 subunit DNA binding (P <0.01) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) phosphorylation was inhibited by HSYA. HSYA attenuated LPS triggered ICAM-1 and E-selectin mRNA levels elevation and phosphorylation of p38 MAPK or c-Jun N-terminal kinase MAPK. HSYA also inhibited LPS-induced cell surface ICAM-1 protein expression P <0.01) and leukocyte adhesion to EC (P <0.05).</p><p><b>CONCLUSION</b>HSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.</p>

Expression of autophagy related gene BECLIN-1 and number of autophagic vacuoles in bone marrow mononuclear cells from 40 myelodysplastic syndromes patients and their significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>The purpose of this study was to detect the expression level of autophagy related gene BECLIN-1 and the number of autophagic vacuoles in bone marrow mononuclear cells (BMMNC) from myelodysplastic syndrome(MDS) patients and to explore their difference in different stage of MDS and relationship between their difference and disease characteristics.</p><p><b>METHODS</b>The BMMNC from 9 normal controls, 19 cases of low-risk MDS, 14 cases of high-risk MDS and 7 cases of MDS-transformed AML were collected. The expression level of BECLIN-1 was detected by real time PCR (RT-PCR) and the amount of autophagic vacuoles was counted by transmission electron microscopy.</p><p><b>RESULTS</b>The expression level of BECLIN-1 in BMMNC from patients with low-risk group was obviously higher than that in BMMNC from normal controls; the expression level of BECLIN-1 in BMMNC from patients of hgh risk group was higher than that in BMMNC of normal group, but there was no statistical significance (P > 0.05); the expression level of BECLIN-1 in BMMNC from patients with MDS-transformed AML group was significanly lower than that in BMMNC of normal group (P < 0.05). Transinission electron microscopy showed that the amount of autophagic vacuoles in BMMNC from patients with low-risk and high-risk MDS groups was more than that in normal control, but there was no stetistcal significance (P > 0.05), while the amount of autopuagic vecuoles in BMMNC from patients of MDS-transformed AML group was significantly less (P < 0.05).</p><p><b>CONCLUSIONS</b>The expression of BECLIN-1 and the number of autophagic vacuoles in BMMNC from patients with MDS progression and patients with MDS-transformed AML are gradually declining. The autophagy may be associated with disease progression.</p>

Effect of safflor yellow injection on inhibiting lipopolysaccharide-induced pulmonary inflammatory injury in mice / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the effect of Safflor Yellow (SY) Injection on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice.</p><p><b>METHODS</b>Seventy-two mice were divided into six groups: control (saline + saline); LPS (LPS + saline); SY Injection [LPS + SY (10, 20 or 40 mg/kg, intravenously)] and anisodamine (AD) (LPS + AD). Thirty minutes after SY or AD administration, 15 mg/kg LPS was given intraperitoneally. All animals were sacrificed 4 h after LPS injection. Arterial blood gas and lung water content index (LWCI) were measured. Lung tissue myeloperoxidase (MPO) activity was assayed. mRNA expression of inflammatory cytokines was assayed by reverse-transcription polymerase chain reaction. Lung morphological and nuclear factor (NF)-κB p65-positive cell changes were observed by HE and immunohistochemical staining. p38 mitogen-activated protein kinase (MAPK) phosphorylation was observed by Western blotting.</p><p><b>RESULTS</b>After LPS administration, all animals displayed increased arterial carbon dioxide partial pressure (PaCO2) and decreased arterial oxygen partial pressure (PaO2), arterial oxygen saturation (SO2), HCO3 (-) concentration and pH, and increased LWCI, MPO activity, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α mRNA expression, NF-κB p65-positive staining and p38 MAPK activation compared with normal controls (all P<0.01). SY Injection significantly mitigated the LPS-induced increase in arterial PaCO and the decreases in arterial PaO2, SO2 and pH, and attenuated increases in LWCI and lung tissue MPO activity (all P<0.01). Moreover, SY Injection inhibited the increases in NF-κB p65 staining and in TNF-α, IL-1β and IL-6 mRNA expression (all P<0.01), and promoted the expression of the antiinflammatory cytokine IL-10 (P<0.05) following LPS injection. LPS-induced pulmonary p38 MAPK phosphorylation was suppressed by pretreatment with SY Injection (P<0.01).</p><p><b>CONCLUSION</b>SY Injection ameliorates inflammatory ALI induced by LPS in mice.</p>

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.

Surgical high ligation of the ovarian vein and preservation of ovarian function for twisted ovarian tumors / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Appendectomy is the traditional surgical procedure for correcting torsion of the adnexa. Although it prevents pulmonary embolism, ovarian necrosis, and secondary infection, it can have critical adverse effects on the ovarian function.</p><p><b>METHODS</b>We performed surgery for adnexal torsion in 12 patients, using high ligation of the ovarian vein, followed by removal of the ovarian tumor.</p><p><b>RESULTS</b>Blood flow in the residual ovary gradually returned to normal within 1 - 3 months, and a dominant follicle could be seen in the residual ovary within 2 - 6 months post-surgery in all the 12 cases. Menstruation recovered in these three cases within 2 - 3 months. Postoperative intrauterine pregnancies occurred in two cases, with a corpus luteum graviditatis in the residual ovary in one case, while the other patient underwent labor after 13 months and a normal ovary on the affected side was seen at cesarean section.</p><p><b>CONCLUSIONS</b>This new surgical technique involving high ligation of the ovarian vein for adnexal torsion allowed successful preservation of the residual ovary and ovarian blood distribution, and can thus be used for the treatment of primary diseases of the ovary. The surgical procedure is simple, safe, and effective, and warrants extensive application in clinical practice.</p>

Characterization of vinflunine tartrate liposomes in vitro and in vivo / 药学学报

Vinflunine tartrate-loaded liposomes (VT-L) with two drug-to-lipid ratios were prepared by pH gradient method. Vesicle size and zeta potential were determined by the Zetasizer Nano ZS. Entrapment efficiency was evaluated by cation exchange resin centrifugalization method. The toxicity and tumor inhibition to nude mouse administrated by VT-L with different drug-to-lipid ratios were investigated and compared with the vinflunine tartrate injection (VT-I). The results showed that the mean particle size, zeta potential and entrapment efficiency of the VT-L with drug-to-lipid ratios of 1 : 5 and 1 : 10 were 124.6 nm and 128.3 nm, -25.3 mV and -22.8 mV, 94.46% and 97.31%, respectively. The VT-L with two different drug-to-lipid ratios has significantly higher anti-tumor effect to nude mouse transplanted human non-small cell lung carcinoma A549 and lower toxicity than VT-I. While there were no significant differences in anti-tumor effect and toxicity between VT-L with two different drug-to-lipid ratios.

Clinical significance of chromosomal abnormalities detected by interphase fluorescence in situ hybridization in newly diagnosed multiple myeloma patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chromosome 13q14 deletion (del13q14), chromosome 1q21 gain (amp1q21) and chromosome 17p13 deletion (del17p13) are the most frequent chromosomal aberrations in multiple myeloma (MM). They play an important role in prognosis. The aim of this study was to investigate the clinical significance of the chromosomal changes in Chinese MM patients.</p><p><b>METHODS</b>Interphase fluorescence in situ hybridization (FISH) on bone marrow (BM) cells was performed in 72 enrolled MM patients. Relationships between chromosomal abnormalities and clinical features, response to therapies and prognosis were analyzed.</p><p><b>RESULTS</b>As a result of interphase FISH, 77.8% (56/72) patients had chromosome changes. The incidences of each probe were RB1 51.4% (37/72), D13S319 47.2% (34/72), 1q21 45.8% (33/72) and p53 22.2% (12/72). Osteolytic lesion, BM plasma cells index, serum calcium and serum M component were significantly correlated to del13q14. BM plasma cells and hemoglobin were correlated to amp1q21. Serum lactate dehydrogenase (LDH) was correlated with del17p13. Patients with del13q14 treated with bortezomib had a notably higher overall response rate than the patients treated with traditional chemotherapies (93% vs. 65%, P = 0.048). Patients carrying amp1q21 or/and del17p13 did not achieve satisfactory response to bortezomib. The median progression-free survival (PFS) for patients with amp1q21 was 5 months and patients without amp1q21 got 9-month PFS (P = 0.001). The median PFS for patients with del13q14 was 5 months (vs. 8 months, P = 0.026). The median PFS for patients with del17p13 was 3 months (vs. 8 months, P = 0.002). Patients with β(2)-microglobulin > 5.5 mg/L also had a worse outcome, whose median PFS was 5 months (vs. 8 months, P = 0.016).</p><p><b>CONCLUSIONS</b>The prevalence of chromosomal abnormalities of MM patients was similar in Chinese and Caucasian people. Genetic changes were associated with patients' responses to therapies and prognosis.</p>

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>

Brain derived neurotrophic factor induces endothelial cells angiogenesis through AKT and ERK1/2 signal pathway / 中国实验血液学杂志

Our previous studies have demonstrated the effects of brain derived neurotrophic factor (BDNF) on promoting proliferation of multiple myeloma (MM) cells and inducing angiogenesis in MM in vitro. To further investigate whether the PI3K/Akt and MEK1/ERK pathway play a role in the BDNF-induced angiogenesis in vitro and to explore the further molecular mechanisms, two ways to establish human myeloma xenograft animal model were developed, their advantages and disadvantages were elucidated. The phosphorylation of AKT and ERK1/2 were detected in human umbilical vein endothelial cells (HUVECs) by Western blot. The angiogenic activity in vitro was evaluated by transwell migration assay and tubule formation assay. Cell proliferation was determined by crystal violet staining. Cell apoptosis was detected by FITC-Annexin-V/PI double staining and flow cytometry. The results showed that the BDNF activated the PI3K/Akt and MEK1/ERK pathway in the time-dependent manner. Ly294002 and PD98059 blocked the activation of Akt and ERK1/2 respond to BDNF. 100 ng/ml BDNF significantly increased HUVEC tube formation, migration and proliferation in vitro at a similar degree of 25 ng/ml VEGF. Furthermore, tube formation of HUVECs toward BDNF was significantly inhibited by 57% and 40% with 20 micromol/L Ly294002 and 20 micromol/L PD98059 treatment, respectively. At the same time, Ly294002 and PD98059 reduced the BDNF-induced migration of HUVECs by 74% and 36%, respectively. While BDNF-induced survival was only blocked by Ly294002 and BDNF-induced proliferation was only inhibited by PD98059. It is concluded that BDNF promotes angiogenesis of HUVECs in vitro. ERK and Akt are two crucial events in BDNF-mediated signal transduction leading to HUVECs angiogenesis by different mechanisms. Moreover, the latter is more important.

Antitumor effect of anti-brain derived neurotrophic factor monoclonal antibody in human multiple myeloma xenograft animal model / 中国实验血液学杂志

This study was aimed to further explore whether brain derived neurotrophic factor (BDNF) pathway is a potential therapeutic target in multiple myeloma (MM) and whether anti-BDNF monoclonal antibody can prevent the development of this disease. The in vivo antitumor effect of anti-BDNF monoclonal antibody (McAb) on a human myeloma xenograft animal model was evaluated. The model of xenograft tumors was established in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice by subcutaneous injection of human myeloma cell line RPMI8226. The antibodies were injected intraperitoneally at a dose of 20 microg/mouse at day 1, 2, 3 after inoculation or at a dose of 100 microg/mouse once a week after tumors were detected. The microvascular densities in tumors were analyzed by immunohistochemistry study. The effect of anti-BDNF McAb on the proliferation of RPMI8226 cells in vitro and on endothelial cells network formation in the co-culture system were determined by using a (3)H-thymidine incorporation assay and a Matrigel network formation assay, respectively. The results showed that multiple injections of anti-BDNF McAb reduced the tumor size, decreased the microvascular density and significantly prolonged tumor-free time and survival time. Moreover, the proliferation of RPMI8226 cells was inhibited in vitro by anti-BDNF McAb, but not by the control IgG. Anti-BDNF McAb also inhibited RPMI8226-induced network formation in endothelial cells in vitro. It is concluded that anti-BDNF monoclonal antibody can inhibit cell growth and angiogenesis in subcutaneous plasmacytoma.

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>

Role of multiple myeloma cells on normal endothelial cells in co-culture system / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of multiple myeloma (MM) cells on normal endothelial cells in co-culture system in vitro.</p><p><b>METHODS</b>A co-culture system of human MM cell line RPMI8226 with human umbilical vein endothelial cells (HUVECs) was established in vitro. Mono-cultured normal endothelial cells were used as control. Light microscopy and transmission electron microscopy were used to observe the morphology of the endothelial cells. The effects of HUVECs co-cultured with RPMI8226 on HUVECs angiogenesis were studied by modified transwell migration assay and net-like formation assay. The protein expression of brain derived neurotrophic factor (BDNF), TrkB, Endoglin, Tie-2, beta 3 integrin and vascular cell adhesion molecule-1 (VCAM-1) in HUVECs were determined by FACS and Western blot analysis, respectively.</p><p><b>RESULTS</b>The morphology of HUVECs co-cultured with RPMI8226 cells became a narrower apart of extended shape as they began to align themselves. The sizes of nucleus and nucleolus were enlarged with an increased ratio of nuclear to nucleoplasm. The endoplasm was lose and distorted and the number of surface microvilli decreased. The RPMI8226 cell stimulated the migration and net-like formation of HUVEC, the number of net-like structure and migration cell being increased by 112% and 136%, respectively, compared with that of mono-cultured HUVECs. The expressions of BDNF, TrkB, Endoglin, Tie-2, beta 3 integrin and VCAM-1 in the ECs co-cultured with RPMI8226 were all up-regulated in comparison with those in the controls.</p><p><b>CONCLUSION</b>The MM cells promote formation of new vessels in co-cultured endothelial cells and the endothelial cells in MM are different from the normal ECs in character, and behavior.</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>

Monoclonal antibody against brain derived neurotrophic factor inhibits myeloma growth and angiogenesis in the xenograft NOD/SCID animal model / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the in vivo antitumor effect of anti-brain derived neurotrophic factor (BDNF) monoclonal antibody (MoAb) on a human myeloma xenograft animal model.</p><p><b>METHODS</b>The xenograft tumor model was established in the nonobese diabetic/severe combined immunodeficiency (NOD/ SCID) mice by subcutaneous injection of human myeloma cell line RPMI 8226. The antibodies were injected intraperitoneally at a dose of 20 microg/mouse at day 1, day 2, day 3 after tumor cell inoculation or at a dose of 100 microg/mouse once a week after tumors were developed. The histologic and cytologic examination were performed to confirm the development of plasmacytomas. The microvascular densities (MVD) in tumors were analyzed by immunohistochemistry. The effect of anti-BDNF MoAb on the proliferation of RPMI 8226 cells in vitro and on endothelial cell network formation in the co-culture system were determined by 3H-thymidine incorporation assay and Matrigel network formation assay, respectively.</p><p><b>RESULTS</b>The xenograft NOD/SCID animal model had high capacity for growth of RPMI 8226 subcutaneous tumors and presented pathologic features of plasmacytomas. After subcutaneous injection of RPMI 8226 cells, all mice developed localized tumors in (20 +/- 2) d. On 20 microg anti-BDNF MoAb 3 consecutive treatment, the mean tumor-free time was extended to (30 +/- 6) d and survival was significantly prolonged compared with IgG-treated group [(57 +/- 7) d vs (48 +/- 4) d, P < 0.05]. When mice died naturally, the tumors size in anti-BDNF MoAb treated ones was also reduced compared with control group [(157.9 +/- 21.6) mm3 vs (405.5 +/- 35.2) mm3, P < 0.05]. When the antibody treatment (100 microg/mouse) underwent from 27 th to 60 day once a week after tumor inoculation, the local tumor growth was inhibited partially and necrosis and infiltration were observed in the tumors. The median MVD in the antibody-treated mice (100 microg/mouse) was 11 vessels/0.216 mm2. The IgG treated mice had no decrease in MVD of subcutaneous tumors compared with untreated mice. In vitro, anti-BDNF MoAb (1.5 microg/ml) significantly but partially inhibited HUVEC network formation induced by RPMI 8226 (68.2% reduction) and significantly inhibited RPMI 8226 proliferation, too. The IgG (1.5 microg/ml) treated mice had no significant effect on both of two assays.</p><p><b>CONCLUSIONS</b>Anti-BDNF monoclonal antibody could inhibit growth and angiogenesis in subcutaneous myeloma tumors. BDNF is a potential therapeutic target in MM.</p>

Study of molecular mechanism of doxorubicin enhancement of TRAIL, inducing apoptosis of myeloma cell line KM3 / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of doxorubicin enhancement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) inducing apoptotic effect on multiple myeloma cell line KM3.</p><p><b>METHODS</b>Apoptosis was studied independently through flow cytometry analysis and TUNEL staining. The expression of death receptor 5 (DR5) and nuclear factor P65 in nuclear was examined by Western blot.</p><p><b>RESULTS</b>The apoptosis ratio of KM3 cells was 20.88%, 40.03%, 57.87%, 60.82% respectively when treated with different concentration of TRAIL (10, 20, 50, 100 ng/ml) combining with doxorubicin. It is markedly higher than the group treated with TRAIL or doxorubicin alone. DR5 expression increased while P65 decreased as the doses of doxorubicin increased when KM3 cells treated with doxorubicin (0.5, 1.0, 2.0 and 4.0 microg/ml) plus 20 ng/ml TRAIL.</p><p><b>CONCLUSION</b>Increasing the expression of DR5 and nuclear transferring of P65 are the important molecular mechanism by which doxorubicin enhances TRAIL-inducing apoptosis of KM3 cells.</p>

Human multiple myeloma cell line RPMI8226 activates brain derived neurotrophic factor autocrine loop of co-cultured endothelial cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of multiple myeloma cells on normal endothelial cells in co-culture system.</p><p><b>METHODS</b>Human multiple myeloma cell line RPMI8226 was co-cultured with human umbilical vein endothelial cells (HUVECs). HUVECs cultured alone were used as control. The expression of brain derived neurotrophic factor (BDNF) and its specific acceptor TrkB mRNA and protein in HUVECs were determined by RT-PCR and Western blot, respectively, BDNF levels in culture supernatant by enzyme-linked immunosorbent assay (ELISA). After transferring the co-culture, the effects RPMI8226 on HUVECs angiogenesis were studied by modified transwell migration assay and net-like formation assay.</p><p><b>RESULTS</b>The median BDNF concentration in culture supernatant was increased in co-cultured HUVECs compared with that in HUVECs cultured alone [(31.6 +/- 7.2) ng/ml vs (12.4 +/- 5.1) ng/ml, P < 0.05]. The expression of BDNF transcript demonstrated by RT-PCR did the same in the two culture systems (1.7 fold increase, P < 0.05). TrkB mRNA was hardly detected in culture of HUVECs alone but was increased in co-cultured HUVECs (4.4- fold increase, P < 0.05). The BDNF and TrkB protein expressions determined by Western blot were similar to that of their mRNAs. On the other hand, the RPMI8226 activated HUVECs showed enhanced migration and net-like formation, being increased by 99% and 72% , respectively. Addition of anti-human BDNF antibody to the culture medium partly reduced these effects.</p><p><b>CONCLUSION</b>Multiple myeloma cells activated BDNF/TrkB autocrine loops in co-cultured endothelial cells and resulted in endothelial self-activating angiogenesis.</p>