Berberine derivative WJCPR11 enhances osteoblast differentiation

Berberine is a phytocompound from plants viz. Phellodendri cortex and Coptis rhizome, used to treat a variety of diseases. It is effective in preventing osteoporosis, but it is less effective than drugs currently used in clinical practice. In this study, we used a novel berberine derivative, WJCPR11, to promote osteoblast differentiation and to investigate its use in the prevention and treatment of osteoporosis. WJCPR11 at a safe concentration without toxicity increased alkaline phosphatase (ALP) activity induced by bone morphogenetic protein 2 (BMP2) dose-dependently. The mRNA expression of ALP, osteocalcin (OC), runt-related transcription factor 2 (Runx2), and osterix was increased, with the ALP level increasing the most. In addition, the protein abundance of bone sialoprotein (BSP), collagen, type I, alpha 1, Runx2, and osterix were also increased. Moreover, the transcriptional activity of ALP, BSP, and OC was increased by WJCPR11, with OC showing the most significant increase. The results indicate that osteoblast differentiation is promoted by WJCPR11, and it could play a role in the prevention of osteoporosis.

Serum levels of bone sialoprotein, osteopontin, and β2-microglobulin in stage I of multiple myeloma

Context: The fluctuations of proteins in multiple myeloma (MM) are well-known markers for checking the status of the patients. Aims: The objective of this study was to examine three proteins that have an important role in disease progression. Subjects and Methods: The study was performed with two groups: 30 MM stage I patients' (14 females/16 males; aged 60.83 ± 12.38 years) as case group and 40 healthy individuals (18 females/22 males; aged 57.65 ± 6.43 years) as control group. Both groups have been matched in gender and age. Bone sialoprotein (BSP), osteopontin (OPN), and β2-microglobulin (β2M) were measured with an enzyme-linked immunosorbent assay. Results: Serum BSP levels of MM-I patients was significantly higher than that of healthy controls (29.24 ± 5.57 vs. 20.89 ± 3.67, P = 0.001). OPN levels of MM-I patients were significantly lower than that of healthy individuals (12.03 ± 3.45 vs. 19.35 ± 4.67, P = 0.001). β2M levels of patients and controls were similar (1.49 ± 0.67 vs. 1.29 ± 0.55, P = 0.193). Conclusions: The results suggested that myeloma cells may affect the production of BSP and OPN, which possibly contributes to osteoclastic bone resorption in MM-I patients. Their levels may be a useful biomarker for assessing bone destruction in MM-I patients and distinguishing MM-I from healthy individuals

Effects of silencing the HIF-1α gene on the expression of BSP and osterix in rat BMMSCs under tension / 口腔疾病防治

Objective@#To explore the effect of hypoxia inducible factor 1α (HIF-1α) gene silencing in rat bone marrow mesenchymal stem cells (BMMSCs) under mechanical distraction on the expression of bone sialoprotein (BSP) and osterix and to provide a new idea for repairing bone defects with BMMSCs.@*Methods @#The shRNA sequence was designed according to the rat HIF-1α gene, and the pGMLV-SC1RNAi lentiviral vector was cloned after PCR amplification. After screening positive clones and identifying competent transformed cells by sequencing, 293T cells were packaged and titered, rat BMMSCs were transfected and cultured in vitro. Clones with stably silenced HIF-1α expression were screened by inverted fluorescence microscopy. The RNAi response experiment was divided into four groups: the blank control group, the HIF-1α shRNA group, the negative control group, and the response group. Western blot was used to detect the expression of HIF-1α protein in the four groups to verify the response of the target genes and exclude off-target effects. A Flexcell FX-5000T cell stress loading system was used to intervene in the mechanical stretch of the cells. qRT-PCR and Western blot were used to detect the expression of BSP and osterix in the blank control group, HIF-1α shRNA group, and negative control group.@*Results@#The HIF-1α shRNA lentiviral vector was successfully constructed. The results of the RNAi response showed no significant difference in the expression of HIF-1α between the response and the blank control group (P > 0.05). The recombinant lentivirus could effectively silence HIF-1α in BMMSCs. After mechanical distraction of the BMMSCs, compared with the blank and negative control groups, the HIF-1α shRNA group showed significantly increased mRNA and protein expression of the bone-related factors BSP and osterix (P < 0.05); there was no significant difference in the mRNA and protein expression of BSP or osterix between the blank and negative control groups (P > 0.05).@*Conclusion @#Silencing HIF-1α in BMMSCs under mechanical distraction can promote the expression of BSP and osterix.

The relationship between the expression of bone sialoprotein and the bone metastasis in women with breast cancer / 安徽医科大学学报

Objective To investigate the relationship between the expression of bone sialoprotein(BSP) and the bone metastasis of breast cancer.Methods 840 cases of female breast cancer patients were retrospectively analyzed.The patients were divided into breast cancer bone metastasis group and no bone metastasis group according to whether the patients had bone metastasis after treatment,BSP expression in paraffin specimens of 840 cases of primary breastcancer was determined by immunohistochemical method,x2 test and COX survival analysis was used in analysis of influencing factors of bone metastasis and 5 years survival after treatment of breast cancer.Results 840 cases of breast cancer postoperatively of single factor analysis showed that age,primary tumor size,clinical stage,TNM and molecular typing were statistically significant(P ＜0.05).BSP in bonemetastasis group was significantly higher than that in patients without bone metastasis(non metastasis group and bone metastasis group)(P ＜ 0.05).However,CerbB-2,lymph node metastasis,whether menopause,pathological type,cell grading showed no correla tion.Multivariate analysis revealed that age,tumor volume,clinicalstage,three negative breast and high expression of BPS were risk factors for bone metastasis.COX survival analysis of breast cancer 5 year survival rate,BSP high expression group and low expression group was statistically significant (P ＜ 0.05).Conclusion The clinical stage,age,primary lesion size and molecular type are the main risk factors for bone metastasis after breast cancer treatment.Bone sialoprotein expression is closely related to breast cancer bone metastasis.

Expression of bone sialoprotein in prostate cancer and its clinical significance / 肿瘤研究与临床

Objective To investigate the expression of bone sialoprotein (BSP) in prostate cancer and its clinical significance. Methods Prostate cancer tissues of different pathological grades (68 cases) and benign prostatic hyperplasia tissues (22 cases) were selected. SP method was used to detect the expression of BSP. Serum total prostate-specific antigen (tPSA) levels of prostate cancer were detected by electrochemiluminescence immunoassay before the operation. Results Compared with no or low expression in the adjacent normal glandular tissues, the detectable levels of BSP were examined in most of the prostate cancer tissues. The expression rate of BSP in prostate cancer tissues was higher than that in benign prostatic hyperplasia tissues [76.47%(52/68) vs 13.64%(3/22),χ2=27.614, P<0.001]. The expression rates of BSP in well differentiated, moderately differentiated and poorly differentiated tissues according to cell differentiating degree (Gleason system) were 75.0 % (12/16), 77.5 % (31/40) and 75.0 % (9/12) respectively. There was no significant difference in various pathological grading (χ2=0.057, P=0.972). The expression rates of BSP in pathological stage pT2, pT3 and pT4 tissues were 62.16%(23/37), 95.24%(20/21) and 90.0%(9/10) respectively. A statistically significant association was found between BSP expression and pathological stage (χ2=9.338, P=0.009). Serum tPSA level of prostate cancer group with BSP expression was higher than that with no BSP expression [(69.06±25.52)μg/L vs (38.00±21.64)μg/L, F=19.355, P<0.001]. Conclusion The high expression of BSP in prostate cancer has a relationship with pathological stage and serum tPSA level, it may play an important role in the biological behaviour of prostate cancer.

Crystal structures of Bbp from Staphylococcus aureus reveal the ligand binding mechanism with Fibrinogen α

Bone sialoprotein-binding protein (Bbp), a MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family protein expressed on the surface of Staphylococcus aureus (S. aureus), mediates adherence to fibrinogen α (Fg α), a component in the extracellular matrix of the host cell and is important for infection and pathogenesis. In this study, we solved the crystal structures of apo-Bbp(273-598) and Bbp(273-598)-Fg α(561-575) complex at a resolution of 2.03 Å and 1.45 Å, respectively. Apo-Bbp(273-598) contained the ligand binding region N2 and N3 domains, both of which followed a DE variant IgG fold characterized by an additional D1 strand in N2 domain and D1' and D2' strands in N3 domain. The peptide mapped to the Fg α(561-575) bond to Bbp(273-598) on the open groove between the N2 and N3 domains. Strikingly, the disordered C-terminus in the apo-form reorganized into a highly-ordered loop and a β-strand G'' covering the ligand upon ligand binding. Bbp(Ala298-Gly301) in the N2 domain of the Bbp(273-598)-Fg α(561-575) complex, which is a loop in the apo-form, formed a short α-helix to interact tightly with the peptide. In addition, Bbp(Ser547-Gln561) in the N3 domain moved toward the binding groove to make contact directly with the peptide, while Bbp(Asp338-Gly355) and Bbp(Thr365-Tyr387) in N2 domain shifted their configurations to stabilize the reorganized C-terminus mainly through strong hydrogen bonds. Altogether, our results revealed the molecular basis for Bbp-ligand interaction and advanced our understanding of S. aureus infection process.

Effect of magnesium and calcium phosphate coatings on osteoblastic responses to the titanium surface

PURPOSE: The aim of this study was to evaluate the surface properties and in vitro bioactivity to osteoblasts of magnesium and magnesium-hydroxyapatite coated titanium. MATERIALS AND METHODS: Themagnesium (Mg) and magnesium-hydroxyapatite (Mg-HA) coatings on titanium (Ti) substrates were prepared by radio frequency (RF) and direct current (DC) magnetron sputtering.The samples were divided into non-coated smooth Ti (Ti-S group), Mg coatinggroup (Ti-Mg group), and Mg-HA coating group (Ti-MgHA group).The surface properties were evaluated using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The surface roughness was evaluated by atomic force microscopy (AFM). Cell adhesion, cell proliferation and alkaline phosphatase (ALP) activity were evaluated using MC3T3-E1 cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed. RESULTS: Cross-sectional SEM images showed that Mg and Mg-HA depositionson titanium substrates were performed successfully. The surface roughness appeared to be similaramong the three groups. Ti-MgHA and Ti-Mg group had improved cellular responses with regard to the proliferation, alkaline phosphatase (ALP) activity, and bone-associated markers, such as bone sialoprotein (BSP) and osteocalcin (OCN) mRNA compared to those of Ti-S group. However, the differences between Ti-Mg group and Ti-MgHA group were not significant, in spite of the tendency of higher proliferation, ALP activity and BSP expression in Ti-MgHA group. CONCLUSION: Mg and Mg-HAcoatings could stimulate the differentiation into osteoblastic MC3T3-E1 cells, potentially contributing to rapid osseointegration.

Expression and significance of bone sialoprotein and matrix metalloproteinase-9 in calcified metral valves of rheumatic heart disease / 中国综合临床

Objective To investigate the expression and significance of bone sialoprotein and matrix metalloproteinase-9 in calcified mitral valves in patients with rheumatic heart disease.Methods A total of 150 mitral valves were divided into the rheumatic group (n =120) and the non-rheumatic group (n =30 ).Expressions of bone sialoprotein and matrix metalloproteinase-9 were determined by immunohistochemistry.Results Expressions of bone sialoprotein ( 91.6％,x2 =56.6354 ) and matrix metalloproteinase-9 ( 90.8％,x2 =59.4272) in the rheumatic group increased significantly than in the non-rheumatic group (P ＜ 0.01).Conclusion Both bone sialoprotein and matrix metalloproteinase-9 are highly expressed in the calcified rheumatic group.This suggests that caficify of rheumatic mitral valves is related with the degradation and remodeling of extra cellular matricx by matrix metalloproteinase-9,as well as osteoblastlike bone formation by bone sialoprotein.

Preparation and identification of the monoclonal antibody against human bone sialoprotein / 肿瘤研究与临床

Objective To prepare the human bone sialoprotein (BSP) monoclonal antibodies (mAb)with high titer and specificity and identify its characterization,which is based on further studying BSP as clinical biomarker for breast cancer metastasizing to bone. Methods BALB/c mice were immunized with purified recombinant BSP protein.Cell fusion was performed between mouse splenic cells and myeloma cells (Sp2/0), and then the hybridoma cell lines secreting mAb against BSP antigen were screened and cloned. The ascites were prepared and purified with Protein G affinity chromatography.The titer and subtypes of mAb against BSP were identified and measured by ELISA and Western blotting analysis. ResultsNine hybridoma cell lines that stably secreted mAb against BSP were successfully obtained.Two of them,D001 and D002,were further identified, which belonged to the subtypes of IgG1 and κ light chain. The two antibodies titers in culture supernatant were 1∶5120 and 1∶10 240, respectively, and those in the ascites fluid were 1∶25 600 and 1∶51 200,respectively.Results of Western blotting analysis and immunohistochemistry showed that the two antibodies could specifically bind with BSP derived from human breast cancer cells.ConclusionNine mAb against BSP have been successfully prepared which can be used for further studying the biological properties of BSP and reveal its relationship with data from clinic patients.

Combined examination of serum cross-linked N-telopeptides of type Ⅰ collagen and bone sialoprotein in detection of bone metastasis of breast cancer and lung cancer / 中国肿瘤生物治疗杂志

Objective:To study the clinical significance of examining serum pyridinoline cross-linked N-telopeptides of type I collagen(NTx) and serum bone sialoprotein(BSP) in diagnosing bone metastasis of lung cancer and breast cancer.Methods:A total of 105 patients treated in the Oncology Department of Changhai Hospital were included in this study.Patients were divided into 2 groups:bone metastasis(n=50) and non-bone metastasis groups(n=55).The levels of serum NTx and serum BSP were measured by ELISA.Results:The levels of serum NTx and serum BSP in patients with bone metastasis were significantly higher than in those without bone metastasis(P

Inhibitory effects of bone sialoprotein monoclonal antibody on bone-seeking breast cancer cells adhering to bone matrix / 中国肿瘤生物治疗杂志

Objective: To determine the inhibitory effects of anti-bone sialoprotein(BSP) antibody on human breast cancer cells adhering to bone matrix in vitro.Methods: Expression of BSP in 3 different cancer cell lines(bone seeking breast cancer cell MDA-MB-231-BO,lung adenocarcinoma cell SPCA-1 and colon carcinoma cell LOVO)was detected immunohistochemically.Cells adhering test was carried out to investigate the adhering of the 3 different cancer cell lines to mouse bone matrix in vitro and the inhibitory effect of anti-BSP antibody on MDA-MB-231-BO cells adhering to mouse bone matrix;Enzyme-linked immunosorbent assay was carried out for quantitative detection of TGF-?.Results: BSP immunostaining was positive in MDA-MB-231-BO cells and negative in SPCA-1 and Lovo cells.The number of MDA-MB-231-F10 cells adhering to mouse bone matrix was significantly more than SPCA-1 or Lovo cells(P

Effects of irradiation on the mRNA expression of osteonectin and bone sialoprotein in MC3T3-E1 osteoblastic cell line / 대한구강악안면방사선학회지

PURPOSE: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-E1 osteoblastic cell line, especially on the osteonectin and bone sialoprotein. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.5, 1, 4 and 8 Gy at a dose rate of 5.38 Gy/min using Cs-137 irradiator. After specimens were harvested, total RNA was extracted on the 3rd, 7th, 14th, 21st day after irradiation. The total RNA was reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR with a pair of primers. RESULTS: The irradiated cells showed a dose-dependent increase in osteonectin mRNA expression when compared with the unirradiated control group. The irradiated cells showed no difference in bone sialoprotein mRNA expression when compared with the unirradiated control group. In accordance with the duration of culture period after irradiation, the level of osteonectin mRNA expression showed no difference, but it increased a little at the 21st day in the 4 and 8 Gy exposure groups. In the case of bone sialoprotein, however, the level of mRNA expression increased significantly at the 3rd and 7th day after irradiation, but it showed no difference at the 14th and 21st day when compared with the control group. CONCLUSION: These results showed that each single dose of 0.5, 1, 4 and 8 Gy influenced the mRNA expression of osteonectin and bone sialoprotein at the calcification stage of osteoblastic cells, suggesting that single dose of irradiation affected the osteoblastic bone formation at the cell level.

Construction of pIRES2-hBSP-EGFP vector and stably expression in MDA-MB-231BO breast cancer cells / 解放军医学杂志

Objective To constitute a higher expression system and to obtain the breast cancer cell strains in which BSP is stably expressed. Methods hBSP gene was subcloned from pB-hBSP vector by PCR. The PCR fragment was inserted into the eukaryon expression vector, pIRES2-EGFP, which allow exogenous protein and EGFP to express respectively. The recombinant vector, pIRES2-hBSP-EGFP, was transfected into human breast cancer cells with Lipofectamine TM 2000. The expression of symbol protein EGFP, could be conveniently observed with fluorescent microscope. Results The recombinant pIRES2-hBSP-EGFP plasmid was constituted and successfully transfected into breast cancer cells. In the breast cancer cell strain hBSP and EGFP were expressed. Conclusion The successful constitution and transfection of hBSP and EGFP nonfusion expression vector laid a foundation for the further study on the effect of BSP on breast cancer metastasizing to bone in vivo or in vitro.