Isoliquiritigenin Inhibits Metastatic Breast Cancer Cell-induced Receptor Activator of Nuclear Factor Kappa-B Ligand/Osteoprotegerin Ratio in Human Osteoblastic Cells

Bone destruction induced by the metastasis of breast cancer cells is a frequent complication that is caused by the interaction between cancer cells and bone cells. Receptor activator of nuclear factor kappa-B ligand (RANKL) and the endogenous soluble RANKL inhibitor, osteoprotegerin (OPG), directly play critical roles in the differentiation, activity, and survival of osteoclasts. In patients with bone metastases, osteoclastic bone resorption promotes the majority of skeletal-related events and propagates bone metastases. Therefore, blocking osteoclast activity and differentiation via RANKL inhibition can be a promising therapeutic approach for cancer-associated bone diseases. We investigated the potential of isoliquiritigenin (ISL), which has anti-proliferative, anti-angiogenic, and anti-invasive effects, as a preventive and therapeutic agent for breast cancer cell-induced bone destruction. ISL at non-toxicity concentrations significantly inhibited the RANKL/OPG ratio by reducing the production of RANKL and restoring OPG production to control levels in hFOB1.19 cells stimulated with conditioned medium (CM) of MDA-MB-231 cells. In addition, ISL reduced the expression of cyclooxygenase-2 in hFOB1.19 cells stimulated by CM of MDA-MB-231 cells. Therefore, ISL may have inhibitory potential on breast cancer-induced bone destruction.

Effects of 137 Cs γ-rays on proliferation, differentiation and mineralization of osteoblastic cells in vitro / 中华放射医学与防护杂志

Objective To evaluate the effect of gamma irradiation on the proliferation,differentiation,and mineralization of murine osteoblastic cells,and to investigate the related molecular mechanism.Methods Osteoblastic cells were irradiated by different doses (0,0.5,1.0,2.0,5.0 Gy)of 137Cs γ-rays.Cell morphology was observed with a microscopy,cell viability was analyzed by MTT assay,and ALP activity was analyzed by the methods of enzyme histochemistry and PNPP.Meanwhile,gene expressions of ALP,osteocalcin (OC),collagen Ⅰ,osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) were measured by semi-quantified RT-PCR.Results Cell viability decreased with the radiation doses over 1.0 Gy ( t =6.197 - 18.677,P ＜ 0.05 ).After radiation with a dose over 2.0 Gy,the cell number and the junctions of cell protrusions decreased,the cells had low refractivity and the activity and mineralization ability of ALP were also inhibited ( t =2.790 -2l.374,P ＜0.05).In addition,the expressions of ALP and OC mRNA were down-regulated significantly (t =3.563 -16.508,P ＜ 0.05) when the radiation dose was higher than 0.5 Gy,and the expressions of OPG,OPG/RANKL mRNA were down-regulated ( t =12.942,4.954,P ＜ 0.05 ) at 5 Gy.But the expressions of collagen Ⅰ and RANKL mRNA were not affected by irradiation.Conclusions The osteoblastic cells were significantly influenced by γ-irradiation,including morphological changes,inhibition of cell proliferation,differentiation and mineralization ability. Meanwhile,mRNA expressions of ALP and OC were downregulated.OPG/RANKL may be a main pathway of osteoblastic cell damage under high dose radiation.

Influence of peroxisome proliferator activated receptor γ2 endogenous ligands on mRNA expression of bone metabolism related genes in osteoblastic cells / 中华内分泌代谢杂志

Objective To observe the effect of oxidized low-density lipoproteins (Ox-LDL),15-Deoxy-△ 12,14-prostaglandin J2 ( 15d-PGJ2 ),leukotrienes B4 ( LTB4 ) on mRNA expressions of peroxisome proliferator activated receptor γ2 ( PPARγ2 ),receptor activator of NF-κB ligand (RANKL),alkaline phosphatase ( ALP),and osteoprotegerin(OPG) in osteoblastic cells of rats; and to investigate the influence of these PPARγ2 endogenous ligands on bone metabolism.Methods Rat osteoblastic cells were cultured in vitro for 24 h in medium with different PPARγ2 endogenous ligands at various concentrations ( the final concentrations of Ox-LDL were 0,12.5,25,50μg/ml; the final concentrations of 15 d-PGJ2 were 0,10,20,30 μmol/L; the final concentrations of LTB4 were 0,0.1,1.0,10 μ mol/L).RT-PCR was performed to determine the mRNA expressions of PPARγ2,RANKL,ALP,and OPG in osteoblastic cells.Results RT-PCR analysis showed that Ox-LDL,15d-PGJ2,and LTB4 all down-regulated the mRNA expressions of RANKL,ALP,and OPG,while up-regulated the mRNA expressions of PPARγ2 in osteoblastic cells in a dose-dependent manner.Significant differences were found in interclass comparisons( P＜0.05 or P＜ 0.01 ).Conclusions These findings suggest that Ox-LDL,15d-PGJ2,and LTB4 suppress the expressions of osteogenic genes through activating the transcription activity of PPARγ2,and this may be a plausible mechanism of senile osteoporosis.

Differential proteomic analysis on osteoblasts stimulated by mechanical strain / 医用生物力学

Objective To identify the differentially expressed proteins and clarify the major proteins involved in the molecular mechanism of osteoblasts under mechanical strain loading. Method Saos 2 osteoblastic cells were subjected to 12% elongation for 24 hours by using Flexcell strain loading system. Proteins extracted from Saos 2 cells were separated by two dimensional electrophoresis (2 DE). Differential expressed protein spots among groups were submitted to matrix assisted laser desorption/ionization time of flight mass spectrometer (MALDI TOF MS) assay and peptide mass fingerprinting (PMF) identification. The Swiss Prot and NCBI databases were used to obtain further information about proteins identified. Results Saos 2 stimulated by mechanical strain showed a significant difference in 2 DE system compared with the control group. A total of (1031±41) or (928±25) protein spots were resolved by 2 DE of controls or experimental groups extractions respectively. 17 significant up regulated proteins were identified. These associated proteins fell into 6 groups, including stress reaction, energy metabolism, cell proliferation, reconstruction of cytoskeleton, signaling and osteogenesis. Conclusions The Saos 2 can express differential proteins stimulated by mechanical strains and these proteins may play an important role in molecular mechanism of osteoblasts under mechanical strain loading.

Effects of 2-deoxy-D-glucose and quercetin on the gene expression of bone sialoprotein and osteocalcin during the differentiation in irradiated MC3T3-E1 osteoblastic cells / 대한구강악안면방사선학회지

PURPOSE: To investigate the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of bone sialoprotein (BSP) and osteocalcin (OC) during the differentiation in irradiated MC3T3-E1 osteoblastic cells. MATERIALS AND METHODS: When MC3T3-E1 osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or 10 micrometer QCT, and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the synthesis of type I collagen, and expression of BSP and OC. RESULTS: The synthesis of type I collagen in cells exposed to 2 Gy of radiation in the presence of 2-DG or QCT showed no significant difference compared with the control group within 15 days post-irradiation. When the cells were irradiated with 8 Gy, 2-DG facilitated the irradiation mediated decrease of type I collagen synthesis, whereas such decrease was inhibited by treating with QCT. During MC3T3-E1 osteoblastic cell differentiation, the mRNA expression of BSP and OC showed the peak value at 14 days and 21 days, respectively. 2-DG or QCT treatment alone decreased the level of BSP mRNA, but increased the OC mRNA level only at early time of differentiation (day 7). In the cells irradiated with 2, 4, 8 Gy, the mRNA expression of BSP and OC decreased at 7 days after the irradiation. The cells were treated with various dose of radiation in the presence of 2-DG or QCT, the mRNA level of both BSP and OC increased although this increase was observed at low dose of radiation (2 Gy) and at the early stage of differentiation. However, when the cells were exposed to 4, 6, or 8 Gy, the increase of BSP and OC mRNAs was detected only in cells co-incubated with QCT. CONCLUSION: This study demonstrates that 2-DG and QCT affect differently the expression of bone formation related factors, type I collagen, BSP, and OC in the irradiated MC3T3-E1 osteoblasic cells, according to the dose of radiation and the times of differentiation. Overall, the present findings suggest that 2-DG and QCT could have the regulatory roles as radiation-sensitizer and -protector, respectively.

Screening for pathogenesis-related genes of osteosarcoma using gene microarray / 中国肿瘤生物治疗杂志

Objective:To screen for the pathogenesis-related genes of osteosarcoma and to assess their roles for the de- velopment of osteosareoma.Methods:Total RNA was extracted from 3 ATCC osteosarcoma cell lines and an osteoblastic cell line and was used to synthesize biotinylated cRNAs;the latter were hybridized to Affymetrix~(?)GeneChip~(?)U133A ar- rays and a gene with more than 2 folds of change was selected.Ten of the differentially expressed genes were chosen and the primers were designed and the synthesized.Then SYBR~(?)Green real-time PCR(RT-PCR)method was used to detect the expression of the 10 genes in 9 fresh osteosarcoma specimens.ABI Prism 7 000 system was used to analyze the differ- ent expression between osteosarcoma cell line and osteoblastic cell line.Results:We identified 58 up-regulated and 142 down-regulated genes in the 3 osteosareoma cell lines.Many of the genes were firstly reported to be related to the patho- genesis of osteosarcoma.These differentially expressed genes were mainly involved in energy and material metabolism,on- cogene,signal transduction gene,transcription- related genes,cell cycle-related genes,cell apoptosis-related gene,im- mune response gene,tumor suppressor genes,etc.The array results of 10 randomly selected genes were further verified by the RT-PCR in 9 fresh osteosarcoma specimens.Conclusion:Many genes are involved in the pathogenesis of osteosarcoma. Gene microarray can help to discover the genes related to the pathogenesis of osteosarcoma,which may lay a foundation for studying the molecular mechanism of osteosarcom.

Effects of Low Intensity Ultrasound on the Co-Culture of Human Osteoblastic Cells (SaOS-2) with Endothelial Cells (HUVEC) / 대한정형외과연구학회지

PURPOSE: We investigated the effects of low intensity ultrasound on the co-culture of human osteoblastic cells with endothelial cells and analysed cell proliferation and growth factors that might be involved in bone formation. METHODS: Cell culture system was established with human osteoblastic cells (SaOS-2) and primary isolated human umbilical vein endothelial cells (HUVEC). Low intensity ultrasound (1 MHz) treatment was administered for 20 minutes per day to each well for 4 consecutive days and its effects were determined by analysing cell proliferation activity, alkaline phosphatse and amount of growth factors such as basic fibroblast growth factor, transforming growth factor beta and transforming growth factor beta in the conditioned medium. RESULTS: Low intensity ultrasound treatment increased cell proliferation activity, level of the basic fibroblast growth factor and transforming growth factor beta in the co-culture system. The levels of alkaline phosphatase and transforming growth factor beta in the conditioned medium were not changed. We could not observe the change of cell proliferation or amount of various growth factors in a culture system of SaOS-2 or HUVEC. CONCLUSION: The low intensity ultrasound treatment can increase osteogenesis by certain interaction between the two cells and increase in bFGF and TGF beta participate in the process.

An experimental study on the effect of cytoskeletal changes on osteoblastic cell activities / 대한치과교정학회지

The cytoskeleton has been shown to form a network, connecting the extracelluar matrix via integrin with the nucleus and the cytoplasmic constituents of the cell It is therefore assumed that the cytoskeleton may mediate signals generated by perturbations originating in the matrix. The purpose of this study is to examine the effect of cytoskeletal change on osteoblastic cell activities. The author cultured osteohinstic cells obtained from neonatal mouse calvaria The cells were treated with cytochalasin B(CB) or colchicine (COL) at four concentrations for 3 hours and after another 24 hours the conditioned media was collected and assayed for prostaglandin E(2) (PGE(2)), interleukin-6(IL-6), tumor necrosis factors-alpha(TNF-alpha) and matrix metalloproteinase-1(MMP-1). In addition, the cytoskeletal protein actin were observed by immunofluorescence. The results were as follows: 1. The production of PGE(2) showed the tendency to be increased in CB-treated group. PGE(2) was increased in COL-treated group dose-dependantly. 2. IL-6 Production, in CB-treated group, was increased, except at 1.0 microgram/ml. IL-6 was induced in COL-treated group. 3. TNF-alpha production was increased in CB-treated group, except at 1.0 microgram/ml, and in COL-treated group, that was increased. 4. The MMP-1 production was decreased in CB-treated goup and was not changed in COL-treated group, which could be selectively visualized by immunoblotting with monospecific antibody. 5. The cytoskeletal actin stress fibers were disappeared and the cells showed to be rounded in CB-treated group. These results indicated that there are a relationship between the cytoskeletal rearrangements and osteoblastic cell activities, especially in release of paracrine/autocrine factors, such as PGE(2), IL-6, and TNF-alpha.

EFFECT OF ALL TRANS RETINOID ACID ON NEWBORN RAT CALVARIAL OSTEOBLASTIC CELLS IN VITRO / 营养学报

Objective: To investigate the effect of all trans retinoid acid (ATRA) on bone cell proliferation and differentiation. Methods: Newborn rat calvarial osteoblastic cells were isolated and the metabolism of the osteoblastic cells were determined by MTT, Goldens method and immunocytohistologic method. Results: 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 mol/L ATRA could increase osteoblastic cells proliferation after 72 h culture; 10 -5 , 10 -6 , 10 -7 mol/L ATRA could increase ALP activity. The expression of cyclin D 1 was decreased. Conclusion: ATRA stimulates the proliferation and differentiation of rat calvarial osteoblastic cells in vitro. Cell cycle relative proteins may play an important role in control of cell proliferation and differentiation induced by retinoic acid and derivatives.