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Background: Osteosarcoma is a malignant cancer that effect bone and metastasizing to many vital organs such as lungs. There are many available drugs to treat the disease including tamoxifen, methotrexate (MTX), and cisplatin which have their own side effects and hurdles to become drugs of choice for the disease. On the other hand, introduction of herbal drugs as chemotherapeutic agents opened up new arena to potentiate the existing treatment by exhibiting synergy. Piperine (PPN) is widely used drug as anti-cancer agent as well as it has anti-inflammatory, analgesic properties, and also used in the treatment of abdominal pains, tuberculosis, arthritis, and respiratory illness. Aims and Objective: Thus, this study was designed to investigate the synergistic inhibitory potential of PPN and MTX on the MG63 osteosarcoma cell lines in vitro. Materials and Methods: The cell lines were cultured on DMEM medium and investigated for cytotoxicity of the drugs using MTT assay at 540 nm in UV. Three groups of cell lines administered with PPN, MTX, and PPN+MTX (1:1) in various concentrations and IC50 values were calculated based on the % cell viability graphs. Results: Results showed that the IC50 of PPN was 38.65, MTX was 123.98, and PPN+MTX was 15.13 proving the significant synergistic cytotoxic effect of PPN and MTX in inhibiting the proliferation of MG63 cell lines. Conclusion: Further research needs to be conducted in this field to elucidate the synergistic pathways in which PPN has shown a better anti-osteosarcoma effect when combined with MTX.
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@#[Abstract] Objective: To analyze the expression of miR-185 and cell division cyclin 42 (CDC42) in osteosarcoma tissues and cells, and to preliminarily explore whether miR-185 affects the proliferation and migration of osteosarcoma MG63 cells by regulating CDC42. Methods: The cancer tissues and para-cancerous tissues of 28 patients with osteosarcoma that pathologically confirmed in the Fourth People's Hospital of Hengshui City from January 2020 to January 2021 were collected for this study. Immunohistochemistry was used to detect the expression of CDC42 in osteosarcoma tissues, and qPCR was used to detect the expression of miR-185 in osteosarcoma tissues. Dual-luciferase reporter gene experiment was applied to verify the targeting relationship between CDC42 and miR-185. According to different transfectants, MG63 cells were divided into miR-185 mimic group, miR-NC group, miR-185 inhibitor group, NC-inhibitor group, CDC42 group (transfected with CDC42 over-expression vector), and negative control (NC) group. The effects of miR-185 and CDC42 expression on the migration, proliferation and cell cycle of MG63 cells were detected by scratch healing assay, CCK-8 method and FCM, respectively. A nude mouse xenograft model was constructed by inoculating osteosarcoma MG63 cells. Immunohistochemistry, qPCR and WB methods were used to detect the effects of over-expression or knock-down of miR-185 on the expression of Ki67 and CDC42 in transplanted tumor tissues. Results: Compared with para-cancerous tissues, the expression of miR-185 in osteosarcoma tissues was significantly decreased, while the expression of CDC42 was significantly increased (all P<0.01). CDC42 was verified to be a target gene of miR-185. Compared with the control group, the migration and proliferation of MG63 cells in the miR-185 mimic group were inhibited (all P<0.01), while the migration and proliferation of MG63 cells in the CDC42 group were increased and the cell cycle was arrested in the S phase (all P<0.01). Compared with the miR-185 group, the migration and proliferation abilities of MG63 cells in the miR-185+CDC42 group were promoted, and the proportion of cells in S phase was increased (all P<0.01). Compared with the control group, the expression of Ki67 and CDC42 in the transplanted tumor tissues of miR-185 mimic group was significantly decreased (all P<0.01), while the opposite results were observed in miR-185 inhibitor group (all P<0.01). Conclusion: miR-185 is lowly expressed while CDC42 is highly expressed in osteosarcoma tissues. miR-185 can inhibit the proliferation and migration of osteosarcoma MG63 cells by negatively regulating the expression of CDC42.
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BACKGROUND: The compounding of RGD polypeptide on the surface of the material can induce the expression of osteoblast integrin gene, promote the adhesion of osteoblasts to the surface of biomaterials and differentiate into mature cells, and promote the formation of new bone. OBJECTIVE: To analyze the effect of domestic porous tantalum modified by RGD polypeptide on integrin/focal adhesion kinase signaling pathway in MG63 cells. METHODS: Porous tantalum material modified by RGD polypeptide was prepared. MG63 cells were inoculated on the surface of porous tantalum and porous tantalum materials modified with RGD polypeptide. MG63 cells cultured alone were used as the blank group. When cultured for 1, 3, 5, and 7 days, the cell proliferation was detected by the CCK-8 method. At 1, 3, and 5 days, the cell growth status was observed under an inverted microscope. At 3, 5 days of culture, cell adhesion was observed with scanning electron microscope. At 5 days of culture, RT-PCR and western blot assay were used to detect type I collagen and integrin β1 and focal adhesion kinase expression. RESULTS AND CONCLUSION: (1) The cell proliferation of the RGD modified group cultured at 3, 5, and 7 days was faster than that of the porous tantalum group and the blank group (P 0.05). (2) Observation by an inverted phase contrast microscope showed that the cells of the porous tantalum group and the RGD modified group were attached to the edge of the material when cultured for 1 day, and the number of cells gradually increased with the extension of the culture time. The number and density of cells in the RGD modified group were better than that of the porous tantalum group. (3) Observation by scanning electron microscope showed that cells adhered to the surface of the porous tantalum group and RGD modified group after 3 days of culture. The cells adhered to the material pore walls and pores, and protruded pseudopods into the pores. When cultured for 5 days, the cells secreted a large amount of extracellular matrix, and the cells were connected to each other through the matrix and gradually covered the surface of the material. The cell growth state, matrix secretion and cell coverage area of the RGD modified group were better than those of the porous tantalum group. (4) Western blot detection results showed that the expressions of type I collagen and integrin β1 protein in the RGD modified group were higher than those in the porous tantalum group and the blank group (P < 0.05). The expression levels of type I collagen, integrin β1, and focal adhesion kinase protein in the porous tantalum group were higher than those in the blank group (P < 0.05). (5) RT-PCR detection showed that the expressions of type I collagen, integrin β1, and focal adhesion kinase mRNA in the RGD modified group were higher than those of the porous tantalum group and the blank group (P < 0.05), and the expression of the porous tantalum group was higher than that of the blank group (P < 0.05). (6) The results showed that porous tantalum modified with RGD polypeptide can up-regulate the expression of type I collagen and integrin β1 on the cell membrane, activate the integrin/focal adhesion kinase signaling pathway, and promote cell adhesion and growth.
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@#[Abstract] Objective: To explore the mechanism of glucose transport protein-1(Glut-1) promoting the migration of osteosarcoma MG63 cells through Wnt/β-catenin pathway. Methods: RNA interference recombinant adenovirus targeting Glut-1 gene (Ad-Glut-siRNA) and control recombinant adenovirus (Ad-GFP) were constructed and transfected into MG63 cells to silence Glut-1 gene expression. The cell migration ability of Blank group, Ad-AFP group, Ad-Glut-siRNA group and AZD2858 (inhibitor of GSK-3) group were detected by Transwell chamber migration assay. Immunofluorescence assay was used to detect the expression of E-cadherin and vimentin in each group and the nuclear translocation of β-catenin. The expression of MMP-2 and MMP-9 in each group and FZD7, β-catenin, Dsh protein in Blank group, Ad-AFP group, Ad-Glut-siRNA group were detected by Western blotting assay. Results: The migration ability of MG63 cells was significantly decreased (P<0.05) after Glut-1 gene silencing, which was restored afterAZD2858 treatment (P <0.05). Compared with Blank group and Ad-GFP group, the E-cadherin level in MG63 cells in Ad-Glut-siRNA group was significantly increased (P<0.05), while the expressions of vimentin, MMP-2, MMP-9, FZD7, β-catenin and Dsh protein were significantly reduced (all P<0.05). Compared with Ad-Glut-siRNA group, E-cadherin expression of AZD2858 group was significantly reduced, while the expressions of vimentin, MMP-2, MMP-9 were significantly up-regulated (P<0.05). Conclusion: The high expression of Glut-1 gene is closely related to the invasion and metastasis of MG63 cells. The possible mechanism is that the high expression of Glut-1 leads to the activation of Wnt/β-catenin pathway, which leads to the decrease of EMT-related protein E-cadherin, and the increase of vimentin and MMP-2, MMP-9, and further promotes the migration of MG63 cells.
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Objective @#To investigate the morphology and proliferation viability in oxidative stress induced damage in human MG63 cells. @*Methods@# The MG63 cells were treated with superoxide anion (O2.') produced by different concentrations of xanthine/xanthine oxidase enzymatic reactions to establish the model of oxidative stress in MG63 cells, using the xanthine oxidase inhibitor oxypurinol to observe the reverse effect of oxypurinol on xanthine/xanthine oxidase induced damage in human MG63 cells. Using the flow cytometry, the production of intracellular reactive oxygen species (ROS) induced by xanthine/xanthine oxidase induced cellular oxidative stress damage was evaluated by the oxidation⁃sensitive fluorescent probe, the 2’7' dichlorofluorescin diacetate. Cellular viability and morphology was evaluated by the MTT assay and the phase contrast microscope.@*Results @#Xanthine/xanthine oxidase induced intracellular ROS production in a dose and time dependent manner (P < 0.05). The cellular viability was reduced and cellular morphology was damaged, too (P < 0.05). Xanthine/xanthine oxidase induced the damage of the cellular morphology. At the same processing time, the higher the xanthine/xanthine oxidase concentration, the higher intracellular ROS fluorescence intensity value, and the lower OD value, the difference was statistically significant (P < 0.05). The intracellular mean ROS fluorescence intensity in xanthine/xanthine oxidase + oxypurinol combined treatment group was significantly lower compared with the same concentration of xanthine/xanthine oxidase (P < 0.05). At the same concentration of xanthine/xanthine oxidase, with the extension of treatment time, the intracellular mean ROS fluorescence intensity gradually increased, the OD value decreased, compared with the control group, the intracellular mean ROS fluorescence intensity of 120 min increased to 345% of the control, was the highest among the xanthine/xanthine oxidase groups. The OD value of 24 h was the 22.9% of the control group, was the lowest among the xanthine/xanthine oxidase groups, cell proliferation activity decreased more obvious. @*Conclusions@#Xanthine/xanthine oxidase could induce oxidative stress damaged the cellular morphology and reduced the cellular viability in MG63 cell lines. The oxypurinol (the inhibitor of xanthine oxidas) could reverse the oxidative stress injury induced by xanthine/xanthine oxidase in human osteoblastic cells.
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Objective:To investigate the effect of blocking the expression of receptor activity modifying protein 1 (RAMP1 )on calcito-nin gene-related peptide(CGRP)-induced MG-63 cell proliferation.Methods:RAMP1 siRNA was synthesized and screened by tran-scription in vitro.The subcultured MG-63 cells were divided into the following groups:RAMP1 siRNA interference group,empty vector group and blank control group.The mRNA expression and the membrane distribution changes of the calcitonin receptor-like receptor (CRLR)and the receptor component protein (RCP)in MG-63 cells were examined by real-time PCR and immunofluorescence method respectively.Results:RAMP1 and CRLR mRNA and the fluorescence intensity of MG-63 cells decreased after transfection by RAMP1 siRNA(P <0.05).In RAMP1 interference group,the expression of RCP mRNA and the fluorescence intensity were higher than those in the other two groups(P <0.05).After RAMP1 siRNA interference,the proliferation of MG-63 cells was inhibited(P <0.05). Conclusion:RAMP1 siRNA transfection may reduce CRLR expression and inhibite the proliferation of MG-63 cell.
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PURPOSE: The effects of water extract and distillate from the mixture of black goat meat and medicinal herb on MG-63 osteoblast proliferation and mouse bone marrow derived osteoclast formation were investigated. METHODS: Proximate composition, volatile basic nitrogen (VBN), mineral content, free amino acid composition and free fatty acid composition in black goat meat were determined. Water extract and distillate were prepared with three groups; goat meat only (BG-E, BG-D), six herbs added group (BG-E6, BG-D6), and eight herbs added group (BG-E8, BG-D8). Osteoblast proliferation, mineralization and calcium uptake activity of MG-63 cells were measured and tartrate resistant acid phosphatase activity of osteoclasts was analyzed. RESULTS: Black goat meat had remarkably low fat and high level of calcium. Glutamic acid was the most abundant amino acid. Herbs added extract groups (BG-E6 and BG-E8) showed increased MG-63 cell proliferation in a concentration dependent manner, while all the distillates did not show the effect. All extracts and distillates showed significantly increased osteoblast mineralization depending on the concentration. In particular, herb added extract, BG-E6, increased 170.3% of control and the distillate of BG-D and BG-D6 increased up to 168.5% and 159.8%, respectively. Calcium uptake activities of all water extracts showed remarkable increase of BG-E6 and BG-E8 up to 615.5% and 628.1% of control, respectively. Ditillates had no effect except BG-D6. All water extracts significantly reduced the activity of tartrate-resistant acid phosphatase (TRAP) in osteoclasts derived from mouse bone marrow. CONCLUSION: Combination of black goat meat and medicinal herb increased the MG-63 cell proliferation and effectively inhibited osteoclast differentiation in both water extracts and distillate of them, which implies that they could be used as potent functional food materials for bone health.
Subject(s)
Animals , Mice , Acid Phosphatase , Bone Marrow , Calcium , Cell Proliferation , Functional Food , Glutamic Acid , Goats , Meat , Nitrogen , Osteoblasts , Osteoclasts , Plants, Medicinal , WaterABSTRACT
PURPOSE: The purpose of this study was to evaluate the surface characteristics and response of osteoblast-like cell at SLA surface treated with NaOH. MATERIALS AND METHODS: Three kinds of specimens were fabricated for the experiment groups. Control group was a machined surface, SLA group was a conventionally SLA treated surface, and SLA/NaOH gorup was SLA surface treated with NaOH. To evaluate the surface characteristics, the surface elemental composition (XPS), surface roughness and surface contact angle were evaluated in each group. And the cytotoxicity, cell adhesion, cell proliferation and ATP activity of osteoblast-like cells (MG-63 cells) were compared in each group for evaluatation of the cell responses. Statistical comparisons between groups were carried out via one-way ANOVA using the SPSS software (SPSS Inc., Chicago, USA), and then performed multiple comparisons. The differences were considered statistically significant at P<.05. RESULTS: SLA surface treated with NaOH (SLA / NaOH group) was changed to hydrophilic surface. All groups did not show the cytotoxicity to the MG-63. In cell adhesion studies, SLA / NaOH group showed the higher degree of adhesion than anothers (P<.05), Up to 7 days of incubation, the proliferation was showed the increasing tendency in all groups but SLA / NaOH group showed the highest cell proliferation between the three groups (P<.05). At 7 days of incubation, there was no difference in ALP activities between the three groups, but at 14 days, SLA / NaOH group showed significant increase in ALP activities (P<.05). CONCLUSION: In this study, SLA surface treated with NaOH promoted cell adhesion, proliferation and differentiation. It means that SLA/NaOH group is possible to promote osseointegration of implants.
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Adenosine Triphosphate , Cell Adhesion , Cell Culture Techniques , Cell Proliferation , OsseointegrationABSTRACT
Osteoprotegerin (OPG) is a secreted glycoprotein and a member of the tumor necrosis factor receptor superfamily. It usually functions in bone remodeling, by inhibiting osteoclastogenesis through interaction with a receptor activator of the nuclear factor kappaB (RANKL). Transglutaminases-2 (Tgase-2) is a group of multifunctional enzymes that plays a role in cancer cell metastasis and bone formation. However, relationship between OPG and Tgase-2 is not studied. Therefore, we investigated the involvement of 12-O-Tetradecanoylphorbol 13-acetate in the expression of OPG in MG-63 osteosarcoma cells. Interleukin-1beta time-dependently induced OPG and Tgase-2 expression in cell lysates and media of the MG-63 cells by a Western blot. Additional 110 kda band was found in the media of MG-63 cells. 12-O-Tetradecanoylphorbol 13-acetate also induced OPG and Tgase-2 expression. However, an 110 kda band was not found in TPA-treated media of MG-63 cells. Cystamine, a Tgase-2 inhibitor, dose-dependently suppressed the expression of OPG in MG-63 cells. Gene silencing of Tgase-2 also significantly suppressed the expression of OPG in MG-63 cells. Next, we examined whether a band of 110 kda of OPG contains an isopeptide bond, an indication of Tgase-2 action, by monoclonal antibody specific for the isopeptide bond. However, we could not find the isopeptide bond at 110 kda but 77 kda, which is believed to be the band position of Tgase-2. This suggested that 110 kda is not the direct product of Tgase-2's action. All together, OPG and Tgase-2 is induced by IL-1beta or TPA in MG-63 cells and Tgase-2 is involved in OPG expression in MG-63 cells.
Subject(s)
Blotting, Western , Bone Remodeling , Cystamine , Gene Silencing , Glycoproteins , Interleukin-1beta , Multifunctional Enzymes , Neoplasm Metastasis , Osteogenesis , Osteoprotegerin , Osteosarcoma , Receptors, Tumor Necrosis FactorABSTRACT
Objective: To observe the effects of different concentrations of glucose on the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), osteoprotegerin (OPG), and the ligand of osteoprotegerin (OPGL) in osteosarcoma MG63 cells, so as to assess the role of glucose in pathogenesis of diabetic osteoporosis. Methods: MG63 cells were incubated with glucose at the concentrations of 5.5,16.7,33.3 mmol/L for 24 h. The expression of TRAIL, OPG and OPGL mRNA were examined by RT-PCR. Expression and distribution of TRAIL in MG63 cells were investigated by immunohistochemical method. Results: The mRNA expression of TRAIL and OPGL in MG63 cells increased in the order of control group, 5.5,16.7,33.3 mmol/L glucose groups(P<0.05), and the expression of OPG mRNA decreased in the same order(P<0.05). TRAIL mRNA level in 33.3 mmol/L glucose group was significantly higher than that in the control group [(1.004±0.070) vs (0.740±0.023), P<0.05] and the expression of TRAIL was stronger than that in the control group. Conclusion: High concentration of glucose can lead to increased expression of TRAIL and OPGL in osteoblasts and decreased expression of OPG, which may be one of the key pathogenetic factors of diabetic osteoporosis.
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Objective To explore the inhibiting effects of arsenic trioxide and cisplatin on MG-63 cells. Methods Using MTT assay,flowcytometry,phase contrast microscopy and electron microscopy methods,the therapeutic effect of arsenic trioxide was studied for the osteosarcoma in the cultured MG-63 cells in vitro,and compared these effects with cisplatin. The inhibitory rotes of cell growth and the effect of apoptosis and cell cycle were compared between arsenic trioxide and cisplatin on MG-63 cells. Results The contrast phase microscope revealed the adhesion ability of normal groups was good and cellular morphology showed epithelium cells. But the celhdar morphology showed irregular arrangement in arsenic trioxide groups and cytoplasmic vacuoles in cisplatin group. Electron microscope revealed the globular plasmalemma ecphymas in cell surface of control groups,the enlarged crista mitochondriales and the double-deck membrane structure appeared clearly. But electron microscope revealed globular plasmalemma processes in cell surface of arsenic trioxide groups,thinned crista mitochondriales and clearly seen karyopycnosis and nuclear membrane of apoptotic cells. The globular plasmalemma processes in cell surface of cisplatin groups were separated,nuclear membrane thickened and chromatin were in sandy shape. Both arsenic trioxide and cisplatin inhibited effectively MG-63 cells growth. There was a significant difference in different groups of inhibition ratios to the growth of cells(all P < 0.05). In 2,4,8,16,32,64,128 hours,the inhibition ratios(%) of arsenic trioxide(56.31±0.03,70.00±0.06,79.84±0.03,87.31±0.13,84.70±0.09,90.68±0.06,91.18±0.05) and cisplatin groups(7.55±0.15,15.70±0.17,30.72±0.07,49.80±0.05,45.11± 0.13,61.62±0.08,93.80±0.12) were obviously increased as compared with those in the control group(2.03± 0.07,2.78±0.08,3.11±0.01,5.67±0.04,12.23±0.04,18.65±0.04,24.45±0.04,all P < 0.05). Moreover the inhibition ratio of arsenic trioxide group in 2 to 32 hour was significantly higher than that of cisplatin group and the effect was more faster(all P < 0.05). Both arsenic trioxide and cisplatin could induce apoptosis MG-63 cells. There was a significant difference in different groups of the inhibition ratio to the growth of cells(F = 13.317,P < 0.05). The inhibition ratios(%) of arsenic trioxide on 24,36,48 hour(20.50±3.78,45.76±9.90,25.16±15.41),and cisplatin groups on 24,36,48 hour(12.55±1.51,18.85±3.40,12.37±5.43),were obviously increased as compared with those in the control group at the same time(6.57±1.48,8.03±2.08,6.54±1.30,P< 0.05 or<0.01). Both arsenic trioxide and cisplatin inhibited MG-63 cells cycle. There was a significant difference in different groups of the inhibition ratio to the growth of cells(F = 54.579,43.429,21.795,P < 0.05 or < 0.01). And the total inhibition ratios(%) in G1 cycle of arsenic trioxide(78.26±5.24) and cisplatin groups(80.48±2.81) were obviously increased as compared with those in the control group(57.49±6.65,all P < 0.05 or < 0.01). Conclusions Arsenic trioxide and cisplatin can effectively inhibit the proliferation of MG-63 cell line and induce the apoptosis of MG-63 cell line. And the effects induced by arsenic trioxide group were faster than that of cisplatin groups. Moreover arsenic trioxide can arrest the cell cycle of MG-63 cell line at G1 phase.
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Statement of problem. The success of osseointegration can be enhanced with an implant that has improved surface characteristics. Anodic oxidation is one of the surface modifying method to achieve osseointegration. Voltage of anodic oxidation can change surface characteristics and cell activity. Purpose. This study was performed to evaluate MG63 cell responses such as affinity, proliferation and to compare surface characteristics of anodic oxidized titanium in various voltage. Material and method. The disks for cell culture were fabricated from grade 3 commercially pure titanium, 1 mm in thickness and 12 mm in diameter. Surfaces of 4 different roughness were prepared. Group 1 had a machined surface, used as control. Group 2 was anodized under 220 V, group 3 was anodized under 300 V and group 4 was anodized under 320 V. The microtopography of specimens was observed by scanning electron microscope (JSM-840A, JEOL, Japan) and atomic force microscope(Autoprobe CP, Park Scientific Instrument, USA). The surface roughness was measured by confocal laser scanning microscope(Pascal, LSM5, Zeiss, Germany). The crystal structure of the titanium surface was analyzed with x-ray diffractometer(D8 advanced, Bruker, Germany). MG63 osteoblast-like cells were cultured on these specimens. The cell morpholgy was observed by field emission electron microscope(Hitachi S-4700, Japan). The cell metabolic and proliferative activity was evaluated by MTT assay. Results and conclusion. With in limitations of this in vitro study, the following conclusions were drawn. 1. In anodizing titanium surface, we could see pores which did not show in contol group. In higher anodizing voltage, pore size was increased. 2. In anodizing titanium surface, we could see anatase. In higher anodizing voltage, thicker oxide layer increased crystallinity(anatase, anatase and rutile mixed). 3. MG63 cells showed more irregular, polarized and polygonal shape and developed more lamellipodi in anodizing group as voltage increased. 4. The activity of cells in MTT assay increased significantly in group 3 and 4 in comparison with group 1 and 2. However, there was no difference between group 3 and 4 at P<0.05. Proliferation of MG63 cells increased significantly in pore size(3-5.5 micrometer) of group 3 and 4 in comparison with in pore size(0.2-1 micrometer) of group 2.
Subject(s)
Cell Culture Techniques , Osseointegration , TitaniumABSTRACT
Phytoestrogens, especially Yak-kong or soybean-derived isoflavones have been traditionally used as a supplement of estrogen for preventing postmenopausal osteoporosis in oriental folk medicine. In a previous study, we demonstrated that as Yak-kong and soybean increased MG-63 human osteoblastic cell proliferation, the expression of estrogen receptor alpha and beta (ERalpha: ERbeta) both were increased. However, the increased level of ERalpha is much higher than that of ERbeta. To determine whether the altered level of ERalpha expression affects Yak-kong or soybean induced MG-63 cell proliferation, we established cell lines stably expressing either ERalpha or antisense ERalpha RNAs. Increased expression of ERalpha in MG-63 cells (ERalpha-MG63) enhanced Yak-kong or soybean induced proliferation which paralleled with the enhanced expression of IGF-I. Inhibition of ERalpha expression by antisense ERalpha RNAs (As-ERalpha-MG63) caused these cells to insensitize Yakkong or soybean induced proliferation and IGF-I expression. Furthermore, the comparable effects between Yak-kong and the combined treatment of genistein and daidzein at 0.5 x 10-8M, which is a concentration of these two isoflavones similar to Yak-kong at 0.001 mg/ml, on cell proliferation and IGF-I expression in ERalpha-MG63 or As-ERalpha-MG63 cells demonstrate that ERalpha plays an important, active role in MG-63 cell proliferation induced by phytoestrogens, especially Yak-kong or soybean derived isoflavones.
Subject(s)
Female , Humans , Cell Line , Cell Proliferation , Estrogen Receptor alpha , Estrogen Receptor beta , Estrogens , Genistein , Insulin-Like Growth Factor I , Isoflavones , Medicine, Traditional , Osteoblasts , Osteoporosis, Postmenopausal , Phytoestrogens , RNA , Glycine maxABSTRACT
PURPOSE: To determine the role of Insulin-like Growth Factor-I (IGF-I) in the regulation of Vascular Endothelial Growth Factor (VEGF) expression in MG-63 cells and then to find the mechanism b which this regulation occurs. MATERIALS AND METHODS: MG-63 cells were grown to confluence in 60-mm dishes. To determine the effects of IGF-I on expression of VEGF mRNA according to time and concentration, the cells were treated with 10 nM IGF-I, following isolation of total RNA and Northern blot analysis after 1, 2, 4, 8, 12, 24 hours and after 2 hours of treatment with 0.5, 2, 10, 25, 50 nM IGF-I respectively, isolation of total RNA and Northern blot analysis were followed. To determine the mechanism of action of IGF-I, inhibitors such as hydroxyurea (76.1 microgram/ml), actinomycin D (2.5 microgra/ml), cycloheximide (10 microgram/ml) were added 1 hour after treatment of 10 nM IGF-I. RESULTS: 1. the expression of VEGF mRNA was increased with treatment of IGF-I. 2. The expression of VEGF mRNA was increased according to time- and concentration dependent manner of IGF-I. 3. The effect of IGF-I was decreased by hydroxyuera, actinomycin D, but not by cycloheximide. CONCLUSION: IGF-I regulate the expression of VEGF mRNA in the level of DNA synthesis and transcription. These results could suggest that IGF-I plays an important role in angiogenesis in the process of new bone formation and remodeling.
Subject(s)
Blotting, Northern , Cycloheximide , Dactinomycin , DNA , Hydroxyurea , Insulin-Like Growth Factor I , Osteogenesis , RNA , RNA, Messenger , Vascular Endothelial Growth Factor AABSTRACT
Objective: To explore the inhibitiory effect of antisense oligodeoxynucleotide(ASODN) of survivin gene on the cultured osteosarcoma cell line MG63 and its sensitization effect to chemotherapy.Methods: survivin phosphorothioate ASODN was synthesized and transfected into MG63 cells by lipofectamine 2000.MTT assay was used to detect cell inhibition ratio.Apoptosis was observed by flow cytometry.Survivin mRNA and protein expression were determined by RT-PCR and Western blot respectively.Results: The proliferation of the cells transfected by lipofectamine 2000 was inhibited by survivin ASODN in a dose and time dependent manner.A higher total apoptosis rate(81.12?3.2)% could be induced in MG 63 cells in group Lip-ASODN than in group Lip-SODN(27.09?2.1)%(P
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Objective: To study the vitro bioactivity of the Ti-24Nb-4Zr-7.9Sn (TNZS) disks modified by anodic oxidation(AD) and its effects on adhesion of human osteoblast-like MG63 cells. Methods:The TNZS titanium alloy disks were treated with anodic oxidation and the bioactivity was assessed by investigating the formation of apatite on the film surface after soaking in simulated body fluids. The surface composition of the specimens after immersion was evaluated by EDX and X-ray diffraction. MG63 osteoblast-like cells were seeded on the Ti6Al4V,TNZS,AD-TNZS disks, and then the number of attached cells were counted and the cell morphology was examined at each given period. Results:The apatite can form on the treated TNZS disks after soaking in SBF for 6 days. The ratio of attached MG63 on AD-TNZS disks after 60 or 120 min seeding were significant higher than that on other samples and the cells on AD-TNZS disks spread better. Conclusion:The TNZS alloy modified by anodic oxidation can induce apatite formation in SBF and increase the early attachment of osteoblasts.
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0.05) among the three groups in cell proliferation in 1~10 d cultures and in total protein content in 4~7 d cultures. At 4 and 7 days, ALP activity of MG63 cells cultivated on AD-TNZS disks was significantly higher than that of cells on the other samples(P