Synergistic effects of methotrexate and piperine on human osteosarcoma cell lines – An in-vitro study

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.

miR-185 inhibits the proliferation and migration of osteosarcoma MG63 cells via regulating CDC42 gene expression / 中国肿瘤生物治疗杂志

@#[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.

Effects of calcium sulfate on proliferation of osteoblast-like MG-63 cells and osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB system / 中华创伤骨科杂志

Objective:To investigate the effects of calcium sulfate on the proliferation of osteoblast-like MG-63 cells and the osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB (OPG/RANKL/RANK) system.Methods:The extract of calcium sulfate was prepared. The osteoblast-like MG-63 cells were cultured for 24 hours in the medium containing calcium sulfate (the calcium sulfate group) and in the normal medium without calcium sulfate (the blank group), respectively. The growth of osteoblast-like MG-63 cells was observed and their proliferation detected by CCK-8. The mRNA and protein expression levels of OPG/RANKL were detected.Results:The growth of osteoblast-like MG-63 cells was fine in both groups. The CCK-8 test showed that the absorbance value at 24 h was 0.997±0.008 for the calcium sulfate group, significantly higher than that for the blank group (0.640±0.003) ( P<0.001). Respectively, the mRNA expression levels of OPG were 2.834±0.176 and 1.005±0.102 and the mRNA expression levels of RANKL 0.355±0.035 and 1.002±0.068 for the calcium sulfate group and the blank control group, showing statistically significant differences ( P<0.001). The results of Western blot showed that compared with the blank control group, the protein expression of OPG in osteoblast-like MG-63 cells was promoted but the protein expression of RANKL inhibited in the calcium sulfate group. Conclusion:Calcium sulfate may have a positive effect on bone formation, because it can promote the proliferation and activity of osteoblast-like MG-63 cells and regulate the OPG/RANKL/RANK system.

Expression of miRNA-95 in osteosarcoma tissues and its effect on proliferation and invasion of MG-63 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To study the expression of miRNA-95 in osteosarcoma tissues and cell lines, as well as to reveal its effect on proliferation, apoptosis, cell cycle and invasion ability of osteosarcoma cells. Methods: Real-time fluorescent quantitative PCR was used to detect the expression of miRNA-95 in 15 pairs of osteosarcoma tissues and their adjacent normal tissues (specimens were collected from patients underwent surgery in Qingdao Haici Medical Group from January 2015 to January 2018), osteosarcoma cell lines (MG-63, U2OS, 143B and HOS) and normal human osteoblast hFOB1.19 cell line. miRNA-95 mimics and miRNA-95 inhibitors were respectively transfected into MG-63 cells by Lipofectamine 2000, and miRNA-NC group was set up as control group. CCK-8 method was used to detect the changes in cell proliferation, Flow cytometry was used to detect the changes in cell cycle and apoptosis, Transwell method was used to detect the changes in cell invasion ability, and Dual luciferase enzyme activity assay was used to detect and validate the target gene of miRNA-95 in osteosarcoma cells. Results: The expression level of miRNA-95 in human osteosarcoma tissues and cell lines (MG-63, U2OS, 143B and HOS) was significantly higher than that in adjacent tissues and normal human osteoblast hFOB1.19 cell line (all P<0.01), with the highest expression in MG-63 cells (P<0.01). Compared with the miRNA-NC group, the proliferation and invasion abilities of MG-63 cells in miRNA-95 mimics group increased significantly, while the apoptosis rate decreased significantly (all P<0.01). However, the proliferation and invasion activities of MG-63 cells in miRNA-95 inhibitor group decreased significantly, while the apoptosis rate increased significantly, and the cell cycle was obviously blocked (all P<0.01). miRNA-95 played a role in targeting the gene of epithelialmembraneprotein1 (EMP-1) in human osteosarcoma MG-63 cells. Conclusion: miRNA-95 is highly expressed in human osteosarcoma tissues and cells; inhibitor of miRNA-95 expression can promote apoptosis and inhibit proliferation, cell cycle and invasion of osteosarcoma cells, which may be related with targeting EMP-1 gene.

Osteosarcoma-derived exosomes induces M2 polarization of macrophages and promotes metastasis of MG63 cells through Tim-3 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of exosomes derived from osteosarcoma on the differentiation of tumor-related macrophages and its mechanism. Methods: From March 2018 to October 2019, tumor tissues and corresponding normal tissues from 18 patients with primary osteosarcoma who underwent osteosarcoma resection and pathological diagnosis in the Departments of Orthopedics and Pediatric Surgery of the Affiliated Hospital of North Sichuan Medical College were collected. The expression level of Tim-3 was detected by Western blotting; Exosomes of osteosarcoma MG63 cells (MG63-Exo) were isolated and identified by transmission electron microscopy and nanoparticle size analysis, and its phagocytosis by macrophages was verified by Dual fluorescent staining; The effects of MG63-Exo on macrophage differentiation and the expression levels of IL-10, TGF-β and VEGF were detected by qPCR; The effects of MG63-Exo induced macrophages on the migration and invasion of MG63 cells and the expression of EMT related proteins were detected by Transwell invasion and migration assay and Western blotting; CRISPR/cas9 was used to knock out Tim-3 in MG63 cells, and its knockout efficiency was verified by Western blotting, and then qPCR, transwell assay and Western blotting were used to detect the effect of MG63-Exo with Tim-3 knock-out on macrophage differentiation, as well as migration, invasion and expression of EMT related proteins in MG63 cells; Finally, the mouse model of osteosarcoma lung metastasis was used to verify the effect of exosomes from different sources on the lung metastasis of osteosarcoma. Results: Transmission electron microscopy and nanoparticle size assay confirmed that MG63-Exo were successfully isolated, and Confocal fluorescence results confirmed that it could be phagocytized by macrophages; qPCR results showed that MG63-Exo significantly promoted M2 differentiation of macrophages compared with PBS (P<0.05); Compared with PBS control group, M2 macrophages induced by MG63-Exo significantly promoted the migration, invasion and EMT of osteosarcoma cells (all P<0.05); The mRNA and protein expressions of Tim-3 in the MG63 cells knocked out by CRISPR/cas9 (Tim-3-KO) were significantly reduced (all P<0.05), and Tim-3 could be transferred into macrophages in the form of exosomes; Compared with MG63-Exo co-cultured macrophages, the M2 type differentiation of macrophages treated with Tim-3-KO-exo was significantly decreased (P<0.05); Compared with the MG63 cells co-cultured with macrophages induced by MG63-Exo, the migration, invasion and EMT were significantly reduced while the lung metastasis was significantly promoted in MG63 cells co-cultured with macrophages induced by Tim-3-KO-Exo (all P<0.05). Conclusion: Exosomes derived from osteosarcoma can induce M2 polarization of macrophages through Tim-3 and promote the invasion and metastasis of tumor.

Porous tantalum coated with RGD polypeptide can activate the integrin/focal adhesion kinase signaling pathway of MG63 cells / 中国组织工程研究

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.

Effect of TSP-1 gene on angiogenesis of osteosarcoma and its mechanism / 实用肿瘤学杂志

Objective The aim of this study was to investigate the effect of TSP-1 gene on angiogenesis in human osteosar-coma and its mechanism of action. Methods MG-63 cells were transfected with constructing pBPLV-shRNA-TSP-1 vector and pBPLV-TSP-1 expression vector. Cell viability was measured by CCK8,and its invasive ability was measured by Transwell assay. The expression of CD36 in intracells was detected by immunofluorescence. The expression levels of TSP-1,CD36,p38MAPK,VEGF, VEGFR-1,EGF and PDGF were detected in MG-63 cells by qRT-PCR and Western blot. Results The cell viability and inva-sion ability were significantly increased after transfected pBPLV-TSP-1 vector compared with the empty vector group(P<0. 05), and significantly decreased after transfected pBPLV-shRNA-TSP-1 vector( P<0. 05). The expression of TSP-1,EGF,P38, PDGF,VEGF and VEGFR -1 at mRNA and protein levels was significantly increased after transfection pBPLV -TSP -1 ( P <0. 05),and significantly decreased after transfection pBPLV-shRNA-TSP-1 vector(P<0. 05). Conclusion TSP-1 gene can promote the proliferation and invasion of MG-63 cells,and promote the formation of human osteosarcoma,indicating its mechanism related to the increase of growth factors EGF,VEGF,PDGF and activation of P38-MAPK pathway.

Effect of miR-129 on proliferation and apoptosis of osteosarcoma MG-63 cells by targeting SEPHS1 / 实用肿瘤学杂志

Objective The aim of this study was to investigate the effect of miR-129 on the proliferative activity and apop-tosis of osteosarcoma MG-63 cells. Methods Thirty cases of osteosarcoma and its adjacent paracancerous tissues were collected. RT-PCR was used to detect the expressions of miR-129 mRNA and SEPHS1 mRNA. Western blot was used to detect the protein level of SEPHS1. After MiR-129 was over-expressed and knocked down,the cell proliferation was detected in MG-63 cells by CCK-8,and apoptosis was detected in MG-63 cells by Westerm blot and hoechest staining. Results In this study,the expression of miR-129 in osteosarcoma tissues was significantly higher than that in para-cancerous tissues(P<0. 05). Through the established model in vitro of miR-129 overexpression or knockdown,it was determined that the miR-129 mimetic and inhibitor concentrations were optimal for overexpression and knockdown at 50 nM and 200 nM, respectively. The possibility of binding between miR -129 and SEPHS1 was predicted by software,and the luciferase reporter assay further confirmed the binding relationship between miR-129 and SEPHS1. Knockdown of miR-129 significantly inhibited the proliferation of MG-63 cells and accelerated the apoptosis of MG-63 cells. Conclusion miR -129 plays a key role in regulating the proliferation and apoptosis of osteosarcoma cells by binding to SEPHS1.

Effect of plasma membrane-associated sialidase NEU3 activity on the proliferation and apoptosis of osteosarcoma MG-63 cells / 国际肿瘤学杂志

Objective To investigate the effect of plasma membrane-associated sialidase 3(NEU3) activity on the proliferation and apoptosis of osteosarcoma MG-63 cells in vitro. Methods MG-63 cells were cultured in vitro. Anti-NEU3 antibody(Ab)immunofluorescent staining was used to indicate the cellular locali-zation of NEU3 in MG-63 cells. The cells treated with 0 nmol/ L 2-deoxy-2,3-didehydro-N-acetyl neuraminic acid(DANA)or 0 μg/ ml anti-NEU3 Ab were used as blank control groups. The cells were treated with 10, 20,50 nmol/ L DANA,or 0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 24 h or 48 h,respectively. The inhibition rates of the cell proliferation and cell apoptosis rates were measured with CCK-8 and flow cytometry. The expression levels of oncogene-related proteins,Ras protein and Bcl-2 protein,were detected by Western blotting. Results The immunofluorescence result showed that NEU3 was located in the cytoplasm of MG-63 cell. After treating with 0,10,20,50 nmol/ L DANA for 48 h,the inhibition rates of cell proliferation were 0, 15. 10％ ± 3. 23％ ,41. 46％ ± 2. 31％ ,64. 68％ ± 4. 12％ ,with significant statistical difference(F = 99. 90, P < 0. 001),and the following contrast between each two groups met the statistical significance(all P < 0. 05). After treating with 0,0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 48 h,the inhibition rates of cell proliferation were 0,9. 34％ ± 1. 53％ ,19. 66％ ± 4. 18％ ,42. 50％ ± 5. 68％ ,and the difference was statistically signifi-cant(F = 25. 67,P < 0. 001),and the following contrast between each two groups met the statistical signifi-cance(P < 0. 05),except the difference between 0. 5 and 1. 0 μg/ ml groups(P > 0. 05). When the MG-63 cells were treated with 0,10,20,50 nmol/ L DANA for 24 h,the cell apoptosis rates were 4. 05％ ± 0. 07％ , 4. 15％ ± 0. 23％ ,12. 85％ ± 1. 48％ ,8. 29％ ± 0. 86％ ,respectively,and the difference was statistically sig-nificant(F = 23. 21,P < 0. 001). And the following contrast between each two groups met the statistical signi-ficance(P < 0. 05),except the differences between 0 nmol/ L and 10 nmol/ L,20 nmol/ L and 50 nmol/ L groups(P > 0. 05). When the MG-63 cells were treated with 0,0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 24 h,the cell apoptosis rates were 4. 05％ ± 0. 07％ ,20. 13％ ± 2. 97％ ,20. 29％ ± 2. 82％ ,20. 58％ ± 0. 70％ ,with statistical significant difference(F = 15. 36,P = 0. 001). And the following contrast between each two groups showed that the differences between 0 μg/ ml and each treated group were statistically signifi-cant(P < 0. 05),while the differences between two treated groups were not statistically significant( P >0. 05). Western blotting results showed that the expression levels of Ras and Bcl-2 decreased with the increasing concentrations of DANA and anti-NEU3. Conclusion Inhibition of NEU3 enzyme activity can suppress the survival rate of MG63 cells and increase the cell apoptosis. The possible mechanism may be related to the declined expression of oncogene-related proteins Ras and Bcl-2,which suggests that NEU3 may be a possible target for treating osteosarcoma.

Effect of laser-etched pure titanium surface on early proliferation of MG63 cells / 口腔疾病防治

Objective @# To investigate the effect of a laser-etched pure titanium surface on proliferation of the human osteosarcoma cell line MG63 and to provide a basis for study of implant surface modification. @*Methods@#The pure titanium plate was cut into titanium pieces by a numerical control machine tool and divided into smooth surface and laser etching groups. The titanium surface of the laser etching group was etched with an Nd:YAG continuous wave laser using predetermined parameters, and the surfaces were observed by scanning electron microscopy (SEM). The surface micromorphology of each titanium sheet was evaluated. The relative element content of the titanium surface was measured by energy dispersive X-ray spectroscopy (EDS). The Ra value of each surface was determined using the Veeco roughness tester. MG63 cells were inoculated on 2 sets of titanium tablets. At 1, 3, and 6 h postinoculation, cell adhesion to the two groups of titanium sheets was observed under the microscope. At 24 h after inoculation, cellular F-actin was directly stained using immunofluorescence, and the morphology of the cytoskeleton was observed by laser confocal microscopy. Cell proliferation was examined at 1, 3, and 5 d using a MTS kit, and the data were analyzed with SAS 9.4.@* Results @#The surface of the smooth surface group was smooth and flat, the element composition was pure titanium, and the roughness Ra was 179.23 nm. The surface of the laser-etched group formed a regular and uniform pore structure. The composition was mainly Ti, O, C, etc, and the surface roughness Ra was 14.11 μm. A large number of cells were uniformly distributed on the two titanium sheets in the observations at 1, 3, and 6 h. At 24 h postinoculation, MG63 cells were completely stretched on the two sets of titanium sheets and had extended a large number of pseudopods and microfilaments to cross-link with peripheral cells; moreover, the cell division phase was observed. The cell proliferation of the two groups at 1, 3, and 5 d showed a significant increase with time, indicating that no cytotoxicity occurred on the surfaces of the two groups. However, the cell proliferation in the laser-etched group was superior to that in the mechanical smooth surface group.@*Conclusion@#The surface morphology of titanium can be controlled by laser etching, which is conductive to increase the microstructure of implants without cytotoxicity and promoting osteoblast proliferation in the early stage.

Study on miR-650 for promoting proliferation of osteosarcoma cells via targeting ING4 / 重庆医学

Objective To investigate the expression of miR-650 in osteosarcoma tissue and cell lines and its action mechanism in the osteosarcoma formation.Methods The realtime fluorescence quantitative PCR was used to detect and compare the expres-sions of miR-650 between osteosarcoma tissue and normal tissue or between osteosarcoma cell lines(MG63)and normal human os-teoblast cell lines(hFOB1.19);the MTT experiment was used to investigate the proliferation situation in different groups;Western blot was used to detect the ING4 protein expression.Results The expression of miR-650 in osteosarcoma tissue and MG63 cells was higher than that in normal tissue and human osteoblast cell;after inhibiting miR-650 expression,the proliferation ability of MG63 was significantly decreased.Moreover the expression of inhibitor of growth 4(ING4)was significantly increased when miR-650 was reduced,ING4 mRNA of negative control group,scramble group and miRNA group were 1.00 ± 0.16,1.08 ± 0.14 and 5.35 ± 0.32 respectively;the ING4 protein expressions were 0.62 ± 0.06,0.59 ± 0.12 and 2.45 ± 0.20 respectively;compared with negative control group,the difference was statistically significant(P<0.05);but after suppressing this elevation,the proliferation a-bility of MG63 cells had a certain recovery.Conclusion MiR-650 promotes MG63 proliferation via reducing ING4 expression,which suggests that miR-650 could be a new target of treating osteosarcoma.

Glucose transporter-1 promotes migration of osteosarcoma MG63 cells via the Wnt/β-catenin pathway / 中国肿瘤生物治疗杂志

@#[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.

Effect of attenuated expression of neuraminidase 3 via RNA interference on the proliferation and apoptosis of osteosarcoma MG-63 cells / 中国修复重建外科杂志

Objective: To investigate the effect of attenuated expression of neuraminidase 3 (NEU3) via RNA interference on the proliferation and apoptosis in human osteosarcoma MG-63 cells. Methods: MG-63 cells were immunostained to observe the expression of NEU3. The cells were then divided into 5 groups: MG-63 cells in normal control group (group A) were not treated; MG-63 cells in 30, 50, and 100 nmol/L NEU3 RNA interference groups (groups B, C, and D) were transfected with 30, 50, and 100 nmol/L of NEU3 small interfering RNA (siRNA); negative control group (group E), MG-63 cells were transfected with different species negative siRNA (actin siRNA of mice, 50 nmol/L). The expression level of NEU3 mRNA was measured with real-time fluorescence quantitative PCR (qPCR). The proliferation of the cells was measured by cell counting kit 8 (CCK-8). The cell apoptosis rate was detected by flowcytometry (FCM). The expressions of cell apoptosis related proteins (Ras and Bcl-2) were detected by Western blot assay. Results: NEU3 expressed in the cytoplasm of MG-63 cells under fluorescence microscope. The qPCR results showed that NEU3 mRNA levels were significantly lower in groups B, C, D than that in groups A and E ( P0.05). The results of Western bolt assay showed that the protein levels of Ras and Bcl-2 in groups B and C were not significantly different from groups A and E ( P>0.05), while the protein levels of Ras and Bcl-2 were significantly decreased in group D ( P<0.05). Conclusion: Attenuated expression of NEU3 could inhibit the survival of MG-63 cells and accelerate its apoptosis. The results suggest that NEU3 could be a possible target for treating osteosarcoma.

Effect of decoction and drug-containing serum of sweated and crude Radix Dipsaci on proliferation of MG-63 cells and osteoblasts / 中草药

Objective To investigate the effect of decoction and drug serum of sweated and crude Dipsaci Radix on the proliferation of human osteoblast-like cells (MG-63) and osteoblasts. Methods MG-63 cells and osteoblasts were co-cultured with decoction and rat serum containing Dipsaci Radix before and after “sweating”. The cell proliferation was detected by MTT method and the ALP activity of osteoblasts was detected by nitrophenyl phosphate method. Results Both decoction and drug-containing serum can significantly promote the proliferation of MG-63 cells and osteoblasts (P < 0.01), and significantly increase the ALP activity of osteoblasts (P < 0.01). Moreover, sweated group were generally better than or non-inferior to crude group at the same dose concentration of two administration methods. Conclusion Combined with component studies, it can be inferred that the changes in composition before and after "sweating" affect its role in promoting cell proliferation and differentiation, with view to laying the foundation for further research.

The influence of STK31 on osteosarcoma's malignant biological behavior / 重庆医学

Objective To observe the expression of serine/threonine kinase 31 (STK31) in osteosarcoma and its effect on the malignant biological behavior of osteosarcoma.Methods Fifteen cases of osteosarcoma specimens and adjacent normal tissue were collected.The expression of STK31 in tumor tissues and normal tissue were detected by immunohistochemistry,real-time quantitative PCR and Western blot.The STK31 knockout plasmids PGenesil-STK31-shRNA or control plasmid pGenesil-1 were transfected into osteosarcoma cell line MG63 cells.The effect of STK31 on the proliferation of MG63 cells was detected by CCK8 cell activity assay.Tanswell experiment was used to observed the effect of STK31 on the migration ability of osteosarcoma cells.Results Immunohistochemical showed that STK31 expressed in the tumor tissue,and it was significantly higher than the adjacent normal tissues;Real time quantitative PCR[(3.65±0.83)vs.(1.05±0.14),P＜0.05] and Western blot also revealed that STK31 expression in tumor tissue were significantly higher than adjacent normal tissues(P＜0.05);CCK8 experiments showed that knockdown STK31 inhibited proliferation of MG63 cell when compared with the control group after 36 h[(1.71±0.17)vs.(1.39±0.11),P＜0.05],72 h[(2.15±0.21)vs.(1.54±0.14),P＜0.05];Tansewell experiments showed that transfection of pGenesil-STK31-shRNA could suppress MG63 cell's migration[(13±4)vs.(55±8),P＜0.05].Conclusion STK31 is overexpression in osteosarcoma with increased biological activity of osteosarcoma cells.

Effect of oxidative stress on the activity of human osteoblastic MG63 cells / 口腔疾病防治

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.

Effects of Baicalin on Human Osteosarcoma MG63 Cells Apoptosis and the Expression of MMPs / 中国药房

OBJECTIVE:To study the effects of baicalin on human osteosarcoma MG63 cells apoptosis and the expression of MMPs. METHODS:Treated with 0(blank control),5,10,20,40,80,160,320 μg/ml baicalin for 24 h,the survival rate,the expression amount of MMP-2 and MMP-9 were detected,and IC50 was calculated. Cell apoptosis was observed. RESULTS:Com-pared with blank control group,after treated with baicalin,survival rate of MG63 cells decreased,while apoptotic amount in-creased,the expression amount of MMP-2 and MMP-9 decreased;there was statistical significance in the expression amount de-crease of MMP-2 and MMP-9 in MG63 cells after treated with 320 μg/ml baicalin(P<0.05);IC50 was(40.21±9.20)μg/ml,all responses were in concentration-dependent manner. CONCLUSIONS:Baicalin can inhibit the proliferation of MG63 cells,induce cell apoptosis,and inhibit the expression of MMP-2 and MMP-9 in cells under high concentration.

Efetividade de scaffolds de poli (butileno adipato-cotereftalato)/ nanohidroxiapatita obtidos por eletrofiação para aplicação biomédica: avaliação in vitro / Effectivenes of novel scaffolds poly (butylene adipate-coterephthalate) / nanohydroxyapatite obtained by electrospinning for biomedical application: In vitro evaluation

A necessidade da fabricação de novos biomateriais que possam, além de mimetizar o tecido ósseo, fornecer resistências mecânicas favoráveis próximas às do tecido ósseo natural têm despertado interesse de pesquisadores com o objetivo de melhorar a qualidade de vida de pessoas que sofreram algum tipo de lesão. Scaffolds de nanofibras poliméricas fabricados por eletrofiação apresentam características tridimensionais (3D) e poros interconectados que permitem a colonização de toda a superfície 3D por células com a consequente formação de tecidos. O scaffold de poli(butileno adipato-co-tereftalato) (PBAT) mostra-se um biomaterial promissor para regeneração óssea, porém tem sido pouco explorado até a data. Embora do uso da HA seja consagrado para uso biomédico, sua utilização em polímeros ainda é pouco estudada, principalmente em associação ao PBAT. Desta forma, o objetivo deste estudo foi avaliar a efetividade in vitro de scaffolds poliméricos (PBAT) com incorporação de nanopartículas de HA (nHAp) em diferentes concentrações, produzidos por eletrofiação, por meio da bioatividade celular e expressão gênica de osteoblast-like MG63. Células (MG63) foram cultivadas sobre scaffolds de PBAT; PBAT/3%nHAp e PBAT/5% nHAp e sem a presença dos mesmos (controle) e avaliadas pelos testes qualitativo (MEV) e quantitativo de adesão e proliferação celular (1 e 7 dias e aos 1, 3, 7, 14 e 21 dias, respectivamente), citotoxicidade celular (1, 3 e 7 dias), corante vermelho de alizarina e formação de mineralização (14 dias)e análise da expressão de genes relacionados à osteogênese por qRT-PCR aos 7, 14 e 21 dias de cultura celular. Os dados foram analisados estatisticamente por variância (ANOVA) e Tukey (p<0,05). Os scaffolds de PBAT e PBAT/nHAp não apresentaram efeito citotóxico e sua arquitetura tridimensional influenciou positivamente na adesão e proliferação celular, formação de matriz mineralizada bem como em alguns períodos na expressão dos genes ALP, Col I, Runx2, OC e OPN em...

The need for the manufacture of new biomaterials that may, in addition to mimic to bone tissue, providing favorable mechanical strength close to natural bone have aroused the interest of researchers in order to improve the quality of life of people who have suffered some kind of injury. Scaffolds polymer nanofibers fabricated by electrospinning have three dimensional features (3D) and interconnected pores that allow the colonization of the entire 3D surface of cells with the consequent formation of tissue. Poly (butylene adipate-co-terephthalate) (PABT) scaffold showed to be a promising biomaterial for bone regeneration, however, has been underexplored todate. Although the use of HA is consecrated to biomedical use, their use in polymers is not well known, especially in association with PBAT. The aim of this study was evaluating in vitro effectiveness of polymeric (PABT) scaffolds with incorporated HA(nHAp) nanoparticles, obtained by electrospinning, through cellular bioactivity and osteoblast-like MG63 gene expression. MG63 cells were grown on PABT; PABT/3%nHAp and PABT/5%nHAp scaffolds and without their presence (control), and evaluated by qualitative (MEV) and quantitative tests of cell adhesion andproliferation (1 and 7 days and at 1, 3, 7, 14 and 21 days, respectively), cell cytotoxicity (1, 3 and 7 days), alizarin red dye and mineralization formation (14 days) and expression of genes related to osteogenesis by qRT-PCR to 7, 14 and 21 days of cell culture. Data were statistically analyzed by variance (ANOVA) and Tukey test (p<0.05). The PBAT and PBAT/nHAp scaffolds showed no cytotoxic effect and its three-dimensional architecture influenced positively in the cell adhesion and proliferation, mineralized matrix formation as well as in some periods the expression of genes ALP, Col I, Runx2, OC and OPN in relation the control group. The osteoconductive and osteoinductive effect of nHAp promoted better cellular response in scaffolds of PABT/nHAp independent.

Efetividade de scaffolds de poli (butileno adipato-cotereftalato)/ nanohidroxiapatita obtidos por eletrofiação para aplicação biomédica: avaliação in vitro / Effectivenes of novel scaffolds poly (butylene adipate-coterephthalate) / nanohydroxyapatite obtained by electrospinning for biomedical application: In vitro evaluation

A necessidade da fabricação de novos biomateriais que possam, além de mimetizar o tecido ósseo, fornecer resistências mecânicas favoráveis próximas às do tecido ósseo natural têm despertado interesse de pesquisadores com o objetivo de melhorar a qualidade de vida de pessoas que sofreram algum tipo de lesão. Scaffolds de nanofibras poliméricas fabricados por eletrofiação apresentam características tridimensionais (3D) e poros interconectados que permitem a colonização de toda a superfície 3D por células com a consequente formação de tecidos. O scaffold de poli(butileno adipato-co-tereftalato) (PBAT) mostra-se um biomaterial promissor para regeneração óssea, porém tem sido pouco explorado até a data. Embora do uso da HA seja consagrado para uso biomédico, sua utilização em polímeros ainda é pouco estudada, principalmente em associação ao PBAT. Desta forma, o objetivo deste estudo foi avaliar a efetividade in vitro de scaffolds poliméricos (PBAT) com incorporação de nanopartículas de HA (nHAp) em diferentes concentrações, produzidos por eletrofiação, por meio da bioatividade celular e expressão gênica de osteoblast-like MG63. Células (MG63) foram cultivadas sobre scaffolds de PBAT; PBAT/3%nHAp e PBAT/5% nHAp e sem a presença dos mesmos (controle) e avaliadas pelos testes qualitativo (MEV) e quantitativo de adesão e proliferação celular (1 e 7 dias e aos 1, 3, 7, 14 e 21 dias, respectivamente), citotoxicidade celular (1, 3 e 7 dias), corante vermelho de alizarina e formação de mineralização (14 dias)e análise da expressão de genes relacionados à osteogênese por qRT-PCR aos 7, 14 e 21 dias de cultura celular. Os dados foram analisados estatisticamente por variância (ANOVA) e Tukey (p<0,05). Os scaffolds de PBAT e PBAT/nHAp não apresentaram efeito citotóxico e sua arquitetura tridimensional influenciou positivamente na adesão e proliferação celular, formação de matriz mineralizada bem como em alguns períodos na expressão dos genes ALP, Col I, Runx2, OC e OPN em ..

The need for the manufacture of new biomaterials that may, in addition to mimic to bone tissue, providing favorable mechanical strength close to natural bone have aroused the interest of researchers in order to improve the quality of life of people who have suffered some kind of injury. Scaffolds polymer nanofibers fabricated by electrospinning have three dimensional features (3D) and interconnected pores that allow the colonization of the entire 3D surface of cells with the consequent formation of tissue. Poly (butylene adipate-co-terephthalate) (PABT) scaffold showed to be a promising biomaterial for bone regeneration, however, has been underexplored todate. Although the use of HA is consecrated to biomedical use, their use in polymers is not well known, especially in association with PBAT. The aim of this study was evaluating in vitro effectiveness of polymeric (PABT) scaffolds with incorporated HA(nHAp) nanoparticles, obtained by electrospinning, through cellular bioactivity and osteoblast-like MG63 gene expression. MG63 cells were grown on PABT; PABT/3%nHAp and PABT/5%nHAp scaffolds and without their presence (control), and evaluated by qualitative (MEV) and quantitative tests of cell adhesion andproliferation (1 and 7 days and at 1, 3, 7, 14 and 21 days, respectively), cell cytotoxicity (1, 3 and 7 days), alizarin red dye and mineralization formation (14 days) and expression of genes related to osteogenesis by qRT-PCR to 7, 14 and 21 days of cell culture. Data were statistically analyzed by variance (ANOVA) and Tukey test (p<0.05). The PBAT and PBAT/nHAp scaffolds showed no cytotoxic effect and its three-dimensional architecture influenced positively in the cell adhesion and proliferation, mineralized matrix formation as well as in some periods the expression of genes ALP, Col I, Runx2, OC and OPN in relation the control group. The osteoconductive and osteoinductive effect of nHAp promoted better cellular response in scaffolds of PABT/nHAp independent.

St. John's Wort (Hypericum perforatum) stimulates human osteoblastic MG-63 cell proliferation and attenuates trabecular bone loss induced by ovariectomy

BACKGROUND/OBJECFTIVES: The effect of St. John's Wort extract (SJW) on MG-63 cell proliferation and trabecular bone loss induced by ovariectomy was examined. MATERIALS/METHODS: Proliferation, expression of estrogen receptor (ER) alpha and ER beta, and gene expressions of osteoprotegerin (OPG), osteocalcin (OC) and alkaline phosphatase (ALP) were examined in MG-63 cells treated with or without SJW. Ovariectomized rats were treated with SJW at the dose of 100 or 200 mg/kg/day, beta-estradiol-3-benzoate (E2), or vehicle only (OVX-C), and sham operated rats were treated with vehicle only (Sham-C). Serum ALP and C-telopeptide (CTX), and femoral trabecular bone loss were examined. RESULTS: SJW increased MG-63 cell proliferation and expression of ER alpha and ER beta, and positive effect was shown on gene expressions of ALP, OC and OPG. SJW also showed estrogen like effect on bone associated with slowing down in trabecular bone loss. Histopathology by H&E showed rats treated with SJW displayed denser structure in metaphyseal region of distal femur compared with rats in OVX-C. SJW was shown to reduce serum CTX in OVX rats. CONCLUSION: The present study provides new insight in preventing estrogen deficiency induced bone loss of SJW and possibility for its application in bone health supplement.