Proliferation Of Osteosarcoma MG-63 Cells Research Articles

Arthpyrone L, a New Pyridone Alkaloid from a Deep-Sea Arthrinium sp., Inhibits Proliferation of MG63 Osteosarcoma Cells by Inducing G0/G1 Arrest and Apoptosis.

Fractionation of the ethanol extract of a marine fungus, Arthrinium sp., afforded a new pyridone alkaloid (arthpyrone L (1)), the structure with absolute configuration of which was established by comprehensive spectroscopic analyses. In vitro cell viability assays revealed that compound 1 showed antiproliferative effects toward human A549 (lung), MG63, U2OS (bone), MCF-7 and MDA-MB-231 (breast) cancer cells. MG63 cell lines were chosen for further biological evaluations and presented apoptosis and cell cycle arrest (G0/G1 phase) upon treatment of 1. Subsequent mechanism studies demonstrated that the growth inhibition of 1 against MG63 cells was via activation of caspase-modulated apoptotic pathway and inhibition of PI3K/Akt pathway.

Three-dimensionally printed polycaprolactone/multicomponent bioactive glass scaffolds for potential application in bone tissue engineering

AbstractOver the last years, three-dimensional (3D) printing has been successfully applied to produce suitable substitutes for treating bone defects. In this work, 3D printed composite scaffolds of polycaprolactone (PCL) and strontium (Sr)- and cobalt (Co)-doped multi-component melt-derived bioactive glasses (BGs) were prepared for bone tissue engineering strategies. For this purpose, 30% of as-prepared BG particles (size <38 μm) were incorporated into PCL, and then the obtained composite mix was introduced into a 3D printing machine to fabricate layer-by-layer porous structures with the size of 12 × 12 × 2 mm3.The scaffolds were fully characterized through a series of physico-chemical and biological assays. Adding the BGs to PCL led to an improvement in the compressive strength of the fabricated scaffolds and increased their hydrophilicity. Furthermore, the PCL/BG scaffolds showed apatite-forming ability (i.e., bioactivity behavior) after being immersed in simulated body fluid (SBF). The in vitro cellular examinations revealed the cytocompatibility of the scaffolds and confirmed them as suitable substrates for the adhesion and proliferation of MG-63 osteosarcoma cells. In conclusion, 3D printed composite scaffolds made of PCL and Sr- and Co-doped BGs might be potentially-beneficial bone replacements, and the achieved results motivate further research on these materials.

Effects of rapamycin on osteosarcoma cell proliferation and apoptosis by inducing autophagy.

To investigate the influences of rapamycin on proliferation and apoptosis of human osteosarcoma MG-63 cells and the mechanisms of action. The human osteosarcoma MG-63 cells were randomly divided into Control group, Rapamycin group, and Rapamycin + Beclin-1 plasmid transfection group. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was adopted to detect the viability of MG-63 cells in each group, and the 5-Ethynyl-2'-deoxyuridine (EdU) staining and Hoechst staining were applied to determine the proliferation and apoptosis, respectively, of MG-63 cells in each group. The levels of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured using enzyme-linked immunosorbent assay (ELISA) kits, and the protein expression levels of Beclin-1 and Vps34 in each group of MG-63 cells were tested using the Western blotting. Compared with the Control group, Rapamycin group, and Rapamycin + Beclin-1 plasmid transfection group had markedly weakened the viability of MG-63 cells, inhibited cell proliferation, remarkably increased cell apoptosis rate, elevated Bax level, notably declined Bcl-2 level, and significantly raised the levels of Beclin-1 and Vps34 proteins in MG-63 cells. Besides, the effects in Beclin-1 plasmid transfection group were stronger. Rapamycin may decrease the viability, inhibit the proliferation, and promote the apoptosis of MG-63 cells by activating autophagy.

Effect of plasma membrane-associated sialidase NEU3 activity on the proliferation and apoptosis of osteosarcoma MG-63 cells

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 localization 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 0.05). 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 significant (F=23.21, 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 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. Key words: Neuraminidase; Osteosarcoma; Cell proliferation; Apoptosis; MG-63 cells

Effects of microRNA-122 on proliferation and invasion of osteosarcoma cells and mechanisms

Objective To investigate the effects of microRNA (miRNA, miR)-122 on the proliferation and invasion of osteosarcoma MG-63 cells and the related mechanisms. Methods The expression of miR-122 in osteosarcoma MG-63 cells was detected by real-time quantitative polymerase chain reaction (Real-time PCR) after miR-122 mimic transfection. After the validation of miR-122 overexpression, cell count kit-8 (CCK-8) assay was used to examine the proliferation after miR-122 mimic transfection; Would scratch assay was performed to evaluate the migration activity of MG-63 cells, and the Transwell assay to determine the invasion activity. Then the expression levels of sex determining region Y-box 2 (SOX2) and matrix metalloproteinase (MMP)-9 were detected by Western blotting. Results After transfection of miR-122 mimic into OS MG-63 cells, the relative expression of miR-122 in mimic group (3.83±0.45) was significantly increased as compared with the negative-control (NC, 1.05±0.09) group (P<0.01). The proliferation at 24 and 48 h after miR-122 mimic transfection was significantly suppressed as compared with the NC group (P<0.05). Would scratch assay showed the migration activity in mimic group (0.47±0.04) was inhibited significantly as compared with the NC group (0.93±0.07, P<0.01). Transwell assay showed the invasive cells were significantly reduced in mimic group (84.01±5.69) as compared with the NC group (122.30±8.11, P<0.05). After the transfection, Western blotting results revealed that the relative expression of SOX2 in mimic group (0.51±0.10) decreased remarkably as compared with NC group (1.10±0.04, P<0.01), and that of MMP-9 in mimic group (0.40±0.06) was significantly lower than NC group (0.99±0.11, P<0.01) as well. Conclusion MiR-122 inhibited the proliferation and invasion of osteosarcoma MG-63 cells, which was probably related with the down-expression of SOX2 and MMP-9. Key words: Osteosarcoma; MicroRNA-122; Proliferation; Invasion

Preparation and Characterization of HAp Coated Chitosan‐Alginate PEC Porous Scaffold for Bone Tissue Engineering

Recently, various scaffolds for bone tissue engineering have been developed and analyzed using polymers and bioactive ceramics. The development of an interconnected bioceramic‐biopolymer scaffold combines the advantage of both the components to meet mechanical and physiological requirements of the host tissue. In the present study, development of 3D porous hybrid scaffold using chitosan‐alginate polyelectrolyte complex (CAPEC) by freeze drying method, has been reported. The CaCl2 cross‐linked CAPEC scaffold was further coated with synthesized HAp (Hydroxyapatite) by dip coating technique. The HAp was synthesized by wet chemical precipitation method. The CAPEC and HAp coated‐CAPEC (CAPEC/HAp) scaffolds were assessed for their porosity, pore size, morphology, mechanical strength, swelling behavior, in‐vitro biodegradation. The pore size and morphology of developed scaffolds were studied by scanning electron microscope (SEM). The pore size ranged from 30 to 280µm for CAPEC scaffolds and no apparent change in pore size was found in case of CAPEC/HAp. Both the scaffolds possessed desired pore size with interconnected pore network. Whereas, the porosity was decreased with the increase in HAp coating, but it is still high enough for bone tissue engineering application. The mechanical strength of CAPEC scaffold increased with the coating of HAp, compressive strength was increased from 0.469 MPa to 0.61 MPa. The CAPEC/HAp scaffolds also exhibited favorable swelling behavior and biodegradation. Further, the developed scaffolds also supported in vitro attachment and proliferation of MG63 osteosarcoma cells. Overall, the study has demonstrated that the developed CAPEC/HAp scaffolds have potential application in non‐load bearing, cancellous bone tissue engineering applications.

吴茱萸碱抑制Wnt/β-catenin信号通路诱导骨肉瘤MG-63细胞凋亡

Objective To investigate the effects and mechanism of evodiamine on the proliferation and apoptosis of osteosarcoma MG-63 cells. Methods MG-63 cells were cultured with evodiamine for 24 hours, and the cell proliferation was evaluated by methyl thiazolyl tetrazolium (MTT) assay. Cell cycle arrest, apoptosis and intracellular Ca2+ accumulation were evaluated by flow cytometry. BALB/C mice model of osteosarcoma was established to investigate the tumor inhibitory effect of evodiamine on human osteosarcoma. Wnt/β-catenin signaling protein expressions in osteosarcoma cells were detected by Western blotting. Results As concentration of evodiamine increasing (0.25 μmol/L, 0.5 μmol/L, 1.0 μmol/L, 2.0 μmol/L and 4.0 μmol/L), the inhibition rate of MG-63 cells increased [(4.18±1.26)%, (15.49±2.26)%, (40.55±6.57)%, (49.87±7.69)% and (60.42±8.29)%]. The difference was statistically significant between 2.0 μmol/L group and the control group (t=-2.66, P<0.05). MG-63 cells were cultured with 2.0 μmol/L evodiamine for 24 hours, and the apoptotic rate was (64.67±8.63)%, the proportion of S phase cells was (85.33±9.31)%, the fluorescence of Ca2+ was 97.33±21.31. The corresponding data of the control group were (4.94±0.81)%, (43.67±8.92)% and 28.67±8.92, the differences were statistically significant (t=-11.90, P<0.05; t=-7.22, P<0.05; t=-6.65, P<0.05). On mice model, the tumor weight of evodiamine group and the control group was (2.15±0.35)g and (4.29±0.49)g respectively, the difference was statistically significant (t=7.95, P<0.05). Comparing with the control group (1.00±0.00), evodiamine decreased the expression of β-catenin protein (0.72±0.36) and increased the expressions of Bax (1.15±0.27) and Caspase-3 (1.46±0.18) protein, and the differences were statistically significant (t=-3.05, P<0.05; t=-6.42, P<0.05; t=-5.85, P<0.05). Conclusion Evodiamine inhibits proliferation and induces apoptosis of human osteosarcoma MG-63 cells by blocking Wnt/β-catenin signaling. Key words: Evodiamine; Osteosarcoma; Cell proliferation; Apoptosis; Wnt/β-catenin

Discovery of human lactate dehydrogenase A (LDHA) inhibitors as anticancer agents to inhibit the proliferation of MG-63 osteosarcoma cells.

Human lactate dehydrogenase A (LDHA) has been identified as a potential therapeutic target in the area of cancer metabolism. Herein, we report the discovery of novel LDHA inhibitors through docking-based virtual screening and biological assays. The primary enzymatic assay suggested that compound 11 targeted LDHA with an IC50 value of 0.33 μM. The in vitro cytotoxic assay demonstrated that compound 11 reduced the growth of MG-63 cancer cells with an EC50 value of 3.35 μM. Finally, we found that compound 11 induced the apoptosis of MG-63 cancer cells in a dose dependent manner, upregulated the oxygen consumption rate (OCR), and decreased the lactate formation and extracellular acidification rate (ECAR) in MG-63 cancer cells. Collectively, our data suggested that compound 11 could be a promising lead for the development of potent LDHA inhibitors.

Crosstalk between Beclin-1-dependent autophagy and caspase‑dependent apoptosis induced by tanshinoneIIA in human osteosarcoma MG-63 cells.

The aim of the present study was to ascertain whether or not autophagy is induced by tanshinone IIA (TanIIA), and to explore the crosstalk between autophagy and apoptosis in regards to the antitumor effects of TanIIA on MG-63 cells and the potential mechanism. MG-63 cells were cultured in vitro with various concentrations of TanIIA (0, 2.5, 5, 10 and 20 mg/l) for 0, 24, 48 and 72 h, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to evaluate the inhibition of the proliferation of osteosarcoma MG-63 cells by TanIIA or in the presence/absence of chloroquine (CQ). Autophagic vacuoles and characteristic autophagosomes were observed by transmission electron microscopy (TEM). TanIIA-induced autophagy in MG-63 cells was confirmed by GFP-LC3 punctate fluorescence. The expression levels of apoptosis-related proteins caspase-3, caspase-8, caspase-9 and cleaved-PARP and autophagy-related proteins LC3II/LC3I and Beclin-1 were detected by western blotting. FITC-Annexin V/propidium iodide (PI) staining, flow cytometry and Hoechst 33258 staining were used to analyze the apoptotic rate. Fluorescence intensity of reactive oxygen species (ROS) was examined under a fluorescence microscope using an analysis software system. Cell proliferation was obviously inhibited by TanIIA in a dose- and time-dependent manner. Generation of autophagy was triggered by TanIIA (0–20 mg/l) treatment, and in a Beclin-1-dependent manner. Compared with the control group, the apoptosis ratio following treatment with 2.5 mg/l TanIIA failed to achieve statistical significance. Expression of caspase-3, -8 and -9, and cleaved-PARP in the other groups was gradually enhanced in dose-dependent manner. Our analysis also suggested that the influence of autophagy on TanIIA cytotoxicity had a phase effect; with low-dose drugs and shorter treatment periods, autophagy functioned as a damage repair mechanism. In conrast, when the cells were treated with higher doses of TanIIA for longer treatment periods, autophagic cell death contributed to apoptosis. Furthermore, generation of ROS occurred in a dose-dependent manner and pretreatment with NAC, a selective ROS scavenger, blocked the coexistence of Beclin-1 autophagy and caspase-dependent apoptosis. In conclusion, our findings provide strong evidence that TanIIA may be a potential therapeutic drug against osteosarcoma. Moreover, its cytotoxity can be enhanced with ROS agonists.

Improvement of poly(ester-urethane) surface properties by RAFT mediated grafting initiated by gamma radiation

RAFT (ang. Reversible Addition–Fragmentation chain Transfer) polymerization has been considered as one of the most successful techniques for the preparation of polymers with predetermined molecular weights, narrow molecular weight distributions and high degree of chain end functionalities. This method was used for grafting of N-isopropylacrylamide onto polyurethane (PUR) surface to improve biocompability of the matrix surface. The pristine and grafted samples were investigated by the following surface characterization techniques: ATR-FTIR spectroscopy and scanning electron microscopy (SEM). The analysis of paramagnetic species was performed by EPR spectroscopy in order to evaluate the ability of PUR to form covalent bonds with monomer molecules. Grafting of PNIPAAm onto PUR surface in controlled manner was confirmed by GPC. The wettability of PUR and PUR-g-PNIPAAm surfaces was evaluated by contact angle (CA) measurements. Preliminary in vitro cytotoxicity experiments were carried out to study biocompability of pristine and modified samples using MTT assay. Attachment and proliferation of MG-63 osteosarcoma cells on polyurethane films before and after radiation grafting was investigated to assess the potential use of this material for biomedical applications.The applied modification resulted in surface changes of poly(ester-urethane). Polyurethane matrix coated by poly(N-isopropylacrylamide), the polymer classified to the group of thermosensitive polymers, exhibited hydrophilic properties in water at 25°C, while at 35°C the surface properties change into hydrophobic. Additionally, narrow molecular weight distribution of the grafted chains was obtained, uniformly covering the surface of the matrix. In vitro studies confirmed that the modified matrices are not cytotoxic when degree of NIPAAm grafting is 14% and number-average molecular weight of the grafted chains (Mn) is about 40,000g/mol.

LY294002 suppresses the malignant phenotype and sensitizes osteosarcoma cells to pirarubicin chemotherapy.

Pirarubicin is frequently used in chemotherapy against tumors. However, clinical use is severely limited by the development of progressive dose-dependent cardiomyopathy and acquired drug resistance. LY294002 is a commonly used pharmacologic inhibitor, which selectively inhibits the phosphoinositide 3-kinase-AKT nexus. The aim of this study was to investigate the combined inhibitory effect of LY294002 and pirarubicin on human osteosarcoma (OS) cells in vitro. The inhibitory effect of LY294002 plus pirarubicin on U2-OS and MG-63 OS cell proliferation, apoptosis, migration and invasion was investigated by cell proliferation, wound healing and Transwell invasion assays. The results revealed that LY294002 and pirarubicin synergistically induced apoptosis, and inhibited the growth, migration and invasion of OS cells. This indicates that LY294002 enhanced the effects of pirarubicin on OS in vitro. LY294002 combined with pirarubicin may thus be a future therapeutic strategy in OS.

Cell adhesion and growth on ultrananocrystalline diamond and diamond-like carbon films after different surface modifications

Diamond and diamond-like carbon (DLC) films possess a set of excellent physical and chemical properties which together with a high biocompatibility make them attractive candidates for a number of medical and biotechnological applications. In the current work thin ultrananocrystalline diamond (UNCD) and DLC films were comparatively investigated with respect to cell attachment and proliferation after different surface modifications. The UNCD films were prepared by microwave plasma enhanced chemical vapor deposition, the DLC films by pulsed laser deposition (PLD). The films were comprehensively characterized with respect to their basic properties, e.g. crystallinity, morphology, chemical bonding nature, etc. Afterwards the UNCD and DLC films were modified applying O2 or NH3/N2 plasmas and UV/O3 treatments to alter their surface termination. The surface composition of as-grown and modified samples was studied by X-ray photoelectron spectroscopy (XPS). Furthermore the films were characterized by contact angle measurements with water, formamide, 1-decanol and diiodomethane; from the results obtained the surface energy with its dispersive and polar components was calculated. The adhesion and proliferation of MG63 osteosarcoma cells on the different UNCD and DLC samples were assessed by measurement of the cell attachment efficiency and MTT assays. The determined cell densities were compared and correlated with the surface properties of as-deposited and modified UNCD and DLC films.

VEGFR, RET, and RAF/MEK/ERK Pathway Take Part in the Inhibition of Osteosarcoma MG63 Cells with Sorafenib Treatment

Osteosarcoma (OS) is the leading primary malignant bone tumor in children and young adults. It is response for a high mortality rate. Nowadays, few researches have been performed on sorafenib against OS and no tools are available to guide the use of sorafenib in the OS treatment. In this study, we aim to investigate the effect of sorafenib on OS cell MG63 and figure the potential effective molecular pathway of its function. In the present study, we performed assays of cell proliferation, RT-PCR, and western blot to investigate the effect of sorafenib on OS MG63 cells and to elucidate the molecular actions of sorafenib against RTKs VEGFR2 and RET, as well as MEK/ERK signaling pathway. The present study confirmed that sorafenib could inhibit the proliferation of OS MG63 cells and caused a series of biomolecule effects, including the change of VEGFR2 and ERK gene expression, and the phosphorylation alteration of VEGFR2, RET, and MEK1 proteins. VEGFR2, RET, and MEK/ERK signaling pathway are involved in the pharmacological mechanism of sorafenib. They are potential candidate targets for OS treatment.

Effects of siRNA-mediated Cdc2 silencing on MG63 cell proliferation and apoptosis

The present study aimed to determine the effect of small interfering RNA (siRNA)‑induced inhibition of cyclin‑dependent kinase 2 (Cdc2) expression on osteosarcoma MG63 cell proliferation and apoptosis. An siRNA expression plasmid, psilencer 2.1‑U6/Cdc2, targeting the Cdc2 gene, and a control psilencer 2.1‑U6/Scramble plasmid were constructed and transfected into MG63 cells using liposomes. Cdc2 expression in the MG63 cells was investigated by western blot analysis and real‑time polymerase chain reaction. Cell morphology was also examined. The effects of psilencer 2.1‑U6/Cdc2 on MG63 cell proliferation and the cell cycle were detected via MTT and flow cytometry, respectively. Expression levels of apoptosis‑related molecules, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X (Bax) were determined by western blot analysis. MG63 cells stably transfected with the psilencer 2.1‑U6/Cdc2 plasmid (MG63‑siRNA/Cdc2) and negative control cells, MG63‑siRNA/Scramble, were successfully obtained. The silencing efficiencies of the Cdc2‑expressing mRNA and protein in MG63‑siRNA/Cdc2 were 86 and 89% of that of the control MG63‑siRNA/Scramble cells, respectively. Interference of Cdc2 expression inhibited MG63 cell proliferation and was demonstrated to significantly increase and decrease cells in the G2/M and S phases, respectively. Cdc2 expression silencing had negligible effects on Bcl‑2 and Bax expression in MG63 cells. In conclusion, silencing of Cdc2 expression suppresses proliferation of osteosarcoma MG63 cells but has negligible effects on apoptosis.

Regulation of Osteoblast Differentiation: A Novel Function for Fibroblast Growth Factor 8

Several members of the fibroblast growth factor (FGF) family have an important role in the development of skeletal tissues. FGF-8 is widely expressed in the developing skeleton, but its function there has remained unknown. We asked in this study whether FGF-8 could have a role in the differentiation of mesenchymal stem cells to an osteoblastic lineage. Addition of FGF-8 to mouse bone marrow cultures effectively increased initial cell proliferation as well as subsequent osteoblast-specific alkaline phosphatase production, bone nodule formation, and calcium accumulation if it was added to the cultures at an early stage of osteoblastic differentiation. Exogenous FGF-8 also stimulated the proliferation of MG63 osteosarcoma cells, which was blocked by a neutralizing antibody to FGF-8b. In addition, the heparin-binding growth factor fraction of Shionogi 115 (S115) mouse breast cancer cells, which express and secrete FGF-8 at a very high level, had an effect in bone marrow cultures similar to that of exogenous FGF-8. Interestingly, experimental nude mouse tumors of S115 cells present ectopic bone and cartilage formation as demonstrated by typical histology and expression of markers specific for cartilage (type II and IX collagen) and bone (osteocalcin). These results demonstrate that FGF-8 effectively predetermines bone marrow cells to differentiate to osteoblasts and increases bone formation in vitro. It is possible that FGF-8 also stimulates bone formation in vivo. The results suggest that FGF-8, which is expressed by a great proportion of malignant breast and prostate tumors, may, among other factors, also be involved in the formation of osteosclerotic bone metastases.