Human Osteosarcoma MG-63 Cells Research Articles

Evaluation of Pulsed Electromagnetic Field Effects: A Systematic Review and Meta-Analysis on Highlights of Two Decades of Research In Vitro Studies.

Pulsed electromagnetic field (PEMF) therapy is a type of physical stimulation that affects biological systems by producing interfering or coherent fields. Given that cell types are significantly distinct, which represents an important factor in stimulation, and that PEMFs can have different effects in terms of frequency and intensity, time of exposure, and waveform. This study is aimed at investigating if distinct positive and negative responses would correspond to specific characteristics of cells, frequency and flux density, time of exposure, and waveform. Necessary data were abstracted from the experimental observations of cell-based in vitro models. The observations were obtained from 92 publications between the years 1999 and 2019, which are available on PubMed and Web of Science databases. From each of the included studies, type of cells, pulse frequency of exposure, exposure flux density, and assayed cell responses were extracted. According to the obtained data, most of the experiments were carried out on human cells, and out of 2421 human cell experiments, cell changes were observed only in 51.05% of the data. In addition, the results pointed out the potential effects of PEMFs on some human cell types such as MG-63 human osteosarcoma cells (p value < 0.001) and bone marrow mesenchymal stem cells. However, human osteogenic sarcoma SaOS-2 (p < 0.001) and human adipose-derived mesenchymal stem cells (AD-MSCs) showed less sensitivity to PEMFs. Nevertheless, the evidence suggests that frequencies higher than 100 Hz, flux densities between 1 and 10 mT, and chronic exposure more than 10 days would be more effective in establishing a cellular response. This study successfully reported useful information about the role of cell type and signal characteristic parameters, which were of high importance for targeted therapies using PEMFs. Our findings would provide a deeper understanding about the effect of PEMFs in vitro, which could be useful as a reference for many in vivo experiments or preclinical trials.

Analysis and Construction of a Competitive Endogenous RNA Regulatory Network of Baicalin-Induced Apoptosis in Human Osteosarcoma Cells.

Background Baicalin is an extract from the traditional Chinese herb Scutellaria baicalensis and has the potential to treat osteosarcoma (OS). However, the transcriptome-level mechanism of baicalin-mediated antitumor effects in OS has not yet been investigated. The aim of this study was to analyze the competitive endogenous RNA (ceRNA) regulatory network involved in baicalin-induced apoptosis of OS cells. Methods In this study, CCK-8 and flow cytometry assays were used to detect the antitumor effects of baicalin on human OS MG63 cells. Furthermore, transcriptome sequencing was employed to establish the long noncoding RNA (lncRNA), microRNA (miRNA), and mRNA profiles. Results Baicalin inhibited MG63 cell proliferation and induced apoptosis. Totals of 58 lncRNAs, 31 miRNAs, and 2136 mRNAs in the baicalin-treated MG63 cells were identified as differentially expressed RNAs compared to those in control cells. Of these, 2 lncRNAs, 3 miRNAs, and 18 mRNAs were included in the ceRNA regulatory network. The differentially expressed RNAs were confirmed by quantitative real-time PCR (qRT-PCR). Conclusions By identifying the ceRNA network, our results provide new information about the possible molecular basis of baicalin, which has potential applications in OS treatment.

Ca2+ -activated K+ channel KCa 1.1 as a therapeutic target to overcome chemoresistance in three-dimensional sarcoma spheroid models.

The large‐conductance Ca2+‐activated K+ channel KCa1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three‐dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). KCa1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. KCa1.1 activator‐induced hyperpolarizing responses were significantly larger in human osteosarcoma MG‐63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of KCa1.1 and its role in chemoresistance using a 3D spheroid model. KCa1.1 protein expression levels were significantly elevated in the lipid‐raft‐enriched compartments of MG‐63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to KCa1.1 protein degradation in breast cancer. The siRNA‐mediated inhibition of FBXW7 in MG‐63 cells from 2D monolayers upregulated KCa1.1 protein expression. Furthermore, a treatment with a potent and selective KCa1.1 inhibitor overcame the chemoresistance of the MG‐63 and human chondrosarcoma SW‐1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP‐binding cassette transporters, the expression of the multidrug resistance‐associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of KCa1.1. Therefore, the pharmacological inhibition of KCa1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of KCa1.1‐positive sarcomas.

Dieckol exerts anticancer activity in human osteosarcoma (MG-63) cells through the inhibition of PI3K/AKT/mTOR signaling pathway.

BackgroundOsteosarcoma (OS) is the most common malignant bone cancer with more metastasis and increased occurrence in children and teen-agers and being responsible for more number of morbidity and mortality worldwide.ObjectiveThe current exploration was planned study the in vitro anticancer actions of dieckol against human OS MG-63 cells via PI3K/AKT/mTOR signaling inhibition.MethodologyThe cytotoxicity of dieckol was scrutinized by MTT assay. Effects of dieckol on the ROS accumulation, apoptotic cell death, and MMP level in the MG-63 cells were studied by respective fluorescence staining assays. The levels of proliferative, inflammatory, and apoptotic markers in the dieckol treated MG-63 cells were scrutinized by marker specific kits. The expressions of PI3K, AKT, and mTOR was assayed by RT-PCR.ResultsThe MTT assay revealed that the dieckol dose dependently prevented MG-63 cells viability and the IC50 was found at 15 µM. Dieckol treatment effectively reduced the MMP level and improved the ROS generation and apoptosis in MG-63 cells. Dieckol also regulated the proliferative (cyclin D1), inflammatory (COX-2, IL-6, TNF-α, and NF-κB), and apoptotic (caspase-3, Bax, Bcl-2) markers in the MG-63 cells. The PI3K/AKT/mTOR signaling in the MG-63 cells were effectively inhibited by the dieckol treatment.ConclusionIn conclusion, our findings from this study recommends that the dieckol could be a talented anticancer candidate for the OS management in the future.

Biocompatibility assessment and antiproliferative activity of Detarium microcarpum Guill. and Perr. fruit pulp extracts.

The consumption of tropical fruits rich in chemopreventive compounds are required to prevent cells carcinogenesis and proliferation. This study was designed to assess de biocompatibility of Detarium microcarpum fruit extract on normal fibroblasts and its antiproliferative potentiality on human osteosarcoma MG-63 cells. Primary dermal fibroblasts and human osteosarcoma MG-63 cells were treated with different concentrations of hexane, chloroform, ethyl acetate and methanol extracts of D. microcarpum fruit pulp for 24h, 48h and 72h. The biocompatibility property of extracts on the normal fibroblasts and its antiproliferative activity on the human osteosarcoma cells were evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay. The biocompatibility study of D. microcarpum fruit pulp showed that the chloroform extract has exhibited the highest cytotoxic effect on normal fibroblasts followed by the ethyl acetate extract. Hexane extract wasn’t cytotoxic at concentrations of 125 and 250 µg/mL but caused more than 80 % of cell death at a concentration of 500 µg/mL. Methanol extract didn’t show a significant cytotoxic effect. Furthermore, chloroform and ethyl acetate extracts showed the best antiproliferative activity on osteosarcoma cells. A complete cell death was observed when osteosarcoma cells were treated with ethyl acetate extract at all concentrations while chloroform extract at concentrations of 250 and 500 µg/mL caused a complete cells death. Methanol extract exhibited any antiprolifarative activity. Chloroform and ethyl acetate extract of fruits pulp of D. microcapum are potent source of anticancer phytomolecules and have potential to be a promising anti-osteosarcoma extract.

GO-Y078, a Curcumin Analog, Induces Both Apoptotic Pathways in Human Osteosarcoma Cells via Activation of JNK and p38 Signaling.

Osteosarcoma is the most common primary bone malignancy in teenagers and continues to confer a generally poor prognosis due to its highly metastatic potential. Poor solubility in water and instability of curcumin limits its bioavailability for use in the adjuvant situation to improve the prognosis and the long-term survival of patients with osteosarcoma. To further obtain information regarding the apoptosis induced by a new curcumin analog, GO-Y078, in human osteosarcoma cells, flow cytometric analysis, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. GO-Y078 dose-dependently decreased viabilities of human osteosarcoma U2OS, MG-63, 143B, and Saos-2 cells and induced sub-G1 fraction arrest and apoptosis in U2OS and 143B cells. In addition to the effector caspase 3 and poly adenosine diphosphate-ribose polymerase, GO-Y078 significantly activated both initiators of extrinsic caspase 8 and intrinsic caspase 9, whereas cellular inhibitors of apoptosis 1 (cIAP-1) and X-chromosome-linked IAP (XIAP) in U2OS and 143B cells were significantly repressed. Moreover, GO-Y078 increased phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2, and p38 in U2OS and 143B cells. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), GO-Y078′s increases in cleaved caspases 8, 9, and 3 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Altogether, GO-Y078 simultaneously induces both apoptotic pathways and cell arrest in U2OS and 143B cells through activating JNK and p38 signaling and repressing IAPs. These findings contribute to a better understanding of the mechanisms responsible for GO-Y078′s apoptotic effects on human osteosarcoma cells.

Coumarins from Angelica dahurica and Their Antitumor Activities in Human MG-63 Osteosarcoma Cells

A new coumarin, angedahurin A (1), and seven known analogues (2−8), were isolated from the roots of Angelica dahurica. Their structures were identified by extensive NMR, IR, and HR-ESIMS spectroscopic analyses. The cytotoxicities of coumarins 1−8 against MG-63 human osteosarcoma cell lines were screened. Compound 1 showed significant cytotoxic effects against MG-63 with an IC50 value of 7.2 uM，for comparison, the positive control, 5-FU, had an IC50 value of 32.4 uM. Morphological features of apoptosis activities were evaluated in 1-induced MG-63 cells and the results confirmed MG-63 cell apoptosis in a dose-dependent manner.

MiR‐124‐3p signaling in the control of metastasis‐related cytoskeleton organization, cell junctions and adhesion

Our recent studies using novel bioengineered miR-124-3p molecules have demonstrated the functions of miR-124-3p in the inhibition of human osteosarcoma cell invasiveness in vitro and lung metastasis in vivo. This study is to investigate the molecular and cellular mechanisms by which miR-124-3p controls tumor metastasis. LC-MS-based proteomics study identified a set of proteins selectively and significantly downregulated by bioengineered miR-124-3p, which were assembled into multiple biological pathways critical for cancer, such as cell junctions and focal adhesion. Among these proteins, we identified and verified plectin (PLEC) as a new direct target for miR-124-3p that links cytoskeleton to intercellular junctions. Associated with significantly lower levels of PLEC, ITGB1, IQGAP1 and CDH2 proteins in miR-124-3p-treated human osteosarcoma 143B and MG63 cells as well as NSCLC A549 cells, cytoskeleton was remodeled and cell-cell junctions were sharply inhibited. Furthermore, miR-124-3p decreased the ability of cells to form focal adhesion complexes and plaques, leading to much lower levels of cell adhesion capacity. Together, our results connect miR-124-3p-PLEC to other known elements in the control of cytoskeleton, cell adhesion and junctions essential for cancer cell invasion and extravasation towards metastasis.

Synthesis, Characterization, Antibacterial Properties, and In Vitro Studies of Selenium and Strontium Co-Substituted Hydroxyapatite.

In this study, as a measure to enhance the antimicrobial activity of biomaterials, the selenium ions have been substituted into hydroxyapatite (HA) at different concentration levels. To balance the potential cytotoxic effects of selenite ions (SeO32−) in HA, strontium (Sr2+) was co-substituted at the same concentration. Selenium and strontium-substituted hydroxyapatites (Se-Sr-HA) at equal molar ratios of x Se/(Se + P) and x Sr/(Sr + Ca) at (x = 0, 0.01, 0.03, 0.05, 0.1, and 0.2) were synthesized via the wet precipitation route and sintered at 900 °C. The effect of the two-ion concentration on morphology, surface charge, composition, antibacterial ability, and cell viability were studied. X-ray diffraction verified the phase purity and confirmed the substitution of selenium and strontium ions. Acellular in vitro bioactivity tests revealed that Se-Sr-HA was highly bioactive compared to pure HA. Se-Sr-HA samples showed excellent antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus carnosus) bacterial strains. In vitro cell–material interaction, using human osteosarcoma cells MG-63 studied by WST-8 assay, showed that Se-HA has a cytotoxic effect; however, the co-substitution of strontium in Se-HA offsets the negative impact of selenium and enhanced the biological properties of HA. Hence, the prepared samples are a suitable choice for antibacterial coatings and bone filler applications.

Apoptotic activity of demethoxycurcumin in MG-63 human osteosarcoma cells

Demethoxycurcumin (DMC), which is a curcuminoid found in turmeric, has anti-proliferative effects on cancer cells. However, the effect of DMC on osteosarcoma has not been established. The aim of this study was to examine the effects of DMC on cell growth and apoptosis induction in MG-63 human osteosarcoma cells. This study was investigated using 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromid assay, Live/Dead cell assay, 4’, 6-diamidino-2-phenylindole staining, and immunoblotting in MG-63 cells. DMC induced MG-63 cell death in a dosedependent manner, with an estimated IC50 value of 54.4 μM. DMC treatment resulted in nuclear condensation in MG-63 cells. DMC-induced apoptosis in MG-63 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting results showed that Bcl-2 and Bcl-xL were downregulated, while Bax and Bad were upregulated by DMC in MG-63 cells. These results indicated that DMC inhibits cell proliferation and induces apoptotic cell death in MG-63 human osteosarcoma cells via the death receptormediated extrinsic apoptotic pathway and mitochondria-mediated intrinsic apoptotic pathway.

Strontium and selenium doped bioceramics incorporated polyacrylamide-carboxymethylcellulose hydrogel scaffolds: mimicking key features of bone regeneration

ABSTRACT Polyacrylamide-carboxymethylcellulose hydrogels lack mechanical strength, uncontrolled rate of degradation, and poor osteogenic properties which can be improved by the incorporation of bioceramics. Strontium and selenium doped bioceramics xSrO.(43-x)CaO.40SiO2.(12-y)P2O5.ySeO3.5MgO (where x = 6 mol%, y = 3 mol%) incorporated polyacrylamide-carboxymethylcellulose hydrogels were synthesized by free-radical polymerization. Above mentioned bioceramics were prepared by the sol-gel route. XRD analysis indicated an increase in crystallinity with the increment of bioceramics content. With the incorporation of bioceramics, the apatite phase formation was observed on 1st day of immersion in simulated body fluid at pH 7.4. Strontium and selenium doped bioceramics impregnated hydrogels exhibited an inhibitory effect on human osteosarcoma MG63 cell line. Moreover, bioceramics incorporated hydrogels provide a conducive environment for MC3T3-E1 osteoblast cell line proliferation, adhesion and also exhibited good alkaline phosphatase activity. Synthesized hydrogels indicated special osteogenic properties and can be potentially utilized in the hard tissue regeneration as well as for recovery of bone cancer patients.

RETRACTED ARTICLE: MiR-151-3p transferred by cancer-associated fibroblast-derived extracellular vesicles promotes osteosarcoma progression through the CHL1/integrin 1\u03b2/TGF-\u03b2 axis

microRNA-151-3p (miR-151-3p) is a widely reported oncogene with a documented role in tumorigenesis and cancer progression. miRNAs are potent regulators of gene expression that are transferred between cells by extracellular vesicles (EVs). However, there is limited data about the role of EV-derived miR-151-3p in osteosarcoma (OS) progression, or the possible effects of miR-151-3p on the paracrine activity of cancer-associated fibroblasts (CAFs) in OS. To fill this gap of knowledge, we isolated CAF-derived EVs (CAF-EVs) from examined their up-take by human MG63 OS cells and their effects on MG63 biology. We found high expression of miR-151-3p in OS tissues, and miR-151-3p derived from CAF-EVs promoted the process of epithelial-mesenchymal transition (EMT), migration, and invasion of MG63 cells. Notably, bioinformatics analysis, RIP, and dual luciferase report test determined that CHL1 was the direct binding target of miR-151-3p. CAF-EVs mediated the CHL1/integrin 1β axis through miR-151-3p to regulate the TGF-β pathway and then promoted the proliferation, migration, invasion, and EMT of OS cells. Our in vivo results confirmed that EV secretion of miR-151-3p activated the TGF-β pathway through the CHL1/integrin 1β axis to promote the proliferation of OS cells and increase tumor volume and weight. Other effects were to upregulate E-Cadherin and downregulate the expression of N-Cadherin and β-catenin. Hence, our data reveal a potential functional axis mediated by EVs in the tumor microenvironment of OS and suggest potential candidate therapy targets.

Assessment of the Nutraceutical Effects of Oleuropein and the Cytotoxic Effects of Adriamycin, When Administered Alone and in Combination, in MG-63 Human Osteosarcoma Cells.

Oleuropein (OLEU) is the most distinguished phenolic compound found in olive fruit and the leaves of Olea europaea L., with several pharmacological properties, including anti-cancer actions. Adriamycin (ADR) is an anthracycline widely used as a chemotherapeutic agent, although it presents significant side effects. The aim of the present study was to investigate the effect of oleuropein alone (20 μg/mL) and in co-treatment with ADR (50 nM), in MG-63 human osteosarcoma cells. Therefore, cellular and molecular techniques, such as MTT assay, flow cytometry, real-time Polymerase Chain Reaction (PCR), western blot and Elisa method, as well as Nuclear Magnetic Resonance (NMR) spectroscopy, were applied to unveil changes in the signal transduction pathways involved in osteosarcoma cells survival. The observed alterations in gene, protein and metabolite levels denote that OLEU not only inhibits MG-63 cells proliferation and potentiates ADR’s cytotoxicity, but also exerts its action, at least in part, through the induction of autophagy.

Selective Estrogen Receptor Modulator-Like Activities of Herba epimedii Extract and its Interactions With Tamoxifen and Raloxifene in Bone Cells and Tissues.

Herba epimedii (HEP), a kidney-tonifying herb, has been commonly used alone or in formula for strengthening kidney function and treating bone disorders. Its bone protective activity has been demonstrated to be via estrogen receptor (ERs). HEP activates the phosphorylation of ERα in an estrogen response element- (ERE-) dependent manner. We examined the bone protective effects of HEP and its potential interactions with Selective Estrogen Receptor Modulators (SERMs, such as tamoxifen and raloxifene) as they act via the same ERs. Six-month-old mature Sprague Dawley sham-operated (Sham) or ovariectomized (OVX) rats were treated with either vehicle, 17ß-estradiol (1.0 mg/kg.day), tamoxifen (Tamo, 1.0 mg/kg.day), raloxifene (Ralo, 3.0 mg/kg.day), HEP (0.16 g/kg.day), or its combinations with respective SERMs (HEP + Tamo; HEP + Ralo) for 12 weeks. HEP and SERMs as well as their combinations significantly restored changes in bone mineral density (BMD), trabecular bone properties, and bone turnover biomarkers induced by ovarian sex hormone deficiency in ovariectomized rats. Besides the increase in serum estradiol, inhibition on follicle stimulating hormone (FSH) might also be involved in the osteoprotective activities of HEP and SERMs. HEP interacted with SERMs to protect bones from ovarian sex hormone deficiency without altering SERMs’ bone protective activities. HEP neither induced changes in uterus weight nor altered the uterotrophic activity of SERMs in OVX rats. In human osteosarcoma MG-63 cells, HEP-treated serum (HEP-Ts) significantly promoted alkaline phosphatase (ALP) activity like the crude HEP extract did but did not stimulate ERE activity. Our study also reported that biologically activated HEP interacted with SERMs to promote ALP activity without altering the action of SERMs at most of the concentrations tested in MG-63 cells. HEP exerted bone protective activity and the use of HEP did not alter the bone protective activities of SERMs when they were used simultaneously in an estrogen-deficient rat model.

Structural and osteoblast cell response of strontium-zinc co-substituted nano-biphasic fluorohydroxyapatite

Strontium(Sr2+) and zinc(Zn2+) co-substituted nanobiphasic fluorohydroxyapatite (Ca10-x-ySrxZny(PO4)6F0.5OH1.5), [x = 5–40 mol% of Sr2+ and y = 5–20 mol% of Zn2+] improved osteoblastic cell response and is investigated in the present study. The structure, phase, thermal studies, morphology and composition were analysed by XRD, TG/DTA/DSC, FESEM and EDAX. The osteoblast cell response was analysed using MG-63 human osteosarcoma cells. The XRD pattern confirmed the presence of the β-TCP phase in nano-Sr/Zn-FHA which induces dissolution of Sr2+, Zn2+ and Ca2+ ions. The β-phase has influence on the osteoblasts for steady and sustained bone formation. The samples 40Sr/10Zn-FHA and 40Sr/5Zn-FHA exhibited sustained cell viability and alkaline phosphatase activity.

VEGF-C/VEGFR-3/iNOS Signaling in Osteosarcoma MG63 Cells Mediates Stimulatory Effects on Human Umbilical Vein Endothelial Cell Proliferation

Objective To assess the effects of vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor-3 (VEGFR-3) signaling on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells (HUVECs). MethodsMG63 cells were treated with VEGF-C alone (VEGF-C group), VEGF-C + iNOS inhibitor aminoguanidine (AG; AG group), and VEGF-C + VEGFR-3 inhibitor MAZ51 (MAZ51 group); untreated MG63 cells were used as controls. NO production was evaluated by a colorimetric method involving nitrate reductase. Meanwhile, mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs, we set up six groups: HUVECs, HUVECs+MG63, HUVECs+VEGF-C, HUVECs+MG63+VEGF-C, HUVECs+MG63+VEGF-C+AG, and HUVECs+MG63+VEGF-C+MAZ51 groups. The proliferation of HUVEC cells was assessed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and proliferating cell nuclear antigen (PCNA) expression quantitation. ResultsVEGF-C treatment enhanced iNOS expression at both gene and protein levels (mRNA: LSD-t=4.152, P<0.01; protein: LSD-t=3.486, P<0.01) and increased NO release of MG63 cells (LSD-t=3.774, P<0.01); treatment with either AG or MAZ51 decreased these effects (mRNA: LSD-t=9.183, P<0.001; LSD-t=8.639, P<0.001; protein: LSD-t=5.170, P<0.001; LSD-t=7.255, P<0.001; NO production:LSD-t=10.326, P<0.001; LSD-t=10.540, P<0.001). Interestingly, co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation (EdU: LSD-t=5.374, P<0.001; LSD-t=2.984, P<0.05; LSD-t=8.526, P<0.001; PCNA: LSD-t=9.267, P<0.001; LSD-t=5.515, P<0.001; LSD-t=14.873, P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG (EdU: LSD-t=10.770, P<0.001; PCNA: LSD-t=19.940, P<0.001) or MAZ51 (EdU: LSD-t=6.950, P<0.001; PCNA: LSD-t=14.001, P<0.001). ConclusionIn human osteosarcoma MG63 cells, activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production, which subsequently induces HUVEC proliferation.

Treatment Depth Effects of Combined Magnetic Field Technology using a Commercial Bone Growth Stimulator

Lumbar spinal fusion is one of the more common spinal surgeries, and its use is on the rise. If the bone fails to fuse properly, then a pseudarthrosis or “false joint” develops and results in pain, instability, and disability. Since1974, three types of electrical stimulation technologies have been approved for clinical use to enhance spinal fusions. One such technology is inductive coupling, which includes combined magnetic fields (CMFs). The purpose of this study was to evaluate the effects of a CMF device known as the Donjoy (SpinaLogic®) on MG-63 (ATCC® CRL1427TM) human osteosarcoma cells at treatment depths ranging from 0.5” to 6.0”. The cells were grown to confluence on 4-well chamber slides that were kept in a nickel-alloy chamber within an incubator to shield the cells from unwanted environmental electromagnetic fields. During treatment, a specially designed apparatus held both the treatment device and the chamber slide. Briefly, the chamber slide was placed inside an acrylic tube at a specific distance from the transducer housing, and the device was turned on for 30 minutes. The chamber slides were then returned to the incubator to be evaluated at 7, 14, and 21 days post treatment for cell viability and bone nodule formation. Our results showed that compared to control cells, the cells located at 3” from the source had the greatest increase in bone nodule formation 7 days post treatment which is the depth at this consistent with manufacturer recommendations.

Roles of endothelial cells in the regulation of cell motility via lysophosphatidic acid receptor-2 (LPA2) and LPA3 in osteosarcoma cells

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of biological responses. In tumor microenvironment, endothelial cells promote cancer cell functions. In this study, we investigated the roles of endothelial cells in the regulation of cell motile activity via LPA2 and LPA3 in human osteosarcoma MG-63 cells. In cell motility assay, the cell motile activity of MG-63 cells was markedly increased by the supernatants of endothelial F2 cells. MG-63 cell motility elevated by the supernatants was enhanced by GRI-977143 (LPA2 agonist) and reduced by (2S)-OMPT (LPA3 agonist). LPAR2 and LPAR3 expressions were increased in highly migratory MG63-CR7(F2) cells, which were generated from MG-63 cells by co-culture with F2 cell supernatants. MG63-CR7(F2) cell motility was stimulated by LPA treatment. In the presence of F2 cell supernatants, MG63-CR7(F2) cell motility was markedly enhanced by GRI-977143 and suppressed by (2S)-OMPT. Autotaxin (ATX) enzymatically converts lysophosphatidylcholine (LPC) to LPA. ATX expression was higher in MG63-CR(F2) cells than in MG-63 cells. MG63-CR7(F2) cell motility was markedly increased by LPC in comparison with MG-63 cells. In addition, MG63-CR(F2) cell motility was significantly stimulated by the supernatants of LPC treated F2 cells. The present results suggest that the activation of LPA signaling via LPA2 and LPA3 by endothelial cells is involved in the modulation of cell motile activity of MG-63 cells.

Curcumin Analogue L48H37 Suppresses Human Osteosarcoma U2OS and MG-63 Cells' Migration and Invasion in Culture by Inhibition of uPA via the JAK/STAT Signaling Pathway.

Osteosarcoma, the most prevalent malignant bone tumor in the pediatric age group, is responsible for the great majority of cancer-associated deaths owing to its highly metastatic potential. The anti-metastatic effects of the new curcumin analogue L48H37 in human osteosarcoma are still unknown; hence, we investigated whether L48H37 represses human osteosarcoma cells’ biological behavior of migratory potential and invasive activities and attempted to delve into its underlying mechanisms. L48H37 up to 5 μM inhibited, without cytotoxicity, the motility, migration, and invasion of human osteosarcoma U2OS and MG-63 cells. In U2OS cells, the human protease array revealed an obvious decrease in urokinase plasminogen activator (uPA) expression after L48H37 treatment, and L48H37 actually reduced the level, protein and mRNA expression, and promoter activity of uPA dose-dependently. L48H37 decreased the phosphorylation of STAT3, JAK1, JAK2, and JAK3 in U2OS cells, but did not affect the phosphorylation of ERK, JNK, p38, and Akt. Using colivelin, an activator of STAT3, the L48H37-induced decrease in uPA and migratory potential could be countered as expected. Collectively, L48H37 represses the invasion and migration capabilities of U2OS and MG-63 cells by the suppression of uPA expression and the inhibition of JAK/STAT signaling. These results suggest that L48H37 may be a potential candidate for anti-metastatic treatment of human osteosarcoma.

Onosma bracteata Wall. induces G0/G1 arrest and apoptosis in MG-63 human osteosarcoma cells via ROS generation and AKT/GSK3β/cyclin E pathway

Onosma bracteata Wall. (Boraginaceae), commonly known as "gaozaban" is a highly valuable medicinal herb, useful in the treatment of body swellings, abdominal pain, eye-related problems, fever, and urinary calculi. The present study was performed to investigate the antioxidant properties of extract/fractions, viz. ethanol (Obeth) extract, hexane (Obhex) fraction, chloroform (Obcl) fraction, ethyl acetate (Obea) fraction, butanol (Obbu) fraction, and aqueous (Obaq) fraction isolated from O. bracteata. Obea fraction showed stronger free radical quenching ability in various antioxidant assays, as compared to the other fractions. Obea fraction with effective free radical-scavenging properties was further evaluated for the antiproliferative activity against human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung cancer A549 cell lines using MTT assay. Obea fraction showed strong cytotoxicity with GI50 value of 88.56, 101.61, and 112.7 μg/ml towards MG-63, IMR-32, and A549 cells respectively. Mechanistic studies revealed that Obea fraction in osteosarcoma MG-63 cells increased reactive oxygen species (ROS) level and reduced mitochondrial membrane potential. In the presence of Obea, the cells were found to be arrested in the G0/G1 phase in a dose-dependent manner which is also confirmed by the enhancement in the early apoptotic cell population in flow cytometer analysis. Western blotting demonstrated the decrease in expression of p-NFκB, COX-2, p-Akt, and Bcl-xL, whereas upregulation was observed in the expression of GSK-3β, p53, caspase-3, and caspase-9 proteins. RT-qPCR studies revealed downregulation of Bcl-2, cyclin E, CDK2, and mortalin gene expression and upregulation in the expression of p53 genes. The antioxidant and cytotoxic potential of Obea was attributed to the presence of catechin, kaempferol, onosmin A, and epicatechin, as revealed by HPLC analysis. This is the first report regarding the antiproliferative potential of O. bracteata against osteosarcoma.