Osteosarcoma MG-63 Research Articles

NDRG1 regulates osteosarcoma cells via mediating the mitochondrial function and CSCs differentiation

BackgroundCancer stem cells (CSCs) are mainly contributed to malignancy metastatic potential and resistant therapy of osteosarcoma (OS). The mitochondria-related apoptosis was generally accepted as the target of tumor therapy. However, the effect of N-myc downstream-regulated gene 1 (NDRG1) on CSCs and mitochondrial health in OS is still unknown.MethodsIn OS cells, MG63 and U2OS, the siRNA of NDRG1 were conducted. Transwell, western blot, RT-qPCR, and mitochondria isolation were used to identify the effect of NDRG on OS cells and mitochondria. Moreover, the differentiation-related factors of CSCs were determined.ResultsAfter downregulation of NDRG1, the cell viability, invasion ability decreased whereas cell apoptosis increased. The expressions profiles of fibronectin, vimentin, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP) 2, MMP9, and MMP13 were downregulated, but E-cadherin expression level was upregulated by NDRG1 siRNA. At the same time, cytochrome (Cyt) C levels were increased in cytosol with the decreasing in mitochondria after siRNA treatment. The mitochondrial membrane potential (MMPs) was declined, and the function of mitochondria was impeded. The expressions of uncoupling proteins (UCP) 2, voltage dependent anion channel (VDAC), peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, and cyclooxygenase (COX) 2 were downregulated by NDRG1 silencing. Moreover, NDRG performed its function primarily through the Wnt pathway and could regulate the differentiation of osteosarcoma stem cells.ConclusionSilencing of NDRG1 could damage the function of mitochondria, promote the CSCs differentiation, alleviating OS progression.

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.

Preclinical evaluation of an engineered oncolytic herpes simplex virus for pediatric osteosarcoma.

10040 Background: Osteosarcoma is the most common primary bone tumor in children. For those with relapsed or metastatic disease, the five-year survival rate is approximately 20%, and survivors often suffer from long-term disability from current therapies. The high morbidity and mortality for these patients highlight a great need for improved therapies. One such novel therapeutic approach is oncolytic herpes simplex virus (oHSV) immunovirotherapy. We previously demonstrated that M002, an engineered oHSV that contains deletions of the neurovirulence gene preventing infection of normal cells, effectively infects and kills neuroblastoma and rhabdomyosarcoma. Currently, similar oHSVs are being evaluated in early phase clinical trials for children and adults with relapsed or refractory brain tumors. To date, there has been limited investigation of oncolytic virotherapy in osteosarcoma. Thus, we sought to examine the ability of oHSV, M002, to infect and kill osteosarcoma cells in vitro. Methods: We evaluated two long-term passaged human osteosarcoma cell lines, U2-OS and MG-63. Flow cytometry was used to assess baseline expression of oHSV viral entry-mediated receptors (CD111, CD112, syndecan, HVEM). Single and multi-step viral recovery experiments measured virus infectivity and replication. Cells were infected with increasing multiplicity of infection (MOI) of M002, and cell viability was measured 72 hours post-infection via alamarBlue assay. Results: Both MG-63 and U2-OS cells expressed HSV entry molecules (Table) including high levels of the primary HSV entry molecule CD111. Single step virus recovery experiments in MG-63 cells infected at a MOI of 10 plaque-forming units (PFU)/cell demonstrated a 3 log-fold increase in virus titer from 12 to 24 hours post-infection. For multi-step experiments, MG-63 cells were infected with a MOI of 0.1 PFU/cell; viral replication significantly increased from 1.1x103 PFU at 6 hours post-infection to 3.8x1010 PFU at 72 hours post-infection. M002 successfully decreased osteosarcoma viability with a lethal dose in 50% of cells (LD50)of 2.82 and0.67 PFU/cell for MG-63 and U2-OS cells, respectively. Notably, at a virus MOI of 5 PFU/cell, viability was decreased by 64% ± 0.1% (p<0.001 vs control) in MG-63 cells and 96% ± 0.1% (p<0.001 vs control) in U2-OS cells. Conclusions: MG-63 and U2-OS osteosarcoma cells express high levels of HSV entry receptors. Virus recovery experiments demonstrated the ability of M002 to infect cells and replicate over time. The viability of osteosarcoma cells significantly decreased following infection with M002. These data suggest M002 may be a promising novel therapeutic option for patients with osteosarcoma and warrant further investigation for translation to the clinical setting.[Table: see text]

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.

Ginsenoside impedes proliferation and induces apoptosis of human osteosarcoma cells by down-regulating β-catenin.

Osteosarcoma (OS) is the most commonly occurred primary bone malignancy with high incident rates among children and adolescents. In pharmacologic treatment, the drug ginsenoside has been shown to exert anticancer effects on several malignant diseases. The purpose of this research was to investigate the effect of ginsenoside on the apoptosis and proliferation of human OS MG-63 and Saos-2 cells by regulating the expression of β-catenin. Human OS MG-63 and Saos-2 cells were assigned into control group, and four groups with treatment by varying concentrations (12.5 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL) of ginsenoside, respectively. Cell growth after treatment was observed through cell slides. The proliferation rate of MG-63 and Saos-2 cells in each group was detected by CCK-8. After cell transfection at 48 h, cell cycle and cell apoptosis were detected by FITC-Annexin V staining and flow cytometry. The protein and mRNA expressions of β-catenin, Cyclin D1, Bcl-2, Bax and cleaved caspase-3 were detected by RT-qPCR and western blot analysis. With increased exposure and concentration of ginsenoside, the cell density, total cell numbers and the absorbance of MG-63 and Saos-2 cells gradually decreased. FITC-Annexin V and FITC-Annexin V/PI staining demonstrated that the cell proportion at S phase decreased, whereas the total apoptotic rate of MG-63 and Saos-2 cells was increased. Furthermore, RT-qPCR and western blot analysis highlighted a gradual decrease in protein and mRNA expressions of β-catenin, Bcl-2 and Cyclin D1, while an elevation in those of Bax and cleaved caspase-3. The results of this study demonstrate that ginsenoside inhibits proliferation and promotes apoptosis of human OS MG-63 and Saos-2 cells by reducing the expressions of β-catenin, Bcl-2 and Cyclin D1 and increasing the expression of Bax and cleaved caspase-3.

The effect of miR-539 regulating TRIAP1 on the apoptosis, proliferation, migration and invasion of osteosarcoma cells

ObjectiveThe purpose of this study is to explore the effect of miRNA-539 on osteosarcoma (OS) and the underlying mechanism, so as to find a new method for early diagnosis and treatment of osteosarcoma.MethodmiRNA-539 mimics was transfected into osteosarcoma cells 143b and MG-63 and upregulated the expression of miR-539. QT-PCR was used to detect transfection efficacy. CCK-8 method was used to detect proliferation of 143b and MG-63 osteosarcoma cells and flow cytometry was used to detect the apoptosis of osteosarcoma cells 143b and MG-63. Wound-healing test and Transwell test were used to detect the migration and invasion ability of osteosarcoma cells. TRIAP1 was found to be the potential target gene of miRNA-539 by online bioinformatics software and the expression level of TRIAP1 in osteosarcoma cells overexpressing miRNA-539 was detected by qT-PCR. Western blot was used to detect the level of expression of TRIAP1 and its downstream genes (p53, p21, apaf1 and caspase9) in osteosarcoma cells 143b and MG63 transfected with miR-539 mimics or miR-539 mimics-NC. A model of osteosarcoma subcutaneously transplanted in nude mice was constructed to observe the effect of miRNA-539 on the growth rate of osteosarcoma in vivo.ResultsAfter transfection of miRNA-539 mimics in osteosarcoma cells 143b and MG63, the proliferation level, migration ability, and invasion ability of the osteosarcoma cells were significantly lower than that in the control group, and the apoptosis level was significantly higher than that in the control group (P < 0.01). The dual luciferase reporter confirmed that TRIAP1 was the target of miR-539, and the expression level of TRIAP1 in 143b and MG63 transfected with miRNA-539 mimics was proved to be significantly lower than that in the control group (P < 0.01).The western blot showed the expression of genes targeted by TRIAP1 was upregulated when the expression of TRIAP1 was downregulated. In vivo, the osteosarcoma growth rate in the miRNA-539 mimics group was significantly slower than that in the control group (P < 0.01).ConclusionsMiRNA-539 may inhibit the cell proliferation, migration and invasion of osteosarcoma cells and promote the apoptosis of osteosarcoma cells by targeting on TRIAP1.

Fraxinellone Has Anticancer Activity by Inducing Osteosarcoma Cell Apoptosis via Promoting Excessive Autophagy Flux.

Osteosarcoma is a malignant bone tumor that is easy to metastasize in the early stage and has a very poor prognosis. Fraxinellone (FRA) is one of the main components isolated from the D. dasycarpus plant. Its anti-inflammatory and neuroprotective effects have been confirmed, but the research on the anti-cancer effect of FRA and its potential mechanism is relatively scarce. In this study, we found that FRA inhibited the proliferation and migration of osteosarcoma cells HOS and MG63 in a dose-dependent manner. Immunofluorescence, fluorescence staining and western blotting analysis showed that FRA could simultaneously induce osteosarcoma cell apoptosis and increase autophagy flux. Subsequent turnaround experiments suggested that the pro-apoptotic effect of FRA was achieved through excessive autophagy flux. The results of the xenograft orthotopic model further supported the anti-cancer effects of FRA, indicating that FRA treatment inhibited the growth of osteosarcoma, and the pro-apoptotic and autophagy effects of FRA were also proved in vivo. These studies provide new ideas for the future treatment of osteosarcoma and offer theoretical support for the anti-cancer mechanism of FRA.

Zinc oxide nanoparticles from Cassia auriculata flowers showed the potent antimicrobial and in vitro anticancer activity against the osteosarcoma MG-63 cells.

Osteosarcoma (OS) is a foremost mesenchymal bone neoplasm and it can occur at any age with survival rate is nearly 2–8 times lesser in elders than in teenagers. The clinical therapies for cancer treatment have gradually becoming outdated because of the developments of nano-medicine and multi-targeted drug-delivery. In this work, we green synthesized the zinc oxide nanoparticles from the Cassia auriculata flower (AS-ZnONPs) extract and evaluated its antimicrobial and in vitro anticancer potential against the OS MG-63 cells. The synthesized AS-ZnONPs were confirmed and characterized by using UV–vis spectroscopy, XRD, FE-SEM, and photoluminescence techniques. The antimicrobial activity of AS-ZnONPs was studied by disc diffusion technique. The viability of AS-ZnONPs treated MG-63 cells were examined by MTT assay. The apoptotic cells in the AS-ZnONPs treated MG-63 cells were assayed by dual staining. The MMP status of AS-ZnONPs treated cells were tested by Rh-123 staining. The cell adhesion assay was performed to detect the anticancer effects of AS-ZnONPs against MG-63 cells. The results of UV–vis spectroscopy, XRD, FE-SEM, and photoluminescence techniques proved the formation of AS-ZnONPs and it has the hexagonal wurtzite structures. AS-ZnONPs displayed the potent antimicrobial activity against the tested microbial strains. The AS-ZnONPs were appreciably inhibited the cell viability of MG-63 cells. The outcomes of fluorescence staining proved that AS-ZnONPs reduced the MMP and prompted the apoptosis in MG-63 cells. In conclusion, our discoveries demonstrated that the formulated AS-ZnONPs has the potent antimicrobial and in vitro anticancer activity against the MG-63 cells. The AS-ZnONPs could be potent chemotherapeutic agent in the future to treat the OS.

Preparation and biological assessment of a ZrO2-based bone scaffold coated with hydroxyapatite and bioactive glass composite

Zirconia (ZrO2) bioceramics has superior stiffness, prominent fracture toughness and fracture strength. However, ZrO2 is almost bioinert material as an alternative scaffolds for repairing bone defects. In this paper, we prepared a porous ZrO2-based bone scaffold coated with hydroxyapatite (HA) and bioactive glass (BG) composite and optimized the quality hybrid proportion of HA and BGs composite coating and sintering temperature. The scaffold has a dense composite coating at the coating sintering temperature of 950 °C and HA content of 70% with Ca2SiO4 and β-TCP, HA and Ca10(PO4)6CO3 phases. The ZrO2 composite scaffold with HA-BG coating was capable of forming rich calcium and phosphorus deposits in the simulated body fluids. Its compression strength was superior to that of the porous ZrO2 scaffold. And the scaffold can promote proliferation of cultured mesenchymal stem cells and the adhesion and propagation of osteosarcoma MG-63 cells. The results exhibited that the excellent mechanical properties, biological activities and good cytocompatibility of the scaffold. Therefore, The composite scaffold represents a potential bone implant for future clinical applications.

Water extract of Er-xian decoction selectively exerts estrogenic activities and interacts with SERMs in estrogen-sensitive tissues

Ethnopharmacological relevanceThe increasing use of “kidney”-nourishing Traditional Chinese Medicine (TCM) like Er-xian decoction (EXD) for management of menopausal symptoms and osteoporosis has aroused concerns about their safety, and whether they interact with prescription drugs as both of them act via estrogen receptors (ERs) and regulate serum estradiol. Aim of the studyThe present study aimed to evaluate whether EXD selectively exerted estrogenic activities and interacted with Selective Estrogen Receptor Modulators (SERMs). Materials and methodsIn vivo, mature ovariectomized (OVX) rats were administrated with EXD or combined treatment of EXD and SERMs for 12 weeks. The tissue-selective effect of EXD and its interaction of SERMs were studied in four estrogen sensitive tissues, bone, brain, breast and uterus. In vitro, the interaction of extracts of EXD-treated serum and SERMs in four ER-positive cell lines. ResultsIn OVX rats, EXD selectively alleviated estrogen deficiency-induced changes in the bone and brain without inducing any estrogenic effects in the breast or uterus. Two-way ANOVA indicated the presence of interactions between EXD and SERMs in OVX rats but EXD did not significantly alter the tissue responses to SERMs in the bone, breast or brain. Indeed, the combined use of EXD and SERMs appeared to suppress the estrogenic effect of raloxifene and tamoxifen in the uterus. Extract of EXD-treated serum directly stimulated cell proliferation or differentiation in human osteosarcoma MG-63, neuroblastoma SHSY5Y, breast cancer MCF-7, and endometrial Ishikawa cells. Two-way ANOVA revealed that EXD-treated serum interacted with SERMs at various concentrations and altered the effects of tamoxifen in MG-63 and MCF-7 cells. ConclusionsEXD exerted estrogenic effects in a tissue-selective manner and interacted with SERMs. Combined treatment of EXD and SERMs did not hamper the beneficial effects of SERMs on the bone or brain but appeared to moderate the estrogenic effect of SERMs in the uterus.

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.

Berberine and Cisplatin Exhibit Synergistic Anticancer Effects on Osteosarcoma MG-63 Cells by Inhibiting the MAPK Pathway.

Berberine (BBR) has been reported to have potent anticancer activity and can increase the anticancer effects of chemotherapy drugs. The present study aims to investigate whether BBR and cisplatin (DDP) exert synergistic effects on the osteosarcoma (OS) MG-63 cell line. In the present study, MG-63 cells were treated with BBR and DDP alone or in combination. The effects of these therapeutics on cell viability, colony formation, migration, invasion, nuclear morphology, apoptosis, and the cell cycle, as well as their role in regulating the expression of proteins related to apoptosis, the cell cycle, and the mitogen-activated protein kinase (MAPK) pathway, were determined. The results demonstrated that BBR or DDP significantly inhibited the proliferation of MG-63 cells in a dose- and time-dependent manner. The combination treatment of BBR and DDP exerted a prominent inhibitory effect on proliferation and colony formation. Furthermore, the results showed that the combination treatment of BBR and DDP enhanced the inhibition of cell migration and invasion and reversed the changes in nuclear morphology. The results showed that the combination treatment of BBR and DDP induced apoptosis and cell cycle arrest in the G0/G1 phase. Mechanistically, the combination treatment of BBR and DDP inhibited the expression of MMP-2/9, Bcl-2, CyclinD1, and CDK4, enhanced the expression of Bax and regulated the activity of the MAPK pathway. Collectively, our data suggest that the combination therapy of BBR and DDP markedly enhanced OS cell death.

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.

Combined bezafibrate, medroxyprogesterone acetate and valproic acid treatment inhibits osteosarcoma cell growth without adversely affecting normal mesenchymal stem cells.

Drug repurposing is a cost-effective means of targeting new therapies for cancer. We have examined the effects of the repurposed drugs, bezafibrate, medroxyprogesterone acetate and valproic acid on human osteosarcoma cells, i.e., SAOS2 and MG63 compared with their normal cell counterparts, i.e. mesenchymal stem/stromal cells (MSCs). Cell growth, viability and migration were measured by biochemical assay and live cell imaging, whilst levels of lipid-synthesising enzymes were measured by immunoblotting cell extracts. These drug treatments inhibited the growth and survival of SAOS2 and MG63 cells most effectively when used in combination (termed V-BAP). In contrast, V-BAP treated MSCs remained viable with only moderately reduced cell proliferation. V-BAP treatment also inhibited migratory cell phenotypes. MG63 and SAOS2 cells expressed much greater levels of fatty acid synthase and stearoyl CoA desaturase 1 than MSCs, but these elevated enzyme levels significantly decreased in the V-BAP treated osteosarcoma cells prior to cell death. Hence, we have identified a repurposed drug combination that selectively inhibits the growth and survival of human osteosarcoma cells in association with altered lipid metabolism without adversely affecting their non-transformed cell counterparts.

Resveratrol is cytotoxic and acts synergistically with NF-κB inhibition in osteosarcoma MG-63 cells

IntroductionOsteosarcoma is the most common primary malignancy of the bone. The existing adjuvant chemotherapy regimens, while improving the overall survival, have been limited by the significant systemic toxicity. Substantial clinical and research efforts are being invested to develop novel pharmaceutical agents. Resveratrol (Res) has been suggested to have a chemopreventive effect. However, the mechanism of Res in osteosarcoma remains to be elucidated.Material and methodsThe MG-63 osteosarcoma cell culture model was used to investigate the chemotherapeutic effect of Res. MTT assay, wound healing assay, and Transwell migration assay were used to document the effect of Res on cell proliferation, migration, and invasion, respectively. Apoptosis in MG-63 cells was quantified with the TUNEL assay. Western blotting analysis was used to examine the molecular changes following Res treatment. Data processing and analysis were conducted using GraphPad Prism 5.0. P < 0.05 was considered statistically significant.ResultsOur data suggested that Res blocks cell proliferation, migration, and invasion, and activates apoptotic cell death in osteosarcoma MG-63 cells. We found that Res potentially down-regulates nuclear factor κB (NF-κB) and Akt intracellular signaling transduction. Moreover, the combination of Res and pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, resulted in synergistic growth inhibition of osteosarcoma.ConclusionsOur in vitro preclinical study in the MG-63 cell line model supports the translation of Res to the clinical management of patients with osteosarcoma.

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.

Bioactive glass doped with noble metal nanoparticles for bone regeneration: in vitro kinetics and proliferative impact on human bone cell line.

This work investigates the bioactivity of novel silver-doped (BG-Ag) and gold-doped (BG-Au) quaternary 46S6 bioactive glasses synthesized via a semi-solid-state technique. A pseudo-second-order kinetic model successfully predicted the in vitro uptake kinetic profiles of the initial ion-exchange release of Ca in simulated body fluid, the subsequent Si release, and finally, the adsorption of Ca and P onto the bioactive glasses. Doping with silver nanoparticles enhanced the rate of P uptake by up to approximately 90%; whereas doping with gold nanoparticles improved Ca and P uptake rates by up to about 7 and 2 times, respectively; as well as Ca uptake capacity by up to about 19%. The results revealed that the combined effect of Ca and Si release, and possibly the release of silver and gold ions into solution, influenced apatite formation due to their effect on Ca and P uptake rate and capacity. In general, gold-doped bioactive glasses are favoured for enhancing Ca and P uptake rates in addition to Ca uptake capacity. However, silver-doped bioactive glasses being less expensive can be utilized for applications targeting rapid healing. In vitro studies showed that BG, BG-Ag and BG-Au had no cytotoxic effects on osteosarcoma MG-63 cells, while they exhibited a remarkable cell proliferation even at low concentration. The prepared bioactive glass doped with noble metal nanoparticles could be potentially used in bone regeneration applications.

A novel redox-responsive ursolic acid polymeric prodrug delivery system for osteosarcoma therapy

Ursolic acid (UA), found widely in nature, exerts effective anti-tumoral activity against various malignant tumors. However, the low water solubility and poor bioavailability of UA have greatly hindered its translation to the clinic. To overcome these drawbacks, a simple redox-sensitive UA polymeric prodrug was synthesized by conjugating UA to polyethylene glycol using a disulfide bond. This formulation can self-assemble into micelles (U-SS-M) in aqueous solutions to produce small size micelles (∼62.5 nm in diameter) with high drug loading efficiency (∼16.7%) that exhibit pH and reduction dual-sensitivity. The cell and animal studies performed using the osteosarcoma MG-63 cell line and MG-63 cancer xenograft mice as the model systems consistently confirmed that the U-SS-M formulation could significantly prolong the circulation in blood and favor accumulation in tumor tissue. Targeted accumulation allows the U-SS-M to be effectively internalized by cancer cells, where the rapid release of UA is favored by a glutathione-rich and acidic intracellular environment, and ultimately achieves potent antitumor efficacy.

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.