Osteosarcoma Cell Line MG-63 Research Articles

MiR-33a Mediates the Anti-Tumor Effect of Lovastatin in Osteosarcoma by Targeting CYR61

Background/Aims: Preventing cell metastasis is an effective therapeutic strategy to treat osteosarcoma and improve prognosis. Statins have been found to have anticancer effects in addition to their cholesterol-lowering action. As a new target of statins, cysteine-rich 61 (CYR61) was recently identified to promote cell migration and metastasis in osteosarcoma. However, the underlying mechanisms mediating the regulation of CYR61 expression by statins remain unknown. Methods: Human osteosarcoma cell lines MG63 and SaOS2 were used to clarify the effect of lovastatin on CYR61 expression. Real-time PCR was performed to detect mRNA or microRNA (miRNA) levels and western blot was performed to detect protein levels. Cell invasive ability was determined using Transwell assays. Lentivirus encoding CYR61 cDNA or sterol regulatory element-binding protein 2 (SREBP-2) shRNA was used to upregulate CYR61 expression or downregulate SREBP-2 expression. Binding of the CYR61 3’ untranslated region (UTR) and miR-33a was analyzed by luciferase reporter assay. Results: We found that lovastatin treatment decreased CYR61 expression, inhibited cell invasion and altered epithelial-to-mesenchymal-transition (EMT)-related protein expression, while CYR61 overexpression abolished the effect of lovastatin. Moreover, lovastatin increased the expression of SREBP-2 and miR-33a, which were then downregulated by SREBP-2 silencing. Bioinformatics analysis indicated that the CYR61 3′UTR harbored a potential miR-33a binding site and luciferase reporter assay demonstrated that CYR61 was a target of miR-33a in osteosarcoma cells. Furthermore, miR-33a could inhibit cell invasion and alter EMT-related protein expression. SREBP-2 silencing or miR-33a inhibitor upregulated CYR61 expression and reversed the effects of lovastatin on cell invasion and EMT-related proteins. Conclusion: Our findings suggest lovastatin suppresses osteosarcoma cell invasion through the SREBP-2/miR-33a/CYR61 pathway.

Induction apoptosis and G2/M phase cell cycle arrest by non-thermal plasma in human osteosarcoma MG-63 cell line

Osteosarcoma is a malignant bone tumor that develops in the osteoblast cells that form the outer covering of bone. It is the most frequent malignant bone tumor found at 10-14 years and at >65 years of age. Atmospheric pressure plasma has advantages of high density and rich chemical agents without elevation of the substrate temperature. These non-equilibrium characteristics show promising applications in the biomedical field, opening a new research area called “Plasma Medicine”, which includes sterilization, coagulation, wound healing, and cancer treatment. However, the mechanism of apoptosis caused by plasma treatment in osteosarcoma is not well understood. In this study, the researchers show that microwave plasma causes selective cell cycle arrest and apoptosis in osteosarcoma cells in vitro. An inhabitation of cell growth of microwave plasma showed that this plasma has selective cytotoxic effects on MG-63 cells in comparison to hFOB 1.19 cells. Also, the resesearchers observed that microwave plasma treatmenst significantly showed disruption and aggregation of F-actin. The results demonstrated that treatment of MG-63 cells with microwave plasma induced cell cycle arrest in the G2/M phase, using flow cytometry and western blot assay. To detect PARP and DFF-45 cleavage or a decrease as a result of caspase-3 activation, MG-63 cells were treated either with various times of microwave plasma. The research studies demonstrated that microwave plasma induced G2/M cell phase arrest and triggered apoptosis.

Salvianolic acid B suppresses cell proliferation and induces apoptosis in osteosarcoma through p38-mediated reactive oxygen species generation

The present study aimed to investigate the potential anticancer effect and mechanisms of salvianolic acid B on osteosarcoma. Salvianolic acid B suppressed osteosarcoma cell proliferation and induced apoptosis in the osteosarcoma MG63 cell line, and activated the expressions of cleaved caspase-3, phosphorylated-tumor protein (p)38 mitogen-activated protein kinase (p-p38 MAPK) and phosphorylated-p53 (p-p53) proteins in the MG63 cells. Additionally, Salvianolic acid B also increased the level of reactive oxygen species (ROS) generation in the MG63 cells. The silencing of p38 expression inhibited the anticancer effect of salvianolic acid B on the levels of cell proliferation, p-p53 protein expression and ROS generation level in the MG63 cells. All these data supported the hypothesis that the anticancer effect of salvianolic acid B includes the suppression of cell proliferation and induces apoptosis in MG63 cells, and that p38 is important in the anticancer effect of salvianolic acid B on osteosarcoma cells due to the direct regulation of ROS generation. These data suggest that salvianolic acid B is important in the proliferation of osteosarcoma cells due to the direct regulation of p38-mediated ROS signaling.

Emodin Impairs Radioresistance of Human Osteosarcoma Cells by Suppressing Sonic Hedgehog Signaling.

BackgroundThis study aimed to investigate the possible involvement of sonic hedgehog (Shh) signaling in the radioresistance of human osteosarcoma cells and the inhibitory effects of emodin on radioresistance.Material/MethodsHuman osteosarcoma (OS) cell line MG63 was employed as parent cells. After 30-repeat low-dose (2 Gy) x-ray irradiation on MG63 cells, radioresistant OS cell MG63R cells were produced. Colony formation assay was used to assess the radioresistance. Cell viability was evaluated by CCK-8 assay. TUNEL assay was used to detect cell apoptosis. Protein expression levels and nuclear translocation were evaluated by western blotting.ResultsMore survival colony formation, elevated cell viability, and less cell apoptosis were found in MG63R cells compared to MG63 cells exposed to irradiation. The nuclear translocation of glioma-associated oncogene homolog (Gli), expression levels of Shh and Bcl2 were increased in MG63R cells compared to MG63 cells. Emodin pretreatment significantly inhibited cell viability and survival colony formation but increased cell apoptosis of irradiation exposed MG63R cells in a concentration dependent manner. Moreover, emodin pretreatment inhibited Shh and Bcl2 expressions as well as Gli1 nuclear translocation but increased C-caspase-3 expression of irradiation exposed MG63R cells in a concentration dependent manner.ConclusionsShh signaling activation was involved in the radioresistance of human OS cells. Emodin impaired the radioresistant capacity of OS cells by inhibiting Shh signaling pathway.

Bifunctional Platinum(II) Complexes with Bisphosphonates Substituted Diamine Derivatives: Synthesis and In vitro Cytotoxicity

A series of N,N'-dibisphosphonate-containing 1,3-propanediamine derivatives (L1 - L6) and their corresponding dichloridoplatinum(II) complexes (1 - 6) have been synthesized and characterized by elemental analysis, 1 H-NMR, 13 C-NMR, 31 P-NMR and HR-MS spectra. The in vitro antitumor activities of compounds L1 - L6 and 1 - 6 were tested by WST-8 assay with Cell Counting Kit-8, indicating that platinum-based complexes 1 - 6 showed higher cytotoxicity than corresponding ligands L1 - L6 against A549 and MG-63, especially complex 2 which displayed comparable cytotoxicity to those of cisplatin and zoledronate after 48 h incubation. In addition, complexes 1 - 6 were more active in vitro on osteosarcoma cell line MG-63 than normal osteoblast cell line hFOB 1.19. The structure-activity relationship has been summarized based on the in vitro cytotoxicity of three series of platinum complexes from this and our previous studies. The in vitro bone affinity of platinum complexes was also tested by hydroxyapatite (HAP) chromatography in terms of capacity factor K'. Besides, in this paper, representative complex 2, which has been proved to be a promising antitumor agent with high cytotoxicity and bone HAP binding property, was investigated for its mechanism of action producing cell death against MG-63.

Low Arid1a Expression Correlates with Poor Prognosis and Promotes Cell Proliferation and Metastasis in Osteosarcoma.

AT-rich interactive domain-containing protein 1A (ARID1A) has been shown to function as a tumour suppressor in various malignancies. However, the biological role of ARID1A in osteosarcoma is not clear. The present study aimed to investigate the expression pattern, prognostic value and the biological role of ARID1A in human osteosarcoma. ARID1A expression in 53 osteosarcoma surgical specimens was examined by quantitative real-time polymerase chain reaction, and its clinical significance was analysed. The role of ARID1A in cell proliferation, apoptosis, and metastasis were examined. ARID1A mRNA expression were significantly down-regulated in osteosarcoma tumours from that in matched adjacent non-tumour tissues. ARID1A expression was significantly inversely correlated with tumour stage and distant metastasis, as well as poor overall survival in patients with osteosarcoma. Furthermore, ARID1A mRNA was down-regulated in four human osteosarcoma cell lines MG-63, U2OS, HOS and Saos-2. Restoring of ARID1A expression in MG-63 and U2OS cells significantly inhibited cell proliferation and metastasis in vitro. Collectively, our data demonstrate that ARID1A may serve as a tumour suppressor in osteosarcoma progression, and represent a valuable prognostic marker and potential therapeutic target for osteosarcoma.

Inhibition of miRNA‑21 attenuates the proliferation and metastasis of human osteosarcoma by upregulating PTEN

The present study aimed to investigate the expression of micro (mi)RNA-21 in osteosarcoma cells, and its role in inhibiting the invasion and metastasis of osteosarcoma. Human osteosarcoma MG-63 cells and osteoblast hFOB1.19 cells were used to compare the expression of miRNA-21 using reverse transcription-quantitative polymerase chain reaction analysis. A miRNA-21 mimic or inhibitor were transfected into the MG-63 cells to upregulate and downregulate the expression of miRNA-21, respectively. The present study investigated the proliferation and invasion of transfected MG-63 cells using MTT and Transwell assays. Western blot analyses were used to investigate the regulation of important proteins in the phosphatase and tensin homolog/phosphoinositide 3-kinase/RAC-α serine/threonine-protein kinase (PTEN/PI3K/AKT) signaling pathway. Compared with hFOB1.19 cells, miRNA-21 expression was significantly upregulated in the MG-63 cells (P<0.01), which lead to increased proliferation. Downregulating miRNA-21 expression reduced the proliferation of MG-63 cells compared with hFOB1.19 cells. Invasion assays and western blot analyses revealed that the overexpression of miRNA-21 significantly enhanced the invasion ability of MG-63 cells and the expression of phosphorylated (p-)AKT, while downregulation of miRNA-21 expression reduced the protein level of AKT and p-AKT. In the MG-63 cells, miRNA-21 upregulation significantly inhibited the protein level of PTEN, resulting in significantly increased AKT and PI3K protein levels (P<0.01). In conclusion, the results of the present study indicate that the expression of miRNA-21, PI3K and AKT is increased in the osteosarcoma cell line MG-63, which results in reduced expression of PTEN and increased expression of proteins in the PI3K/AKT signaling pathway, and thus increases the aggressiveness of osteosarcoma cells.

Brain type glycogen phosphorylase small interfering RNA inhibits proliferation of human osteosarcoma cell lines

Objective To observe the effect of brain type glycogen phosphorylase (PYGB) small interfering RNA (siRNA) on proliferation, apoptosis, distribution of cell cycle in human osteosarcoma cell line MG63 with significant expression level of PYGB. Methods Thirty-five samples of osteosarcoma and 15 samples of bone cysts were detected for the PYGB expression level using real-time quantitative polymerase chain reaction(Real-time PCR). Western blotting was used to select osteosarcoma cell lines with high expression level of PYGB. MG63 cells were taken out for the following study. Cell counting kit-8 (CCK-8) assay was preformed to evaluate the cell proliferation. Cell apoptosis and cell cycle of MG63 cells were identified using flow cytometry. Besides, cell invasion and migration were evaluated by Transwell assay. In addition, cell apoptosis and metastasis related proteins B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2) were measured by Western blotting. Results Expression of PYGB exhibited a higher level in osteosarcoma samples (0.105±0.945) than that in bone cyst (0.062±0.038, P=0.006). The cell proliferation of MG63 cells were inhibited by PYGB siRNA. PYGB knockdown in MG63 cells increased the proportion of G1 phase cells from (49.74±1.54)% to (69.41±2.35)% (P=0.004), while decreased the proportion of S phase cells from (21.04±2.59)% to (14.15±0.98)% (P=0.028) and decreased the proportion of G2 phase cells from (25.62±0.64)% to (8.19±0.11)% (P=0.002). The expression of bax significantly reduced in PYGB siRNA group while the expression of bcl-2 significantly reduced in PYGB siRNA group. Low expression of PYGB reduced G1 phase of the cell cycle transition. Conclusion PYGB siRNA inhibited proliferation of MG63 cells by targeting cysteinyl aspartate-specific protease/bcl and cyclin-dependent kinase (CDK)1 signaling pathway. Key words: Brain type glycogen phosphorylase; Small interfering RNA; Osteosarcoma; Cell cycle

Screening for bioactivity of Mutinus elegans extracts

Mutinus elegans is a species of fungi that is commonly called as Elegant Stinkhorn. The aim of this study was to screen the crude extracts of the fungus for phytochemical analysis, antimicrobial activity, antioxidant assay and anticancer activity. Extraction of the fungal sample in Soxhlet apparatus was done with n-hexane and methanol as the solvent. Stock solutions of the crude methanol extract were prepared and used for microbiological assay. Thin layer chromatography was performed in order to determine the number of active components in n-hexane, and methanol solvent system for the fungus Mutinus elegans. Further, antioxidant assay was performed using DPPH radical scavenging assay. The fungal sample was then tested for cytotoxicity assay against MG63 osteosarcoma cell lines. The antimicrobial assay of Mutinus elegans extract exhibited activity against five pathogens. The zone of inhibition was measured with respect to standard antibiotics. Gas chromatography and Mass spectrometry (GC/MS analysis), revealed the presence of dibromo-tetradecan-1-ol-acetate, 2-myristynoyl-glycinamide, fumaric acid, and cyclohexylmethyldecyl ester compounds were presented in methanol and n-hexane extract of Mutinus elegans. The present study concludes the presence of bioactive compound in the extract which exhibited antimicrobial and antioxidant activity in Mutinus elegans.

Establishment and characterization of human osteosarcoma cells resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy.

The present study was performed to establish and characterize new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT). MPPa-PDT-resistant cells are isolated from the human osteosarcoma MG63 and HOS cell lines and two resistant populations were finally acquired, including MG63/PDT and HOS/PDT. Cell Counting Kit-8 (CCK-8) assay was used to determine the MPPa-PDT, cisplatin (CDDP) resistance and proliferation of MG63, MG63/PDT, HOS and HOS/PDT cells. The intracellular ROS were analyzed using DCFH-DA staining. The colony formation, invasion and migration of parental and resistant cells were compared. FCM was employed to examine the cell cycle distribution, the apoptosis rate and the proportion of CD133+ cells. The fluorescence intensity of intracellular MPPa was observed by fluorescence microscopy and quantified using microplate reader. The protein levels were assessed by western blotting (WB). Compared with two parental cells, MG63/PDT and HOS/PDT were 1.67- and 1.61-fold resistant to MPPa-PDT, respectively, and also exhibited the resistance to CDDP. FCM assays confirmed that both MG63/PDT and HOS/PDT cells treated with MPPa-PDT displayed a significantly lower apoptosis rate in comparison with their corresponding parental cells. The expression of apoptosis-related proteins (i.e. cleaved-caspase 3 and cleaved-PARP), intracellular ROS and the antioxidant proteins (HO-1 and SOD1) in MG63/PDT and HOS/PDT cells was also lower than that in parental cells. Both MG63/PDT and HOS/PDT cells exhibited changes in proliferation, photosensitizer absorption, colony formation, invasion, migration and the cell cycle distribution as compared to MG63 and HOS cells, respectively. Compared to MG63 and HOS cells, both resistant cell lines had a higher expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1 and ABCG2, but a lower expression of Bax. The present study successfully established two resistant human osteosarcoma cell lines which are valuable to explore the resistance-related mechanisms and the approaches to overcome resistance.

Inhibition of autophagy attenuated curcumol-induced apoptosis in MG-63 human osteosarcoma cells via Janus kinase signaling pathway.

The present study aimed to investigate whether autophagy was triggered by curcumol and to explore the association between autophagy and apoptosis of MG-63 cells and the underlying mechanism. MG-63 cells were cultured in vitro. An MTT assay was performed to evaluate the proliferation inhibition of the MG-63 osteosarcoma cell line by curcumol. Fluorescein isothiocyanate-Annexin V/propidium iodide staining flow cytometry was performed to analyze the apoptotic rate of cells. The morphological alterations of cell nuclei were evaluated by Hoechst 33258 viable cell staining. The effects of autophagy in cells was investigated by green fluorescent protein (GFP)-light chain 3 (LC3) transfection and using a fluorescence microscope. The expression levels of LC3II, LC3I and cleaved caspase-3 and Janus kinase (JNK) signaling pathway activation were determined by western blot analysis. Cell proliferation was inhibited by curcumol in a dose- and time-dependent manner. Curcumol induced apoptosis by the caspase-dependent signaling pathway in MG-63 cells. The present study demonstrated that curcumol could induce autophagy of MG-63 cells, which was evaluated by transmission electron microscopy. Compared with the curcumol treatment alone group, the GFP-LC3-transfected green fluorescence plasmids and the LC3II/LC3I levels in cells of the curcumol and chloroquine (CQ) treatment group were upregulated, and the apoptotic ratio was downregulated following pretreatment with autophagy inhibitor CQ for 1 h. Furthermore, curcumol treatment induced phosphorylation of the JNK signaling pathway. Of note, pretreatment with the JNK inhibitor, SP600125, decreased the rates of autophagy and apoptosis, suggesting a crucial role served by the JNK signaling pathway in the activation of autophagy by curcumol. Taken together, the results of the present study suggested that activation of the JNK signaling pathway was involved in curcumol-induced autophagy. Curcumol is a novel drug for chemotherapeutic combination therapy. Curcumol demonstrated potential antitumor activities in MG-63 cells and may be used as a novel effective reagent in the treatment of osteosarcoma.

Correlation between osteosarcoma and the expression of WWOX and p53.

The objective of this study was to analyze the effect of the expression of WWOX and p53 on the growth of MG-63 osteosarcoma cells and to explore the correlation between osteosarcoma and the expression of WWOX and p53. WWOX and p53-overexpressing MG-63 osteosarcoma cell lines were established by transfection and named the MW and MP cell lines, respectively. Untransfected MG-63 cells (blank control) were used as control. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of WWOX and wild-type p53 mRNA and protein, respectively. The effects of WWOX and p53 (wild-type) on the activity of MG-63 cells were determined by MTT assay and flow cytometry. The expression of mutant p53 protein in 65 cases of osteosarcoma was detected by immunohistochemistry to analyze the correlation between p53 and the development of osteosarcoma. qPCR showed that WWOX and p53 mRNA was overexpressed in MW and MP cells, respectively. Western blot analysis showed that the levels of WWOX and p53 protein in MW and MP cells were higher than in the blank control group. MTT assay showed that the cell proliferation ability of MW and MP cells was significantly lower than in the blank control group. Flow cytometry showed that 78.49% of MW and 66.76% of MP cells were arrested in the G0/G1 phase. Immunohistochemistry showed that mutant p53 was highly expressed in osteosarcoma, with a positive expression rate of 47.7%. The expression rate was positively correlated with the pathological grade of cancer. In conclusion, WWOX can affect the cell cycle of MG-63 osteosarcoma cells to inhibit cell proliferation, which provides new insights into gene therapy for osteosarcoma. The two types of the p53 gene have different functions in the development of osteosarcoma. Wild-type p53 acts as a tumor suppressor, while mutant p53, which is overexpressed in malignant osteosarcoma, has a carcinogenic effect associated with the degree of osteosarcoma.

Towards modular bone tissue engineering using Ti-Co-doped phosphate glass microspheres: cytocompatibility and dynamic culture studies.

The production of large quantities of functional vascularized bone tissue ex vivo still represent an unmet clinical challenge. Microcarriers offer a potential solution to scalable manufacture of bone tissue due to their high surface area-to-volume ratio and the capacity to be assembled using a modular approach. Microcarriers made of phosphate bioactive glass doped with titanium dioxide have been previously shown to enhance proliferation of osteoblast progenitors and maturation towards functional osteoblasts. Furthemore, doping with cobalt appears to mimic hypoxic conditions that have a key role in promoting angiogenesis. This characteristic could be exploited to meet the clinical requirement of producing vascularized units of bone tissue. In the current study, the human osteosarcoma cell line MG-63 was cultured on phosphate glass microspheres doped with 5% mol titanium dioxide and different concentrations of cobalt oxide (0%, 2% and 5% mol), under static and dynamic conditions (150 and 300 rpm on an orbital shaker). Cell proliferation and the formation of aggregates of cells and microspheres were observed over a period of two weeks in all glass compositions, thus confirming the biocompatibility of the substrate and the suitability of this system for the formation of compact micro-units of tissue. At the concentrations tested, cobalt was not found to be cytotoxic and did not alter cell metabolism. On the other hand, the dynamic environment played a key role, with moderate agitation having a positive effect on cell proliferation while higher agitation resulting in impaired cell growth. Finally, in static culture assays, the capacity of cobalt doping to induce vascular endothelial growth factor (VEGF) upregulation by osteoblastic cells was observed, but was not found to increase linearly with cobalt oxide content. In conclusion, Ti-Co phosphate glasses were found to support osteoblastic cell growth and aggregate formation that is a necessary precursor to tissue formation and the upregaulation of VEGF production can potentially support vascularization.

CXCL8 promotes the invasion of human osteosarcoma cells by regulation of PI3K/Akt signaling pathway.

Chemokine cysteine-X-cysteine motif ligand 8 (CXCL8) is up-regulated in many malignancies, indicating that CXCL8 takes part in tumor progression. However, the expression and function of CXCL8 in osteosarcoma remained not fully elucidated. In this study, expressions of 12 cytokines and chemokines were measured in the serum from 12 of normal controls (NCs) and 25 of osteosarcoma patients. The human osteosarcoma cell line MG-63 was stimulated by recombinant CXCL8 to further analyze invasion, proliferation, apoptosis, cell cycles, cytokine secretions, and signaling pathways. We found that serum concentrations of CXCL8 and vascular endothelial growth factor were elevated in osteosarcoma patients in comparison with those in NCs. CXCL8 stimulation led to enhancement of invasion and suppression of late stage apoptosis in MG-63 cells. Moreover, secretions of MMPs by MG-63 cells were also increased upon stimulation. However, early stage apoptosis, proliferation, and cell cycles were not affected by CXCL8 treatment. Furthermore, CXCL8 stimulation induced elevations of phosphorylated PI3K and Akt, but not PKC or FAK. In conclusion, our findings suggested that CXCL8 enhanced the invasion and suppressed late stage apoptosis of osteosarcoma cells probably via influencing PI3K/Akt signaling pathway and elevating the expression of MMPs. CXCL8 may promote disease progression of osteosarcoma as a protumorigenic molecule, and may be served as a new therapeutic target for osteosarcoma.

Effect of microRNA-422a expression on proliferation of osteosarcoma cells

Objective To observe the effect of microRNA (miRNA, miR)-422a expression on the proliferation of osteosarcoma cells. Methods 40 healthy male Sprague-Dawley (SD) rats were randomly divided into two groups: osteosarcoma MG63 and U2OS cell line bearing mice. Annexin V/propidium iodide (PI) double staining was used to transfect miR-422a primers and inhibitors to regulate miR-422a, the proliferation and apoptosis of osteosarcoma cells were analyzed under overexpression and miR-422a expression, and the inhibitory effect of miR-422a expression on different cell cycle osteosarcoma cells was examined. Results The proliferation of MG63 and U2OS cells was significantly inhibited at 48 h after transfection of miR-422a, and that of MG63 and U2OS was promoted at 48 h after transfection of miR-422a inhibitor with the difference being statistically significant (P=0.068, 0.035, 0.016, 0.004; 0.072, 0.028, 0.019, 0.003). The apoptosis rate of MG63 cells in the control group at 0 and 96 h was (5.3±0.1)% and (5.2±0.1)% respectively, and that in miR-422a group was (5.3±0.5)% and (5.4±0.4)% respectively. The apoptosis rate of U20S in the control group at 0 and 96 h was (5.2±0.2)% and (5.3±0.2)% respectively, and that in miR-422a group was (5.5±0.1)% and (5.2±0.1)% respectively. After starvation for 48 h, the apoptosis rate of MG63 cells in control group at 0 and 96 h was (5.2±0.4)% and (28.6±2.7)% respectively, and that in miR-422a group was (5.5±0.2)% and (48.5±2.4)% respectively; the apoptosis rate of U20S in control group at 0 and 96 h was (6.2±0.3)% and (18.7±3.1)% respectively, and that in miR-422a group was (6.2±0.2)% and (39.8±3.2)% respectively. As compared with negative control group, the apoptosis rate of osteosarcoma cells had no significant changes in miR-422a group at 96 h. Howdever, the combination of miR-422a transfection and apoptosis induction for 48 h, the number of apoptotic osteosarcoma cells was significantly increased as compared with negative control group. After transfection of miR-422a, the number of cells in G0/G1 phase was significantly increased, and that in G2/M was significantly reduced. Conclusion The high expression of miR-422a can effectively inhibit the proliferation of osteosarcoma cells and increase the apoptosis rate of osteosarcoma cells in harsh environment. Key words: Osteosarcoma; MicroRNA-422a; Cell proliferation; Apoptosis

Chemosensitive effects of Astragaloside IV in osteosarcoma cells via induction of apoptosis and regulation of caspase-dependent Fas/FasL signaling.

The response of conventional chemotherapy for osteosarcoma treatment is usually poor, and chemotherapy-related severe side effects and drug resistance remain a problem. Abundant evidence has shown that Astragaloside IV, extracted from Astragalus membranaceus Bunge, strongly inhibits the growth of many carcinomas. We aimed to investigate the chemosensitive effects of Astragaloside IV in osteosarcoma in vitro and in vivo. Human osteosarcoma cell lines MG-63 and 143B, and BALB/c nu/nu mice xenograft were used. MTT, Clonogenic assay, Annexin V/PI assay and Western bloting analysis were carried out. Our present study found that Astragaloside IV was a critical chemosensitizing agent for osteosarcoma treatment. Astragaloside IV suppressed cell proliferation and enhanced chemosensitivity in osteosarcoma cell lines and xenograft. Caspase-dependent Fas/FasL signaling was involved in cisplatin-induced apoptosis which was enhanced by Astragaloside IV. It indicated that Astragaloside IV might be a promising therapeutic agent for osteosarcoma treatment.

Abstract 2552: MDM2 induces cell fusion in breast cancer and osteosarcoma

Abstract Cell fusion is a physiological process utilized by a variety of biological systems during fertilization, embryogenesis, and cellular differentiation and morphogenesis. During the malignant transformation of breast and bone tissues, normal and malignant cells become fused to form hybrid structures. These enhanced phenotypes acquire genetic characteristics which support initiation, progression, and oncogenesis. In breast cancer and osteosarcoma cell lines, cell fusion generates chromosomal instability, aneuploidy, and DNA damage while maintaining the characteristics of enhanced migration, invasion, and chemoresistance. We have found that mouse double minute 2 (Mdm2) significantly induces cell fusion and the generation of large multinucleated cells in MDA468, MDA231, MDA157, T47D breast cancer and MG63 osteosarcoma cell lines under normoxia when compared to silenced Mdm2 controls (P&amp;lt;0.0005). Interestingly, silencing mutant p53 in the MDA468 cell line resulted in significantly higher fusion events (P&amp;lt;0.0004) suggesting mutant p53 may play a regulatory role in reducing Mdm2-associated cell fusion. Silenced Mdm2 MDA231 cells showed a three-fold increase under hypoxia whereas MG63 cells showed similar levels of cell fusion events as in normoxic conditions suggesting cell specific dependency to environmental stimulus. The variation in proportions of cell fusion among each cell type suggests this phenomenon is dependent on Mdm2 expression and cell type. CD133+ MDA468 cells have increased fractions of polyploidal cell events suggesting stemness may influence the polyploidal giant cancer cell (PGCC) dynamics. The mechanism by which cell fusion occurs in breast and osteosarcoma is poorly understood. Collectively these data suggest the interplay of Mdm2, p53, and stemness in supporting the formation of fused polypoidal cells and may serve as relevant targets for cancer therapy. Citation Format: David J. Olivos, Mikhail Chtcherbinine, Eric R. Wolf, Ciarán McAtarsney, Lindsey D. Mayo. MDM2 induces cell fusion in breast cancer and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2552. doi:10.1158/1538-7445.AM2017-2552

Involvement of c-Fos in cell proliferation, migration, and invasion in osteosarcoma cells accompanied by altered expression of Wnt2 and Fzd9.

Osteosarcoma (OS) is an aggressive bone tumor, and proto-oncogene c-Fos is involved in this lethal disease. However, the role and molecular mechanism of c-Fos in the development and progression of OS remain enigmatic. As one of the Wnt family members, Wnt2 is closely associated with the development of several malignant tumors. In the present study, the expression of c-Fos, Wnt2, and its receptor Fzd9 in human OS tissues, MG63 OS cell line, and human osteoblast hFOB 1.19 cell line was detected by Western blot analysis, immunohistochemical staining, or reverse transcription-polymerase chain reaction. The role of c-Fos in the OS was clarified by treating MG63 cells with small interfering RNA to knockdown c-Fos. Then, cell migration and invasion were assayed by transwell assays and wound healing assay; cell proliferation was assayed by MTS method and 5-ethynyl-2'-deoxyuridine DNA proliferation in vitro detection; cell apoptosis was assayed by flow cytometric method. Co-immunoprecipitation kit was used to confirm the relationship between c-Fos and Wnt2/Fzd9. We found that the expression of c-Fos, Wnt2, and Fzd9 protein was distinctly higher in human OS tissues than that in the adjacent non-cancerous tissues, and their expression in the MG63 OS cell line was markedly increased compared with that in the human osteoblast hFOB 1.19 cell line. Knockdown of c-Fos inhibited the proliferation, migration, and invasion of MG63 cells, and promoted the apoptosis of MG63 cells. Moreover, knockdown of c-Fos inhibited the expression of Wnt2 and Fzd9 mRNA and protein. Our data enforced the evidence that knockdown of c-Fos inhibited cell proliferation, migration, and invasion, and promoted the apoptosis of OS cells accompanied by altered expression of Wnt2 and Fzd9. These findings offer new clues for OS development and progression, and c-Fos may be a potential therapeutic target for OS.

Crude Fucoidan Extracts Impair Angiogenesis in Models Relevant for Bone Regeneration and Osteosarcoma via Reduction of VEGF and SDF-1.

The marine origin polysaccharide fucoidan combines multiple biological activities. As demonstrated by various studies in vitro and in vivo, fucoidans show anti-viral, anti-tumor, anti-oxidant, anti-inflammatory and anti-coagulant properties, although the detailed molecular action remains to be elucidated. The aim of the present study is to assess the impact of crude fucoidan extracts, on the formation of vascular structures in co-culture models relevant for bone vascularization during bone repair and for vascularization processes in osteosarcoma. The co-cultures consisted of bone marrow derived mesenchymal stem cells, respectively the osteosarcoma cell line MG63, and human blood derived outgrowth endothelial cells (OEC). The concentration dependent effects on the metabolic activity on endothelial cells and osteoblast cells were first assessed using monocultures of OEC, MSC and MG63 suggesting a concentration of 100 µg/mL as a suitable concentration for further experiments. In co-cultures fucoidan significantly reduced angiogenesis in MSC/OEC but also in MG63/OEC co-cultures suggesting a potential application of fucoidan to lower the vascularization in bone tumors such as osteosarcoma. This was associated with a decrease in VEGF (vascular endothelial growth factor) and SDF-1 (stromal derived factor-1) on the protein level, both related to the control of angiogenesis and furthermore discussed as crucial factors in osteosarcoma progression and metastasis. In terms of bone formation, fucoidan slightly lowered on the calcification process in MSC monocultures and MSC/OEC co-cultures. In summary, these data suggest the suitability of lower fucoidan doses to limit angiogenesis for instance in osteosarcoma.

Cytotoxic withanolides from Physalis angulata

A new withanolide (1), physagulide P, together with five known withanolides (2–6), was isolated from the aerial parts of Physalis angulata L. The structure of new compound was elucidated on the basis of extensive spectroscopic techniques, including 1D, 2D NMR and HRESIMS. The activity screening indicated that compound 1 showed significant cytotoxicities against the human osteosarcoma cell line MG-63, HepG-2 hepatoma cells and breast cancer cells MDA-MB-231 with the IC50 value of 3.50, 4.22 and 15.74 μM.