Human Osteosarcoma Cell Line Saos-2 Research Articles

The Curcumin Analog CH-5 Exerts Anticancer Effects in Human Osteosarcoma Cells via Modulation of Transcription Factors p53/Sp1.

Curcumin is a potential anticancer drug with poor bioavailability, which limits its clinical use as a therapeutic agent. The aim of this study was a preliminary evaluation of the curcumin analogue CH-5 as a cytotoxic agent in human osteosarcoma cell lines U2OS, MG-63, and Saos-2. CH-5 inhibited cell viability at lower concentrations than curcumin, leading to the induction of apoptosis. The cellular levels of the transcription factors p53 and Sp1 affect the expression of cellular pathways that lead to apoptosis. CH-5 increased p53 protein levels in U2OS cells and reduced Sp1 levels, with a consequent effect on the expression of their target genes DNA methyltransferase 1 (DNMT1) and growth arrest and DNA damage-inducible 45 alpha gene (Gadd45a). CH-5 repressed DNMT1 and increased Gadd45a mRNA expression, which was dependent on p53, as this effect was only observed in the colorectal cancer cell line HCT116 with active p53, but not in the isogenic p53-deficient HCT116 cells. CH-5 also reduced the protein levels of DNMT1, which led to the upregulation of Gadd45a. These results suggest that CH-5 has potentially higher anticancer activity than curcumin, which is associated with the expression of apoptosis-associated genes regulated by the transcription factors Sp1 and p53. Future work on CH-5 will define the therapeutic potential of this compound in vivo.

The effects of cobalt and chromium ions on transforming growth factor-beta patterns and mineralization in human osteoblast-like MG63 and SaOs-2 cells.

Bone homeostasis, the balance of bone formation and resorption is affected by numerous influences, such as, hormones, inflammation, mechanical load, and external stimuli. The transforming growth factor-beta (TGF-β), which exists in three isoforms in humans, is a major factor in the maintenance of this balance by regulating osteoblast and osteoclast maturation, development, and function. In artificial joint replacements, release of particles or ions from arthroplasties may exert local effects on the periprosthetic tissue and modulate the expression of bone specific genes and functions. Therefore, the influence of cobalt (II) and chromium (III) ions on the expression levels of the three TGF-β isoforms in human osteosarcoma cell lines MG63 and SaOs-2 was analyzed and the impact on mineralization was studied. The osteosarcoma cell lines expressed all three TGF-β isoforms, with TGF-β1 being the most abundant isoform. A dose dependent reduction of all TGF-β isoforms by Co2+ ions was observed, the strongest effect was found on TGF-β2. The effect was lesser pronounced in SaOs-2 cells. However, the Cr3+ ions had no significant effect on the expression of all TGF-β isoforms. In contrast, Co2+ ions in a concentration range of 50-250 µM did not impair the mineralization, but Cr3+ exerted a strong inhibitory effect on the mineralization in a dose dependent fashion. These data suggest that the influence of cobalt ions on bone homeostasis may in part result from the inhibitory effect on the transcription of the bone regulating cytokines TGF-β1-3 whereas the chromium ions affect the process of mineralization. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2105-2115, 2018.

Eukaryotic translation initiation factor 3H suppression inhibits osteocarcinoma cell growth and tumorigenesis.

Eukaryotic translation initiation factor 3H subunit (EIF3H) is a member of the EIF3 family and exhibits a central role in translation initiation in higher eukaryotes. Although EIF3H expression is upregulated in numerous tumour types, its potential role in human osteosarcoma (OS) has not yet been investigated. In the present study, it was demonstrated that EIF3H mRNA expression was upregulated in the human OS cell lines Saos-2 and U2OS. A recombinant lentivirus harbouring short hairpin RNA targeting EIF3H was constructed and successfully infected human OS Saos-2 and U2OS cells, resulting in 95% downregulated EIF3H expression compared with the respective control groups. Knockdown of EIF3H significantly inhibited the proliferation and colony formation of OS cells in vitro, and tumour growth in nude mice in vivo. Flow cytometry analysis revealed cell cycle arrest and promotion of apoptosis in OS cells with EIF3H knocked down. In conclusion, the results strongly suggested that EIF3H is a critical factor mediating the growth of OS cells and may represent a novel therapeutic target.

Silicate-doped nano-hydroxyapatite/graphene oxide composite reinforced fibrous scaffolds for bone tissue engineering.

In this study, novel graphene oxide-incorporated silicate-doped nano-hydroxyapatite composites were prepared and their potential use for bone tissue engineering was investigated by developing an electrospun poly(ε-caprolactone) scaffold. Nanocomposite groups were synthesized to have two different ratios of graphene oxide (2 and 4 wt%) to evaluate the effect of graphene oxide incorporation and groups with different silicate-doped nano-hydroxyapatite content was prepared to investigate optimum concentrations of both silicate-doped nano-hydroxyapatite and graphene oxide. Three-dimensional poly(ε-caprolactone) scaffolds were prepared by wet electrospinning and reinforced with silicate-doped nano-hydroxyapatite/graphene oxide nanocomposite groups to improve bone regeneration potency. Microstructural and chemical characteristics of the scaffolds were investigated by X-ray diffraction, Fourier transform infrared spectroscope and scanning electron microscopy techniques. Protein adsorption and desorption on material surfaces were studied using fetal bovine serum. Presence of graphene oxide in the scaffold, dramatically increased the protein adsorption with decreased desorption. In vitro biocompatibility studies were conducted using human osteosarcoma cell line (Saos-2). Electrospun scaffold group that was prepared with effective concentrations of silicate-doped nano-hydroxyapatite and graphene oxide particles (poly(ε-caprolactone) - 10% silicate-doped nano-hydroxyapatite - 4% graphene oxide) showed improved adhesion, spreading, proliferation and alkaline phosphatase activity compared to other scaffold groups.

Effect and Mechanism of EGFL7 Downregulation in Human Osteosarcoma Cells on the Biological Function of Co-cultured HUVEC.

Background:Even though epidermal growth factor-like domain 7 is known to be overexpressed in osteosarcoma and is associated with poor clinical outcome, few reports are available regarding its mechanism.Aims:The objective of this study was to explore the effect and mechanism of downregulating epidermal growth factor-like domain 7 expression in a human osteosarcoma cell line on the biological function of co-cultured human umbilical vein endothelial cells.Study Design:Cell study.Methods:In the present study, human osteosarcoma cell lines U2OS, Saos-2, HOS, and MG63, and normal human osteoblasts were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 1x antibiotics at 37 °C and 5% CO2 in an incubator. Of the four osteosarcoma cell lines, U2OS expresses the highest level of epidermal growth factor-like domain 7 mRNA as determined using quantitative reverse transcription polymerase chain reaction. With the knockdown of epidermal growth factor-like domain 7 in U2OS and human umbilical vein endothelial cells by lentivirus, the proliferation and apoptosis of U2OS and human umbilical vein endothelial cells were investigated using MTT and flow cytometry assays. After the co-culture of human umbilical vein endothelial cells and epidermal growth factor-like domain 7-knockdown U2OS, the in vitro effects on cell proliferation, apoptosis, adhesion, migration, and the angiogenic ability of human umbilical vein endothelial cells were detected using MTT, flow cytometry, Transwell, and tube formation assays, respectively. The expressions of phosphoinositide 3-kinase, phospho-Akt, total Akt, and vascular endothelial growth factor in human umbilical vein endothelial cells were detected using western blot assay.Results:Lentivirus with epidermal growth factor-like domain 7 shRNA could not significantly affect the proliferation and apoptosis of both U2OS and human umbilical vein endothelial cells, whereas the knockdown of epidermal growth factor-like domain 7 in U2OS could significantly inhibit the migration, adhesion, and angiogenic ability of co-cultured human umbilical vein endothelial cells. In addition, the expressions of phosphoinositide 3-kinase, phospho-Akt, and vascular endothelial growth factor in human umbilical vein endothelial cells decreased after co-culturing with epidermal growth factor-like domain 7-knockdown U2OS.Conclusion:Epidermal growth factor-like domain 7-knockdown U2OS cells inhibit the migration, adhesion, and angiogenesis of co-cultured human umbilical vein endothelial cells by diminishing phosphoinositide 3-kinase, Akt signaling pathway activity and vascular endothelial growth factor expression.

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6–18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. β-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.

Abstract A23: Pterostilbene (3',5'-dimethoxyresveratrol) inhibits cell growth and augments the efficacy of doxorubicin through stem cell phenotype suppression in osteosarcoma cells in vitro

Abstract Objective: Osteosarcoma (OS) is the most common primary malignant neoplasm of bone and mainly affects adolescents and young adults. Intensive multiagent chemotherapy and definitive surgery have dramatically improved its prognosis as well as functional outcome of affected limbs since the 1980s. However, survival rate of patients has remained around 70% for more than 30 years, and the prognosis remains especially poor in patients with metastatic disease or unresectable lesions. Thus, additional or alternative treatment strategy has long been desired for patients with such poor prognosis. Pterostilbene (PTE), a methyl ether of resveratrol derived from plants like grapes and blueberries, has recently attracted attention as a growing number of reports describe promising pharmacologic properties of antioxidative and antiaging effects and have shown its inhibitory effects against cancers including colon, oral cancer, and malignant lymphoma. However, there are very scarce reports about the effects of PTE against OS. Here, we show a very characteristic efficacy of PTE on human OS cells and propose its potential as a novel therapeutic intervention against OS. Materials and Methods: Effects of PTE on cellular growth were evaluated in human OS cell lines SaOS2, U2OS, and MG63 treated at concentrations ranging from 2 μM to10 μM (2, 5, 10 μM) for 48 hrs compared to nontreated cells by MTS assay. Additionally, we evaluated the effects of cotreatment of PTE with low-dose (0.2 μg/ml) of doxorubicin (DXR) against SaOS2 and U2OS. To investigate the efficacy of PTE on stemness, ability of spheroid formation in anchorage-independent 3D culture condition was evaluated in SaOS2 and U2OS cells treated by 10 μM PTE for 48 hrs and mRNA expression of stemness markers of Oct3 and CD44 was evaluated by RT-PCR at the same condition. Results: Treatment of PTE showed dose-dependent growth inhibitory effects on all cell lines and was especially significant on MG63 cells at the highest inhibition rate of 65% at the concentration of 10 μM for 48 hrs treatment. In contrast, PTE did not show growth inhibition on normal mesenchymal stromal cells, one of the candidates for OS cell of origin. Cotreatment of PTE with DXR indicated that PTE augmented the growth suppressive effects of DXR lifting the growth inhibition rate up to 515% from DXR alone against SaOS2 and U2OS (62-67% and 25-40%, respectively). PTE treatments decreased sphere-forming ability in both SaOS2 and U2OS cells in terms of sphere numbers and sphere sizes, and also decreased mRNA expression of stemness markers of Oct3 and CD44 in both cell lines. Discussion: Current results suggest that PTE not only inhibits cellular growth of OS but also suppresses stem cell phenotype which was defined by sphere-forming ability and mRNA expression of stemness markers. Mechanisms of stem cell phenotype suppression by PTE are still unknown, but it might be caused by either decrease or differentiation of stem cell subpopulation. Suppression of stem cell phenotype could lead to enhancing the efficacy of cytotoxic chemotherapeutic agents such as DXR. Previous reports suggested that antitumor activity of PTE against OS cells targeted the JAK2/STAT3 signaling pathways. The efficacy of PTE is cell-type dependent, possibly due to the mechanisms of cell death or cell metabolism. The molecular pathways involved in growth inhibition and stem cell phenotype suppression caused by PTE are under investigation using pathway inhibitors and metabolism modulators. Conclusion: PTE inhibits cellular growth and augments the efficacy of DXR against OS cells possibly through the suppression of stem cell characteristics represented by sphere-forming ability and mRNA expression of stemness markers. The current study suggests that PTE could be a possible candidate for a novel therapeutic intervention against OS. Citation Format: Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Akira Kido, Toshifumi Tsujiuchi, Hiroki Kuniyasu, Yasuhito Tanaka. Pterostilbene (3',5'-dimethoxyresveratrol) inhibits cell growth and augments the efficacy of doxorubicin through stem cell phenotype suppression in osteosarcoma cells in vitro [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A23.

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.

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.

MicroRNA-21 Inhibits the Apoptosis of Osteosarcoma Cell Line SAOS-2 via Targeting Caspase 8.

Currently, multiple microRNAs (miRNAs) have been found to play vital roles in the pathogenesis of osteosarcoma. This study aimed to investigate the role of miR-21 in osteosarcoma. The level of miR-21 in 20 pairs of osteosarcoma and corresponding adjacent tissues was monitored by qPCR. Human osteosarcoma cell line SAOS-2 was transfected with either miR-21 mimic or miR-21 inhibitor, and then cell viability, survival, and apoptosis were measured by MTT, colony formation assay, and flow cytometry. A target of miR-21 was predicted by the microRNA.org database and verified in vitro by using luciferase reporter, qPCR, and Western blot analyses. Finally, cells were cotransfected with siRNA against caspase 8 and miR-21 inhibitor, and the apoptotic cell rate was determined again. Results showed that the mRNA level of miR-21 was highly expressed in osteosarcoma tissues compared with adjacent tissues. Overexpression of miR-21 improved cell viability and survival but suppressed apoptosis. Caspase 8 was a direct target of miR-21, and it was negatively regulated by miR-21. Moreover, miR-21 suppression attenuated caspase 8 silencing and induced the decrease in apoptosis. In conclusion, overexpression of miR-21 suppressed SAOS-2 cell apoptosis via directly targeting caspase 8.

MicroRNA-208b inhibits human osteosarcoma progression by targeting ROR2.

MicroRNAs are widely involved in cancer progression by inhibiting the expression levels of oncogenes or tumor suppressor genes, and dysregulation of microRNAs may contribute to tumorigenesis. Here, we found that overexpressed miR-208b can reduce the proliferation of human osteosarcoma cell lines U-2OS and Saos-2 by arresting cell cycle progression. The in vivo xenograft tumors induced by Saos-2 cells overexpressing miR-208b had smaller size and grew more slowly than those induced by the control cells. The mobility of U-2OS or Saos-2 cells was also downregulated by miR-208b. MiR-208b targeted a site in the 3' untranslated region of receptor tyrosine kinase-like orphan receptor 2. Inhibition of receptor tyrosine kinase-like orphan receptor 2 suppresses osteosarcoma metastasis in vitro. Recovering the expression levels of receptor tyrosine kinase-like orphan receptor 2 in miR-208b-overexpressed U-2OS or Saos-2 cells attenuated the inhibitory effects of miR-208b. In addition, the expression levels of miR-208b are significantly reduced in human osteosarcoma tissue samples compared to normal tissue samples, and miR-208b levels correlated inversely with receptor tyrosine kinase-like orphan receptor 2 levels. On these bases, we identified that miR-208b targets receptor tyrosine kinase-like orphan receptor 2 gene by which miR-208b can regulate the development of osteosarcoma.

Different Expression and Localization of Phosphoinositide Specific Phospholipases C in Human Osteoblasts, Osteosarcoma Cell Lines, Ewing Sarcoma and Synovial Sarcoma

Background: Bone hardness and strength depends on mineralization, which involves a complex process in which calcium phosphate, produced by bone-forming cells, was shed around the fibrous matrix. This process is strictly regulated, and a number of signal transduction systems were interested in calcium metabolism, such as the phosphoinositide (PI) pathway and related phospholipase C (PLC) enzymes. Objectives: Our aim was to search for common patterns of expression in osteoblasts, as well as in ES and SS. Methods: We analysed the PLC enzymes in human osteoblasts and osteosarcoma cell lines MG-63 and SaOS-2. We compared the obtained results to the expression of PLCs in samples of patients affected with Ewing sarcoma (ES) and synovial sarcoma (SS). Results: In osteoblasts, MG-63 cells and SaOS-2 significant differences were identified in the expression of PLC δ4 and PLC η subfamily isoforms. Differences were also identified regarding the expression of PLCs in ES and SS. Most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS. The most relevant difference observed in ES compared to SS regarded PLC ε and PLC η isoforms. Conclusion: MG-63 and SaOS-2 osteosarcoma cell lines might represent an inappropriate experimental model for studies about the analysis of signal transduction in osteoblasts

Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus

Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway.IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1.

MicroRNA-93 promotes cell proliferation by directly targeting P21 in osteosarcoma cells.

MicroRNAs (miRNAs) are small, non-coding RNAs that are key regulators of gene expression by directly binding to the 3'-untranslated region of their target mRNAs, resulting in translational repression or degradation of mRNA. It has been demonstrated that miRNAs have key roles in a variety of human malignancies, including osteosarcoma. The present study aimed to assess the molecular mechanism of miR-93 in the regulation of osteosarcoma cell proliferation. Reverse-transcription quantitative PCR and western blot assays were used to examine mRNA and protein expression. An MTT assay and flow cytometry were performed to determine the cell proliferation and cell cycle distribution. A luciferase reporter assay was performed to confirm the direct targeting of cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as P21, by miR-93, which was suggested by a bioinformatics analysis. The results showed that the expression of miR-93 was frequently and significantly increased in a total of 19 osteosarcoma tissues compared to their matched adjacent non-tumor tissues, and the upregulation of miR-93 was associated with the malignant progression of osteosarcoma. Furthermore, miR-93 was also upregulated in the human osteosarcoma cell lines Saos-2, U2OS, SW1353 and MG63 when compared with that in the human osteoblast cell line hFOB1.19. Transfection with miR-93 inhibitor significantly reduced the miR-93 levels and inhibited the proliferation of U2OS and MG63 osteosarcoma cells. The protein levels of P21 were negatively regulated by miR-93 in U2OS and MG63 cells. Knockdown of miR-93 caused cell cycle arrest at G1 stage in U2OS and MG63 cells, identical to the effect of P21 overexpression. Finally, P21 was found to be significantly downregulated in osteosarcoma tissues compared to their matched adjacent non-tumor tissues, suggesting that the inhibition of P21 may be due to increased miR-93 expression in osteosarcoma tissues. In conclusion, the present study demonstrated that miR-93 enhances the proliferation of osteosarcoma cells, at least in part via inhibiting P21 expression and thus promoting cell cycle progression.

Effects of Placental Extract on Cell Proliferation, Type I Collagen Production, and ALP Secretion in Human Osteosarcoma Cell Line Saos-2

Effects of Placental Extract on Cell Proliferation, Type I Collagen Production, and ALP Secretion in Human Osteosarcoma Cell Line Saos-2

Reduced cell proliferation and promotion of apoptosis by Leu5AMD in osteosarcoma cells

Leu 5 AMD is an actinomycin D (AMD) analog obtained by replacing both N-methylvalines present in AMD with N-methylleucines. The potential of Leu 5 AMD for the treatment of osteosarcoma cells and its mechanism of action are not clearly understood. In this study, we examined the potential use of Leu 5 AMD in halting the proliferation of the human osteosarcoma cell line Saos-2. Saos-2 cells were treated with different concentrations of Leu 5 AMD, and proliferation, cell viability, apoptosis and the cell cycle were examined. Among the various concentrations used, 1.2 μM and 6 μM Leu 5 AMD exhibited a significant effect on Saos-2 cells compared with untreated cells. The proliferation assay and Hoechst staining indicated that Leu 5 AMD treatment of Saos-2 human osteosarcoma cells resulted in a significant reduction in cell proliferation and enhanced apoptosis. Western blot analysis revealed that Leu 5 AMD-treated cells had significantly reduced cyclin expression compared with the control. Leu 5 AMD is responsible for inducing cell cycle arrest and apoptosis. These results support the development of a new drug for the treatment of osteosarcoma. https://doi.org/10.2298/ABS160712001W Received: July 12, 2016; Revised: December 2, 2016; Accepted: December 2, 2016; Published online: August 9, 2017 How to cite this article: Wu X, Zhou Z. Reduced cell proliferation and promotion of apoptosis by Leu5AMD in osteosarcoma cells. Arch Biol Sci. 2017;69(4):577-83.

Targeted drug delivery of near IR fluorescent doxorubicin-conjugated poly(ethylene glycol) bisphosphonate nanoparticles for diagnosis and therapy of primary and metastatic bone cancer in a mouse model.

BackgroundMost primary and metastatic bone tumors demonstrate increased osteoclast activity and bone resorption. Current treatment is based on a combination of surgery, radiotherapy and chemotherapy. Severe side effects are associated with chemotherapy due to use of high dosage and nonspecific uptake. Bisphosphonates have a strong affinity to Ca2+ ions and are widely used in the treatment of bone disorders.ResultsWe have engineered a unique biodegradable bisphosphonate nanoparticle (NPs) bearing two functional surface groups: (1) primary amine groups for covalent attachment of a dye/drug (e.g. NIR dye Cy 7 or doxorubicin); (2) bisphosphonate groups for targeting and chelation to bone hydroxyapatite. In addition, these engineered NPs contain high polyethyleneglycol (PEG) concentration in order to increase their blood half life time. In vitro experiments on Saos-2 human osteosarcoma cell line, demonstrated that at a tenth of the concentration, doxorubicin-conjugated bisphosphonate NPs achieved a similar uptake to free doxorubicin. In vivo targeting experiments using the NIR fluorescence bisphosphonate NPs on both Soas-2 human osteosarcoma xenograft mouse model and orthotopic bone metastases mCherry-labeled 4T1 breast cancer mouse model confirmed specific targeting. In addition, therapeutic in vivo experiments using doxorubicin-conjugated bisphosphonate NPs demonstrated a 40% greater inhibition of tumor growth in Saos-2 human osteosarcoma xenograft mouse model when compared to free doxorubicin.ConclusionsIn this research we have shown the potential use of doxorubicin-conjugated BP NPs for the targeting and treatment of primary and metastatic bone tumors. The targeted delivery of doxorubicin to the tumor significantly increased the efficacy of the anti-cancer drug, thus enabling the effective use of a lower concentration of doxorubicin. Furthermore, the targeting ability of the BP NPs in an orthotopic xenograft mouse model reinforced our findings that these BP NPs have the potential to be used for the treatment of primary and metastatic bone cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0233-6) contains supplementary material, which is available to authorized users.

Evaluation of CD146 as Target for Radioimmunotherapy against Osteosarcoma.

BackgroundOsteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo.Methods and ResultsA murine monoclonal anti-CD146 IgG1 isotype antibody, named OI-3, was developed along with recombinant chimeric versions with human IgG1 or human IgG3 Fc sequences. Using flow cytometry, selective binding of OI-3 to human osteosarcoma cell lines OHS, KPDX and Saos-2 was confirmed. The results confirm a higher expression level of CD146 on human osteosarcoma cells than HER2 and EGFR; antigens targeted by commercially available therapeutic antibodies. The biodistribution of 125I-labeled OI-3 antibody variants was compared with 125I-labeled chimeric anti-EGFR antibody cetuximab in nude mice with subcutaneous OHS osteosarcoma xenografts. OI-3 was able to target CD146 expressing tumors in vivo and showed improved tumor to tissue targeting ratios compared with cetuximab. Subsequently, the three OI-3 variants were conjugated with p-SCN-Bn-DOTA and labeled with a more therapeutically relevant radionuclide, 177Lu, and their biodistributions were studied in the nude mouse model. The 177Lu-labeled OI-3 variants were stable and had therapeutically relevant biodistribution profiles. Dosimetry estimates showed higher absorbed radiation dose to tumor than all other tissues after administration of the chimeric IgG1 OI-3 variant.ConclusionOur results indicate that CD146 can be targeted in vivo by the radiolabeled OI-3 antibodies.

Wetspun poly-L-(lactic acid)-borosilicate bioactive glass scaffolds for guided bone regeneration

We developed a porous poly-L-lactic acid (PLLA) scaffold compounded with borosilicate bioactive glasses (BBGs) endowing it with bioactive properties. Porous PLLA-BBG fibre mesh scaffolds were successfully prepared by the combination of wet spinning and fibre bonding techniques. Micro-computed tomography (μCT) confirmed that the PLLA-BBG scaffolds containing ≈25% of BBGs (w/w) exhibited randomly interconnected porous (58 to 62% of interconnectivity and 53 to 67% of porosity) with mean pore diameters higher that 100μm. Bioactivity and degradation studies were performed by immersing the scaffolds in simulated body fluid (SBF) and ultrapure water, respectively. The PLLA-BBG scaffolds presented a faster degradation rate with a constant release of inorganic species, which are capable to produce calcium phosphate structures at the surface of the material after 7days of immersion in SBF (Ca/P ratio of ~1.7). Cellular in vitro studies with human osteosarcoma cell line (Saos-2) and human adipose-derived stem cells (hASCs) showed that PLLA-BBGs are not cytotoxic to cells, while demonstrating their capacity to promote cell adhesion and proliferation. Overall, we showed that the proposed scaffolds present a tailored kinetics on the release of inorganic species and controlled biological response under conditions that mimic the bone physiological environment.

Comparison of Phosphoinositide-Specific Phospholipase C Expression Panels of Human Osteoblasts Versus MG-63 and Saos Osteoblast-Like Cells

Background: A large number of phospholipase C (PLC) enzymes, both mRNA transcripts and proteins, have been detected in osteoblasts, corroborating the importance of calcium regulation in bone tissue. MG-63 and SaOS-2 human osteosarcoma cell lines are actually considered osteoblast-like cells, and are therefore widely used as experimental models for osteoblasts. Objectives: Our aim was to verify whether MG-63 or SaOS-2 cells might also represent appropriate experimental osteoblast models for signal transduction studies, with special regard to the phosphoinositide (PI) pathway. We analyzed the expression and the subcellular distribution of enzymes related to calcium signal transduction (the PI-specific PLC family), which are known to possess high cell/tissue specificity. Materials and Methods: The expression of PLC genes was analyzed by performing RT-PCR experiments. The presence of PLC enzymes and their subcellular distribution within the cells was analyzed with immunofluorescence experiments. Conclusions: MG-63 and SaOS-2 osteosarcoma cell lines might not represent appropriate experimental models for studies that aim to analyze signal transduction in osteoblasts.