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Background: Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N 6 -methyladenosine (m 6 A) modification plays a critical role in influencing RNA fate. However, the biological significance of m 6 A modification and its potential regulatory mechanisms in the development of OS remain unclear., Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m 6 A levels. Western blotting, quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) were used to investigate METTL14 expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL14. RNA pull-down and RNA immunoprecipitation (RIP) assays were conducted to explore the specific binding of target genes and relevant m 6 A "readers". RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of the downstream genes of METTL14. IHC and clinical data were applied to explore the clinical correlations of METTL14 and its downstream target genes with the prognosis of OS., Findings: We observed the abundance of m 6 A modifications in OS and revealed that METTL14 plays an oncogenic role in facilitating OS progression. MeRIP-seq and RNA-seq revealed that MN1 is a downstream gene of METTL14. MN1 contributes to tumor progression and all-trans-retinoic acid (ATRA) chemotherapy resistance in OS. Mechanistically, MN1 is methylated by METTL14, specifically in the coding sequence (CDS) regions, and this modification is recognized by the specific m 6 A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to prevent MN1 mRNA degradation and promote it translation efficiency. IHC showed that MN1 expression was positively correlated with METTL14 and IGF2BP2 expression in OS tissues. The METTL14-IGF2BP2-MN1 panel demonstrated more promising prognostic value for OS patients than any of these molecules individually., Interpretation: Our study revealed that METTL14 contributes to OS progression and ATRA resistance as an m 6 A RNA methylase by regulating the stability and translation efficiency of MN1 and thus provides both an underlying biomarker panel for prognosis prediction in OS patients., Funding: This work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864)., Competing Interests: Declaration of interests No potential conflicts of interest were disclosed., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Numerous patients with osteosarcoma either are not sensitive to chemotherapy or develop drug resistance to current chemotherapy regimens. Therefore, it is necessary to develop several potentially useful therapeutic agents. Dihydromyricetin is the major flavonoid component derived from Ampelopsis grossedentata, which has a long history of use in food and medicine. The present study examined the antitumor activity both in vitro and in vivo without noticeable side effects and the underlying mechanism of action of dihydromyricetin in osteosarcoma cells. We found that dihydromyricetin induced increased p21 expression and G2-M cell-cycle arrest, caused DNA damage, activated ATM-CHK2-H2AX signaling pathways, and induced apoptosis in osteosarcoma cells as well as decreasing the sphere formation capability by downregulating Sox2 expression. Mechanistic analysis showed that the antitumor potential of dihydromyricetin may be due to the activation of AMPKα and p38(MAPK), as the activating AMPKα led to the inactivation of GSK3β in osteosarcoma cells. Moreover, GSK3β deletion or GSK3β inhibition by LiCl treatment resulted in increased p21 expression and reduced Sox2 expression in osteosarcoma cells. Taken together, our results strongly indicate that the antitumor potential of dihydromyricetin is correlated with P38(MAPK) and the AMPKα-GSK3β-Sox2 signaling pathway. Finally, immunohistochemical analysis indicated that some patients had a lower p-AMPK expression after chemotherapy, which supports that the combination of dihydromyricetin and chemotherapy drug will be beneficial for patients with osteosarcoma. In conclusion, our results are the first to suggest that dihydromyricetin may be a therapeutic candidate for the treatment of osteosarcoma., (©2014 American Association for Cancer Research.)

Objective: To study the apoptosis inducing effects of bufalin on various human osteosarcoma cells and the concerning molecular mechanisms., Method: MTT assay was used to detect the growth inhibition rates of osteosarcoma cells U-20S, U-20S/MTX300, SaOS-2, IOR/OS9 treated with bufalin in different concentrations and times. The apoptosis of cells was observed flow cytometry 48 h following bufalin treatment. The proteomic techniques were used to separate and compare the treated and control groups 48 h after bufalin-incubation. Then, the proteomic results were validated by western blot., Result: Bufalin inhibited the growth of human osteosarcoma cells U20S, U20S/MTX300 (methotrexate resistant cells), SAOS2, IOR/OS9 in a dose- and time-dependent manner. The 72 h IC50 were (37.43 +/- 4.1), (32.24 +/- 5.3) nmol x L(-1) in U20S,U20S/MTX300 cells,respectivly. Flow cytometry showed that the apoptosis cells were increased following bufalin treatment. The protein expression profile showed 24 differentiated expression proteins. Among these proteins, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27) decreased significantly and the result was then validated by western blot. Ectopic expression of Hsp27 could reduce the bufalin-induced apoptosis remarkably in U20S and U20S/MTX300 cells., Conclusion: Bufalin could inhibit the cell growth and induce apoptosis on human osteosarcoma cells. The effect of bufalin may be related to the joint intervention with multiple protein targets. Among them, downregulation of Hsp27 plays a critical role in the bufalin-induced apoptosis in human osteosarcoma cells.

Background: Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3β in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent., Methods: We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3β expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5-8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3β in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3β activity with overall survival., Results: Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3β formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3β had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3β resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3β resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3β inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3β, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3β and NF-κB (109.2 months)., Conclusion: GSK-3β activity may promote osteosarcoma tumor growth, and therapeutic targeting of the GSK-3β and/or NF-κB pathways may be an effective way to enhance the therapeutic activity of anticancer drugs against osteosarcoma.

Bufalin is the primary component of the traditional Chinese herb "Chan Su". Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s) is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX) sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27), decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27) were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.

Osteosarcoma is a life-threatening malignancy that most often occurs in teenagers. Ezrin is a cytoskeleton linker protein that is actively involved in the metastatic capacity of cancer cells. In this study, we investigated the expression change of ezrin after preoperative chemotherapy and its prognostic value in patients with primary osteosarcoma. Ezrin mRNA expression level of initial biopsy specimens and resected tumor specimens after preoperative chemotherapy of 25 patients who had primary osteosarcoma with ezrin expression was detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) to evaluate the expression change of ezrin. In addition, the prognostic factors and the relationship between the expression change of ezrin and the clinical characteristics were analyzed. Expression change of ezrin was found in 64% of all 25 patients. The expression change of ezrin had good relation with histology (P = 0.037), grade (P = 0.006), chemotherapy response (P = 0.017), and metastasis or recurrence (P = 0.041). The 2-year overall survival and event-free survival were associated with expression change of ezrin (P = 0.001 and P = 0.002, respectively) and response to preoperative chemotherapy (P = 0.001 and P = 0.012, respectively). Our findings suggest that expression change of ezrin is an independent positive prognostic factor in non-metastatic osteosarcoma.

Osteosarcoma is the most common primary bone tumor in children and adolescents and is typically associated with a poor prognosis. Tumor stem cells (TSCs) are presumed to drive tumor initiation and tumor relapse or metastasis. Hence, the poor prognosis of osteosarcoma likely results from a failure to target the osteosarcoma stem cells. Here, we have utilized three different methods to enrich TSCs in osteosarcoma and further evaluated whether salinomycin could selectively target TSCs in osteosarcoma. Our results indicated that sarcosphere selection, chemotherapy selection and stem cell marker OCT4 or SOX2 over-expression are all effective in the enrichment of TSCs from osteosarcoma cell lines. Further investigation found that salinomycin inhibited osteosarcoma by selectively targeting its stem cells both in vitro and in vivo without severe side effects, and the Wnt/β-catenin signaling pathway may be involved in this inhibition of salinomycin. Taken together, we have identified that salinomycin is an effective inhibitor of osteosarcoma stem cells, supporting the use of salinomycin for elimination of osteosarcoma stem cells and implying a need for further clinical evaluation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Osteosarcoma is the most common primary malignant bone cancer in children and adolescents. Emerging evidence has suggested that the capability of a tumor to grow is driven by a small subset of cells within a tumor, termed cancer stem cells (CSCs). Although several methods have been explored to identify or enrich CSCs in osteosarcoma, these methods sometimes seem impractical, and chemotherapy enrichment for CSCs in osteosarcoma is rarely investigated. In the present study, we found that short exposure to chemotherapy could change the morphology of osteosarcoma cells and increase sarcosphere formation in vitro, as well as increase tumor formation in vivo. Furthermore, methotrexate (MTX)-resistant U2OS/MTX300 osteosarcoma cells were larger in size and grew much more tightly than parental U2OS cells. More importantly, U2OS/MTX300 cells possessed a higher potential to generate sarcospheres in serum-free conditions compared to parental U2OS cells. Also, U2OS/MTX300 cells exhibited the side population (SP) phenotype and expressed CSC surface markers CD117 and Stro-1. Notably, U2OS/MTX300 cells showed a substantially higher tumorigenicity in nude mice relative to U2OS cells. Therefore, we conclude that chemotherapy enrichment is a feasible and practical way to enrich osteosarcoma stem cells.

Objective: To study the growth inhibition and apoptosis induction effects of bufalin on human osteosarcoma cell lines in vitro., Methods: U-2OS and U-2OS/methotrexate (MTX) 300-resistant cell lines were treated with bufalin. Cell viability was assessed by MTT assay. Cell-cycle status, apoptosis-inducing effects, and the expression of apoptosis-related proteins were evaluated by flow cytometry, fluorescent staining, DNA fragmentation assay, and Western blotting., Results: Bufalin inhibited cell growth in both U-2OS and U-2OS/MTX300 cells. The IC(50) values of bufalin for U-2OS and U-2OS/MTX300 cells were (8.49 ± 2.1) ng/ml and (10.19 ± 1.7) ng/ml, respectively. The induction of G(2)/M cell-cycle arrest was also seen in the bufalin-treated cells. The bufalin-induced apoptosis was confirmed by increased expression of tumor suppressor protein p53, bax and decreased expression of bcl-2., Conclusion: Bufalin inhibits the growth of and induces apoptosis in both MTX-sensitive and MTX-resistant human osteosarcoma U-2OS cell lines. The apoptosis-inducing effect of bufalin is not influenced by the presence of high levels of DHFR.

C-myc is an oncogene with the important role of cell proliferation controller. It has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation and induce metastatic features. Some studies showed that overexpression of c-myc could induce resistance in response to antineoplastic agents. Currently, we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin(CDDP). The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Western Blot, MTT assay, RT-PCR, flow cytometry (FCM), and transmission electron microscopy (TEM) were used to study expression of c-myc and caspase-3 protein, tumor cell proliferation in vitro, cell apoptotic morphology and cell cycle change. Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2.0 x 10(9) pfu/ml. Ad-Asc-myc downregulated the expression of c-myc protein after transfected MG-63 cells for 48 hours, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 hours can inhibited tumor cells proliferation in vitro by 33.4 and 54.2 percent, respectively, which had significant difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). RT-PCR revealed that Ad-Asc-myc downregulated expression of bcl-2 and upregulated expression of Bax, and no appreciable changes were observed in the expression of E2F-1. Detection of caspase-3 protein TEM, and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin. Cell cycle analysis showed that obvious G(2)/M phase arrested in transfected cells. In conclusion, Ad-Asc-myc increased the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

Objective: To construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin., Methods: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 720 base pairs in a reverse direction into adenovirus vector, then undergoing recombination, amplifying and being complemented in vivo. The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Wright staining, Acridine Orange staining, Western Blot, MTT, Flow Cytometry (FCM) were used to study cell morphology, expression of c-myc protein, tumor cell proliferation in vitro, apoptosis and cell cycle change., Results: Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2 x 10(9) pfu/ml. Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 h could inhibit tumor cells proliferation in vitro by 33.4% and 54.2% respectively, which were significantly difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). Acridine Orange staining and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin cell. Cycle analysis showed that obvious G2/M phase arrested in transfected cells., Conclusion: Ad-Asc-myc increases the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

Background & Objective: c-myc, an oncogene, plays an important role in regulation of cell proliferation, and has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation, and induce metastasis. This study was to construct recombinant adenovirus encoding antisense c-myc, and to investigate its effects on osteosarcoma cell lines MG-63 (p53-deficient) and U2OS (with wild type p53)., Methods: Recombinant adenovirus Ad-As-c-myc encoding antisense c-myc was constructed by gene reconstruction technique, defined by polymerase chain reaction (PCR), and transfected into human osteosarcoma cell lines MG-63 and U2OS. Western blot, acridine orange staining, reverse transcription-PCR (RT-PCR), and flow cytometry (FCM) were used to detect expression of c-myc, proliferation, apoptosis, and cell cycle of MG-63 and U2OS cells., Results: Ad-As-c-myc encoding antisense c-myc was obtained with the titer of 2x10(9) pfu/ml. Ad-As-c-myc significantly inhibited proliferation of both cell lines, while U2OS with wild type p53 was more susceptible to Ad-As-c-myc. Expression of c-myc mRNA was down-regulated in 2 cell lines 48 h after transfection of Ad-As-c-myc. Acridine orange staining and FCM analysis showed that Ad-As-c-myc induced apoptosis of both cell lines, cell cycle analysis showed obvious G(2)/M phase arrest in MG-63 cells,and G1 phase arrest in U2OS cells after transfection of Ad-As-c-myc., Conclusion: Ad-As-c-myc could induce apoptosis through both P53-dependent and P53-independent pathways, and inhibit proliferation of osteosarcoma cells.

Background Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3β in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent. Methods We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3β expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5–8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3β in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3β activity with overall survival. Results Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3β formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3β had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3β resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3β resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3β inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3β, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3β and NF-κB (109.2 months). Conclusion GSK-3β activity may promote osteosarcoma tumor growth, and therapeutic targeting of the GSK-3β and/or NF-κB pathways may be an effective way to enhance the therapeutic activity of anticancer drugs against osteosarcoma. [ABSTRACT FROM PUBLISHER]