| Osteosarcoma (OS) is the most common primary bone tumor in children andadolescents, comprising approximately20%of all bone tumors and about5%of pediatrictumors overall. OS is high-grade malignant characterized by high-invasion and earlydevelopment of distant metastasis to lung. Up to20%of OS patients present withradio-graphically detectable lung metastases, whereas80%of patients with localized OSdevelop metastases after surgical resection alone. Death from OS is usually caused byprogressive pulmonary metastasis with respiratory failure. Our current understanding of OSetiology is rather limited. Exposure to chemical factors, radioactive substances, and viralinfection cause OS in animal model, but their roles in human OS are unknown. Although OSdevelopment is associated with several genetic and epigenetic predisposition conditions, mostOS tumors are sporadic without consistency. The incidence of OS is involved with multipleprocesses and genes, but the exact pathogenesis has not yet been clarified. With thedevelopment of etiology research, recent studies suggest that cancer appears to be a processthat is fuelled not only by genetic alterations, but also by epigenetic mechanism. It wasreported that the expression of imprinting genes are abnormal in almost all cancer. Imprintinggenes involved in tumorigenesis and tumor progression as proto-oncogenes oranti-oncogenes.Recently, with the development of cancer research and the emerging of "cancer stemcells" theory, more and more researches revealed that cancer was initiated and maintained bya subset of cells, which was termed cancer stem cells (CSCs) or tumor initiating cells (T-ICs).Compared with normal cancer cells, CSCs have self-renew ability, multi-differentiationability, and tumorigenicity ability. Since Lapidot and colleagues first identified asubpopulation of CSCs in leukemic cells by specific surface markers, CSCs were identified from diversity cancers, including brain tumor, breast cancer, alimentary canal cancer,melanoma, lung cancer, and prostatic cancer.In mesenchymal tumor, Gibbs et al. first identified a small subpopulation ofself-renewing bone sarcoma cells that are capable of forming sarcospheres inanchorage-independent, serum-starved conditions. These sarcosphere cells express activatedSTAT3and the marker of pluripotent embryonic stem (ES) cells, Oct3/4and Nanog,implying these cells are stem-like cells. After that the existence of CSCs in OS has beenconfirmed in different cell lines and human specimens by various means. Although OS CSCshave huge cure implications, it brings new challenges for us. Because of CSCs are resistanceto traditional chemo and radio-therapies, there's little ways to cure cancer at present. Newstrategies targeting CSCs are the key problems we must resolve first.Previously we found that imprinting genen TSSC3(tumor-suppressing STF cDNA3) isassociated with OS development, progress and anoikis resistance. TSSC3, also known as IPL(imprinted in placenta and liver), the first apoptosis-related gene found to be imprinted, islocated on11p15.5chromosom. Methylating detection revealed that the promoter region ofTSSC3is highly methylated in OS cell line SaOS2, while TSSC3was overexpression aftertreated with demethylation reagent, and apoptosis rate was increased significantly.Overexpression TSSC3by vector was associated with growth inhibition and apoptoticinduction in human OS; however, whether TSSC3plays a role in OS CSCs remains unknown.Therefore this study focused on the effects and molecular mechanisms of TSSC3in OS CSCs.To this end, we planted to first isolated and identified CSCs from MG63, SaOS2andmalignant transformed human osteoblast hFOB1.9(MThFOB1.19) cell lines, and detected theexpression of TSSC3in these CSCs, and then upregulated or downregulated the expression ofTSSC3in OS cells, and finally detected the effects of TSSC3in sustaining the stemness andinducing apoptosis of CSCs.Our study contained three parts. The aim of the first part was to isolated and identifiedCSCs from MG63, SaOS2and MThFOB1.19cell lines by stem cell medium culture and flowcytometry sorting. And evaluated the tumor initiating cells in malignant transformed humanosteoblast hFOB1.19cells by clonal and population analyses, and detected the self-renew,multi-differentiation, tumorigenesis abilities and the expression of transcriptional factors ofMThFOB1.19cells. The second section was to detect the expression of TSSC3in CSCs by RT-PCR,western-blot, immunocytochemistry and immunofluorescence, and then upregulated theexpression of TSSC3in MThFOB1.19cells, detecting the effects of TSSC3in sustainingCSCs' stemness.The third section was to evaluate the effects of TSSC3in inducing CSCs apoptosis inOS cell lines, and its possible mechanisms.The main results are as follows:1. OS CSCs were successfully isolated and identified.(1) By stem cell medium culture: cultured with stem cell medium, MG63, SaOS2andMThFOB1.19cells formed sarcospheres, the sarcosphere formation rates were5.40±1.06%,8.60±1.16%and67.09±9.28%, respectively. Immunofluorescence showed thatsarcospheres from MThFOB1.19expressed high level of Nanog, Oct4and Sox2.(2) In MThFOB1.19cells: Nearly every MThFOB1.19cell could generate primaryclonal and subclonal populations. The primary clone and subclone formation rates were100%and92.7±4.29%, respectively. All clones and subclones could generate tumors whenxenotransplanted with5×104cells into nude mice. MThFOB1.19cells hadmulti-differentiation abilities and expression transcriptional factors Nanog, Oct4and Sox2.(3) By flow cytometry: the rates of CD133+, CD117+and Stro-1+in SaOS2were0.4%,4.8%and3.3%, respectively, revealing that CSCs are a small sub-population in SaOS2cellline.2. CSCs have a low TSSC3expression. Overexpression TSSC3reduces stemness andinhibits OS tumorigenesis.(1) TSSC3is lowly expressed in CSCs: MG63, SaOS2and MThFOB1.19sarcosphereshad a lower TSSC3expression than non-sphere cells in transcription and translation levels. InSaOS2cell line, CD133+, CD117+and Stro-1+cells had a lower TSSC3expression thanCD133-, CD117-and Stro-1-cells.(2) The Gene SwitchTM system was used to upregulate TSSC3in MThFOB1.19cells,which was confirmed by RT-PCR and Western blot, respectively.(3) Overexpression TSSC3reduces stemness and inhibits tumorigenesis inMThFOB1.19cells: overexpression TSSC3cells had a low sarcosphere formation rate (46.82±8.37%) relative to the control (66.12±9.36%). And the colony formation rate of overexpression TSSC3cells was57.57±2.16%, significantly lower than that of control cells(95.32±4.58%, P <0.05), as well as the tumorigenesis was reduced. In addition,Overexpression TSSC3reduced the expression of stem cell markers Nanog, Oct4and Sox2.3. Overexpression TSSC3promotes apoptosis by activating the intrinsic cell apoptoticpathway(1) TSSC3has an effect on CSCs ratio: In SaOS2cell line, overexpression TSSC3decreased CSCs ratio while downregulated TSSC3expression increased CSCs ratio in vitroand in vivo.(2) Overexpression TSSC3promotes CSCs apoptosis: overexpression TSSC3increasedCD133+, CD177+and Stro-1+cells apoptosis in SaOS2cell line, as well as increasedMThFOB1.19sarcosphere cells apoptosis. In TSSC3overexpressed sarcosphere cells, theexpression of Bak, Bim, Cyt c, Apaf-1, Noxa, Puma, indicators of intrinsic pathway activation,as well as the Bax: Bcl-2ratio was increased. Pro-Caspase3and pro-Caspase9were decreased,while no significant changes in pro-Caspase8, in addition the mitochondrial ΔΨ was disrupted.Further more, TSSC3increased CSCs apoptosis induced by cisplantin.In summary, our study provides preliminary evidence for the first time that OS CSCscan originate from more differentiated cells, while overexpression of TSSC3can efficientlydecrease tumorigenesis by downregulating stem cell-like features and promoting cellapoptosis though intrinsic apoptosis pathways. Moreover, TSSC3overexpression associatedwith enhances chemotherapeutic effects. Additional studies to clarify the exact mechanism ofTSSC3reduces stemness and induces apoptosis will be highly valuable for finding newtherapeutic strategies to treat OS.
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