sphingosine-kinase and Osteosarcoma

Osteosarcoma (OS) is one of the most common and aggressive malignancies in children and adolescents worldwide. Sphingosine kinase 1 (SphK1) has recently been reported to serve a role in OS progression. The present study aimed to investigate the role of SphK1 in the development of chemoresistance and glycolysis in OS cell lines. SphK1 expression levels in OS cell lines (U2OS, MG63 and SaoS2) were analyzed using western blotting and reverse transcription‑quantitative PCR (RT‑qPCR). A cell survival assay was conducted to determine doxorubicin‑resistance in OS cells, and glycolysis was also evaluated. SphK1 expression was increased in the U2OS and SaoS2 cell lines, and both cell lines were more resistant to doxorubicin when compared with the MG63 cell line. SphK1 knockdown or overexpression altered doxorubicin resistance and the viability of OS cell lines. In addition, hypoxia inducible factor‑1α (HIF‑1α) expression was positively associated with SphK1 expression, and partly mediated SphK1‑induced effects on doxorubicin resistance and glycolysis. The present study suggested that SphK1 participated in the development of doxorubicin resistance and contributed to glycolysis in OS cells by regulating HIF‑1α expression. However, further studies investigating the application of SphK1 associated therapies for patients with OS are required.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Doxorubicin; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Osteosarcoma; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction

Osteosarcoma (OS) is the most common malignant bone tumor. In cancer cells, autophagy is related to epithelial-to-mesenchymal transition (EMT). Although microRNA (miR)-506-3p has been demonstrated to act as a tumor suppressor in OS, its role in regulating the EMT process and autophagy remains unknown. The results showed that miR-506-3p directly inhibited the expression of sphingosine kinase 1 (SPHK1) in 143B and SaOS-2 cells. The invasive capability of OS cells was reduced following miR-506-3p mimics transfection, and restored when SPHK1 was overexpressed simultaneously. Further, miR-506-3p mimics initiated mesenchymal-to-epithelial transition (MET) - E-cadherin expression was upregulated, whilst vimentin and fibronectin were downregulated. The basal autophagy flux (LC3II/I) was suppressed by miR-506-3p mimics. The alterations induced by miR-506-3p mimics were partly reversed by SPHK1 overexpression or treatment of rapamycin. Meanwhile, treatment of SPHK1-transfected cells with 3-methyladenine inhibited EMT. The data suggest that miR-506-3p initiates MET and suppresses autophagy in OS cells by targeting SPHK1.

Topics: Autophagy; Bone Neoplasms; Cadherins; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Fibronectins; Humans; MicroRNAs; Osteosarcoma; Phosphotransferases (Alcohol Group Acceptor); Up-Regulation; Vimentin

Osteosarcoma (OS) is one of the most common malignant bone tumors among children and young adults. Furowanin A (Fur A), one of the active ingredients of Millettia pachycarpa Benth, has been found to exert pro-apoptotic activity in human leukemia cells. This study is designed to evaluate the efficacy of Fur A against OS. The effect of Fur A on cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Western blotting and quantitative real-time PCR (qRT-PCR) were performed to determine the protein and mRNA level of sphingosine kinase 1 (SphK1), respectively. To validate the role of SphK1 in the pro-apoptotic activity of Fur A, overexpressing SphK1 vector and siRNA targeting SphK1 were utilized to transfect OS cells. Moreover, an OS xenograft murine model was used to analyze the therapeutic efficacy of Fur A in vivo. Fur A treatment led to a dose-dependent decrease in the number of viable cells. It also exhibited antiproliferative activity and significantly promoted apoptotic cell death in OS cell lines. Our results showed that the anticancer activity of Fur A was associated with downregulation of SphK1 and inactivation of its downstream signaling. The mediatory role of SphK1 was validated when the pro-apoptotic activity of Fur A was significantly blocked by SphK1 overexpression, while SphK1 knockdown sensitized the OS cells to Fur A. We concluded that Fur A can exhibit anti-growth and pro-apoptotic activities in vitro and in vivo in OS by downregulating SphK1. Our study highlights the possibility of utilizing Fur A as a chemotherapeutic agent in the treatment of OS. Anat Rec, 302:1941-1949, 2019. © 2019 American Association for Anatomy.

Topics: Animals; Apoptosis; Bone Neoplasms; Cell Proliferation; Female; Flavonoids; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Osteosarcoma; Phosphotransferases (Alcohol Group Acceptor); Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Increasing evidence has confirmed that the dysregulation of microRNAs (miRNAs) contributes to the proliferation and invasion of human cancers. Previous studies have shown that the dysregulation of miR-124 is in numerous cancers. However, the roles of miR-124 in human osteosarcoma (OS) have not been well clarified. Therefore, this study was to investigate the biological functions and molecular mechanisms of miR-124 in OS cell lines, discussing whether it could be a therapeutic biomarker of OS in the future. In this study, our results demonstrated that miR-124 was down-regulated in OS cell lines and tissues. Furthermore, the low level of miR-124 was associated with increased expression of Sphingosine kinase 1 (SPHK1) in OS cells and tissues. Up-regulation of miR-124 significantly inhibited cell proliferation, invasion, and MMP-2 and -9 expressions of OS cells. Bioinformatics analysis predicted that the SPHK1 was a potential target of miR-124. Further study by luciferase reporter assay demonstrated that miR-124 could directly target SPHK1. Overexpression of SPHK1 in OS cells transfected with miR-124 mimic partially reversed the inhibitory of miR-124. In conclusion, miR-124 inhibited cell proliferation and invasion in OS cells by downregulation of SPHK1, and that downregulation of SPHK1 was essential for the miR-124-inhibited cell invasion and in OS cells.

Topics: Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gene Expression; Humans; MicroRNAs; Molecular Targeted Therapy; Neoplasm Invasiveness; Osteosarcoma; Phosphotransferases (Alcohol Group Acceptor); Up-Regulation

Elucidation of the mechanisms of chemo-resistance and implementation of strategies to overcome it will be pivotal to improve the survival for osteosarcoma (OS) patients. We here suggest that sphingosine kinase-1 (SphK1) might be the key factor contributing to chemo-resistance in OS. Our Western-blots and immunohistochemistry results showed that SphK1 is over-expressed in multiple clinical OS tissues. Over-expression of SphK1 in OS cell line U2OS promoted its growth and endorsed its resistance against doxorubicin, while knocking-down of SphK1 by shRNA inhibited U2OS cell growth and increased its sensitivity to doxorubicin. Co-administration phenoxodiol with doxorubicin synergistically inhibited SphK1 activity to trigger cellular ceramide accumulation, and achieved synergistic anti-OS growth effect, accompanied with a significant increased of apoptosis and cytotoxicity. Increased cellular level of ceramide by the co-administration induced the association between Akt and Protein Phosphatase 1 (PP1) to dephosphorylate Akt, and to introduce a constitutively active Akt (CA-Akt) restored Akt activation and diminished cell growth inhibition. Further, phenoxodiol and doxorubicin synergistically activated apoptosis signal-regulating kinase 1(ASK1)/c-jun-NH2-kinase (JNK) signaling, which also contributed to cell growth inhibition. Significantly, the role of SphK1 in OS cell growth and the synergistic anti-OS effect of phenoxodiol and doxorubicin were also seen in a mice OS xenograft model. In conclusion, our data suggest that SphK1 might be a critical oncogene of OS and co-administration phenoxodiol with doxorubicin synergistically inhibited the activity of SphK1 to suppress osteosarcoma cell growth both in vivo and in vitro.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Ceramides; Doxorubicin; Drug Synergism; Enzyme Activation; Humans; Intracellular Space; Isoflavones; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, SCID; Mitochondria; Oncogenes; Osteosarcoma; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Xenograft Model Antitumor Assays