In high-dose group, metastasis nodules were hardly observed in lungs (Figure 5c, left panel)

In high-dose group, metastasis nodules were hardly observed in lungs (Figure 5c, left panel). pectolinarigenin (20?mg/kg/2 days and 50?mg/kg/2 days) blocked STAT3 activation and impaired tumor growth and metastasis with superior pharmacodynamic properties. Taken together, our findings demonstrate that pectolinarigenin may be a candidate for osteosarcoma intervention linked to its STAT3 signaling inhibitory activity. Osteosarcoma is the most common malignant bone tumor in children and adolescents and arises from cells of mesenchymal osteoblast origin.1, 2 Despite NB001 advances in surgery and multiagent chemotherapy, nearly 30% of patients still die from osteosarcoma.2 And the survival rates for osteosarcoma remain relatively low over the past two decades.3 Therefore, it is necessary to develop novel therapeutic approaches for osteosarcoma treatment. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that involves in proliferation, survival, apoptosis, angiogenesis and metastasis.4, 5 Upon stimulation by cytokines (interleukin-6 (IL-6), IL-11 and etc.) and growth factors (EGF, PDGF and NB001 etc.), STAT3 can be phosphorylated at tyrosine residue 705. STAT3 phosphorylation facilitates its homo- and heterodimerization, and the dimer then enters the nucleus where it regulates transcription, leading to increased downstream gene transcription such as and etc.6 Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1) belongs to a family of non-receptor protein tyrosine phosphatases (PTP) and acts as a negative regulator of numerous signaling pathway.7 Previous studies reported SHP-1 tyrosine phosphatase inhibited JAK/STAT3 signaling and contributed to antitumor activity in a wide variety of tumor.8, 9 Recent studies have indicated that STAT3 is constitutively activated in many cancers, including, but not limited to, head and neck squamous cell carcinoma (HNSCC),10 breast cancer,11 NB001 ovarian cancer,12 lung cancer13 and leukemia.14 With respect to osteosarcoma, the expression level of p-STAT3 is strongly associated with its prognosis and approximately 20% osteosarcoma was shown to express high levels of p-STAT3Tyr705.15 The activated STAT3 pathway is vital for cell growth and metastasis of human sarcoma.16 Consequently, STAT3 pathway may represent a target for therapeutic intervention in osteosarcoma. A variety of inhibitors of STAT3 have shown to inhibit tumor cell growth and metastasis both and has been shown to possess numerous biologic activities such as anti-inflammation and anti-allergy.22, 23 Some research also reported pectolinarigenin repressed cancer growth during tumor cell invasion, we developed a 3D culture model. In control group, osteosarcoma cells formed 3D clusters with cells protruding into the surrounding matrix, whereas treatment with pectolinarigenin resulted in the opposite phenotypes (Figure 4c). EpithelialCmesenchymal transition (EMT) is considered to be a critical mechanism regulating the initial steps in metastatic progression.28 Previous studies reported STAT3 may directly mediate EMT in cancer progression.29 To investigate the effect of pectolinarigenin on osteosarcoma EMT, we examined EMT-associated markers. We found pectolinarigenin could significantly downregulate the expression of mesenchymal markers (N-cadherin, fibronectin and zinc-finger E-box binding homeobox 1 (ZEB1)) and upregulate epithelial cell marker E-cadherin (Figure 4d). In line with this result, an immunofluorescence (IF) assay indicated exposure CDC25B to pectolinarigenin resulted in a reverse of EMT, as indicated by the decreased membrane-located N-cadherin and increased E-cadherin (Figure 4e). These results suggested that pectolinarigenin showed metastasis inhibitory effects antimetastasis efficacy of pectolinarigenin in osteosarcoma. Open in a separate window Figure 4 Pectolinarigenin inhibits adhesion, migration, invasion and reversed EMT phenotype in osteosarcoma cells. (a) Left panel, adhesion assay. 143B and MG63.2 cells were pretreated with various concentrations of pectolinarigenin for 12?h. Cells were trypsinized, and seeded on a fibronectin coated 96-well plate. After 15?min, non-adherent cells were removed and adherent cells were stained with 0.1% crystal violet. The precipitates were dissolved in 30% acetic acid, and the absorption at 590?nm was acquired. Middle panel, wound-healing migration assay. 143B and MG63.2 cells were seeded NB001 into six-well plates and left to grow to full confluence. Cells were scratched to create a wound and exposed to different concentrations of pectolinarigenin. Images were acquired after 12?h. Cell migration was quantified manually. Right NB001 panel, invasion assay. 143B and MG63.2 cells were resuspended in serum-free medium and seeded into the upper chamber of the transwell inserts.

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