Data Availability StatementData posting is applicable to this article. mucosal tissues (PFK15 significantly reduced the glucose uptake, lactate production and ATP generation in HNSCC cell lines. PFK15 suppressed cell proliferation, halted cell cycle progression and induced cell apoptosis. The invadopodia of HNSCC cells was markedly reduced after 4-Demethylepipodophyllotoxin PFK15 treatment, thereby 4-Demethylepipodophyllotoxin impairing cell motility and extracellular matrix degradation ability. The in vivo data from the xenograft mice models proved that PFK15 administration suppressed the tumor growth. And the results from the metastatic mice models showed administration of PFK15 alleviated the lung metastasis of HNSCC and extended the life expectancy of mice. Conclusions The pharmacological inhibition of PFKFB3 PFK15 suppressed tumor growth and alleviated metastasis in HNSCC, offering a promising strategy for cancer therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0481-1) contains supplementary material, which is available to authorized users. the tail vein. Two weeks after injection, mice were randomly divided into two groups and received intraperitoneal injection of normal saline (vehicle, 100?l; flow cytometric analysis (Fig.?3g). Although more apoptotic cells were detected in PFK15 treated group than in charge group, PFK15 demonstrated a weaker efficiency in inducing cell apoptosis than in suppressing cell proliferation. TUNEL Apo-Green recognition assays had been used to research apoptotic cell loss of life by determining fragmented DNA in Cal27 cells using the condensed green fluorescence in cell nuclei. As proven in Fig.?3h, the TUNEL positive staining of Cal27 cells increased after treatment with various PFK15 concentrations for 24?h. The appearance degrees of cell-proliferation- and apoptosis-related genes had been examined by traditional western blots (Fig.?3i). PFK15 decreased the expressions of pRb considerably, cyclin Bcl2 and D1, and upregulated the appearance of cleaved caspase3 (CL-caspase3). In amount, concentrating on PFKFB3 by its selective suppressant PFK15 suppressed cell proliferation and induced cell apoptosis in HNSCC significantly. Open in another home window Fig. 3 PFK15 suppresses cell proliferation, halts cell cycle and induces cell apoptosis in HNSCC cells. a PFK15 suppressed the colony formation of Cal27 cells in 2?weeks. b EdU incorporation assays indicated PFK15 inhibited the cell proliferation of Cal27 cells. c The quantitative data of the EdU incorporation assays. d PI staining revealed that PFK15 halted cell cycle progression and induced G2 phase arrest. e The quantitative data of cell cycle analysis based on Dean-Jett-Fox model. f Tumor sphere formation assays indicated PFK15 decreased the malignancy stem cell populace in Cal27 cells. g Annexin V-FITC/PI double staining exhibited PFK15 induced moderate cell apoptosis of Cal27 cells. h TUNEL assays indicated PFK15 induced apoptotic cell death of Cal27 cells. i Protein expressions of phosphor-Rb (pRb), cyclin D1, Bcl2 and cleaved caspase3?(CL-caspase3) in PFK15 treated Cal27 cells were measured 4-Demethylepipodophyllotoxin by western blots. Mean??S.E.M.; **tail vein. Two weeks after injection of tumor cells, 10?mg/kg PKF15 was administrated intraperitoneal injection (every other day, 3?days/week for 2?weeks). Fifty days 4-Demethylepipodophyllotoxin after the first PFK administration, the mice were euthanized and their lungs were harvested. The representative photos of the lungs harvested from your mice suggested that this metastasis nodules were dramatically decreased in mice 4-Demethylepipodophyllotoxin with PFK15 treatment compared with those of the mice in the control group (Fig.?7a). Rabbit Polyclonal to DNAL1 Microscopic metastases to the lung were confirmed H & E staining and pan-CK immunohistochemistical staining. As shown in Fig.?7b, the compact cell aggregations, which were further evidenced as tumor cells because of their positive staining of human pan-CK, were frequently observed in the lungs of the mice without any treatment, and the cell aggregations were much less and smaller in the lungs of the mice treated with PFK15. The lungs of each mouse from your control group experienced approximately 6 to 15 micrometastatic foci. By contrast, less than three micrometastatic foci were found in the lungs of three mice treated with PFK15, while the lungs of the other mice were free from metastasis. The incidence of metastasis was measured by the number of pulmonary metastatic clones (Fig.?7c), which confirmed the reduced metastatic ability of Cal27 cells in the model after PFK15 treatment. The survival curves exhibited that PFK15 treatment extended the life expectancy of the mice suffering from the metastasis of Cal27 cells (Fig.?7d). In sum, the administration of PFK15 significantly prevents the distant metastases formation of HNSCC cells, increasing the life span expectancy of the mice thereby. This finding is in keeping with the invasion and migration suppressive effects in the in vitro assays. Open in another home window Fig. 7 PFK15 stops HNSCC faraway metastasis within a HNSCC metastasis nude mice model. a Consultant photos from the lung gathered in the mice bearing HNSCC metastasis treated with or without PFK15. b The metastatic nodules.