MicroRNA-196a-5p targeting LRP1B modulates phenotype of thyroid carcinoma cells

Introduction: Thyroid cancer (TC) is a common endocrine malignancy, comprising nearly one-third of all head and neck malignancies worldwide. MicroRNAs (miRNAs) have been implicated in the malignant progression of multiple cancers; however, their contribution to thyroid diseases has not been fully explored. Material and methods: This study aimed to illustrate the regulatory mechanism of microRNA-196a-5p in TC progression and to investigate whether microRNA-196a-5p affects progression of TC cells by targeting low-density lipoprotein receptor-associated protein 1B (LRP1B). MicroRNA-196a-5p and LRP1B expression status in TC cells and normal human thyroid cells was detected by quantative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Dual-luciferase reporter assay, cell counting kit-8 (CCK-8) assay, scratch healing assay, and Transwell assay were also performed. Results: The results showed that microRNA-196a-5p expression was up-regulated and LRP1B expression was down regulated in TC cells. In addition, the upregulation of microRNA-196a-5p facilitated progression of TC cells. Silencing microRNA-196a-5p led to the opposite results. Dual-luciferase reporter assay offered evidence for microRNA-196a-5p targeting LRP1B in TC. MicroRNA-196a-5p could target LRP1B to facilitate proliferation, invasion, and migration of TC cells. Conclusion: Overall, this study revealed that microRNA-196a-5p may be a cancer-promoting microRNA that plays an important role in TC progression

NSK-01105, a Novel Sorafenib Derivative, Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

<div><p>The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i> models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited <i>ex vivo</i> angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.</p></div

NSK-01105 inhibited VEGF secretion and the activation of VEGFR2-mediated signaling pathways in prostate cancer cells.

<p>(a) NSK-01105 inhibited VEGF secretion in prostate cancer cells. VEGF levels were estimated by ELISA method. NSK-01105 and sorafenib suppressed VEGF secretion in both cell lines at concentrations of 2.5, 5 and 10 µM. Columns, mean; bars, SD (n = 3). *, compared with vehicle controls, <i>P</i><0.05. (b) NSK-01105 and sorafenib suppressed the activation of VEGFR2 and its downstream key kinases FAK and eNOS at 5 µM in both prostate cancer cells. NSK-01105 suppressed the phosphorylation of EGFR, while sorafenib had little effect on EGFR activation at 5 µM in both prostate cancer cell lines. (c) The ratios of the optical density between target molecules and β-actin. The optical density was quantified by IPP software. Columns, mean; bars, SD (n = 3). *, compared with vehicle controls, P <0.05.</p

NSK-01105 inhibited cell viability and invasion in LNCaP and PC-3 cells.

<p>(a) NSK-01105 inhibited cell viability in prostate cancer cells. Both LNCaP and PC-3 cell viability was significantly inhibited by NSK-01105 and sorafenib in a dose dependent manner. NSK-01105 was more active against both prostate cancer cells compared with sorafenib for 24 h, with the IC50 values of 5.92 and 5.38 µmol/L, respectively. Points, mean; bars, SD (n = 6). (b, c) NSK-01105 inhibited cell invasion in LNCaP and PC-3 cells. Invasion assay in matrigel-coated transwell chamber was performed to confirm the invasion effects of NSK-01105 in both prostate cancer cell lines. NSK-01105 suppressed the invasion of both cell lines at non-toxic concentrations of 0.5 and 1 µM. Columns, mean; bars, SD (n = 4). *, compared with vehicle controls, <i>P</i><0.05.</p

NSK-01105 inhibited VEGF-induced cell viability in HUVECs.

<p>(a) Chemical structures of the NSK-01105 and Sorafenib. (b) NSK-01105 inhibited the VEGF-induced viability of endothelial cells. Stimulated with VEGF (10 ng/mL), HUVECs showed a high rate of viability. VEGF-induced viability of HUVECs was significantly inhibited by NSK-01105 and sorafenib at concentrations of 5, 10 and 20 µM for 24 h. Columns, mean; bars, SD (n = 6). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls,<i>P</i><0.05.</p

The effect of NSK-01105 and sorafenib in prostate cancer xenograft models.

<p>*, <i>p</i><0.05, compared with control. Significant difference was calculated by one-way analysis of variance.</p><p>The effect of NSK-01105 and sorafenib in prostate cancer xenograft models.</p

NSK-01105 inhibited VEGF-induced migration of HUVECs.

<p>(a, b) NSK-01105 inhibited HUVECs migration by wound-healing assay. Cells were starved to inactivate cell proliferation and then wounded by pipette tips. Stimulated with VEGF, large numbers of HUVECs migrated into the clear area, whereas, as potent as sorafenib, NSK-01105 significantly inhibited the VEGF-induced migration at 0.5, 1 and 2.5 µM. Migration distances were calculated by IPP software. (c, d) NSK-01105 inhibited HUVECs migration by transwell cell migration assay. Cells were cultured in serum-free medium along with the indicated concentrations of VEGF, NSK-01105 and/or sorafenib in the upper chambers and the lower chambers were filled with 10% FBS medium. Stimulated with VEGF, large numbers of HUVECs migrated into the lower surface of the polycarbonate, whereas NSK-01105 and sorafenib significantly inhibited the VEGF-induced migration. Cells were counted under a microscope. Columns, mean; bars, SD (n = 4). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls, <i>P</i><0.05.</p

NSK-01105 inhibited the tube formation of HUVECs and <i>ex vivo</i> angiogenesis in matrigel plug assay.

<p>(a, b) NSK-01105 inhibited the tube formation of HUVECs. HUVECs were seeded on the matrigel bed and treated with various dilutions of NSK-01105 with or without VEGF (0.3 nmol/L) for 24 h. Tubular structures were photographed and the tube lengths were quantified by IPP software. (c, d) NSK-01105 inhibited <i>ex vivo</i> angiogenesis by matrigel plug assay. The matrigels, which contained VEGF (500 ng/mL), and NSK-01105 or sorafenib, were inoculated subcutaneously into the right flank of mice. After 10 days, matrigel plugs were removed and hemoglobin content was determined according to the Drabkin's method. Columns, mean; bars, SD (n = 4). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls, <i>P</i><0.05.</p

NSK-01105 inhibited the activation of VEGFR2-mediated signaling pathways in HUVECs.

<p>VEGF stimulated VEGFR-2 autophosphorylation in HUVECs. (a) NSK-01105 and sorafenib suppressed the activation of VEGFR2 and its downstream key kinases FAK and eNOS at 5 µM in HUVECs. (b) The ratios of the optical density between target molecules and β-actin. The optical density was quantified by IPP software. Columns, mean; bars, SD (n = 3). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls, <i>P</i><0.05.</p