| Objective: To explore the effects of Angelicin on PC-3 proliferation,migration,invasion and stem cell properties in a hypoxic environment;and the effects of Angelicin on angiogenesis and osteoclast differentiation in PC-3 conditioned medium to provide an experimental reference for its anti-tumor effects.Methods:(1)CCK-8 assay was performed to determine the effect of different concentrations of Angelicin solution on the proliferation of PC-3 cells under hypoxic conditions.(2)Cell adhesion assay,cell scratching assay and Transwell assay were performed to evaluate the effects of different concentrations of Angelicin solution on the adhesion,migration and invasion of PC-3 cells under low oxygen conditions.(3)Sphere formation assay and colony formation assay were performed to detect whether different concentration gradients of Angelicin solution affected the tumor stem cell properties of PC-3 cells under hypoxic environment,and relevant stemness markers were detected by Western blot.(4)PC-3 cells were treated with different concentrations of Angelicin solution,and PC-3 cell culture medium supernatant was analyzed by ELISA assay to assess the neuroendocrine function of PC-3 cells.(5)BM-EPCs were cultured using prepared PC-3 cell hypoxic CM with different concentrations of Angelicin solution,and the differentiation ability of BM-EPCs into blood vessels was assessed by angiogenesis assay,and VEGF secretion was detected by ELISA assay.(6)RAW 264.7 cells were cultured using prepared PC-3 cells with hypoxic CM,and the effect of different concentrations of Angelicin solution on osteoclast differentiation was examined by osteoclast differentiation assay.Results:(1)The CCK-8 results showed that in the hypoxic environment,the proliferation ability of PC-3 cells decreased with increasing concentration of Angelicin solution,and the proliferation of PC-3 cells was significantly inhibited by Angelicin solution at 15 μM and higher concentrations compared with the control group(P<0.01).(2)The results of cell adhesion assay suggested that the adhesion ability of PC-3 cells under hypoxia decreased as the concentration of Angelicin solution kept increasing.The results of cell scratching assay indicated that the migration ability of PC-3 cells under hypoxia was significantly inhibited with the increasing concentration of Angelicin solution,among which the inhibitory effect of 60 μM Angelicin solution was the most significant.The results of Transwell assay suggested that with the increasing concentration of Angelicin solution,the invasion ability of PC-3 cells under hypoxic conditions was significantly inhibited,and the difference was statistically significant compared with the control group(P<0.01).(3)The results of spheroid formation test and colony formation test showed that the spheroidization and colony formation of PC-3 cells were obviously inhibited by Angelicin under hypoxic conditions,and the inhibitory effect of 60 μM Angelicin solution was the most obvious,and the difference was statistically significant compared with the control group(P<0.01);the results of Western blot test showed that the expression of CD44,OCT4 and AGO2,the relevant stemness markers of PC-3 cells,were inhibited after Angelicin solution treatment.(4)ELISA results showed that PC-3 cells in hypoxic environment promoted the secretion of MMP-2/9,IL-6/8,IL-1β and TNF-α,while the secretion was reduced after Angelicin treatment,and the difference was statistically significant compared with the control group(P<0.01).(5)The results of angiogenesis experiment showed that the number and thickness of angiogenesis in the experimental group decreased significantly with the increase of Angelicin concentration;the results of ELISA experiment showed that BM-EPCs secreted the least VEGF after treatment with Angelicin 60 μM concentration,and the difference was statistically significant compared with the control group(P<0.01).(6)The results of osteoclast assay showed that Angelicin-treated group inhibited osteoclast differentiation to varying degrees with increasing concentrations,most significantly at 60μM compared to the control group(P<0.01).Conclusion: Angelicin inhibited the proliferation,migration,invasion,tumor stem cell properties and neuroendocrine-related factor expression of PCa bone metastases cells under hypoxic conditions,and also inhibited angiogenesis and osteoclast differentiation in the tumor microenvironment in vitro. |
PDF Full Text Request