Impacts of Atractylonin on Proliferation, Apoptosis and Vasculogenic Mimicry of Bladder Cancer Cells by Regulating RhoA/ROCK1 Signal Pathway
LAI Haiyan1*, LI Qian1, LU Yiling1, LIU Ying1, QIN Xiaoli1, WEI Renbo2
The aim of this study was to investigate the impacts of ATR (atractylonin) on the proliferation, apoptosis and VM (vasculogenic mimicry) of BC (bladder cancer) cells by regulating the RhoA (Ras homologous gene family member A)/ROCK1 (Rho kinase 1) signaling pathway. CCK-8 method was used to detect the effect of different concentrations (0, 10, 20, 40, 80, 160 mg/L) of ATR on the proliferation rate of T24 cells to screen for ATR intervention concentrations. On this basis, T24 cells were separated into NC group (normal culture), L-ATR group (20 mg/L ATR), M-ATR group (40 mg/L ATR), H-ATR group (80 mg/L ATR), and H-ATR+Ang II group (80 mg/L ATR+100 nmol/L RhoA/ROCK1 pathway activator angiotensin Ang II). MTT detection and Edu assay were applied to measure T24 cell proliferation. Flow cytometry and TUNEL assay were applied to determine T24 cell apoptosis. Matrigel matrix gel method was applied to measure angiogenesis in T24 cells. Phalloidin staining method was used to observe the cytoskeleton of T24. Western blot was applied to determine the expression of RhoA/ROCK1 pathway proteins. With the increase of ATR concentration, the proliferation rate of T24 cells decreased in a dose-dependent manner (P<0.05), and the ATR concentration was selected as 20 mg/L, 40 mg/L, 80 mg/L for follow-up studies. Compared with the NC group, the D450 value, Edu positive cell rate, and lumen number of T24 cells in the L-ATR group, M-ATR group, and H-ATR group were greatly reduced, while the apoptosis rate and fluorescence intensity of F-actin were greatly increased, showing a dose-dependent change in ATR (P<0.05). Compared with the H-ATR group, the D450 value, Edu positive cell rate, and lumen number of T24 cells in the HATR+Ang II group were greatly increased, while the apoptosis rate and fluorescence intensity of F-actin were greatly reduced (P<0.05). Compared with the NC group, the expression of RhoA and ROCK1 proteins in T24 cells in the L-ATR group, M-ATR group, and H-ATR group were greatly reduced, and showed a dose-dependent change in ATR (P<0.05). Compared with the H-ATR group, the expression of RhoA and ROCK1 proteins in T24 cells in the H-ATR+Ang II group was greatly increased (P<0.05). ATR may inhibit proliferation of T24 cells, affect VM formation, and induce cell apoptosis by inhibiting the activation of the RhoA/ROCK1 signaling pathway.
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