| Objective:To investigate the effect of Paris forrestii total saponin on the proliferation, migration and invasion of human bladder carcinoma EJ, BIU-87 and EJ-M3 cell lines in vitro, and explore their underlying mechanism.Methods:Human bladder carcinoma EJ, BIU-87 and EJ-M3 cell lines with different invasion ability were cultured in vitro, and treated with paris forrestii total saponin. MTT assays were used to assess the influence of paris forrestii total saponin on the proliferation activity of the three human bladder carcinoma cell lines. Migration and invasion of cells were measured using Wound healing assay and Transwell invasion assay, respectively. Western blots were performed to investigate BECN1 and LC3 protein expression level. The relative expression of Beclinl and ATG5 autophagy genes were examined using real-time quantitative (RT-PCR).Results:The human bladder carcinoma cell lines were treated with different concentration (10μg/ml,5μg/ml.2.5μg/ml,1.25μg/ml,0.625μg/ml and 0.3125μg/ml) of paris forrestii total saponin for 24 hours. MTT assay showed that the inhibition rates were 31.2%,19.1%,15.9%,12.1%,2% and 1% for BIU-87 cells, and 31.2%, 16.7%,11.3%,8.1%,1.5% and 0.2% for EJ cells, and 44.8%,29.9%,15.8%,14.8%, 2.2% and 2.0% for EJ-M3 cells, respectively.9.5μg/ml of paris forrestii total saponin for 24 hours Wound healing assay showed that the migration distance of cells in the control group and experimental group were 2.15±0.10 versus 1.53±0.11 for EJ-M3 cells.1.40±0.14 versus 1.08±0.09 for EJ cells, and 1.15±0.10 versus 0.71±0.09 for BIU-87 cells, respectively. Transwell chamber invasion assay showed, compared with the control groups, the invasion cell number in the experimental groups were significantly decreased as follows:146.65±6.18 versus 219.56±7.85 for EJ-M3 cells (p<0.05),106.00±5.24 versus 136.73±4.86 for EJ cells (p<0.05), and 49.98±4.17 versus 88.08±3.96 for BIU-87 cells (p<0.05), respectively. Compared with the control groups, Western blots analysis revealed both LC3 and Beclinl protein expression level in the experimental groups were significantly increased (For LC3, 2.6691±0.0768 versus 1.4489±0.1345 in EJ-M3 cells,2.8997±0.1104 versus 1.3659±0.2552 in EJ cells, and 1.5620±0.1630 versus 0.9108±0.0521 in BIU-87 cells, respectively. For BECN1,2.8700V0.1071 versus 1.0001±0.0169 in EJ-M3 cells, 2.7139±0.0428 versus 1.2145±0.1645 in EJ cells, and 1.9008±0.0784 versus 0.8095±0.0475 in BIU-87 cells, respectively.) (p<0.05). RT-PCR results were consistent with above analyses. Compared with the control groups, both Beclin-1 and Atg5 mRNA expression levels were significantly increased in those three experimental groups (For Beclin 1,4.02±0.17 versus 2.51±0.23 in EJ-M3 cells, 6.23±0.14 versus 2.65±0.32 in EJ cells, and 6.83±0.16 versus 3.84±0.21 in BIU-87 cells, respectively. For Atg5,0.79±0.04 versus 0.36±0.07 in EJ-M3 cells,8.52±0.22 versus 2.75±0.10 in EJ cells, and 6.58±0.25 versus 2.35±0.09 in BIU-87 cells, respectively.) (p<0.01).Conclusions:Paris forrestii total saponin (PFTS) could effectively inhibit the proliferation, migration and invasion of human bladder carcinoma cells in vitro, up-regulate the expression of Beclin-1 and LC3 gene, and increase both LC3 and Beclinl protein expression level. The results suggested that the biological behavior changes of bladder cancer cells may be correlated with the enhancement of LC3 and Beclinl expression level, and Paris forrestii total saponin may have a considerable potential value for further research. |
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