The Anticancer Activities Of DT-13 On Prostate Cancer Cells And The Underlying Molecular Mechanism

Objective:DT-13,a saponin monomer 13 from the dwarf lilyturf tuber,was demonstrated to exhibit multiple pharmacological activities,such as anti-inflammatory,antithrombotic,immunoregulation and anti-tumor.DT-13 was reported to inhibit the growth,migration and adhesion of breast cancer cell MDA-MB-435 and melanoma cell B16.This article was aimed to investigate the anticancer activities of DT-13 on prostate cancer cells and its molecular mechanism,which will provide a theoretical basis for prostate cancer therapy.Method:1.The effects of DT-13 on the proliferation of prostate cancer cells PC3,DU145,PBMC were tested by MTT assay.Soft-agar clone formation assay was used to evaluate the effect of DT-13 on the proliferation inhibitory activity in PC3 and DU145 cells;2.The effect of DT-13 on the cell cycle and apoptosis of prostate cells were analyzed by flow cytometry;The Hoechst 33342 staining was used to observe the changes of nuclear morphology in prostate cells;The effect of DT-13 on reactive oxygen species and mitochondrial membrane potential in prostate cells were analyzed by flow cytometry;3.Western blot was used to analyze the expression level of apoptosis regulating factors and the expression level of the key factors in PI3K/Akt pathway in prostate cells;4.Wounding healing assay and transwell migration assay were used to assay the invasion and migration of PC3 cells;5.The gelatin zymography was used to examine the expression of MMP2 and MMP9 in PC3 cell and the phosphorylation of integrinβ1 with or without DT-13treatment were detected by Western blot.Results:1.DT-13 inhibited the proliferation of prostate cancer cells PC3 and DU145.The IC₅₀0 values were calculated to be 4.825μM and 5.102μM.Also,DT-13 has a lower2.cytotoxic effect on PBMCs.Soft agar cloning experiments further proved that DT-13 could effectively inhibit the proliferation of PC3 and DU145 cells;3.After 48 hours treatment,DT-13 showed no significant effect on cell cycle distribution in PC3 and DU145 cells;but DT-13 induced obviously apoptosis.The apoptotic rates of prostate cancer PC3 and DU145 cells after 2.5μM-10μM DT-13treatment were 8.21%-25.6%and 7.67%-25.4%,respectively.The apoptotic bodies were observed through Hoechst33342 staining in PC3 and DU145 cells.DT-13 had no significant effect on ROS production in both cells,but it could significantly reduce the mitochondrial membrane potential in PC3 and DU145 cells.4.The results of Western blot showed that the expression of Bax and Bad increased and the expression of Bcl-2 decreased after treated of DT-13 in PC3 and DU145 cells.Cytc was released into the cytoplasm from mitochondria,further increased the cleavage of Caspase-3,Caspase-9 and PARP Fragment expression.In addition,DT-13inhibited the phosphorylation level of PDK-1,Akt,mTOR and p70S6K in PI3K/Akt signaling pathway while the phosphorylation of JNK,ERK and P38 in PC3 and DU145 cells were not changed.5.After 24 hours treatment,DT-13（1,2,4μM）could significantly inhibit PC3 cell metastasis but had less significant effect on DU145 cells.DT-13 inhibited the metastasis on PC3 cell by inhibiting the expression of Integrinβ1 and decreasing the levels of MMP2 and MMP9.Conclusions:1.DT-13 inhibited the proliferation of prostate cancer cells PC3 and DU145,and induced apoptosis by activating the mitochondrial apoptosis pathway in prostate cancer cells.2.DT-13 could inhibit prostate cancer PC3 cell migration and invasion;3.DT-13 inhibited the proliferation and metastasis of prostate cancer cells by inhibiting PI3K/Akt signaling pathway.