Effects And Mechanisms Of Diosgenin Regulating The Expression Of NEDD4 Protein On The Proliferation And Migration Of Prostate Cancer Cells

Part Ⅰ Diosgenin inhibits the growth,induces apoptosis and stops the cell cycle of prostate cancer cellsObjective To investigate cells were treated with 0,25,50,75,or100μM of diosgenin.After 48 and 72 h of diosgenin exposure,MTT assay was performed to measure PC-3 cell proliferation as described previously.Cell apoptosis analysis,Cell cycle assay.Methods 1.To seed PC-3 cells in the plates of 96-wells(5×10 3 cells/well).,to culture overnight Then,the cells were treated with 0,25,50,75,or 100 μM of diosgenin.After 48 and 72 h of diosgenin exposure,MTT assay was performed to measure PC-3 cell proliferation as described previously.2.The PC-3 cells were cultured overnight.,which were seeded(1μ105 cells/well)in 6well plates.Then,the cells were treated with 0,50 or 75 μM of diosgenin for 48 h.Cells were then collected and apoptosis was analyzed using Flow cytometric with the Annexin ⅤFITC/PI Kit,as described previously.3.with a density of 1×105 cells/well,the PC-3 cells were seeded in 6-well plates to be cultured overnight.Cells were then treated with different concentrations of diosgenin for 48 h.Flow cytometry was used to analyze the cell cycle stage,as describe previously.Reasuls 1.Diosgenin inhibits cell growth in PC-3 cells.To investigate whether diosgenin could inhibit the cell growth in prostate cancer cells,the MTT assay was used to evaluate PC-3 cell proliferation after diosgenin exposure.PC-3 cells were treated with 0,25,50,75,or 100 μM diosgenin for 48 h and 72 h.We found that diosgenin treatment inhibited cell growth in a dose-dependent manner.Specifically,a 50 μM diosgenin treatment for 72 h induced approximately 50%cell growth inhibition.Moreover,exposure to 75 μ M diosgenin for 72 h induced 70%cell growth inhibition2.Diosgenin induces cell apoptosis in PC-3 cells.In order to determine the impact of diosgenin on apoptosis death in prostate cancer cells,flow cytometry analysis was performed following diosgenin treatment.PC-3 cells were exposed to 50 μM or 75 μM of diosgenin for 48h,following by double staining with Annexin V-FITC and PI.We found that diosgenin treatment induced cell apoptosis.Indeed,50 μM and 75 μM of diosgenin increased the number of apoptotic cells from 3.25%in control group to 8.72%and 18.81%,respectively.3.Diosgenin induces G2/M cell cycle arrest.Next,we investigate the effects of diosgenin on the cell cycle in prostate cancer cells.PC-3 cells were exposed to different concentrations of diosgenin for 48 h followed by labeling with PI.Our data indicated that diosgenin induced cell cycle arrest at G2/M phase.More specifically,50 μ M and 75 u M diosgenin treatment increased the G2/M phase cell population from 12.84%in the control group to 17.21%and 29.67%,respectively.Conclusions 1.These findings demonstrated that diosgenin inhibited cell growth in prostate cancer cells.On the basis of these data,50 μ M and 75 μ M diosgenin treatments were used for the remaining experiments.2.Our data suggest that diosgenin promotes cell apoptosis in prostate cancer cells.3.These results indicate that diosgenin exposure led to G2/M cell cycle arrest in prostate cancer cells,which could contribute to diosgenin-induced cell growth inhibition.Part Ⅱ Diosgenin inhibited the metastasis and invasion of prostate cancer cells and significantly down-regulated NEDD4 levelsObjective To explore the inhibitory effects of diosgenin on prostate cancer cell migration and invasion in prostate cancer cells.To investigate the role of diosgenin on NEDD4 expression in prostate cancer cells.The expression of two downstream targets,p73 andp-Akt,regulated by the diosgenin treatment.To measure the LATS1 and TAZ in PC-3 cells after diosgenin treatment.Methods 1.Wound healing assay PC-3 cells were incubated in 6-well plates until cells were more than 90%confluer.A scratch wound was then made in the cell monolayer using a sterile pipette tips.After 20 h,the wound area was photographed with an inverted microscope and analyzed,as reported previously.2.Transwell cell invasion assay The treated PC-3 cells were cultured in 24-well Transwell chambers(8 uM pore size,Corning,NY,USA)that had a Matrigel coating.The FBS-free RPMI medium was added in the upper chamber of the inserts,and complete RPMI medium was added in the lower chamber.After 20 h,the invasive cells that had attached to the lower membrane surface were stained with Calcein-AM for 20 min and counted under a light microscope.3.Western blot analysis PC-3 cells(1×10 6 cells/well)were seeded in 6-well plates and cultured..The sample protein concentrations were measured with the BCA protein assay.The protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane.The western blotting analysis was completed as described previouslyResults 1.Diosgenin decreases cell migration and invasion The data revealed that diosgenin inhibited cell migration in a dose-dependent manner.We observed that diosgenin exposure led to decreased cell invasion in PC-3 cells.Moreover,cell invasion was impeded in a concentration-dependent manner.2.Diosgenin inhibits NEDD4 expression The data of western blotting indicated that diosgenin exposure inhibits NEDD4 expression in PC-3 cells.Moreover,the expression of two downstream targets,p73 and p-Akt,was also regulated by the diosgenin treatment.The expression of p73 was significantly increased in PC-3 cells after exposure to diosgenin,but the p-Akt levels were remarkably decreased in diosgenin-treated PC-3 cells.Conclusions 1.Diosgenin could impede cell migration and invasion in prostate cancer cells.Especially cell migration and invasion was impeded in a concentration-dependent manner.2.The diosgenin exposure inhibits NEDD4 expression in PC-3 cells,and the expression of two downstream targets,p73 and p-Akt,was also regulated by the diosgenin treatment.The expression of p73 was significantly increased in PC-3 cells after exposure to diosgenin.Conversely,p-Akt levels were remarkably decreased in diosgenin-treated PC-3 cells.The diosgenin also increased LATS1 and inhibited TAZ expression.Part Ⅲ Mechanism of NEDD4 in diosgenin against prostate cancer cellsObjective:To further confirm if that diosgenin exerted its anti-tumor activity via inhibition of NEDD4 expression in PC-3 cells.To determine the underlying mechanism of impaired cell motility induced by diosgenin.To further investigate the role of NEDD4 in diosgenin-mediated anti-tumor effects.Methods:1.PC-3 cells were transfected with a NEDD4 cDNA construct or an empty vector.2.Using western blotting to measure the expression of NEDD4,p73 and LATS1 after PC-3 cells were transfected with NEDD4 cDNA vector.Using MTT assays to explore the growth of PC-3 cells.3.wound healing assay was performed to measure in PC-3 cell migration after diosgenin exposure and in combination with NEDD4 cDNA transfection.NEDD4 siRNA was transfected into PC-3 cells that were also treated with diosgenin.Results:1.The results from western blotting indicated that NEDD4 expression was increased after PC-3 cells were transfected with NEDD4 cDNA vector.2.NEDD4 cDNA transfection abolished NEDD4 inhibition that was induced by the diosgenin treatments.Furthermore,NEDD4 overexpression also abrogated diosgeninmediated p73 and LATS1 inhibition and p-Akt activation in PC-3 cells.Over expression of NEDD4 decreased p73 and LATS1 expression,and increased p-Akt expression.3.These results suggest that diosgenin inhibits cell motility inhibition partly by downregulation of NEDD4 in prostate cancer.Downregulation of NEDD4 enhanced diosgenininduced tumor suppressive activity.Conclusion:1.Overexpression of NEDD4 rescues diosgenin-induced cell growth inhibition and apoptosis.2.Overexpression of NEDD4 abolishes diosgenin-induced motility inhibition.3.Downregulation of NEDD4 enhanced diosgenin-induced tumor suppressive activity.4.These data demonstrate that diosgenin exerts its tumor suppressive effects in prostate cancer through downregulation of NEDD4,which is involved in the NEDD4/P73/PAkt/LATS1 pathways.Part Ⅳ A preliminary study on the expression of E3 ubiquitin ligase in proteomicsObjective:To explore the expression of the E3 ubiquitin ligase family protein by using proteomic analysis.Methods:6 fresh samples of high-risk prostate cancer(3 samples,S1,S2,S3)and paracancer tissues(3 samples,C1,C2,C3)were obtained for proteomic detection and bioinformatics analysis of human cancer tissues.Based on the lab-free Quantification technology,protein enzymatic peptide segments were analyzed by mass spectrometry with liquid mass spectrometry to select differentially expressed proteins.Clustering analysis based on functional enrichment of the two groups of differential proteins(or differential proteins with different differential multiples)was used to study their potential connections and differences in specific functions(GO,KEGG pathways,protein domains,etc.).The entire network diagram of proteins was presented through the protein network diagram.Each node represented a protein and submitted the name of each group of different proteins to the official website of STRING.Results:1.A total of 5255 Protein groups(card value standard:Peptide Threshold 1.0%FDR,1 Unique Peptide)were identified,and 28 proteins with normal difference in Protein quantity were identified,among which 16 were down-regulated and 12 were up-regulated.2.Both APC4 and FOXB2 of the ubiquitin E3 protein family were highly expressed in high-risk prostate cancer specimens,with significant differences.3.NEDD4 and FOXB2 are submitted to the Official website of STRING to show the relevant effectsConclusion:1.Ubiquitination ligase E3 protein FOXB2 is highly expressed in highrisk prostate.2.FOXB2 interacts with NEDD4;3.Ubiquitinated protein E3 family plays an important role in advanced prostate cancer.