The Molecular Mechanism Of Two Mushrooms Extracts Regulating The Apoptotic Signaling Pathways And SUMOylation System Against Prostate Cancer

Prostate cancer is the second most common malignant tumor in the world,and the search for natural anti-tumor products is a new strategy for its treatment.A recent study has found that SUMO modification is a new protein modification method of ubiquity,mediated by SUMO molecules.The imbalance of the SUMO-chemical system of the new modification of protein is closely related to the development of tumor.In this paper,we chose the phenolic compound Hispolon,extracted from mushroom,as the representative of edible mushrooms,and Illudin S extract of the toxic mushroom,chose to explore their possible anti-tumor and anti-apoptotic-related signaling pathways.On the one hand,we studied the molecular mechanisms of edible Hispolon to regulate apoptosis signal and SUMO system against prostate cancer.First,we analyzed the cytotoxic and apoptotic effects of Hispolon on three prostate cancer cell lines(DU145,LNCa P and PC3)and after 24 h,cytotoxic values of Hispolon on three tumor cells were 40μM,40μM and 43μM,respectively.Analyzing the cell cycle by Annexin/PI flow cytometry showed that Hispolon arrest the DU145 cells at S-phase of cell cycle.In the untreated DU145 cell,the number of cells at S-phase were 14.34%,while in Hispolon treated group(20,40 and 60μM),the number of cells at S-phase increased to 38.45%,42.49% and 44.53%,indicating that Hispolon induced S-phase cell cycle arrest.At the same time,Western Blot results showed that Hispolon adjusted the cell cycle by enhancing expression of the p21,Cyclin E and decreasing expression of the Cyclin B1,and Cyclin D1 proteins.Hispolon down-regulated Bcl-2 and upregulated Bax,thereby down-regulating the mitochondrial membrane potential(MMP),promoting the release of cytochrome c from mitochondria,and significantly starting the cleavage of Caspase9 and Caspase3.We found that Hispolon promoted DU145 apoptosis by the intracellular granulocytes(in-source pathways).STAT3 signal is an important tumor proliferation and anti-apoptosis signal.We also found that Hispolon increased the transcription level of PIAS3(STAT3 inhibitory protein)and increased its protein level,which inhibited STAT3 phosphorylation and STAT3 signal transduction.On the other hand,we studied the molecular mechanism of Illudin S,a toxic fungus extract,that regulated apoptosis signals and the SUMO system against prostate cancer.We found that Illudin S had a stronger cytotoxic effect on three different prostate cancer cell lines(DU145,LNCa P and PC3).The IC50 at 24 h was 5μM,6μM and 8μM,respectively.Further,we studied the Illudin S apoptotic effect and signaling pathways in DU145 cells.Annexin/PI flow cytometry analysis found that Illudin S(2.5,5 and 10μM)promoted DU145 apoptosis in dose dependent manner.Flow cytometric analysis of cell cycle suggested that Illudin S regulated the cell cycle in DU145 cells.As number of cells at S-phase of cell cycle in untreated DU145 cell was 8.22%,while number of cell at S-phase increased to 25%,28.48% and 41.87% with increase concentration of Illudin S(1.5,5 and 10 μM).So Illudin S induced S-phase cell cycle arrest in DU145 cells.At the same time,Western Blot results showed that Illudin S adjusted the cell cycle by enhancing expression of p21,Cyclin E and decreasing expression of the Cyclin B1,Cyclin D1 proteins.We found Illudin S down-regulated Bcl-2 and up-regulated Bax,thereby down-regulating the mitochondrial membrane potential(MMP),promoting the release of cytochrome c from mitochondria,and significantly starting the cleavage of Caspase9 and Caspase3.We found that Illudin S regulated DU145 apoptosis by the intracellular granulocytes(in-source pathways).STAT3 signal is an important tumor proliferation and anti-apoptosis signal.We also found that Illudin S increased the transcription level of PIAS3(STAT3 inhibitory protein)and increased its protein level,which inhibited STAT3 phosphorylation and STAT3 signal transduction.The modification imbalance of SUMO1 and SUMO3 protein systems is closely related to tumor occurrence.We further used Auto Dock 4.2.6 to simulate the molecular docking of Hispolon and Illudin S compounds with the main SUMO proteins(SUMO1and SUMO3),revealing their potential for binding to SUMO molecules.The results show that the binding energy of Hispolon with SUMO1 was-5.27kcal/mol and Illudin S with SUMO1 was-5.36kcal/mol.While the value of inhibition constant(KI)of Hispolon against SUMO1 was 138.15μM and Illudin S against SUMO1 was 117.91μM.It suggests Hispolon or Illudin S can be used as ligands for SUMO1 protein.The residues of Ala-6,Lys-7,Pro-8,Gln-53,Pro-58 and Met-59 were similar in the docking processes of SUMO1 with Hispolon and Illudin S,involved in the formation of docking pockets.Meanwhile the binding energy of Hispolon with SUMO3 was-5.00 kcal/mol and Illudin S with SUMO3 was-5.96kcal/mol.While the value of inhibition constant(KI)of Hispolon against SUMO3 was 217.27μM and Illudin S against SUMO3 was42.61μM.It suggested Hispolon or Illudin S can be used as ligands for SUMO3 protein.The residues of Met-54,Gln-56,Ile-57,Asn-67 and Glu-68 were similar in the docking processes of SUMO3 with Hispolon and Illudin S,involved in the formation of docking pockets.In order to further confirm the docking results,we carried out TSA experiment and found that SUMO1 and SUMO3 proteins become more stable with Hispolon and Illudin S treatment and resulted in increased melting curve temperature of SUMO1 and SUMO3.Our findings concluded that Hispolon as well as Illudin S function as small molecular ligands which bind with SUMO1/SUMO3 proteins.Then,we demonstrated that SUMO1 gene silencing can significantly enhance Hispolon or Illudin S induced the DU145 cell proliferation inhibiting,cell cycle S-arrest and promoting cell apoptosis.On the other hand,SUMO3 silencing attenuated Hispolon or Illudin S induced cell proliferation inhibition,cell cycle arrest and apoptosis.These results suggested that SUMO3 play a synergistically role,whereas SUMO1 play an antagonist role in Hispolon or Illudin S induced cell cycle arrest and apoptosis.Meanwhile Hispolon or Illudin S altered exogenous SUMO1 protein intracellular distribution in different ways from SUMO3.Interestingly,without treatment,SUMO1 protein distributed in the nucleus in dots,and SUMO3 predominantly found in the nucleus in diffuse,suggesting an activated SUMO1-mediated SUMOylation and an inactive SUMO3-mediated SUMOylation in DU145 cells.However,Hispolon or Illudin S altered each exogenous SUMO proteins intracellular distribution.These provided another evidence for the different roles of each SUMO proteins in Hispolon or Illudin S induced DU145 cell apoptosis.In addition,we screened the possible substrate proteins of the SUMO system in Hispolon and Illudin S-regulated DU145 cells.The DU145 cells of prostate cancer were treated two concentrations of Hispolon(10 μM and 40 μM)and two concentrations of Illudin S(1.5 μM and 5 μM)by using the immunoprecipitation experiments of SUMO1,SUMO3-specific antibodies,and analyzing the possible substrate proteins of SUMOmodified modifications by mass spectrometry.Seven proteins interact directly with the SUMO1 or SUMO3 response to Hispolon,including five Hispolon-regulated SUMO1 modified proteins(PKM2;GAPDH,HSP90AB1,FAM184 A and CARS)and two Hispolon-regulated SUMO3 modified proteins(HSP90AB1 and ALB).While two Illudin S-regulated SUMO1-modified proteins(HSP90AB1and HSPA2)and two Illudin S-regulated SUMO3-modified proteins(KRTII and KIF5B)were detected in DU145 cells.Based on this founding,HSP chaperon proteins(HSPAB1 and HSPA2),glycolysis proteins(PKM2 and GAPDH),and cancer related proteins(FAM184A,KRTII,CARS,ALB and KIF5B)as SUMO1/SUMO3 interaction proteins and play a significant role as prediction of the alternative SUMO machinery in Hispolon or Illudin S induced prostate cancer cell apoptosis.Through the above research,we found that Hispolon and Illudin S in prostate cancer cells promote apoptosis,and regulate the SUMO modification system.In summary,this paper finds the effect of two fungal compounds Hispolon and Illudin S on prostate cancer and related signaling pathways,and provides the mechanism of Hispolon and Illudin S for the apoptosis of prostate cancer.In particular,we found that both Hispolon and Illudin S were used as ligand molecules to regulate the SUMO protein,and initially identified its base protein for regulating SUMO signals,providing evidence of antagonist tumor resistance in the Hispolon and Illudin S in regulation of SUMO protein modification system.This paper provides basic data for the application of bacterial components and the discovery of new ligands in SUMO-modified system.