Isolation Of Secondary Metabolites From Macrofungi Hericium Alpestre And Anti-tumor Effects And Mechanism Of 4-hydroxy-2-pyridone Alkaloid Sambutoxin

BackgroundMalignant tumors are common in many clinical cases and have few effective treatments.It has led to a high mortality rate and is becoming one of the most threatening diseases for human health,second only to cardiovascular and cerebrovascular diseases.Drug therapy is still one of the main methods of clinical treatment of various tumor diseases.Although chemical synthetic drugs have achieved certain therapeutic effects,they are more likely to be unaccepted by patients because they have more harmful effects on the normal tissues of the human body.In recent years,a class of major or all components derived from anti-tumor drugs of animals or plants or microorganisms,that is,natural drugs having anti-tumor effect,have attracted more and more attention due to their relatively high efficiency and low toxicity.Microorganisms are the main source of most natural medicines.For a long time,in the field of natural anti-tumor drugs,people mainly focus on the active natural products derived from actinomycetes,but they pay insufficient attention to a wide variety of macrofungi,which also contain natural product resources.As a series of natural products with anti-tumor activity have been obtained from Hericium erinaceus,other fungi of Hericium(such as Hericium alpestre)have received increasing attention as resources for anti-tumor natural products.In the second chapter of this thesis,4-hydroxy-2-pyridones and other types of compounds were isolated from the crude fermentation from Hericium alpestre,which laid a material foundation for the evaluation of anti-tumor activity.Tumor growth inhibition involves very complex and multi-level biological processes,including cell cycle arrest,apoptosis,oxidative stress,DNA damage response,MAPK system regulation,and dysfunction of the thioredoxin redox regulatory system,etc.These factors have a lot to do with each other.In the thesis,LN1,LN2,LN3 and sambutoxin(LN14)were evaluated the antitumor pharmacodynamic activity,and the best compound sambutoxin was screened out.MDA-MB-231 and MCF-7cells were used as research models to further explore the possible anti-tumor mechanism of sambutoxin.The target of sambutoxin on tumor cells was clarified,and the experimental results of anti-tumor activity and related mechanisms of sambutoxin in vitro were verified by in vivo test.1.Isolation and identification of secondary metabolites from Hericium alpestre and cytotoxicity of some compounds in vitroMethods and results:The macrofungi Hericium alpestre was subjected to solid fermentation of PDA medium,and the fermentation products were extracted multiple times through the extract and successively subjected to multiple extractions of ethyl acetate/water and methanol/petroleum ether extraction system to obtain a crude methanol phase extract.The crude extract was subjected to medium pressure liquid phase reversed phase silica gel column chromatography,dextran gel column chromatography,normal phase silica gel column chromatography,thin layer chromatography and recrystallization separation technology,combined with nuclear magnetic resonance spectroscopy,mass spectrometry,X-ray single crystal diffraction,infrared spectroscopy,ultraviolet absorption wavelength detection,optical rotation analysis and circular dichroism analysis.A total of 14 compounds(LN1 to LN14)were obtained,and two of them were new structural compounds(LN2,LN3).Human lung cancer cell A549,human cervical cancer cell Hela and human colon cancer cell HT-29 were used to evaluate the cytotoxicity of LN1,LN2 and LN3 by MTT assay in vitro.DMSO(0.05%)was used as a blank control and vincristine was used as a positive control.Cytotoxicity test results showed that LN1 had relatively better cell growth inhibitory activity to A549 and HT-29 than LN2 and LN3;LN2 and LN3 had lower sensitivity to A549,Hela and HT-29 cells;the cell growth inhibitory activity of LN1,LN2 and LN3 to these three kinds of cells was weaker than that of the positive control vincristine.Conclusions:The pyrone[3,4-g]chromene-4,6-dione group may be a pharmacophore of LN1.2.Antitumor activity and mechanism of sambutoxin(LN14)Methods:Human lung cancer cell A549,human cervical cancer cell Hela,human colon cancer cell HT-29,human breast cancer cell MDA-MB-231,human breast cancer cell MCF-7,human prostate cancer cell PC-3,human gastric cancer cell SGC-7901 and human umbilical vein endothelial cells HUVECs were used to detect cytotoxic activity of sambutoxin by MTT assay with DMSO(0.05%)as a blank control and doxorubicin as a positive control in vitro.Through the cell growth inhibition screening,the most sensitive cell lines-breast cancer cells MDA-MB-231 and MCF-7 were used for subsequent antitumor effects and mechanism research of sambutoxin.The inhibitory effect of Sambutoxin on the proliferation of breast cancer cells was tested by CCK-8 assay with DMSO(0.05%)as a blank control to detect cytotoxic activity in vitro when the time gradient design was added based on the concentration gradient design.The cell cycle distribution was detected by PI staining combined with flow cytometry,and the expression of cycle-associated proteins was detected by Western blot.Hoechst 33258 staining assay and flow cytometry Annexin V/PI double staining assay were used to detect the apoptosis-inducing effect of sambutoxin,and the expression of apoptotic-related proteins was detected by Western blot.Changes in reactive oxygen species(ROS)level in cells induced by sambutoxin were determined by DCFH-DA staining and quantified by flow cytometry.The effect of sambutoxin on DNA damage marker molecule ?-H2AX was detected by immunofluorescence staining.The expression of ?-H2AX and key proteins in DNA damage response ATM/Chk2 pathway was detected by Western blot.NAC(a ROS inhibitor)was introduced to examine its effect on sambutoxin-induced cell cycle arrest and DNA damage response.The effect of sambutoxin on the expressions of key proteins of exogenous apoptotic pathway and endogenous apoptosis pathway was detected by Western blot.Mitochondrial membrane potential in cells were determined by JC-1 staining and quantified by flow cytometiy.Ac-LEHD-FMK(a caspase 9 inhibitor)was introduced to examine its effect on sambutoxin-induced apoptosis by flow cytometry Annexin V/PI double staining and the expression of PARP by Western blot.NAC was introduced to examine its effect on sambutoxin-induced apoptosis by flow cytometry Annexin V/PI double staining and the expression of endogenous apoptosis proteins Bax and Bcl-2 by Western blot.The expression of sambutoxin-induced MAPK family member proteins,including JNK,phospho-JNK,Erk1/2,phospho-Erk1/2,p38 and phospho-p38 was detected by Western blot.SP600125(a JNK inhibitor)was introduced to examine its effect on sambutoxin-induced apoptosis by flow cytometry Annexin V/PI double staining and the expression of JNK,phospho-JNK,Bcl-2 and Bax by Western blot.NAC was introduced and its effect on the expression of JNK and phospho-JNK induced by sambutoxin was detected by Western blot.Female nude mice bearing MDA-MB-231-luc-D3H2LN xenografts were used to detect the inhibitory effect of sambutoxin on tumor growth in vivo.The expression of tumor extract proteins,involving in cell cycle,DNA damage response and apoptosis,was detected by Western blot.Result:MTT results showed that sambutoxin significantly inhibited the proliferation of MDA-MB-231 and MCF-7 cells with IC50 values of 7.96 and 9.36 ?mol/L,respectively;and human normal cells HUVEC were less sensitive to sambutoxin than to the positive drug doxorubicin.CCK-8 results showed that sambutoxin inhibited the growth of MDA-MB-231 and MCF-7 cells in a time-and dose-dependent manner.PI staining flow cytometry analysis showed that sambutoxin induced G2/M phase cell cycle arrest in MDA-MB-231 and MCF-7 cells.Western blot results showed that sambutoxin dose-dependently decreased the expression of cdc25C,cdc2 and cyclin B1,and increased the expression of phospho-cdc25C(Ser216)and phospho-cdc2(Tyr15).NAC reversed the induction of sambutoxin on cell cycle arrest.Hoechst 33258 staining showed a significant increase in the number of cells with chromatin condensation and nuclear fragmentation in the sambutoxin-treated MDA-MB-23 1 and MCF-7 cell populations.Flow cytometry Annexin V/PI double staining showed that the apoptosis rate of MDA-MB-231 and MCF-7 cells increased significantly after sambutoxin was added to cells in a dose-dependent manner for 36 h.Western blot results showed that the expression of pro-caspase-3 and pro-PARP was significantly down-regulated,and the expression of cleaved caspase-3 and cleaved PARP was significantly up-regulated.DCFH-DA staining showed that both green fluorescence foci and fluorescence intensity increased significantly in sambutoxin-treated MDA-MB-231 and MCF-7 cells,indicating that the levels of ROSincreased in cells;quantitative analysis by flow cytometry showed that after treated with 20 ?mol/L sambutoxin for 24 h.the levels of ROS in MDA-MB-231 and MCF-7 cells increased by 28-fold and 13-fold,respectively.Immunofluorescence staining and Western blot showed that y-H2AX foci increased and the expression of y-H2AX was significantly up-regulated in MDA-MB-231 and MCF-7 cells after the treatment of sambutoxin.Western blot showed that sambutoxin significantly up-regulated the expression of phospho-ATM and phospho-Chk2 to activate the ATM/Chk2 pathway DNA damage response.NAC reversed ?-H2AX foci formation and the expression of y-H2AX and phospho-Chk2 induced by sambutoxin,indicating that sambutoxin triggered DNA damage response by inducing ROS production.Western blot showed that sambutoxin had no significant effects on the expression of DR5,caspase-8,Fas,Fas-L and FADD in MDA-MB-231 and MCF-7 cells,indicating that sambutoxin-induced apoptosis was not dependent on exogenous apoptotic pathway.The results of JC-1 staining showed that sambutoxin caused a significant conversion from red fluorescence to green fluorescence in MDA-MB-231 and MCF-7 cells,which was also confirmed by flow cytometry analysis,indicating that sambutoxin caused a significant decrease in mitochondrial membrane potential.Western blot showed that sambutoxin induced significantly up-regulation of Bax,Bim,Bad,cleaved caspase-9 and cytoplasmic Cyt c,while significantly down-regulation of Bcl-2 and Bcl-xL in MDA-MB-231 and MCF-7 cells,indicated that sambutoxin-induced apoptosis was dependent on endogenous apoptotic pathway.The reversal effects of Ac-LEHD-FMK and NAC further demonstrated that sambutoxin caused apoptosis by activating endogenous apoptotic pathway and was controlled by ROS generation.Western blot showed that sambutoxin significantly up-regulated the expression of phospho-JNK in MDA-MB-231 and MCF-7 cells,and had no significant effects on the expression of JNK,Erk1/2,phospho-Erk1/2,p38 and phospho-p38.SP600125 significantly reversed the proapoptotic effects of 20?mol/L sambutoxin in MDA-MB-231 and MCF-7 cells and significantly reversed the up-regulation of Bax and the down-regulation of Bcl-2 induced by sambutoxin.These results indicated that JNK activation played a decisive role in sambutoxin-induced apoptosis.NAC effectively reversed the sambutoxin-induced JNK phosphorylation in MDA-MB-231 and MCF-7 cells,indicating that sambutoxin-induced ROS generation was an early event followed by induction of JNK activation.Sambutoxin had a significant growth inhibitory effect on MDA-MB-231-luc-D3H2LN xenografts in nude mice,and it was confirmed by small animal imaging technique in vivo.The tumor growth inhibition rate of 20 mg/kg sambutoxin was 61.9%without significant decrease of mice's body weight.The cell-cycle proteins phospho-cdc2(Tyr 15)and cyclin B1,the DNA damage signaling protein phospho-Chk2,and the apoptosis signaling proteins cleaved PARP,Bax,Bcl-2,and phospho-JNK were analyzed by Western blot.These results were consistent with the results in vitro.Conclusions:Sambutoxin showed potent inhibitory effects on the proliferation of human breast cancer cells in vitro and in vivo.The effects of sambutoxin were associated with ROS generation,which triggered oxidative DNA damage and ATM-Chk2-cdc2 pathway-dependent G2/M arrest.Moreover,ROS/JNK played a key role in sambutoxin-induced cellular endogenous apoptosis.Both cell-cycle G2/M arrest and apoptosis induction by sambutoxin were partly dependent on the stimulation of oxidative stress.3.The mechanism of sambutoxin-induced abnormal upregulation of ROS levels in breast cancer cellsMethods:The effects of sambutoxin on the expression of Trx-ASK1 signaling pathway-related proteins phospho-ASK1,ASK1 and Trx-1 in human breast cancer cells MDA-MB-231 and MCF-7 were detected by Western blot.The thioredoxin reductase(TrxR)activity assay kit was used to detect the effects of sambutoxin on thioredoxin reductase activity in tumor cells.The effects of sambutoxin on the activity of glutathione reductase(GR),thioredoxin peroxidase(TPX)and glutathione peroxidase(GPX)in tumor cells were detected by GRactivity assay kit,TPX activity assay kit and GPX activity assay kit.The effects of sambutoxin on the total sulfhydryl contents and the contents of reduced glutathione(GSH)in tumor cells were examined by total sulfhydryl contents detection kit and GSH assay kit.Result:Sambutoxin significantly upregulated the expression of phospho-ASK1 in MDA-MB-231 and MCF-7 cells in a dose-dependent manner,and also significantly downregulated the expression of ASK1 and Trx-1 in both cell lines in a dose-dependent manner.Sambutoxin significantly reduced TrxR,GPX activity and total sulfhydryl contents in MDA-MB-231 and MCF-7 cells in a dose-dependent manner.However,it did not cause significant changes in GR activity,TPX activity and GSH contents in tumor cells.Conclusions:TrxR might be a target for sambutoxin-induced abnormal upregulation of reactive oxygen species levels.Sambutoxin reduced the expression of the electron donor Trx-1 by inhibiting TrxR activity,so that intracellular ROS was difficult to be sufficiently reduced in time,resulting in the accumulation of ROS in tumor cells to cause oxidative stress.