Two Compounds From Mushrooms Play Anticancer Effects By Targeting Apoptotic Signaling Pathways

The global morbidity and mortality of tumors is very high,which remain serious hazard to human healthiness.Currently,among cancer treatments are chemotherapies,surgery,radiotherapies,immunotherapies,among which the chemotherapy method has significant toxicity,drug resistance and other adverse reactions.Therefore,the discovery of new anti-tumor components from natural plant products,especially fungi,has become a new strategy for cancer medicine development.Mushroom includes most of three kinds as,edible fungi,medicinal fungi and toxic fungi.A common edible fungus includes Ganoderma lucidum,Cordyceps sinensis,Poria cocos,etc,which can be eaten.Toxic fungi commonly known as“poisonous mushrooms”,contains toxic components to humans or animals,such as poisonous umbrellas,cup umbrellas,orange umbrellas and so on.Medicinal fungi include Ganoderma lucidum,Cordyceps,etc.Firstly,we compared the cytotoxic of Cordyceps,Illudin S,and Lentinan,which comes from Cordyceps sinensis,Poria cocos,and Shiitake?Lentinusedodes?respectively,on different tumor cells.We found that SGC-7901 cells were sensitive to cordycepin with the IC₅₀?concentration estimate toxic for 50%of cells?of 40?M,SGC-7901 and SW480 cells were sensitive to Illudin S with IC₅₀ of 0.35?M and 0.6?M respectively,whereas SGC-7901 and SW480 cells were not sensitive to Lentinan.As the block of apoptosis signal was the basis of tumor cells with long-term survival and unlimited growth,in this article,we selected cordycepin,and Illudin S which were representative extracts of medicinal fungi,and toxic fungi,to explore their possible anti-tumor effects and to reveal the apoptosis related signaling pathways.Firstly,we systematically studied the cytotoxic property as well as the regulation mechanism of apoptosis signal of Cordycepin on gastric cancer SGC-7901 cells.The cytotoxic effect of Cordycepin on gastric cancer cells?SGC-7901?was analyzed by MTT,the IC₅₀ were40?M,32?M,and 7?M after 24 h,48 h,and 72 h treatment correspondingly.The result of DAPI staining and AnnexinV/PI flow cytometry for apoptosis assays showed that the apoptosis rate of Gastric cancer cells?SGC-7901?with 40?M and 80?M Cordycepin treatment,reached50%and 90%after 48h respectively.We found that Cordycepin up-regulated the expression of death receptor DR3 and activated shearing of caspase-8 by DR3,then the caspase-3 and PRAP were further activated,which initiated cells apoptosis by the way of death receptor pathway?extrinsic pathway?.We found that following the initiation of death receptor pathway,Cordycepin initiated the extrinsic pathway by regulating the reactive oxygen species?ROS?,p38 and other signal molecules,which functions as necessary apoptotic factors,to further turn on intracellular mitochondrial apoptotic pathways?endogenous pathways?and to regulate the cell cycle meanwhile.We found that by increasing intracellular ROS levels,reducing the protein expression of Bcl-2 while increasing the protein expression of Bax,Cordycepin sequentially decreased mitochondrial trans-membrane potential?MMP?,which controls cytochrome c release in mitochondria.Therefore Cordycepin promoted the cell apoptosis of intrinsic pathway.At the meantime,ROS release induced by Cordycepin could damage DNA,and activate p53signal that consequently induce SGC-7901 cells cycle arrest at S phase,which is likewise demonstrating with the increase of p53/p21 as well as decrease of Cdk1/2 and CyclinE1/D1.The above results suggested that Cordycepin promoted SGC-7910 tumor cells apoptosis through mitochondrial extrinsic pathways.We also explored the regulatory effects of Cordycepin on cell proliferation signals.The PI3K/AKT and JAK/STAT3 pathways were important in cell survival signals that were abnormally activated in tumor cells,which blocked apoptosis signals and regulated metabolism through the regulation of ROS and Bcl-2.We found that Cordycepin inhibited the phosphorylation of PI3K and AKT,therefore to regulate ROS/Bcl-2 to promote the process of apoptosis.Meanwhile inhibited the phosphorylation of JAK2 and STAT3 and decreased the levels of Bcl-2 and tumor metabolism associated protein,PKM2 and A₃A_R.This showed that Cordycepin inhibited cell proliferation signals PI3K/AKT and JAK/STAT3 and down-regulated tumor metabolism.These results demonstrated Cordycepin inhibited cell proliferation signals,modulated tumor metabolism,and promoted apoptosis on gastric cancer SGC-7901cells.In the other hand,we also explored the cytotoxic effects and mechanism of Illudin S on gastric cancer SGC-7901cells and colon cancer SW480cells.The Illudin S was extracted from toxic fungi and had strong cytotoxic effects.The MTT experiments showed that the IC₅₀ of Illudin S on SW480 cells were 0.2?M,0.6?M,and 1.7?M after 12 h,24 h,and 48 h treatment respectively,while the IC₅₀ of Illudin S on SGC-7901 were 0.25?M,0.35?M,and 0.58?M after 12h,24h and 48h treatment respectively.The results of DAPI staining with Annexin V/PI flow cytometry showed that the apoptosis rate of gastric cancer SGC-7901cells with 0.6?M of Illudin S reached 60%and 80%after 12h and 24h respectively;while the apoptosis rate of colon cancer SW480 cells with 0.35?M reached 50%and 70%after 12 h and 48 h respectively.We found that Illudin S drive the activation of both caspase-8 and caspase-9 then further activate caspase-3 in the process of inducing SGC-7901 and SW480 cell apoptosis.Interestingly,the activation of caspases by Illudin S was reverse by caspase inhibitor of ZVAD-FMK.Regarding these finding we might hypothesis that Illudin S induce apoptosis in SGC-7901 and SW480cells through mitochondrial extrinsic and intrinsic pathways.Furthermore,we provided that Illudin S increased the death receptor DR3,decreased NF-Kb and cIAP,thus to activate the apoptosis of the extrinsic pathway in both tumor cells.Meanwhile,Illudin S also decreased the MMP and the Bcl-2/Bax ratio,and increased intracellular cytochrome C to promote the apoptosis of the intrinsic pathway.Through NAC blocking experiments,it demonstrated that these effects were achieved due to the ROS release from mitochondria by Illudin S.It showed that after the release of ROS,the p53 pathway was activated,which was manifested as the enhancement of p53/p21 signal and the down regulation of CyclinE1/D1.At this point,the cell cycle was suspended in S phase.It is suggested that S activation p53 signal promotes the DNA damage process of tumor cells induced by ROS,thus realizing the s-phase cell block.We detected the regulation of Illudin S on the cell proliferation signals of PI3K/AKT and JAK/STAT3,and found that Illudin S inhibited the phosphorylation of PI3K and AKT,suggesting its inhibition against proliferation signaling.At the same time,it was found that Illudin S inhibited another proliferation signaling of JAK/STAT3 pathway.The results were demonstrated with Illudin S inhibiting the phosphorylation of JAK2 and STAT3.Real-time and WB experiments also found that Illudin S up-regulated the level of PIAS3 significantly,reversed the STAT3/PIAS3 ratio,thus to prevent the activation of STAT3 reporter gene,and inhibition of STAT3 downstream genes activation of ERK phosphorylation or c-myc expression.These results proved that Illudin S can inhibit the cell proliferation signals of PI3K/AKT and JAK/STAT3 strongly to further promote the apoptosis colorectal as well as gastric cancer cells.Cordycepin and Illudin S are extracted from fungi and have similar structures.Through death receptor signaling,they activate the mitochondrial pathway and promote tumor cell apoptosis.Therefore,we simulated the molecular binding of these two fungi components,Cordycepin and Illudin S,to death receptors FasL or DR3?TNF?R?by AUTO-DOCK calculations,meanwhile,we chose another mushroom compound,lentinan,as a control,which has difference.Structure completely compared with these two.We found that Cordycepin and Illudin S were able to dock to the death receptor Fas and DR3 protein structures.Totally seven amino acids docking sites of Fas binding to Cordycepin were obtained,with lowest binding energy of-4.4Kcal/mol.Illudin was surrounded with seven amino acids of Fas and the lowest binding energy of-4.83Kcal/mol was gained.Furthermore,both Cordycepin and Illudin S can also be able to dock to DR3?TNF?R?protein structure.Totally eight amino acids docking sites of TNF?R binding to Cordycepin were obtained,with lowest binding energy of-4.02Kcal/mol.Illudin S was surrounded with five amino acids of Fas and the lowest binding energy of-5.05Kcal/mol was gained.And the control group of lentinan was able to docking to neither Fas nor DR3,and there was no binding site and binding energy calculated.The above results suggest that Cordycepin and Illudin S function as novel death ligands to bind and activate the death receptor,thereby initiate the death receptor pathway and activate the endogenous pathway through the exogenous pathway,which opens the cell apoptotic system.In summary,this thesis found that fungi components of Cordycepin and Illudin S induced cancer apoptosis and revealed the related signaling pathways and molecular mechanisms in tumor cells,and also provide evidence of Cordycepin and Illudin S function as a novel ligand for the binding and activation of the cell death receptors.This thesis provides academic basis for future clinical application of the component of fungi against gastric cancer and colon cancer.