Study On The In Vitro Anti-cancer Activity Of Natural Products Guxi-2 And Tricycloalternarene 3a From Special Eco-environment Microorganisms

Chemotherapy is one of the three major therapies for cancer treatment in clinical.However,most existing chemotherapeutic drugs show drug resistance and serious side effects.Therefore,it has been the researchers' purpose to find out a cheap,safe and effective anti-cancer drug with less side effects continually.Microorganisms in special eco-environment(such as high salt,high alkali,high temperature,etc)had developed special metabolic pathway to adapt to the special environment.They are most likely to produce secondary metabolites with novel structures and significant bioactivities,which has become an important area for natural product research and new drug development.Our previous screening research found that cyclo-decapeptide compound guxi-2 produced by the endophyte Myrothecium roridum IFB-E091 from Artemisia annua and tricycloalternarene 3a produced by the endophyte Alternaria sp.W-1 from Laminaria japonica both had good in vitro cytotoxic activity.Then,in vitro anti-cancer effect of these two compounds had been investigated further in this dissertation.Cyclo-decapeptide compound guxi-2 had been found to inhibit the proliferation of human gastric cancer cell SGC-7901 and induce the cells apoptosis in the previous experiments.A more in-depth study of the mechanism was conducted in this dissertation.The result showed that guxi-2 had significant inhibitory activity to the human gastric cancer cells SGC-7901,AGS and MGC-803(equal to the positive control cisplatin)with dose-and time-dependence,and the most obvious growth inhibitory effect had been observed in SGC-7901 cells.The results also showed that guxi-2 could remarkably increase the expression of the pro-apoptotic protein Bad,up-regulate the expression of caspases-3,-8,-9 and cleaved caspases-3,-8,-9,increase the expression of PARP and cleaved PARP,and decrease the expression of Akt and p-AktS473 in SGC-7901 cells.These results indicated that the induced-apoptosis in SGC-7901 cells by compound guxi-2 might be involved both the mitochondrial pathway and the death receptor pathway,and regulated by the PI3K/Akt signaling pathway.Recent research discovered that tumor development is closely related to miRNA.Therefore,the relationship between cyclo-decapeptide guxi-2 and miRNA in gastric cancer cells was studied in this dissertation.It was found that guxi-2 could up-regulate the expression of miR-451 in human gastric cancer cells SGC-7901,AGS and MGC-803 Overexpression of miR-451 reduced the proliferation rate of SGC-7901 cells,while guxi-2 could reverse the proliferation promotion effect to SGC-7901 cells caused by down-regulation of miR-451 to some extent,implying that the anti-gastric cancer effect of guxi-2 was related to miR-451.It was also found that guxi-2 had changed the miRNA expression profile in SGC-7901 cells significantly.Compared with the negative control group,a total of 164 differentially expressed miRNA molecules were screened out in the 10?g/mL guxi-2 treatment group.149 of miRNA molecules were up-regulated and 15 of miRNA molecules were down-regulated.Among them,the most outstanding up-and down-regulations were miR-6813-5p(FC=25.99)and miR-125b-1-3p(FC=2.77),respectively.Above-mentioned results indicated that guxi-2 might exert anti-cancer effect by regulating the expression of miRNA.The miRNA molecules with significant expression differences,such as miR-6813-5p and miR-125b-1-3p,are expected to be molecular markers for the diagnosis and treatment of gastric cancer.MiRNA is a promising new target for drug development.It is of great significance to investigate the in vitro anti-cancer activity of natural products based on miRNA in discovering new anti-cancer drugs and lead compounds,which has a broad application prospect.Tricycloalternarene 3a is one of tricycloalternarene(TCA)compounds.Proliferation inhibition effect of TCA compounds against the tumor cells had been reported,but the anti-cancer mechanism had not been reported yet.Cell proliferation is precisely regulated by the cell cycle,and disorder of cell cycle is the internal factor of tumorigenesis.In addition,there is a balance between apoptosis and cell proliferation.As a programmed cell death manner,apoptosis plays a negative regulatory role in the formation and development of tumor.Therefore,the action and mechanism of compound tricycloalternarene 3 a focused on cell cycle and cell apoptosis had been explored in this dissertation.It was found that tricycloalternarene 3a could induce G1 phase arrest in SMMC-7721 cells in a dose-dependent manner and up-regulate the expression of cyclin dependentkinase inhibitor p27.The results suggested that the inhibition effect of tricycloalternarene 3a on the growth of SMMC-7721 cells might be related to the induction of expression of protein p27,preventing cell cycle from G1 phase to S phase.It was also found that tricycloalternarene 3a could increase the apoptosis rate of SMMC-7721 cells,down-regulate the expression of anti-apoptotic protein Bcl-2 and up-regulate the expression of pro-apoptotic protein Bax,and the ratio of Bcl-2/Bax was significantly reduced.At the same time,the expression of caspases-3,-8,-9 and cleaved caspases-3,-8,-9 were up-regulated.These results indicated that tricycloalternarene 3a could promote the apoptosis of SMMC-7721 cells,which was depended on the activation of caspases and involved in both the death receptor pathway and the mitochondrial pathway.In conclusion,the in vitro anti-cancer activity of tricycloalternarene 3a to SMMC-7721 cells was related to not only the induced G1 phase cell cycle arrest but also the induced cell apoptosis.This is the first report on the anti-cancer mechanism for TCA compounds.