Study On Apoptotic Mechanisms Of Tumor Cells Induced By Tricholoma Triterpenoids

Higher fungi are organisms with higher creativity.The compounds separated from their sporophore and zymotic fluid have diverse chemical structures and abundant biological activities.A series of triterpenoids from Tricholoma pardinum were isolated.Previous studies found that these compounds generally have high cytotoxicity,but the mechanism is not clear.Therefore,we chose a novel triterpenoid compound LFTP-85 of Tricholoma orientalis as a representative to research the anti-tumor mechanism.Human cervical cancer cell line Hela was used to research the effects of compounds on cytotoxicity,apoptosis,intracellular reactive oxygen species?ROS?level,cell cycle and mitochondria.We also study the effects on the expression of apoptotic related proteins and genes,cell cycle and mitochondrial apoptosis.Finally,we determined the anti-tumor mechanism.Firstly,MTT assay was used to evaluate cell toxicity.It was found that the half inhibitory concentration?IC50?is 16.34?M.Secondly,the changes of cell apoptotic rate,intracellular reactive oxygen species?ROS?level and cell cycle were investigated through flow cytometry.It was found that LFTP-85 induce Hela cell apoptosis.With the increase of drug concentration,the number of early apoptotic cells and late apoptotic cells increased gradually.At the same time,the compound induced the level of ROS increased and cell cycle disorder,the cell cycle arrest at G2/M phase.Then,It was found that LFTP-85 activated procaspase 9 and initiated caspase cascade connection reaction.The cleaved caspase 9 activated the self-cleavage of procaspase 3,and the cleaved caspase 3 activated the self-cleavage of PARP to produce cleaved PARP fragments.Meanwhile,LFTP-85 induces the mutation of wild type p53 gene,lead to the phosphorylation of wild p53 protein and overexpression of mutant p53 protein.We also considered that the protein expression of cyclin B1 and CDK1 was affected in G₂/M phase.Next,the protein expression of cyclin B1 and CDK1 was affected in consistent with the cell cycle was blocked in G₂/M phase.And also the mRNA expression of cyclin B1,P21and P27 gene was evaluated through q-PCR.It was found that the mRNA expression of cyclin B1,P21 and P27 genes was affected in G₂/M phase.Therefore,it is speculated that the apoptotic pathway may be related to mitochondrial apoptosis.Flow cytometry was used to detect the changes of mitochondrial membrane potential?MMP?,and it was shown that the MMP decreased after drug treatment for 24 h.The expression of mitochondrial apoptotic-related Bcl-2 family protein which laies on the surface of mitochondria,revealed that the expression of pro-apoptotic protein Bax increased and the expression of anti-apoptotic protein Bcl-2 decreased.Meanwhile,It was discovered that the cytochrome c was released from mitochondria into cytoplasm.In summary,LFTP-85 could activate the mitochondrial apoptotic pathway through regulating the expression of Bcl-2 family protein and reducing the mitochondrial membrane potential.Then,the cytochrome c which released from the apoptotic mitochondrial membrane activated the caspase cascade reaction and the self-cleavage of PARP,finally inducing apoptosis.At the same time,the copound activated the over-expression of mutant p53,which resulted in the cell cycle arrest at G₂/M phase.Moreover,the overexpression of mutant p53protein and loss of mitochondrial function induced the increasing level of reactive oxygen species,which indirectly leads to apoptosis.