Study On The Effect And Action Mechanism Of Inotodiol On HeLa Cell Migration,invasion And Apoptosis

Inonotus obliquus is a traditional edible and medicinal fungus,which is rich in a variety of chemical components and has significant anti-inflammatory,anti-tumor,anti-mutation,anti-lipid and anti-hypertensive,anti-virus and other biological activities.Inotodiol is a special natural lanostane-type triterpenoid isolated from Inonotus obliquus,which biological activity is extremely rich.Inotodiol inhibited the proliferation of various tumor cells and had different mechanisms in various tumor cells.The present study,it firstly investigated the inhibitory effect of Inotodiol on the proliferation of human cervical cancer cell line HeLa cells,and the effects of various concentrations of Inotodiol on the HeLa cells migration,invasion and apoptosis also were analyzed,which is further clarified that p53 signaling pathway is the key molecular mechanism of Inotodiol against HeLa cells of cervical cancer.The main research results as follows:1.Inotodiol inhibited HeLa cells growth and proliferation.The cytotoxicity assay showed that Inotodiol inhibited the HeLa cell viability in a concentration-dependent manner,and IC50 values(58.9 and 39.8 μM,respectively)were observed at 48 h and 72 h.At the same time,5-FU was selected as the positive control,and the results showed that the anticancer effect of Inotodiol was consistent with that of 5-FU.and the concentration of 50 M was used as the subsequent experimental condition within the effective treatment range.In order to further evaluate its effectiveness,the effects of Inotodiol on HeLa cell morphology and proliferation were investigated.The results showed that the Inotodiol under the concentration of 25 μM had no significant effect on the morphology of HeLa cells.Moreover,when the concentration of Inotodiol was higher than 25 μM,the morphology of HeLa cells could be induced to shrink,become round and undergo apoptosis in a dose-dependent manner.In addition,the similar crystal violet staining results further showed that Inotodiol effectively inhibited the proliferation of HeLa cells,and the inhibitory effect was significant when the concentration was higher than 25 μM.2.Inotodiol inhibited HeLa cells migration,invasion and induced apoptosis.The wound-healing analysis and trans-well invasion assay confirmed that Inotodiol(5,10,25 μM)could effectively inhibit the migration and invasion of HeLa cells,and reduced the expression of MMP2 and MMP9 proteins in HeLa cells at the molecular level.Meanwhile,flow cytometry assay showed that Inotodiol(30,40,50 μM)could induce HeLa cells apoptosis and stimulate the activity of proteolytic enzyme Caspase-3 during HeLa cells apoptosis process,then Inotodiol further up-regulated the expression of pro-apoptotic proteins Cleaved PARP,Cleaved Caspase-3 and Bax,and reduced the expression of anti-apoptotic protein Bcl-2.In addition,the transcription of tumor suppressor p53 was activated during the action of HeLa by Inotodiol treatment,which leaded to the increase of p53 and p21 protein levels.Therefore,it is preliminarily speculated that the tumor suppressor p53 is closely related to the process of inhibition of HeLa cell migration,invasion and induction of apoptosis of cervical cancer by Inotodiol.3.Inotodiol inhibited cells migration and invasion and induces apoptosis via p53-dependent pathway in HeLa cells.Pretreatment of HeLa cells with negative control group(si-NC)and small interfering RNA of specific p53 gene(si-p53),the result showed that compared with si-NC group,si-p53 inhibited the transcriptional activity of p53 gene in HeLa cells and decreased the content of mRNA,Meanwhile,western blotting analysis showed that the expressions of proteins p53 and p21 were also significantly decreased,indicating that the experiment successfully silenced p53 gene.In addition,compared with control group,the data showed that Inotodiol could effectively inhibit the migration and invasion of HeLa cells,and proved the effectiveness of the inhibition by down-regulating the expression levels of MMP2 and MMP9 proteins in the si-NC group.However,when the p53 gene was silenced,Inotodiol could not effectively inhibit the migration and invasion of HeLa cells in the si-p53 group,and its down-regulation of MMP2 and MMP9 protein expression was reversed.Meanwhile,the results also showed that in the si-NC group,Inotodiol induced apoptosis by regulating the expression of pro-apoptotic protein and anti-apoptotic protein,and activated the activity of caspase-3.Conversely,in the si-p53 group,when the p53 gene was silenced,the apoptotic effect induced by Inotodiol was also reversed.The above results showed that p53 was a key regulatory gene in inhibiting the proliferation,migration,invasion and induction of HeLa cells.In summary,Inotodiol could inhibit the HeLa cells proliferation,migration,invasion and induce apoptosis,then further revealed the potential of p53 gene in preventing metastasis and invasion of malignant tumors,which provided scientific basis of Inotodiol for treatment of cervical cancer and other tumor diseases.