Mechanism Of Epoxycytochalasin H Promoting Apoptosis In Human Ovarian Cancer A2780 Cells

Background:Ovarian cancer ranks seventh among the most common malignant tumors in women,seriously threatening women’s reproductive health.It is characterized by hidden pathogenesis,missed diagnosis,high recurrence rate and poor prognosis.Clinically,paclitaxel-based chemotherapy is preferred in first-line treatment for tumor reduction surgery.The harsh truth is that female patients are prone to relapse due to the spread of tumor cells and drug resistance.In this case,the development of new treatment strategies combined with traditional methods can help improve the quality of treatment.Among the numerous drug resources,plant compounds have unique advantages such as multi-target potential,long application history and wide application.Previous studies have revealed the therapeutic role of bioactive plant ingredients in ovarian cancer.These natural ingredients can be used as part of initial therapy or as an adjunctive option for maintenance therapy to further reduce the tumor and metastasis burden.A metabolite extracted from the endophytic fungus Phomopsis sp(pseudostemspot fungus)was speculated to have significant anti-tumor activity due to its nonenolactone structure,and identified as Epoxycytochalasin H.In this study,the antitumor effects of Epoxycytochalasin H on human ovarian cancer cell line A2780 were investigated.Our data suggest that Epoxycytochalasin H significantly reduces ovarian cancer cell proliferation and induces apoptosis.Objective:Compared with traditional chemotherapy drugs,natural compounds from plant and microbial sources have good antitumor activity while reducing genotoxicity.Exploring the antitumor mechanisms of natural compounds may provide new strategies for treating cancer and overcoming drug resistance.Method:1.A2780 cells were treated with Epoxycytochalasin H at the concentration of 0,6.25,12.5,25,50,100 μg/m L.Cell viability was detected by MTT assay.2.A2780 cells were treated with Epoxycytochalasin H at the concentration of 0,20,40,60μg/m L,and cell proliferation was observed by clonal formation assay.3.A2780 cells were treated with Epoxycytochalasin H at the concentration of 40μg/m L,and the nuclei were stained by Hoechst 33342 to observe the nuclear morphology.4.A2780 cells were treated with Epoxycytochalasin H at the concentration of 0,20,40,60μg/m L,and cell apoptosis was detected by flow cytometry.5.A2780 cells were treated with Epoxycytochalasin H at the concentration of 0,20,40,60μg/m L.Mitosox staining was used to detect mitochondrial ROS levels.6.A2780 cells were treated with Epoxycytochalasin H at the concentration of 0,20,40,60μg/m L.Mitochondrial membrane potential was detected by flow cytometry.7.Western blot was used to detect the expressions of apoptosis-related proteins,autophagy/mitochondrial autophagy related proteins,and er stress-related proteins.Results:1.The results of MTT assay showed that Epoxycytochalasin H decreased the viability of A2780 cells in a dose-dependent manner,with IC50=51μg/m L.The results showed that Epoxycytochalasin H decreased the colony number of A2780 cells in a dose-dependent manner,suggesting that Epoxycytochalasin H inhibited the proliferation of A2780 cells.2.Epoxycytochalasin H induces apoptosis of OVARIAN cancer cell A2780.Hoechst33342 was used to stain the nuclei of cells in each group.After Epoxycytochalasin H treatment,chromatin shrinkage was obvious.Cell apoptosis was detected by flow cytometry.It was found that the apoptosis rate of A2780 cells increased in a dose-dependent manner after treatment with Epoxycytochalasin H.Western Blot analysis of apoptosis-related protein expression showed that Cleaved caspase-3 expression was increased in A2780 cells treated with Epoxycytochalasin H.3.Epoxycytochalasin H can induce mitochondrial dysfunction and apoptosis in A2780 cells.Epoxycytochalasin H increased ROS level of human ovarian cancer cells A2780 by detecting Mito ROS level.The mitochondrial membrane potential of A2780 cells treated with Epoxycytochalasin H was measured by flow cytometry with JC-1 fluorescent probe.It was found that the monomer morphology of JC-1 cells increased and the aggregation morphology decreased,and the mitochondrial membrane potential decreased in a dose-dependent manner.Western Blot analysis of mitochondrial apoptosin-related proteins showed that Bax/Bcl-2 ratio in A2780 cells increased after treatment with Epoxycytochalasin H.4.Epoxycytochalasin H can activate autophagy and increase autophagy flux of A2780 cells.Western Blot analysis of p62 and LC3 protein expression showed that the ratio of P62 and LC3II/LC3-I increased gradually after treatment with Epoxycytochalasin H in A2780 cells.CQ combined with Epoxycytochalasin H increased lc3-II /LC3-I significantly.5.Epoxycytochalasin H can activate mitochondrial autophagy in A2780 cells.Western Blot analysis of mitochondrial autophagy related protein expression showed that the protein expression of Pink and Parkin increased after treatment with Epoxycytochalasin H in A2780 cells.6.Overexpression of Parkin can promote apoptosis induced by Epoxycytochalasin H.Western blot analysis of apoptosis-related proteins in overexpressing Parkin cells showed that the expression of anti-apoptotic protein Bcl-2 was decreased and the ratio of Bax/Bcl-2 was increased.7.Epoxycytochalasin H activates endoplasmic reticulum stress in human ovarian cancer cells A2780.Western Blot analysis of Grp78 and Cleaved caspase-4 expression showed that Epoxycytochalasin H induced a time-dependent increase in Grp78 protein expression,and Cleaved caspase-4 expression increased.Conclusion:1.Epoxycytochalasin H activates PINK1/ Parkin-mediated mitochondrial autophagy,resulting in mitochondrial dysfunction of human ovarian cancer A2780 cells,and enhances apoptosis of human ovarian cancer A2780 cells induced by Epoxycytochalasin H.2.Epoxycytochalasin H induces endoplasmic reticulum stress and further induces apoptosis of human ovarian cancer A2780 cells.