Grifolin Induces Autophagic Cell Death In Human Ovarian Cancer Cells And The Underlying Mechanism

Background:Ovarian cancer(OC)is the leading cause of mortality from gynecological tumors with the high morbidity lower than cervical cancer and endometrial cancer,which results in being the third most common cancer in gynecologic tract tumors.The epithelial ovarian cancer occupies 85%-90%.A majority of ovarian cancer patients have already suffered advanced cancer presenting with ascitic fluid and distant metastasis owing to the vague early stage symptoms.Traditional therapeutic schedule of epithelial ovarian cancer is giving priority to the primary cytoreductive surgery and combination chemotherapy as the auxiliary pole.Despite advances in treatment during the last decades,traditional methods with chemotherapy after surgery may cause unwanted side-effects for example nausea and vomiting and drug resistance in patients and the prognosis of patients with ovarian cancer has not substantially improved since most would suffer from distant metastasis unfortunately.So the overall 5-year survival rate remains approximately 30%～40%in recent decades and the recurrence rate and death rate were on the top and remain high though.Therefore,it is of a great necessity to develop more novel and effective drugs with little toxicity against ovarian cancer.In recent years,natural agents have been widely used for cancer therapy.Grifolin(2-trans,trans-famesyl-5-methylresorcinol),a secondary metabolite extracted from the mushroom Albatrellus confluens,is an antibiotic belonging to basidiomycota.The first effect of grifolin is lowering plasma cholesterol concentration while its anti-inflammatory effect,anti-virus effect and anti-tumor activity were testified in recent decades.The mechanisms of anti-tumor activity including suppressing the cell proliferation,arresting the cell phase,promoting cell apoptosis,inhibiting cancer cells adhesion,invasion and migration have been proved in several carcinomas.However,the other mechanisms of the natural agent named Grifolin’s antitumor effects have remained unclear.Objective:Our research aimed to investigate the therapeutic potential of Grifolin for ovarian cancer,especially its effect on autophay,and to clarify the underlying mechanism.Methods:Cell viability of the A2780 and SK-OV-3 cells was measured by MTT assay.Flow cytometric analysis of acidic vesicular organelles(AVOs)using acridine orangeand(AQ).Electron microscopy and GFP-LC3 puncta formation assay were performed to evaluate the autophagic abilities.Western blot analysis and the technique named immunofluorescence staining were used for detect the vehicle-or drug-treated cells of the expressions of autophagic protein markers and associated autophagic signal pathway proteins.Reverse verification methods were used to test the results again.The autophagy inhibitor chloroquine(CQ)was carried out to detect the autophagic effect of Grifolin on ovarian cancer cells.ShRNA was used to silence LC3B protein expression in A2780 and SK-OV-3 cells by establishing stable cell lines.T-test and one-way ANOVA were performed to determine significance by using the software SPSS 18.0.Statistical significance was determined at P<0.05.All the experiments were performed in triplicate.Results:1.Grifolin represses the proliferation of ovarian cancer cells.The effect of grifolin on cell viability of A2780 and SK-OV-3 cells was measured by MTT assay with a range of concentration of Grifolin for various days and found that Grifolin could attenuate the ability of proliferation of ovarian cancer cells in a time-and dose-dependent manner.(P<0.05)2.Grifolin induces autophagy in ovarian cancer cell lines.The induction of autophagy of the vehicle-or grifolin-treated cells was confirmed by flow cytometry using acridine orange staining and GFP-LC3 puncta formation assay and electron microscopy.It revealed that grifolin-treated cells increased formation of AVOs in a dose-dependent manner and increased number of GFP-LC3 puncta.(P<0.05)Finally,we also observed the characteristics of cells undergoing autophagy in grifolin-treated cells.Western blot analysis and immunofluorescence staining were used to detect well-known autophagy markers.We found a significant increase in LC3B,Atg7,Beclin-1 and a decrease in P62 in a dose-dependent manner(P<0.05)while the number and intensity of punc-tuate LC3B fluorescence increased(P<0.05).Next reverse tests were used to verify the effect of grifolin in ovarian cancer cell lines and proved again above results.3.Grifolin inhibits the activity of the Akt/mTOR/S6K pathway.The results of western blot analysis demonstrated that grifolin caused a decrease in levels of the phosphorylated form of Akt,mTOR,p70S6K,S6 and 4E-BP1(P<0.05)while the total of these proteins remained unaffected by the treatment.Those results suggested that grifolin could induce autophagic cell death by inhibiting the activity of Akt/mTOR/S6K pathway.Conclusions:1.Grifolin could inhibit the proliferation and induce the autophagic cell death.2.The inhibitive effect of Grifolin on autophagic cell death in ovarian cancer cells via inhibiting the Akt/mTOR/S6K signal pathway.