Ellagic Acid Exerts Antitumor Effects Via The PI3K Signaling Pathway In Endometrial Cancer

Objective:Endometrial cancer is one of the three most common female reproductive system malignant tumor,accounting for 20%-30%of all female reproductive system malignant tumor.The morbidity of endometrial cancer is increasing gradually worldwide.In U.S.A,the morbidity of endometrial cancer was ranging from49,560 in 2013 to 63,230in 2018.Operation is the main treatment approach for early-stage endometrial cancer,and the prognosis was relatively good.However,it is difficult to treat recurrence or metastasis,moreover,the overall survival time of late-stage is not improved during the last decades.Meanwhile,most recent research reveals that it is important to treat the patients based on the different risk factors,physiopathology,histopathology and genetic patterns.Hence,it is vital to find new target drugs or combined with conventional chemotherapy for treating late-stage endometrial cancer.Ellagic acid is a kind of polyphenols compound,a dimeric derivative of gallic acid,existing widely and rich in many kinds of nuts,strawberry,blueberry,and pomegranate.Currently,many results manifested that ellagic acid exhibits an antitumor effect for chemical-induced cancer and many types of cancers,for example,colorectal cancer,breast cancer,prostate cancer,lung cancer,bladder cancer,liver cancer and ovarian cancer.However,it is still unclear that the function of ellagic acid in endometrial cancer.This study makes use of many bioinformatic methods to predict the signaling pathway of ellagic acid effects,and the results reveal that ellagic acid could inhibit the endometrial cancer cell function through regulating PI3K signaling pathway.This study offers a new horizon for researching the anti-tumor effects in treating endometrial cancer,meanwhile,it presents an orientation for relating targets for ellagic acid.Materials and methods:1.Analysis of target genes and network based on Drugbank and STRING database Ellagic acid is input into the Drugbank database(www.drugbank.ca/)and the potential target genes are detected.All the potential genes then are input into STRING database(https://string-db.org),and the species is set as homo sapiens,the predictive factor is set as 0.9.All the relating genes are constructed as a network,and all the relating genes are exported for next step analysis.2.Hub gene analysis based on Web Gestalt and c Bio PortalAll the selected genes are input into the Web Gestalt database(http://www.webgestalt.org/),the background blast databse is KEGG pathway,and the corresponding signaling pathway is analysed.All the cancer-related pathways are selected,and the overlapped genes are seemed as Hub genes.Hub genes are input into the c Bio Portal database to inquire the mutation situation.3.Cell lines purchase and cultureThe endometrial cancer cell lines KLE and AN3CA were bought from the Cellular Resource Center of Shanghai Institutes for Biological Sciences.All cells were cultured in condition of 37?and 5%CO2.4.IC₅₀ of ellagic acid for endometrial cancer cellThe KLE and AN3CA endometrial cancer cells were incubated in different level of ellagic acid for 48h.Microplate reader measured absorbance value at 450 nm.The IC₅₀curve was drawn by using Graph Pad Prism 5.5.Cell proliferation assayCell Counting Kit-8 reagent was used to conduct the cell proliferation assay.The endometrial cancer cells were seeded into the 96-well plate,and 0?M and 20?M of ellagic acid were added into goups.The absorbance at 450nm was detected at 0h,24h,48h and 72h.Based on the absorbance data,the growth curves were conducted.Each experiment was repeated for at least 3 times.6.The cell cycle assay and the cell apoptosis assayAccording to the manufacturer's protocol,Keygen cell cycle assay was conducted to measure the cell cycle assay.Collection of the digested cells,and the cells were fixative by pre-cold 70%ethanol and then cells were stained with PI,and finally detected using flow cytometry.The cell apoptosis assay was conducted according to the manufacturer's protocol.Annexin V-APC and PI were performed to fix the cell and to stain the cells,and finally detected using flow cytometry.Each experiment was repeated for at least 3 times.7.Migration and invasion assay Transwell kit was used to explore the migration and invasion of ellagic acid on endometrial cancer cell.The invasion assay was performed by adding the Matrigel gel to the upper compartments.The numbers of immigrant cells were calculated.Each experiment was repeated for at least 3 times.8.Total RNA extraction,Reverse transcriptase reaction and Real-time PCRTotal RNA of KLE and AN3CA were extracted from the control group and experimental group which were treated by ellagic acid.The concentration and purity of total RNA was detected by spectrophotometer.The RNA purity was seemed as high if the A260/A280value was between 1.9 and 2.0.Takara reverse transcription kit was used to conduct the reverse transcriptase reaction,and RT-PCR was processed by Light Cycler(?)480-Real-time PCR equipment.The data were analyzed by 2^-??CT method.9.Western blottingUsing the protein extraction kit(Keygen biology),the whole protein from the endometrial cancer cells which were treated with ellagic acid and the control group was extracted.The concentration of the protein was measured by BCA method,all the samples were balanced.The electrophoresis was conducted to examine the expressing level of PI3K,p-PI3K and MMP9.10.Model of lung metastasis in nude miceFive-week-old female BALB/c nude mice were divided into four groups,and 2×10⁶ KLE or AN3CA cells were injected into the tail veil of them.Two weeks later,the mice that was injected with KLE cells and AN3CA cells were treated with ellagic acid by intraperitoneal injection daily for three weeks.The control groups were injected with saline solution.Positron Emission Tomography(PET)scans were used to measure the maximum standardized uptake value(SUVmax).After PET scan,all the lung tissues were resected from the mice.Furthermore,lung tissue sections were stained with hematoxylin and eosin(HE),the metastatic cancer spots were observed and calculated by microscope.Results:1.Ellagic acid may regulate the PI3K signaling pathwayWith the prediction of Drugbank and STRING databse,furthermore,the Web Gestal database analyze that the potential Hub genes were EGFR,AKT1,PIK3CA,PIK3CB,PIK3CD,PIK3CG,PIK3R1,PRKCA and MAP2K1.With the help of c Bio Portal,we found that PIK3CA and PIK3R1 were the two most mutant frequency,41%and 24%,respectively.They are in charge of coding the active subunits.Hence,ellagic acid might exert a regulating function on PI3K signaling pathway.2.The results of cell proliferation,cell cycle and cell apoptosis detection experiment The results of cell proliferation assay showed that ellagic acid could inhibit the KLE and AN3CA cell proliferation.The cell cycle was arrested in G₁ phase according to the cell cycle assay.Moreover,ellagic acid could induce early and late apoptosis.Hence,ellagic acid may inhibit the cell cycle and induce the apoptosis,and finally cause the inhibition of cell proliferation.3.Ellagic acid could suppress the migration and invasion of endometrial cancer cell The migration and invasion of KLE and AN3CA were inhibited by ellagic acid.Moreover,the results manifested that the m RNA expression of PIK3CA and PIK3R1were decreased.The corresponding protein expression of p-PI3K was suppressed,and the expression of MMP9 was finally inhibited.4.Ellagic acid can decrease the lung metastasis in nude mice.The SUVmax values were significantly decreased in the ellagic acid treatment group.HE stain results manifested that the number of lung metastasis in nude mice were suppressed,which showed ellagic acid inhibit the metastatic ability of endometrial cancer cell in vivo.Conclusion:1.Ellagic acid can inhibit the cell proliferation of endometrial cancer cell,inhibition of cell cycle,inducing the apoptosis and decreasing the ability of migr ation and inhibition.2.Ellagic acid may inhibit the function of endometrial cancer cell through regulating the PI3K signaling pathway.3.Ellagic acid can inhibit the metastatic ability of endometrial cancer cell in vivo.