Screening Of Anti-Cancer Ingredients Of Toad Skin On Ovarian Cancer And The Mechanisms Study

Ovarian cancer(OC)is a common gynecologic malignant tumor in women,and ranks the first cause of death of women from gynecologic malignant tumors.It is often detected in late stages.Apithelial ovarian cancer is the most common histological type.Platinum is widely applied to chemotherapeutic regimens for OC.However,the tolerance and resistance to platinum cause the recurrence and metastasis of OC.It is important for us to look for anti-cancer drugs that canbe used to improve the efficacy of platinum.Many Chinese anti-tumor herbs can play a role in synergism and attenuationin cancer chemotherapy,improving the quality of life,and prolonging survival time.Traditional Chinese medicine as a supplemental treatment is positive for improving thetolerance to chemotherapy and the effect of chemotherapy,so integration medicine has advantages in the treatment of malignant tumors.Toad skin,a Chinese medicinal,has the functions of clearing heat-toxicity,inducing diuresis to alleviate edema,containing active ingredientssuch as anti-tumor,anti-hepatitis viruses,and is clinically used for primary liver cancer,lung cancer,colon cancer and chronic hepatitis.In anti-ovarian cancer,we found that it has good clinical efficacy,especially to recurrent patients or patients resistant to chemotherapeutic regimens.However,gastrointestinal side-effects are problems after taking the herb drug,for example,it make some patients feel sick and even vomit,which patientscan nottolerate them.In addition,the medicinal ingredients derived from Toad skin have toxic effects.The toxicity and gastrointestinal side-effects limitedits clinical applications.Therefore,we planned to screening the effective anti-cancer ingredients from Toad skin that can help us to make the most of the advantage of it in ovarian cancer therapies and increas its acceptance of clinical applications and safety.In further study in vitro and in vivo culture model,we observed the effective anti-cancer ingredients could inhibit the proliferation ofovarian cancer cells and invest-igatethe underlying mechanisms,providing a theoretical basis for clinical treatment of ovarian cancer in the future integration medicine.These experiment were divided into 4 parts,as lollows: 1、Screening the Toad skin’s effective ingredients of proliferation inhibition on ovarian cancer cells.2、Proliferation repressive effects of resibufogenin on Ovarian cancer SKOV3 cells and Resibufogenin impact on cisplatin repressive effects on proliferation of ovarian cancer SKOV3 cells.3、The effects of resibufogenin on human ovarian xenografts tumor growth in mice.4、 The underlying mechanisms study of resibufogenin drug action.Chapter One Screening the Toad skin’s effective ingredients of proliferation inhibition on ovarian cancer cellsMethods:Ovarian cancer SKOV3 cells were treaded with different concentrations of 5 kinds of active ingredients of Toad skin,resibufogenin,bufalin,bufotalin,cinobufagin and bufothionine,at indicated time,SKOV3 cells proliferation was evaluated by counting the number of surviving cells with In cell 2000 analyzer method.Two dominant ingredients with outstanding inhibition of cell proliferation were selected.Primary ovarian cancer cells were isolated from cancerous pleural effusion of two ovarian cancer patients and were cultured and treaded with the two dominant ingredients for 72 hours to determine whether the two dominant ingredients could inhibit the growth of primary ovarian cancer cells.Results:The two lipid-soluble ingredients,resibufogenin and bufotalin,have a outstanding inhibitory action on the ovarian cancer SKOV3 cells by calculation and comparison of half inhibitory concentration(IC50)values for the 5 kinds of active ingredients,resibufogenin IC50 48.62μM and bufotalin IC50 86.86 μM,which was positively dependent on the time and dosage of the treatment when the concentration of ingredients above 20μM.When the concentration of ingredients above 20 μM,resibufogenin and bufotalin significantly inhibited the proliferation of the primary ovarian cancer cells similarly with inhibition ratio of above 50%.Conclusion:The two lipid-soluble ingredients,resibufogenin and bufotalin,have a outstanding inhibition of cell proliferation on ovarian cancer SKOV3 cells and the primary ovarian cancer cells.Resibufogenin and bufotalin were effective ingredients of the Toad skin on ovarian cancer cell proliferation.Chapter Two Proliferation repressive effects of resibufogenin on Ovarian cancer SKOV3 cells and Resibufogeninimpact on cisplatinrepressive effects on proliferation of ovarian cancer SKOV3 cellsMethods:Ovarian cancer SKOV3 cells were treaded with different concentrations(20,100,and 500 μM)of resibufogenin alone or resibufogenin combination of cisplatin for 24,48,and 72 hours.SKOV3 cells proliferation was evaluated by MTS assay.cell apoptosis was assessed by flow cytometry and Hoechst fluorescence staining.Results:Compared with control group(0 μM concentration of resibufogenin),cell proliferation in 20,100 and 500 μM concentration groups were significantly inhibited and the proportion of early apoptotic cells(LR),late death(UR)and death cells(UL)increased significantly after avariable period of time,which was positively dependent on the time and dosage of the treatment.With the increase of resibufogenin concentration,number of apoptosis body in treated cells increased.After 72 hours,the proliferation activity of SKOV3 cells was different after treatmengt of combination of cisplatin and different concentrations(20,100,and 500 μM)of resibufogenin,compared with cisplatin group(5μM cisplatin)(P<0.05).More specifically,the cells proliferation activity elevated and the proportion of early apoptotic cells(LR)reduced significantly when cisplatin combined with 20 μM resibufogenin,compared with cisplatin group(5μM cisplatin).The cells proliferation activityweakened and the proportion of early apoptotic cells(LR)increased significantly when compared with 20 μM resibufogenin group.The changes of apoptosis body in treated cells was relevant to cell apoptosis by flow cytometry.Conclusion:Resibufogenin could inhibit the proliferation and induce the apoptosis of SKOV3 cells,which was positively dependent on the time and dosage of the treatment.The proliferation activity of SKOV3 cells was different after treatmengt of combination of cisplatin and different concentrations of resibufogenin.The combination of different concentrations of resibufogenin and cisplatin may positively and negatively impact on the inhibition effect of cisplatin on the proliferation of SKOV3 cells.Chapter Three The effects of resibufogenin on human ovarian xenografts tumor growth in miceMethods:SKOV3 cells(1×106)were injectedsubcutaneously into the right subaxillary of each nude mouse.When model of human ovarian xenografts tumor building Successfully,the mice were divided into 8 groups(n=6)to receive either(1)saline solution as a control(Con);(2)cisplatin(5mg/Kg bodyweight,PT);(3)low dosage resibufogenin(0.25mg/Kg bodyweight,L);(4)Middledosage resibufogenin(1mg/Kg bodyweight,M);(5)high dosage resibufogenin(4mg/Kg bodyweight,H);(6)low dosage resibufogenin(0.25mg/Kg bodyweight)pius cisplatin(5mg/Kg bodyweight);(7)Middledosage resibufogenin(1mg/Kg bodyweight)pius cisplatin(5mg/Kg bodyweight);(8);high dosage resibufogenin(4mg/Kg bodyweight)pius cisplatin(5mg/Kg bodyweight).Resibufogenin was administered by intraperitoneal injection every other day.Cisplatin was injected once a week.Tumor volμMes were measured once every three days and at the end of in vivo studytumor weight were measured When the mice were sacrificed.Results:The growth of subcutaneous xenografts in nude mice was inhibited in diffirent dosage resibufogenin group.Tumor inhibition rates between the diffirent dosage resibufogenin groups was is significantly different(P<0.05)and in order of increasing was L,M and H group.Tumor inhibition rates between the diffirent dosage resibufogenin plus PT and PT was is significantly different(P<0.05)and in order of increasing was L+PT,PT,M+PT and H+PT group.Conclusion:Resibufogenin inhibited human ovarian xenografts tumor growth in mice.Low-dose resibufogenin reversed the inhibition effect of cisplatin on the growth of ovarian xenografts tumor in mice.Middle and high-dose resibufogenin enhanced the inhibition effect of cisplatin t on the growth of ovarian xenografts tumor in mice.Chapter Four The underlying mechanisms study of resibufogenin drug action Methods:Affymetrix Prime View TMHuman Gene Expression Array was used to detect and make comparison of the expression of genes,then raw data was processed by bioinformatic analysis including GO,KEGG and DAVID to ascertain the key factors and key signaling pathway related to regulation of medicinally action of resibufogenin(20、100μM)or resibufogenin(20、100μM)combined with cisplatin(5 μM).The pression of key genes and key proteins were measured by western blot.Results:Bioinformatic analysis showed that PI3K/AKT/CREB signaling pathway with AKT、CREB and Caspase3 were potentially the key of underlying mechanism of inhibition on the proliferation of SKOV3 cells by resibufogenin and resibufogenin combined with cisplatin.The result of western blot showed that resibufogenin down-regulated the pression of AKT,up-regulated the pression of Caspase3 and p-CREB on SKOV3 cells.20μM resibufogenin combined with cisplatin(5 μM)up-regulated the pression of AKT,down-regulated the pression of Caspase3 and p-CREB on SKOV3 cells,compared with cisplatin(5 μM)(P<0.05).100μM resibufogenin combined with cisplatin(5 μM)down-regulated the pression of AKT,up-regulated the pression of Caspase3 and p-CREB on SKOV3 cells,compared with cisplatin(5 μM)(P<0.05).Conclusion:Resibufogenin could potentially regulate the pression of AKT、CREB and Caspase3 via PI3K/AKT/CREB pathway,involvedin the process of apoptosis to inhibit cell proliferation in ovarian cancer and impact inhibition efficacy ofcisplatin on ovarian cancer cells.CREB may be a tumor suppressor transcription factor in ovarian cancer SKOV3 cells.