Research On Sutherlandia Frutescens Extract Resistance To Prostate Tumor

Prostate cancer is one of the malignant tumor of male urogenital system. In the westerncountries, it is the second leading cause of cancer-related deaths in men，only behind lungcancer. Disseminated cancer can be treated by hormone antagonist and chemotherapy.However, more aggressive tumor recurs and acquires resistance to the drugs. Therefore, novelapproaches that inhibition of prostate tumor growth via adjustment of abnormal signalingpathway(s) in prostate cancer are needed.Gli/Hedgehog(Hh) signaling pathway is required by the cancer cell proliferation andtumor growth. It also plays an important role in the regeneration of the prostate epithelium;enhanced signaling pathway transforms progenitor cells into tumorigenic cells, and promotesthe transition of localized cancer into metastatic prostate cancer. Blocking the Gli/Hhsignaling by cyclopamine could suppress the growth of prostate cancer cell in vitro and invivo. However, cyclopamine is not a favorable agent because of its rapid clearance,non-specific toxicity and off-target effects at high concentrations. Therefore, screening novelinhibitors against Gli/Hh pathway may be an effective way to inhibit prostate cancerproliferation and tumorigenesis.Sutherlandia Frutescens（S.Frutescens）is a medicinal plant traditionally used to treatmany human diseases, including fever, cough, infection, stomach disease, diabetes and cancer.Research have shown that S.Frutescens extract could inhibit the growth of human prostatecancer cell, but its mechanism is not very clear. Previous studies in our lab demonstratedbotanical compounds potentially prevent prostate cancer via inhibition of the Gli/Hhsignaling pathway. In this study, we firstly test the anti-cancer effects of S. frutescens weredue to the inhibition of the Gli/Hh signaling pathway. In addition, inhibitors of Gli/Hhsignaling pathway in SLE were screened by the stable transfection of Gli-reporter cell ShhLight II. Secondly, we observed the effect of S. frutescens on prostate tumor in TRAMPmouse.1. Multiple concentrations of SLE treated human prostate cancer cell lines PC3andLNCaP and mouse prostate cancer cell line TRAMP-C2for24h,48h and72h, we foundSLE could inhibit the growth of human and mouse prostate cancer cell lines in a dose-andtime-depending manner. After72h, these prostate cancer cells significantly lost theproliferation ability by approximately50%with IC50167μg/ml for PC3,200μg/ml for LNCaP and100μg/ml for TRAMP-C2. However, it did not show any significant growthinhibition in human normal prostate cell line RWPE-1.2. Stable transfection Gli-reporter cell lines Shh Light II and mouse prostate cancer cellline TRAMP-C2QGli were used to test the effect of SLE on Gli/Hh signaling pathway. Thedata of luciferase assay showed that SLE could decrease the activity of Gli-reporter in adose-depending manner, regardless of CM or SAG. However, cyclopamine (5μM) could notinhibited Gli-reporter activity in Shh Light II cells without stimulation. Therefore, SLE maynot only inhibit tumors with elevated Gli/Hh signaling, but also have potential to killpre-neoplastic cells with basal Gli/Hh activity. Western-blotting assay indicated that SL couldgreatly decrease the expression of Gli1in RMS13cells.3. Real-time qPCR assay were used to test the inhibition of SLE on Gli/Hh signalingpathway in human prostate cancer cell line PC3, and mouse prostate cancer cell lineTRAMP-C2. SLE and cyclopamine could significantly suppress the expression of Gli1andPtch1in TRAMP-C2cells stimulated with CM containing Shh N+; but in absence of CM, itdid not show any inhibition on Gli1and Ptch1. However, cyclopamine could not have anysuppression on Gli1and Ptch1even PC3cells were stimulated with CM, but SLE still had asignificant inhibition on the expression of Gli1and Ptch1.4. High-performance liquid chromatography-evaporative light scattering detector(HPLC-ELSD) was used to get purified cycloartane glycosides: Sutherlandiosides A-D, andflavonoids: Sutherlandins A-D. Compared the inhibition on Gli-reporter activity,Sutherlandioside D had the greatest inhibition with IC501.8μg/ml, but SLE was30μg/ml.Meanwhile, SLE contained approximately0.3%of Sutherlandioside D. It might be as themost potent compound in the SLE, which could regulate the Gli/Hh signaling pathway.5. Diet containing0.05%、0.25%and1%of S. frutescens treated TRAMP (Transgenicadenocarcinoma of the mouse prostate) mice, compared to control group fed casein-baseddiet,0.05%of S. frutescens treated group significantly decreased the incidence of grosspoorly-differentiated carcinomas (gross-PDC) which match with the lowest average ofprostate and prostate tumor size. However，Cyclopamine were subcutaneously injected toTRMAP mice model. The data showed that cyclopamine both neither could significantlyinhibit the incidence of prostate cancer, can not delay the occurrence of prostate cancer, evenfor the size of the prostate tumors also no significant role.6. Immuno-histochemical assay tested the expression of Gli1in differentiated prostatetumor in TRAMP mouse. The expression of Gli1did not significantly change with tumorprogression in TRAMP mouse model.7. In summary, S. frutescens extract could inhibit human and mouse prostate cancer cells via the inhibition of Gli1and ptch1gene in Gli/Hh signaling pathway in vitro, and in vivo,diet containing0.05%S. frutescens reduced the incidence of gross-PDC in TRAMP mouse.