Sonic Hedgehog Pathway Mediates Genistein Inhibition On Renal Cancer Stem Cells

Background: Renal carcinoma is one of the most common malignant tumors of the urinary system and extremely resistant to both traditional chemotherapy and radiotherapy.Emerging evidence has shown that renal cancer stem cells(RCSCs)are implicated in tumorigenesis,progression,chemo-resistance and relapse of renal carcinoma.Making research on renal cancer stem cells might provide valuable avenues in search for potential effective treatment on renal carcinoma.Genistein,a major isoflavone component extracted from soybeans and soy products,has been shown to prevent cardiovascular diseas and possess anticancer activity.Genistein exerts anticancer effects on various cancer types,including breast cancer,colon cancer and ovarian cancer.However,the anticancer property of genistein on renal CSCs remains to be elucidated.Objective: In the present study,renal cancer stem cells were isolated and enriched from786-O and ACHN cells through the tumor sphere formation assay.The current study aimed to evaluate the anticancer effects of genistein on renal CSCs and whether the Shh pathway was involved in the genistein-induced anticancer activity.Methods: In the present study,we enriched three-dimensional RCSCs tumorspheres from adherent renal cancer cell lines 786-O and ACHN with serum-free-medium(SFM)stem cell culture system.FCM analysis was used to analyze the cell cycle distribution of RCSCs and adherent renal cancer cells.Then the RCSCs were treated with various concentrations of gensitein and purmorphamine.Western blot and quantitative PCR analysis were used to determine the expression levels of related molecules,such as CD133,CD44,ALDH1A1,Oct-4,Nanog,Shh,Smo,Gli1 and Gli2.Immunofluorescent staining technique was used to detect the the number of CD44-positive 786-O sphere-forming cells.Results:Our results showed that levels of renal CSCs markers were remarkably up-regulated on m RNA and protein levels,and more cells were mainly arrested at G0/G1 phase instead of S1 phase in sphere-forming cells,which were isolated and enriched from 786-O and ACHN cells through the tumor sphere formation assay.We further demonstrated that genistein could effectively suppress the Shh signal way and diminish the activity of renal CSCs by suppressing tumor sphere formation,decreasing renal CSCs markers,inhibiting proliferation and inducing apoptosis.The suppressive effects of genistein on sphere formation and the Shh pathway could be reversed by purmorphamine,an activator of Smo,was used to activate the Shh pathway.In conclusion,these results revealed the suppressive effect of genistein on renal CSCs through inhibiting the Shh pathway.