Curcumin Inhibits Bladder Cancer Stem Cells By Suppressing Sonic Hedgehog Pathway

Background: Bladder cancer is the most common malignant tumor in the genitourinary system in China.Cancer stem cells(CSCs)are the root cause of human tumor recurrence.Some studies have shown that bladder cancer stem cells(BCSCs)play an important role in the occurrence and development of human bladder cancer.Thus,in order to treat human bladder cancer,it may be a valid way to target BCSCs.Curcumin,extracted from the rhizomes of plants such as the family Curcumae,is one of the most important component of phytochemicals.Curcumin is valuable,and has the quality of anti-inflammatory,anti-oxidant,anti-cancer and so on.Curcumin can be used in the treatment of coronary heart disease,Alzheimer ’s disease,cancer and other diseases.Yet,the effect of curcumin on BCSCs has not been explored.Sonic Hedgehog(Shh)pathway plays an crucial role in stem cell pathway,which is supposed to be related to the existence and development of CSCs.Purmorphamine(PM)is an Shh pathway activator that can specifically activate the Shh signaling pathway.Objective: In this study,serum-free medium culture method was used to enrich BCSCs from UM-UC-3 and EJ cell lines.Subsequently,we explored how curcumin affected the growth of BCSCs and the effect of curcumin on Shh pathway.Methods:In this experiment,we studied the mechanism of curcumin inhibiting BCSCs.Firstly,two bladder cancer cell lines,UM-UC-3 and EJ were cultured in serum-free medium to form three-dimensional cell spheres that we call it BCSCs.Then the BCSCs were treated with various concentrations of purmorphamine and curcumin.Flow cytometry was used to determine the percentage of cells in different cell cycles.The expression levels of relative molecules,including CD44,CD133,ALDH1-A1,OCT-4,Nanog and so on,were detected by Western blot and quantitative PCR analysis.The expression level of CD44 protein was analyzed by immunofluorescence staining technique.Results: The results showed that stem cell markers such as OCT-4,CD133,ALDH1-A1,CD44,and Nanog,were highly expressed in cells cultured in serum-free culture medium.Furthermore,we found that curcumin down-regulated the expression of Shh pathway of BCSCs and inhibited the formation of BCSCs.And it can reduce the expression of relevant stem cell markers(such as OCT-4,CD133,ALDH1-A1,CD44,and Nanog),and inhibits the proliferation of BCSCs,and promote its apoptosis.At the same time,the inhibition of curcumin on BCSCs and Shh pathway can be reversed by purmorphamine.Overall,these data indicated that curcumin can inhibit the activity of BCSCs via Shh pathway,which provides a new idea for the development of new chemotherapeutic methods for bladder cancer.