Identification And Characterization Of Natural Anti-bladder Cancer Compounds:Costunolide And Dracorhodin Perchlorate

Cancer is the leading cause of death and represents one of the most threatening diseases worldwide.There is no effective treatment is available to cure cancer.Current medical therapy consists of surgery,chemotherapy,radiation therapy,immunotherapy,and vaccine therapy but have a high recurrence rate and significant side effects.Thus,natural products from traditional Chinese medicines have been proven to be an important source of novel agents with a pharmaceutical potential.Therefore,in the present study,compound library was screened against urinary bladder cancer T24 cells.The objectives of present study were two-fold;to explore the effects of natural compounds on the proliferation of T24 cells and to determine the role of ROS in costunolide and dracorhodin perchlorate-induced apoptosis in bladder cancer cells with a therapeutic potential.The objectives of present study were two-fold;to explore the effects of natural compounds on the proliferation of T24 cells and to determine the role of ROS in natural compounds-induced apoptosis in bladder cancer cells with a therapeutic potential.Results showed that all natural compounds screened,possess potent anticancer properties against urinary bladder cancer T24 cells.In this study,for the first time we investigated the effects six natural compounds from different groups of natural products including,sesquiterpene lactone(costunolide,isoalantolactone,and altholactone),alkaloids(evodiamine),triterpenoid saponins(tubeimoside-1),and dracorhodin perchlorate on the cell viability and apoptosis in human bladder cancer T24 cells.MMT assay was used to determine the anti-proliferative affects of selected natural compounds(costunolide,isoalantolactone,altholactone,evodiamine,tubeimoside-1,and dracorhodin perchlorate).For the first time,we reported that treatment of T24 cells with these compounds resulted in a dose-dependent inhibition of cell viability in T24 cells.Evodiamine was observed as strong anti-proliferative agent with IC50 15 μM,following tubeimoside-1 IC50 22.5 μM,isoalantolactone IC50 28.8 μM,altholactone IC50 43.5μM,costunolide IC50 46.5 μM,and dracorhodin perchlorate IC50 80 μM.Among these six potent anti-cancer compounds costunolide and dracorhodin perchlorate were selected for further studies.The anti-proliferative effect of costunolide and dracorhodin perchlorate on T24 cells was confirmed by live/dead assay using fluorescent probes calcein AM/PI.The results demonstrated that treatment of cells with costunolide and dracorhodin perchlorate decreased the viability of T24 cells in a dose-dependent manner.To determine the costunolide and dracorhodin perchlorate-induced apoptosis,flow cytometric analysis was carried out.The results showed that costunolide and dracorhodin perchlorate induced apoptosis in a dose-dependent manner in urinary bladder cancer T24 cells.ROS are well known mediators of intracellular signaling of cascades.The excessive generation of ROS can induce oxidative stress,loss of cell functioning,and apoptosis.In the present study,we assumed that costunolide might arouse ROS level,which could be involved in induction of apoptosis.Therefore,the intracellular ROS level was measured using the ROS-detecting fluorescence dye 2,7-dichlorofluorescein diacetate(DCF-DA).For the first time we demonstrated that costunolide and dracorhodin perchlorate induced-apoptosis in bladder cancer T24 cells was associated with the generation of ROS and disruption of mitochondrial membrane potential(△ψm).Interestingly these effects were significantly abrogated when the cells were pretreated with N-acetyl-cysteine(NAC),a specific ROS inhibitor.Interplay between pro-apoptotic(Bax)and anti-apoptotic(Bcl-2)members of the Bcl-2 family drives the mitochondrial apoptotic pathway.Bcl-2 family proteins are essential for increasing the permeability of mitochondrial membranes and the release of cytochrome c,which activates caspases and in turn mobilizes apoptotic cell death.To investigate the effect of costunolide and dracorhodin perchlorate on expression of Bcl-2,Bax and caspases-3 western blotting was done.For the first time,our.results demonstrated that exposure of T24 cells to costunolide and dracorhodin perchlorate was also associated with increased expression of Bax,down-regulation of Bcl-2,survivin,and significant activation of caspase-3,and its downstream target PARP.Therefore,in this study for the first time,we demonstrated anti-proliferative effect of six natural compounds against urinary bladder cancer T24 cells,and for the first time,we revealed the molecular mechanism of costunolide and dracorhodin perchlorate-induced apoptosis in bladder cancer cells.Our results demonstrated that costunolide induced ROS-dependent apoptosis in human bladder cancer T24 cells,while Dracorhodin perchlorate-induced apoptosis was regulated by activation of caspase-3.These findings provide the rationale for further molecular mechanistic and in vivo investigation of these natural compounds(costunolide,isoalantolactone,altholactone,evodiamine,tubeimoside-1,and dracorhodin perchlorate)against human bladder cancer.