Aspirin Attenuates Cancer Stem Cell Properties In Colorectal Cancer

1.BackgroundColorectal cancer is one of the most common malignancies.The treatment of colorectal cancer usually relies on surgery and radiotherapy or chemotherapeutic drugs.The existence of CSCs in colon cancer cells has been demonstrated in experimental and clinical studies.CSCs is responsible for chemoresistance and tumor relapse.Clinical trials have showed that Aspirin reduces the incidence of and mortality from several cancers,particularly colorectal cancer(CRC).The anticancer activity of Aspirin is thought to be linked to their ability of inhibiting cell proliferation and inducing apoptosis,while more recent studies have indicated that aspirin can attenuate colorectal cancer stem cell.We investigated the CSC-suppressing effect of aspirin and the relevant mechanisms in CRC.2.Methods(1)① Using CCK8 assay to detect the cell viability,calculate the inhibition of the colorectal carcinoma cell lines after treat with aspirin or not.②Plate colony formation assay were performed to explore whether aspirin could affect cell proliferation in vitro.(2)① Sphere formation were performed to compare the effect of sphere formation ability after treat with aspirin or not.② Using flow cytometry to detect the ALDH positive cells after treat with aspirin for a long time.(3)Using western blot to detect the expression of some stemness relative factors.(4)Detect the effect of aspirin in vivo:① After the administering the aspirin,we measure the volume and the weight of tumor;②Tumor cells were digested and extracted tumor tissues.Sphere formation assay was performed in vitro to test stemness changes.③ Tumor tissue were collected and subjected to western blot assay to detect the protein expresstion of the sternness relative factors.(5)RT-PCR and Western blot assays were performed to detect whether aspirin could affect the transcriptional and translational level of Nanog.3.Results(1)Aspirin inhibits the proliferation of colorectal cancer cells.① Using CCK8 assay to calculate the IC50 and sensitivity of aspirin in colorectal cancer cells,the cell viability declined in a time and concentration dependent manner;②Aspirin decreases the plate colony numbers in HCT116 and LoVo cells,identify that aspirin can inhibit the proliferation of colorectal cancer cells.(2)Aspirin inhibits the sternness properties of colorectal cancer.① Aspirin inhibits the sphere formation ability(sphere number and diameter)in time and concentration manner;② Using flow cytometry to detect the positive radio of ALDH1,demonstrate aspirin decreases the positive radio of ALDH1 obviously.(3)Aspirin declines the expression of sternness relative factor.Using western blot to detect the expression level of sternness relative factor,demonstrate that aspirin can decrease the expression of stemness relative factor in time concentration manner.(4)Aspirin inhibits the proliferation of colorectal cancer in vivo.① HCT116 cells were injected into subcutaneous of nude mice,then mice were treated intra-gastrically with either a vehicle control or with aspirin,the tumor volumes and tumor weight were measured and calculated,demonstrate that aspirin can inhibit the tumor growth in vivo;② Primary culture the tumor tissue,isolate the tumor tissue cells were performed the sphere formation assay,identify that aspirin decreases the sphere formation ability in vivo;③The whole protein extraction from tumor tissue were used for western blot,found that the sternness relative factor expression were decreased.(5)Aspirin declines the expression of important sternness factor Nanog.① Western blot and RT-qPCR showed that aspirin can decrease Nanog in protein level but not the transcription level.②Treat HCT116 with the combination of aspirin and CHX,we find that aspirin can decrease the half life of Nanog;③ Treat HCT116 with the combination of aspirin and MG 132,we find the level of Nanog still was decreased by aspirin.4.Conclusions(1)Aspirin can inhibit the proliferation of colorectal cancer cell lines;(2)Aspirin can inhibit the colorectal cancer sternness properties;(3)Aspirin decrease the expression of sternness relative factor;(4)Aspirin inhibit the colorectal cancer growth and sternness in vivo;(5)Aspirin decrease the expression of sternness factor Nanog.