Anti-cancer Effects Of Salinomycin On Canine Mammary Cancer Cell Lines

Introduction:Mammary gland tumor is the most common tumor in canine patient,50%are malignancy,it is a critical disease in veterinary clinical practice.Unfortunately,after common cancer therapy(surgery,chemo-and radio-therapy or their combinations),high risk canine mammary tumor still develop recurrence and metastasis.Same to human,presence of cancer stem cells(CSCs)with in the tumor mass is a possible explanation for this clinical clues.Salinomycin(SAL),a polyether ionophore antibiotic isolated from Streptomyces albus,widely used in economic animals as anti-coccidial drug.It has been proved that sal is able to inhibit different types of human cancers in vitro and in vivo.In this study,we focus on the anti-tumor effect of salinomycin in canine mammary cancer cell lines and their cancer stem cells.Methods:1.Spheres(CMT7364S or CIPpS)derived from parental cells(CMT7364 or CIPp)under anchorage-independent conditions and the absence of FBS.CD44+ CD24-/low phenotype,IC50 of DDP and DOX,abilities of clone formation and invasion,the expression of ?-catenin,Cyclin D1,Oct-4,CD44 and the abilities of tumorigenicity were preformed to analyze the different biological features between spheres and their parental cells.2.To proved the anti-cancer effects of SAL on canine cancer cells,the changes of cells vitality,and the proportion of apoptosis cells,cell cycle in CMT7364 and CIPp after exposed to SAL were analysised,in vitro.Tumorigenicity,lung metastasis and expression ?-catenin,CyclinDl in tumor tissues of CMT7364 after treated with SAL were tested,in vivo.3.Investigate the change of cell vitalities,CD44+ CD24-/low phenotype,the ability of spheres formation,clone formation,invasion,expression of ?-catenin,Cyclin D1,Oct-4 and CD44 of CMT7364S and CIPpS after exposured to different concentrations of SAL,to tested the effects of SAL on canine mammary cancer stem cells.Relsults:1.Spheres(CMT7364S and CIPpS)are cancer stem-like cells.Spheres showed high levels of CD44+ CD24-/low phenotype.IC50 of DDP and DOX,ability of clone formation and invasion in vitro,were higher in CMT7364S and CIPpS as compared with their parental cells.Stem marker Oct-4 and CD44,Wnt signaling related proteins ?-catenin and Cyclin D1 were over-expressed,in spheres as compared with their parental cells.Xenograft tumor assay showed CMT7364S had higher ability of tumorigenicity and lung metastasis in BALB/c mice,as compared to CMT7364.2.SAL inhibited proliferation of CMT7364 or CIPp in a time-and concentration-dependent manner,and its mechanism may be related to the blocking of cell cycle at G0/G1 phase and inducing cell apoptosis.SAL inhibited CMT7364 growth and lung metastasis in BALB/c mice,reduced expression of Wnt signaling related proteins ?-catenin and Cyclin D1 in tumor tissue.3.Cancer stem-like ability of CMT7364S and CIPpS were reduced after exposed to SAL.There is no differents between 48 h IC50 in CMT7364S and CMT7364 or between CIPpS and CIPp.Exposure to different concentrations of SAL result in varying degrees of sphere-formation,clone-formation and invasion inhibition in canine mammary cancer stem cells.Meanwhile,SAL reduced the percentage of CD44+ CD24-/low population,down-regulated Wnt signaling related protein(?-catenin and Cyclin Dl)and stem maker Oct-4 and CD44 in spheres.Conclusion:There are cancer stem-like cells in canine mammary cancer.SAL can deplete canine mammary cancer in vivo and in vitro through inhibited proliferation,blocking of cell cycle,inducing cell apotosis,inhibited invasion and metastasis of canine mammary cancer,reudced canine mammary cancer stem cells,interrupted Wnt/?-catenin pathway,supporting SAL is a very promising canine mammary tumor chemotherapeutic agent.