Study On The Anti-tumor Mechanism Of Salinomycin Derivative SAL-98

Malignant tumor is a common disease that threatens human health and life,and its morbidity and mortality are on the rise.It is estimated that more than 1.8 million new cancer cases will be diagnosed in 2020.In recent years,although good progress has been made in diagnosis and tumor treatment,how to fight tumor accurately and efficiently is still a big problem.Salinomycin（Salinomycin,SAL）is a carboxyl polyether potassium ion carrier antibiotic extracted from Streptomyces albicans.It was often used as an antiparasitic drug in the past.Recent studies have shown that salinomycin can selectively kill human breast cancer CSC,in mice and its efficacy is100 times higher than that of paclitaxel.The targeting effect of salinomycin CSC promotes the further development of salinomycin as a new type of antineoplastic drug.In order to improve the selectivity of salinomycin anti-tumor effect,improve its solubility and reduce side effects,it is necessary to develop new salinomycin derivatives to improve anti-tumor activity.In this study,a series of salinomycin derivatives were synthesized by Professor Wu Song group,Union Institute of Medicine,Chinese Academy of Medical Sciences.Firstly,we used MTT method to screen salinomycin and its salinomycin derivatives SAL-77,SAL-79,SAL-81,SAL-96,SAL-98,SAL-106 and SAL-107 on a series of tumor cell lines such as human colon cancer cell lines SW620,SW480,HT29,HCT116,DLD-1 and LOVO,.Human liver cancer cells Huh-7,Hep G2,MHCC97H and HCCLM3,human gastric cancer cells KATOIII,MKN-45,BGC-823,NCI-N87 and SGC-7901,human breast cancer cells MDA-MB-231,MDA-MB-468 and MCF-7,human prostate cancer cells PC-3 tumor cells and normal cells W2 have anti-tumor effects.It is found that some salinomycin derivatives have higher anti-tumor activity than salinomycin,among which SAL-98 has better anti-tumor activity.Especially,it has obvious inhibitory effect on human colon cancer cell line HT29 and human hepatoma cell line Huh-7,whose IC₅₀values are 0.039μM and 0.042μM respectively,while the IC₅₀ values of salinomycin on HT29 and Huh-7 tumor cells are 0.283μM and0.36μM,respectively.The activity of SAL-98 is significantly higher than that of salinomycin,about 7 times,so SAL-98 is selected to further study the anti-tumor mechanism.We examined the cell cycle of HT29 and Huh-7 cells treated with salinomycin derivative SAL-98.It was found that G1 phase arrest occurred in HT29and Huh-7 cells treated with 0.05μM and 0.1μM SAL-98 for 6h,12h and 24h.The expression of apoptosis-related proteins Caspase-9,Bax increased after SAL-98treatment,Bcl-2 is reduced and Annexin-V results showed that SAL-98 could induce apoptosis in HT29 and Huh-7 cells.After HT29 cells were treated with 0.05μM and0.1μM SAL-98 for 3h,6h and 12h,the mitochondrial membrane potential decreased gradually.At the same time,HT29 cells treated with 0.05μM and 0.1μM SAL-98 for6h,12h and 24h could produce ROS in HT29 cells.Then,after HT29 and Huh-7 cells were treated with 0.05μM and 0.1μM SAL-98 for 6h,12h and 24h,it was found that the expression of autophagy-related proteins LC-3 and p62 increased.We found that the expression of endoplasmic reticulum stress proteins such as PERK,CHOP and ATF4 increased in HT29 and Huh-7 treated with 0.05μM and 0.1μM SAL-98 for 6h,12h and 24h,indicating that SAL-98 can produce endoplasmic reticulum stress.In order to further study whether SAL-98 induced autophagy through endoplasmic reticulum stress,we found that the expression of autophagy-related proteins LC-3 and p62decreased after cells were treated with endoplasmic reticulum stress inhibitor 4-PBA,indicating that SAL-98 induced endoplasmic reticulum stress-mediated autophagy in HT29 cells.It has been reported that salinomycin can selectively kill breast cancer stem cells.Therefore,we found that 0.1μM SAL-98 could reduce the ball-forming ability of HT29cells,and the expression of related stem cell markers CD24,CD133,ABCG2 and KLF4decreased,it is suggested that SAL-98 has an effect on tumor stem cells.Tumor microenvironment is the site of tumor occurrence and development（TME）,including cancer stem cells,tumor-associated macrophages（TAM）and mesenchymal stem cells.TAM is a member of inflammatory cells and an important part of tumor microenvironment.M2 macrophages play an important role in tumor angiogenesis,tumor growth and metastasis.We studied the effect of SAL-98 on TAM.When different concentrations of（0.05μM and 0.1μM）SAL-98 were added,the expression of CCL5,CCL22 and CD206 in M2 macrophages decreased significantly,indicating that SAL-98 can inhibit the polarization of M2 macrophages.At the same time,TAM plays an important role in the metastasis of cancer,while the conditioned medium of M2macrophages can promote the proliferation of tumor cells.After HT29 cells were treated with conditioned medium and 0.05μM SAL-98,it was found that SAL-98 could reduce the migration and invasion ability of HT29 cells.In order to verify the anti-tumor effect of SAL-98 in vivo,we used the transplanted tumor model of HT29 cells in nude mice,which were subcutaneously injected according to 1*10⁶ cells,and then injected intraperitoneally with 1mg/kg SAL-98,5mg/kg salinomycin and 5mg/kg 5-Fu respectively 24 hours after injection.After 30days,it was found that 1mg/kg SAL-98 could inhibit tumor growth,and the tumor inhibition rate was 32.1%.The results of WB showed that the expression of LC-3,p62,ATF4,CHOP,PERK,Bip and Beclin-1 increased after SAL-98 administration,indicating that SAL-98 can promote autophagy and endoplasmic reticulum stress in tumor tissue.At the same time,the results of immunohistochemistry showed that the expression of LC-3B in tumors increased significantly after SAL-98 administration,which further proved that SAL-98 could cause autophagy.In addition,the expression of CD44 in tumor tissue was detected,and it was found that the expression of CD44decreased after administration of SAL-98,which proved that SAL-98 could affect the expression of cancer stem cells.To sum up,we found that SAL-98 has spectral anti-tumor effect,which can induce cell cycle arrest,ROS production,mitochondrial membrane potential decrease and apoptosis,autophagy and endoplasmic reticulum stress.SAL-98 can reduce the tumor sphere ability and the expression of cancer stem cell factor.At the same time,it can inhibit the polarization of M2 macrophages and reduce the migration and invasion of HT29 cells after incubation with M2 macrophage conditioned medium.Furthermore,this topic has a certain guiding significance for the development of the anti-tumor effect of salinomycin derivatives.