| Research background and purposeProstate cancer is the most common malignance in men in developed countries,and the incidence of prostate cancer has been rapidly increasing in developing countries as PSA tests become more and more accessible.Mortality rate of prostate cancer ranks second among all cancers in men closely following lung cancer and imposes a great burden on the aged and society.Prostate cancer is prone to bone metastasis and nearly 60%-80% of patients who die of prostate cancer were demonstrated to harbor bone metastases during autopsy.Due to the happening of skeletal-related events(SREs)such as intractable bone pain,fractures,compression of spinal nerves and hematopoietic dysfunction,patients with bone metastatic prostate cancer have poorer quality of life and lower survival rates.At present,the main aim of the treatment of bone metastasis from prostate cancer is to alleviate bone-related symptoms,commonly using drugs such as bisphosphonates and RANK ligand neutralizing antibodies,which inhibit osteoclasts function,but these strategies can not improve the survival rates of prostate cancer patients effectively.In addition,radiotherapy and chemotherapy bring great side effects and reduce the quality of life of patients.Therefore,it is significant to find a new drug that can not only control the symptoms of bone metastasis but also control the progression of the tumor cells.However,the development of new drugs takes a long time and requires great labor and material resources,with huge barriers in clinical transformation.On the contrary,the drug reposition strategy,also known as developing old drug to new functions,exploits the unknown efficacy of existing drugs in a short period of time and introduce old drugs in the treatment of uncorrelated diseases,which is recognized as a fast and efficient strategy in drug development.Salinomycin is one kind of ionophore antibiotics commonly used in the livestock industry to control parasite and infectious diseases.But in recent years,studies have found that Salinomycin has great anti-tumor activity specifically eliminating breast cancer stem cells,and similar anti-cancer effects were found in many tumors such as prostate cancer,ovarian cancer and hepatic carcinoma.Further in-depth studies found that salinomycin could play an important role in regulating some biological processes such as oxidative stress,mitochondrial biosynthesis and autophagy in tumor cells.However,there is not any rational or comprehensive mechanism that can explain the effect of salinomycin on tumor cells.The interaction between drugs and its targets or ligands is one of the basis of therapeutic effect of drugs.Drugs bind their targets through electrostatic bonds,including ionic bonds,hydrogen bonds and van der Waals forces,and covalent bonds and hydrophobic bonds.The specific mechanism by which salinomycin exerts its efficacy is yet unknown.In addition,among the tumor types sensitive to salinomycin,breast cancer and prostate cancer share a common characteristic that they are all hormone responsive and bone targeting.Salinomycin is currently known to inhibit the expression of androgen receptor in prostate cancer,but whether there is therapeutic effect of salinomycin on bone metastasis prostate cancer is still unknown.Therefore,the purpose of this study is to investigate the effect of salinomycin on bone metastasis of prostate cancer and its biochemical and molecular mechanisms.With the strategy of drug reposition,we explored salinomycin as as a new approach in clinical treatment of bone-related event of prostate cancer.Experimental methodCCK8 test,clone formation essay,flow cytometry,wound healing test,transwell chamber shuttle test and some other experiments were used in the cell related experiments in which the effect of salinomycin on cancer cell biological phenotype was tested.WB,q PCR,Immunohistochemistry,immunofluorescence,immunoprecipitation and other molecular experiments were employed to detect pathway or molecular changes in cells after salinomycin treatment.Intracardiac and intratibial injection were used to construct bone metastatic prostate cancer models and tumor size was evaluated and calculated by fluorescence from tumor-derived luciferin.We used Micro CT to detect the micro-structure of bone such as bone trabecula,and calculated parameters reflecting bone function.Cheminformatic methods were used to predict the possible targets of salinomycin.RNA sequencing was used to evaluate the transcriptional change caused by salinomycin.GO analysis and KEGG pathway analysis were performed aiming to find the mechanism of drug effect of salinomycin.Surface Plasmon Resonance technology was used to determine the combination of salinomycin and 14-3-3 ζ protein.Experimental resultsPart 1: Effect of salinomycin on prostate cancer cells.Salinomycin induce ferroptosis of cancer cell which block cell cycle and suppress metastasis of tumor cells.RNA sequencing showed that numerous of gene expression were affected by salinomycin.GO analysis and KEGG pathway analysis listed main biochemical changes in cells treated with salinomycin among which ferroptosis ranked first.Cancer cells were killed by salinomycin in the presence of high concentration of glutamine.Iron and lipid peroxides accumulated in salinomycin treated cancer cells.Protein of CD71 was upregulated by salinomycin.Proliferation and metastasis of cancer cells were inhibited by salinomycin.Part 2: Effect of salinomycin on tumor-induced bone formation.Prostate cancer cells could induced osteoblast differentiation and calcium deposition which could be counteracted by salinomycin.TGF-b1 was upregulated by prostate cancer cells and released increasingly under the stimulation of pre-osteoblasts.But this process was suppressed by salinomycin.In addition,salinomycin had no effect on osteoblast alone.Part 3: Effect of salinomycin on osteoclasts and bone absorbtionSalinomycin killed osteoclasts at higher concentration and block osteoclasts differentiation from bone marrow-derived macrophages at relatively low concentration.Bone absorption by osteoclasts was significantly suppressed by salinomycin.Part 4: Mechanism of drug effect and possible targets of salinomycinHippo,MAPK,Wnt and PI3K/ATK/GSK3 b and some other pathways were dramatically suppressed by salinomycin and five possible targets of salinomycin were predicted.14-3-3 ζ was confirmed to be one of the targets of salinomycin.salinomycin might act as a “double-sided tape” or “scaffold” between salinomycin and its downstream targets including YAP-1,b-Catenin,C-RAF and Tristetraprolin.Knockdown of 14-3-3 ζ showed a protective effect to salinomycin in prostate cancer cells.ConclusionThis study used the strategy of drug reposition to investigate the function of salinomycin in protecting bone from metastatic prostate cancer.Salinomycin exerts its therapeutic function by putting effect on prostate cancer cells,osteoclasts,and interactions between prostate cancer and osteoblasts.For tumor cells,salinomycin can block its cell cycle and induce ferroptosis,thereby inhibiting its ability to metastasize.For osteoclasts,salinomycin inhibits its differentiation and hinders bone resorption.For osteoblasts,Salinomycin does not inhibit its proliferation or differentiation,but blocks tumors from secreting TGF-β1,thereby blocking tumor-mediated osteoblast differentiation and ectopic osteogenesis.Chemical informatics analysis found five potential binding targets of salinomycin.We experimentally verified that 14-3-3 ζ protein can be one of the binding targets of salinomycin. |
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