Cathelicidin Suppresses Colon Cancer Metastasis Via A P2RX7-dependent Mechanism

Objective:Colorectal cancer is the third most common malignancy in the world.Metastasis is more common in colorectal cancer.Approximately 20%of the colorectal tumors have metastasized remotely by the time of initial diagnosis.About 60%of patients diagnosed with advanced colorectal cancer are predicted to have distant metastasis within5 years.Despite advances in surgery and drug therapy,cure rates and long-term survival rates have not changed significantly over the past few decades.The 5-year survival rate for patients with primary colon cancer is 80-90%,and the progression rate is 40-60%.If metastatic cancer is present,the 5-year survival rate is only 5-10%.Therefore,the low survival rate of advanced colorectal cancer and the high proportion of chemoradiotherapy resistance promote the research of new drug treatment targets and treatment methods for colorectal cancer.Cathelicidin?human gene CAMP,human protein LL-37,mouse gene CAMP,mouse protein mCRAMP?is an antimicrobial peptide that has therapeutic effects on inflammatory bowel disease,clostridium difficile infection and obesity.Recent studies have shown that the antimicrobial peptide Cathelicidin is involved in the development of malignant tumors.The expression of LL-37 in colon cancer,stomach cancer,liver cancer and oral squamous cell carcinoma is lower than that in normal colon tissues,indicating that LL-37 plays an anti-tumor role in these tumors.Cathelicidin and its analogue,FK-16,induce p53-dependent apoptosis in human colon cancer HCT116 cells.Other analogs?FF/CAP18 and Ceragenin CSA13?inhibit the proliferation of HCT116 via p53-independent.Our pevious studies also found Cathelicidin does not directly inhibit the activity of colon cancer HT29 cells.Instead,it reduced the proliferation of colon cancer cells by inhibiting tumours associated fibroblasts via EMT and destroying cytoskeletal structures.The biological functions of Cathelicidin are regulated by its receptors.The receptors of Cathelicidin includes at least 4 G protein-coupled receptors,3 receptor tyrosine kinases,1ligand gated ion channel,and toll-like receptors.Studies have shown that FPRL1 and P2RX7 receptors interact directly with Cathelicidin.Metastatic colon cancer cells often possess mesenchymal characteristics as exemplifed by?III-tubulin expression,leading us to hypothesize that Cathelicidin regulates colon cancer cytoskeleton through its receptor,thereby reducing colon cancer cell migration.Methods:1.Human colon cancer cell line SW620 was treated with LL-37?1?M,5?M,10?M?at different concentrations and the control group.MTS and Body Chamber approaches were used to evaluate the effects of LL-37 on the viability,migration and invasion of colon cancer cells.2.A nude mouse model of liver and lung metastasis of colon cancer was established.The adenovirus overexpressed with cathelicidin was injected into the tail vein of the mouse,and the expression of HA-AAV and CRAMP in the liver and lung was detected by immunohistochemistry and RT-PCR,which proved the success of the modeling.Immunohistochemistry and RT-PCR were used to detect the expression of Cytokeratin18 and Keratin20 in liver and lung,and to evaluate whether cathelicidin can inhibit colon cancer metastasis.3.Human colon cancer cell lines ht-29 and SW620 were selected and treated with different concentrations of LL-37?1?M,5?M,10?M?and the control group.Tubulin tracker staining and RT-PCR were used to evaluate the effect of LL-37 on TUBB3 expression.4.Human colon cancer cell line SW620 was treated with5?M LL-37 and control group,and transfected with TUBB3 overexpression and control lentivirus,respectively.Boyden chamber and RT-PCR were used to evaluate whether LL-37 inhibited colon cancer cell migration by reducing TUBB3.5.Human colon cancer PCR Arrays were used to detect the expression of CAMP mRNA,P2RX7 m RNA and FPRL1mRNA in colon cancer tissues at different stages.6.Human colon cancer cell lines ht-29and SW620 were treated with 5?M LL-37 and the control group,and then P2RX7 and FPRL1 antagonists KN62,WRW4 and control were respectively treated.Boyden chamber,RT-PCR and tubulin tracker were used to explore which receptor in P2RX7 or FPRL1 LL-37 inhibited colon cancer cell migration.7.A nude mouse model of colon cancer liver and lung metastasis was established.After intravenous injection of adenovirus overexpressed with cathelicidin in the tail of the mouse,P2RX7 antagonist KN62 and the control group were treated respectively.8.Human colon cancer cell lines SW620 were selected and treated with 5?M LL-37 and control group,then transfected with P2RX7 shRNA and control group,respectively.Relevant micrornas were screened by miRNA Arrays,and human colon cancer cell lines HT-29 and SW620 were selected and verified by RT-PCR.9.Human colon cancer cell line SW620 was treated with 5?M LL-37 and control group,and then mir200c-3p inhibitor and control group,respectively.RT-PCR,Boyden chamber and tubulin tracker were used to evaluate whether LL-37 regulated the expression of TUBB3 and inhibited the migration of colon cancer cells through mir200c-3p.Results:1.LL-37 inhibited the migration of colon cancer cells SW620 at 5?M and 10?M but did not affect their viability and invasiveness.2.Human colon cancer cell line HT-29?1*10⁶/L?was injected into 100ul HBSS solution to prepare cell suspension,and a human colon cancer cell line HT-29 with the same number of cells was injected into the tail vein of 8-week-old nude mice to establish a nude mouse model of colon cancer liver and lung metastasis.The nude mice were divided into two groups and injected HA-AAV and CAMP-HA-AAV(4×10¹² gene copies per 100?L)into the tail vein of the mice on the same day.cathelicidin?CAMP?mRNA expression in liver and lung tissues of the overexpressed group was significantly higher than that of the control group.The ha-tag staining of the two groups was basically the same,indicating that the AAV load was basically the same.Positive expression of cytokeratin 18 was observed in liver and lung tissues of nude mice,while the expression of cytokeratin 18 in liver and lung tissues of the group with overexpression of Cathelicidin was significantly decreased compared with the control group,and keratin 20 mRNA was significantly decreased.3.The tubulin tracker staining showed that LL-37 destroyed the cytoskeleton of colon cancer cells at 5?M and10?M in a dose-dependent manner.Ll-37 did not affect TUBB1 mRNA expression in SW620 and HT-29 cells,while LL-37 significantly inhibited TUBB3 expression in SW620and ht-29 cells at 5?M.4.TUBB3 mRNA expression level was significantly increased in the TUBB3-transfected lentivirus group,and SW620 cell migration was significantly increased in the TUBB3-transfected lentivirus group whether or not exposed to LL-37.5.Reduced expression of Cathelicidin in stage II colon cancer,but no significant change was observed in stage III and IV.The expression of P2RX7 mRNA and FPRL1 mRNA can be seen in normal tissues and in all stages of colon cancer.6.KN62,the antagonist of P2RX7,reversed the inhibition of LL-37 on SW620 cell migration,but WRW4,the antagonist of FPRL1,could not reverse the inhibition,and N62 also prevented the LL-37-mediated disruption of tubulin structure in SW620 cells.Transient transfection of P2RX7 shRNA significantly reduced P2RX7 mRNA,and transfection of P2RX7 shRNA group increased TUBB3m RNA expression which is decreased by LL-37?5?M?.Similarly,P2RX7antagonist KN62 can also prevent LL-37-mediated inhibition of TUBB3 mRNA expression.7.To establish a nude mouse model of liver and lung metastasis of colon cancer.The nude mice were divided into two groups.The same method was used to inject HA-AAV and CAMP-HA-AAV into the tail vein of the mice,and the same method was used to inject P2RX7 antagonist KN62 into the tail vein of the mice and the control group.Cathelicidin inhibited colon cancer metastasis via P2RX7.Cathelicidin overexpression significantly reduced human TUBB3 mRNA expression in the lungs and liver of HT-29-loaded nude mice,which was reversed by KN62 administration.8.LL-37 increased the expression of miR200c-3p in SW620 and HT-29 cells.Transfection of P2RX7 shRNA reversed the LL-37-mediated increase in miR200c-3p.KN62 can also inhibit the expression of miR200c-3p induced by LL-37.The miR200c-3p inhibitor reverses LL-37-mediated TUBB3 inhibition,cell migration,and cytoskeleton destruction.Conclusion:1.Cathelicidin inhibited the migration of colon cancer cells SW620 in a dose-dependent manner.2.Cathelicidin inhibit colon cancer cell metastasis.3.Cathelicidin inhibits of TUBB3 mRNA expression,destroys the cell cytoskeleton and inhibits the migration and metastasis of colon cancer cells.4.The expression of Cathelicidin was found in normal intestinal tissues and in colon cancer tissues at all stages,but significantly decreased in stage II colon cancer tissues.5.P2RX7 and FPRL1 receptors were expressed in normal intestinal tissues and colon cancer tissues at all stages,but there was no significant difference between groups.6.P2RX7 antagonist KN62,but not FPRL1antagonist WRW4,prenvented the inhibition of LL-37 on colon cancer cell migration and TUBB3 mRNA expression.7.Cathelicidin inhibits colon cancer cell metastasis via P2RX7receptor 8.Cathelicidin increases miR200c-3p mRNA expression via P2RX7 receptor.9.Antimicrobial peptides inhibit tubulin expression and colon cancer cell migration via miR200c-3p.