Melatonin Inhibits Proliferation And Metastasis Of LPS-stimulated Prostate Cancer Cells Through Blocking Multiple EMT-relative Pathways

Background:Prostate cancer is one of the leading causes of cancer deaths in men,especially in western developed countries.In recent years,the relationship between infection and tumor occurrence and development has become the focus of tumor research.Studies have shown that about 20%of all types of tumors are caused by microbial infections.More and more evidences show that infection is also a risk factor for prostate cancer.Toll-like receptors（TLRs）play a key role in mediating the host’s innate immunity against infection.Different TLRs specifically recognize different pathogenic microbial components,among which TLR4 recognizes bacterial lipopolysaccharide（LPS）,a gram-negative bacterial component.An earlier study investigated the effect of LPS on tumor growth by constructing a subcutaneous xenograft model of prostate cancer.It was found that LPS injection in the tumor resulted in an increase in tumor weight by inducing TLR4 expression.A recent study reported that there is a large amount of LPS in colorectal cancer tissues.After blocking the LPS in colorectal cancer tissues with LPS targeting fusion protein,the liver metastasis of colorectal cancer was inhibited.Previous studies on the mechanism of LPS promoting prostate cancer progression mainly focused on inflammatory signals（NF-κB,MAPKs,etc.）.However,the non-inflammatory mechanism by which LPS promotes prostate cancer progression is far from elucidated.Epithelial-mesenchymal transition（EMT）plays a key role in tumor metastasis.Several key EMT-related pathways,including IL-6/STAT3,AKT/GSK-3βand Wnt/β-catenin,are over-activated in prostate cancer cells.Pineal gland is a hormone mainly synthesized by the pineal gland and is known for regulating the circadian rhythm.Melatonin exerts a variety of physiological functions through G protein coupling MT1 and MT2 receptors in a dependent and independent manner,such as antioxidant,anti-inflammatory,anti-tumor and immune regulation.More and more evidences indicate that the pharmacological effects of melatonin have multiple tumor suppressor effects at all stages of tumorigenesis and development.However,whether pineal gland can inhibit the proliferation,migration and invasion of PCa cells stimulated by LPS remains to be determined.Objective:This study aims to investigate the effects of lipopolysaccharide（LPS）,the main component of Gram-negative bacteria,on the proliferation,migration and invasion of prostate cancer cells,in order to provide new insights into the mechanism of bacterial infection in the progression of prostate cancer,and evaluate the protective effects of melatonin,looking for a potential therapeutic drug.Methods:The human prostate cancer cell lines PC-3 and DU145 were obtained from the Cell Bank of the Chinese Academy of Sciences（Shanghai,China）.PC-3 cells were cultured in F-12 HAM’S medium,and DU145 cells were cultured in MEM medium.The cells were divided into four groups according to the treatment method:Control（DMSO）,Melatonin（Melatonin 2 m M）,LPS（LPS 2μg/m L）,Melatonin+LPS（Melatonin 2 m M+LPS 2μg/m L）.Flow cytometry was used to detect the effects of the treatments on the cell cycle;wound healing experiments and Transwell experiments were used to analyze the migration and invasion capabilities of prostate cancer cells;RT-PCR and Western blot experiments were used to evaluate related m RNA and protein levels,respectively;ELISA measures the level of IL-6 in culture supernatant;Co-IP is used to analyze the interaction between Axin and p-GSK3β,and the ubiquitination ofβ-catenin.Results:Our results showed that LPS promoted proliferation,migration and invasion of PCa cells.In addition,LPS stimulated inflammatory reaction and induced epithelial-mesenchymal transformation（EMT）in PCa cells by activating several TLR4 downstream pathways.Specifically,LPS promoted NF-κB/IL-6/STAT3 signal transduction.In addition,LPS upregulated phosphorylation levels of cytoplasmic AKT^Ser473 and GSK-3β^Ser9.Moreover,LPS induced phosphorylation of GSK-3β^Ser9 in the"disruption complex",and then inhibited phosphorylation and ubiquitination of cytoplasmicβ-catenin,leading toβ-catenin nuclear translocation.Interestingly,melatonin inhibited proliferation,migration and invasion not only in LPS-stimulated but also in LPS-unstimulated PCa cells.Melatonin suppressed PCa cells proliferation,migration and invasion by blocking EMT mediated by IL-6/STAT3,AKT/GSK-3βandβ-catenin pathways.Conclusions:This study provides evidence that melatonin inhibits proliferation,migration and invasion through blocking multiple TLR4 downstream EMT-associated pathways both in LPS-stimulated and-unstimulated PCa cells.Our results provide new insights into the role of bacterial infection in PCa metastasis and a potential therapeutic agent.