Role Of Hydroxychloroquine For Prevention And Therapy Of Colitis-associated Colorectal Cancer

Background:Comparing to healthy individuals,patients with inflammation bowel disease(IBD),including Crohn’s disease(CD)and ulcerative colitis(UC)have higher risk to have colorectal cancer,which is named as colitis-associated colorectal cancer(CAC).In IBD patients,intestinal flora can reach mucosal lamina propria through epithelial hyperpermeability,inducing the activation of Toll-like receptors(TLRs)in innate immune cells.TLR4,as a critical member of TLRs family,once is activated on macrophages,resulting in the release of pro-inflammatory cytokines and reactive oxygen species(ROS),which act as DNA-mutagen.Indeed,intestinal epithelial cells would experience malignant transformation with time attributed to DNA repair deficiency and DNA mutation,eventually,leading to tumorigenesis.Given the pivotal role of inflammation in colitis-associated tumorigenesis,inhibition of inflammation could be a promising target to prevent tumorigenesis in patients with IBD.Chloroquine(CQ)and its hydroxyl analog,hydroxychloroquine(HCQ),are well known as anti-malarial agents.They have also been used in the management of several inflammatory diseases,such as bacterial infection,systemic lupus erythematosus,rheumatoid arthritis.CQ/HCQ possesses several mechanisms of action.For example,their accumulations in lysosomes and autophagic vacuoles inhibit the growth of intracellular bacteria and target them for degradation in intracellular organelles,down-regulate pro-inflammatory cytokines and control TLR4 and NF-κB activation.Accumulating evidences from both animal studies and clinical trials indicate that CQ/HCQ could be used as effective agents in anti-cancer therapies.The functions of CQ/HCQ include inhibition of tumor cell proliferation and induction of cell death via apoptosis and necrosis.Therefore,CQ/HCQ can be used as anti-inflammatory drugs in the treatment of immune mediated inflammatory disorders and various tumors.It has been reported that CQ has therapeutic potential in patients with mild active UC,however,little is known regarding the effects of CQ/HCQ on colitis-associated tumorigenesis.It’s worthy to study the effect of CQ/HCQ on CAC.Aim:To investigate the effect of HCQ on murine acute colitis,early and late stage of CAC models,and further analyze the potential mechanisms for targeting both inflammation and tumor.Methods:1.Mice were given 3.5%dextran sodium sulfate(DSS)in autoclaved drinking water for 19 days to induce severe colitis,HCQ was given every day during the whole experimental period.The numbers of mice were recorded every day.2.Azoxymethane(AOM)plus DSS were used to induce early stage of CAC in mice,HCQ was given every day during the whole experimental period.The severe of colitis was assessed.Lamina propria mononuclear cells(LPMCs)were isolated from proximal colon,LPMCs were used for the following experiments.The protein expression levels of NF-κB,STAT3 and MAPK were detected by Western blot,mRNA expression levels of pro-inflammatory cytokines were analyzed by quantitative RT-PCR.,including IL1β,IL6,TNFa,COX2,IFNβ,interferon-inducible protein 10(IP10),regulated on activation normal T cell expressed and secreted(RANTES)and monocyte chemoattractant protein 1(MCP1).Intracellular ROS was detected by flow cytometry.3.Calculating intensity values of TLR4 expression in healthy individuals and patients with IBD by analysis of microarray data from the Gene Expression Omnibus(GEO).4.In vitro,macrophages were activated by TLR4 ligand,lipopolysaccharide(LPS).The effect of HCQ on macrophage activation was assessed by the protein expression levels of TLR4 downstream molecules,mRNA expression levels of pro-inflammatory cytokines,as well as intracellular ROS levels.5.AOM plus DSS were used to induce late stage of murine CAC model,HCQ was given every day during the whole experimental period.The numbers and diameters of tumors were assessed.LPMCs were used to detect the expression levels of inflammatory proteins by Western blot.Intestinal tumor cells were isolate and used to assess the proliferation and apoptosis levels,including immunohistochemical staining of Ki-67 and c-PARP,Western blot detection of cell cycle associated proteins and apoptotic proteins,as well as Tunel staining.Results:1.HCQ significantly reduces the mortality of mice with DSS-induced acute colitis.2.HCQ suppresses AOM/DSS-induced murine colitis,as HCQ decreases body weight loss,reduces disease activity index(DAI)and histologic activity index(HAI),as well as alleviates colitis-induced colonic shortening relative to AOM/DSS-treated mice.HCQ suppresses inflammatory protein expression(NF-κB,MAPK and STAT3),pro-inflammatory cytokines release(IL1β,IL6,TNFa,COX2,IFNβ,IP10,RANTES and MCP1)and ROS generation in LPMCs.3.TLR4 is over-expressed in IBD patients compared to healthy controls.In vitro,HCQ inhibits the production of inflammatory cytokines and ROS in response to TLR4 activation in macrophages.4.HCQ prevents colitis-associated tumorigenesis and suppresses growth of CAC in mice,as HCQ decreases the numbers and diameters of intestinal tumors.HCQ suppresses inflammatory protein expression(NF-κB,MAPK and STAT3)in LPMCs.In addition,HCQ inhibits proliferation,and induces cell cycle arrest and apoptosis in tumor cells.Conclusion:In this study,mice treated with HCQ showed a significant reduction in early-stage colitis following AOM/DSS administration,as well as a remarkable inhibition of colonic tumorigenesis and tumor growth at late stages of CAC.Mechanistically,the preventive and therapeutic effects of HCQ were attributed to inhibition of inflammatory responses and production of mutagenic ROS in immune cells and subsequent promotion of apoptosis and cell cycle arrest in tumor cells.Furthermore,we found that HCQ inhibited the production of inflammatory cytokines and ROS in response to TLR4 activation in macrophages.Our data presented herein may help guide the clinical use of HCQ as a prevention and treatment strategy for CAC.