Alcohol Consumption Promotes Colorectal Carcinoma Metastasis Via CCL5 Inducing Autophagy In AMPK Pathway

Research background:Colorectal cancer(CRC)is a malignant disease of high incidence and mortality,which causes about 600,000 deaths each year worldwide,accounting for nearly 8% of total cancer deaths.All over the world,among all the male incidence of cancer,colorectal cancer ranks the the third,and for female cancer patients,colorectal cancer ranks the second.Earlier studies show that incidence of CRC is higher in developed countries,where the onset age ranges from 50 to 60 years old,however,recent survey show an increasing trend presenting in developing countries for the incidence of CRC,especially among young patients.Pathogenic factors of CRC is complex and still unknown for definite causes.It has been considered that factors,which induces CRC,may include an environmental factor and a geneticone.15% to 30% CRC patients have specific family genetic history.Genetic factors such as familial adenomatous polyposis(FAP)or hereditary nonpolyposis colorectal cancer(HNPCC)have been found in about2% to 5% of all CRC patients.The role of environmental factors in the pathogenesis of CRC should be paid more attention to,such as geographical distribution of human population,excessive intaking of nutrients,obesity,and imbalance of intestinal flora,smoking,and drinking problems and etc.,which probably play important roles in progression of CRC.How the relationship between alcohol drinking habits and diseases develops with each other is quite sophiscticated.Although studies have shown that moderate alcohol consumption is beneficial to human health,mealwhile a growing number of studies have confirmed that alcohol consumption is associated with a variety of disease development.Over the past 40 years,the global capita alcohol consumption has doubled,especially in Asia.Statistics shows that about 3.6% of cancer is relevant to chronic alcohol intaking.Alcohol enhances the effect of precarcinogens,such as alcoholic beverages,smoking,high fat diets and chemical carcinogens,by increasing the level of cytochrome enzymes.Alcoholic metabolite is acetaldehyde,which causesdistortion to DNA by bringing methylation to DNA.Alcohol can also causes oxidative stress reaction,which produces active oxygen substances that causes the liposome peroxidation,whoseproducts,such as trans-4-hydroxy-2-nonenal(4HNE),can causes DNA mutations.Recent studies show that alcohol consumption accelerates the progress of cancer.One of the important reasons that causes death of cancer patients is tumor metabasis.Statistics show CRC patients' post-op survival rate within 5 years is around66%,but once metabasis occurs the rate is around 20%.Therefore,identify whether alcohol promotes the metastasis of CRC,and investigate its pathogenesis is particularly important.It is very critical to make sure whether alcohol consumption promotes metabasis in CRC and further inspect the pathogenesy.Chemokine is a family of protein molecular and cellular factors whose common characteristics include small molecular weight(about 8-10 KD),stable chemical structure and the tertiary structure composed by four conserved cysteine.Since 1987,the first chemokine CXCL8(IL-8)was found,about 50 different chemokines and more than 20 chemokine receptors have been found.A variety of cells,such as macrophages,fibroblasts,T cells,B cells,and some specific tumor cells can secrete chemokine,in which surface expression of chemokine receptors have been found.C-C chemokine ligand 5(CCL5)is a kind of chemokines,which could be secreted by T lymphocytes,macrophages,platelets,synovial fibroblasts,tubular epithelial cells or some tumor cell.The exact biological function of CCL5 in tumors has not been fully elucidated.CCL5 can cause adequate immune response to inhibit the formation of tumor,mealwhile,do participate in tumor progression and metastasis.Present studies show that after interaction with its receptors CCL5 can promote tumor cell migration,invasion,directly or indirectly recruit T cells,inhibit the killing effect of CD8 cells,and promote the formation of tumor microvascular formation and bone metastases,which eventually promotes the course of diseaseThe metastasis of cancer cells(lymph nodes adjacent to organs or growing distant from organs)is an important characteristic in tumor development.Tumor metastasis requires the tumor cells' mobility to increase and get more energy to support the progress.Colorectal cancer cells are of epithelial origins mostly.Tumor cells gain more ability to transfer once epithelial-mesenchymal transition(EMT)occurrs.Thisphenomenon has already been widely reported.In more and more researches for the past few years,we found that autophagy could support cancer cell survival and metastasis by recycling cellular components.Autophagy is an evolutively highconservative regulated cellular process that serves to remove damaged proteins and organelles from the cells.The role of autophagy in cancer development and progression is complicated.In normal tissue,autophagy performs homeostatic functions such as organelle and protein quality control.Once autophagy is suppressed in tissues,normal homeostasis is disrupted,leading to inflammation increasing,genomic instability,and aneuploidy,and all these changes together can promote tumor initiation and early tumorigenesis.However,if deficiency of autophagy continues,tumor progression will not proceed either.Alternatively,if autophagy is activated in formed tumor,cells can keep up with their metabolic demand as well as regulate oxidative stress,to develop progression to more advanced malignancy(tumor breaks through the basement membrane)and cause tumor continual growth.In our previous studies,we found that alcohol could promote malignant biological behaviors of many cancer cells,such as lung cancer,liver cancer and colorectal cancer cells.Other studies have shown that alcohol could increase secretion of many types of cytokines and chemokines.Therefore,we set about inspecting whether there is relevance between alcohol consumption and progression of CRC.In addition,we researched on the function of CCL5 secretion in CRC progression of alcoholic patients to figure out whether CCL5 relates to autophagy.Moreover,we further inspected the role of autophagy in regulation of migration of CRC cells and its molecular mechanism.In this issue,we investigated the relationship between alcohol consumption and CRC progression based on drinking history of CRC patients.We investigated the relationship between alcohol and CCL5 expression by investigating CCL5 expression in clinical cases of drinking and non-drikning CRC patients and by vitro alcohol stimulation on CRC cell lines.By changing CCL5 expression level in vitro experiment,we observed changes of malignant biological behaviors in CRC cells and verified whether CCL5 can cause autophagy.Furthermore,we investigated the relationship between autophagy and migration and found underlying molecular mechanism in autophagy which induced by CCL5 through bioinformatics prediction experiment.Exploring the role of CCL5 in alcohol promote CRC progression may provide new clues for the clinical treatment of CRC.Research objective:The aim of this tissue is to investigate the role of CCL5 in progression of alcoholinduced colorectum carcinoma and to make further investigation on the underlying mechanism.Research contents:1.Through collecting 102 cases of colorectal cancer patients with surgical treatment from the NO.1 Affiliated Hospital,Anhui Medical University during 2012 to investigate the relationship between alcohol consumption and CRC progression.The relationship beteween CCL5 expression and alcohol consumption in CRC patients' pathological tissues was detected by immunohistochemical.2.The expression of CCL5 in HT29,DLD-1,RKO and SW480 cell lines was detected by RT-PCR.3.The effect of CCL5 on angiogenesis of Human Umbilical Vein Endothelial Cells(HUVEC)by CCL5 was detected by capillary tube-like formation4.The secretion of CCL5 was detected by ELISA in CRC cells which were stimulated with alcohol.The secretion of CCL5 in HT29 and DLD cells raised obviously after alcohol stimulated,so we chose HT29 and DLD-1 cells for subsequent research.5.The expression and secretion of CCL5 were detected by RT-PCR,Western bolt and ELISA in HT29 and DLD-1 cells which were stimulated with alcohol for two weeks.6.HT29 and DLD-1 cells were stimulated by CCL5 or not,and then:(1)The proliferation of HT29 and DLD-1 cells was detected by MMT assay.(2)The cell cycle of HT29 and DLD-1 cells was detected by flow cytometry.(3)The apoptosis of HT29 and DLD-1cells was detected by Annexin V/PI assay.(4)Plate clone forming assay detected the single cell colon formation of HT29 and DLD-1 cells.(5)Soft agar colony forming assay detected the capacity of anchor independent growth in HT29 and DLD-1 cells.(6)Transwell detected the migration ability in HT29 and DLD-1cells.7.The autophagy formation was detected by TEM in HT29 and DLD cell which was stimulated by CCL5.The autophagy flux was confirmed by Western blot and immunofluorescence with autophagy inhibitor.8.The relationship between autophagy and migration ability that increased by CCL5 was investigated by blocking autophagy in HT29 and DLD-1 cells9.In order to reveal the underlying molecular mechanism in CCL5 inducing atutophagy,we used High throughput sequencing to investigate a signaling pathway.In addition,we demonstrated signaling pathway by Western blot.More ever,we blocked the pathway to find whether the autophagy was reversed.10.Blocking the pathway to investigate the effect of increased ability of cell migration induced by CCL5.Research results:1.We found a positive relationship between alcohol consumption and tumor malignant degree classification,TNM stage and metastasis.Furthermore,we found postoperative 5-year survival rate was lower in CRC patients with drinking with alcohol consumption.2.Alcohol increased CCL5 expression and seceration in HT29 and DLD-1 cells.CCL5 expression was higher in tumor and para-tumor tissues of CRC patients with alcohol consumption.3.CCL5 had no obvious influence on the angiogenesis of HUVEC.4.CCL5 had no effect on proliferation,clone formation,cell cycle and apoptosis of HT29 and DLD-1 cells.5.CCL5 could enhance the migration of HT29 and DLD-1 cells directly.Neutralized effect of CCL5 or knockdown CCL5 of HT29 and DLD-1 cells that were stimulated by ethanol,increased migration by ethanol was abolished significantly.These data shown CCL5 play an important role in alcohol-stimulated migration.6.CCL5 could induce autophagy flux in HT29 and DLD cells efficiently.7.The migration that increased by CCL5 woud be abolished when autophagy was blocked.These data show autophagy may involve in regulation of CCL5 enhanced migration.8.We found CCL5 effectively activated AMPK phosphorylation in HT29 and DLD cells and then induced autophagy through High throughput sequencing technology.9.Blocking AMPK phosphorylation,the migration which increased by CCL5 in HT29 and DLD-1 cells was abolished partly.These data shown AMPK signaling play a crucial role in migration of CRC cells under CCL5 induced autophagy.Conclusion:1.Alcohol consumption promotes CRC progression.2.CCL5 plays an important role in alcohol stimulated migration3.CCL5 induces autophagy is involved in regulation of CRC cells migration through AMPK signaling pathway.