Functional And Mechanism Study Of Metabolic Reprogramming Induced By Chronic Colitis

Colorectal cancer is one of gastrointestinal malignant tumors with the highest incidence of human beings.According to the latest statistical analysis of global malignancy,its morbidity and mortality are ranks third among all types of cancer,which in men are higher than in women.The morbidity and mortality rates in our country is slightly lower than the world average level,but they still have a high incidence.Inflammatory bowel diseases,including ulcerative colitis and Crohn's colitis,is a recurrent episode of unknown cause chronic inflammation,whose mainly clinical manifestations are usually following with chronic diarrhea,abdominal pain,and even hemafecia.With the course of inflammation extension and progression,the risk for developing cancer is increasing.There are accumulating evidences suggested that patients suffering from IBD developing colorectal cancer only accounts for 1%~2% of whole population of colon cancer,but it is the main cause of their death.In 1920 s,German scientist Otto Warburg suggested that cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present.Reflecting this fundamental distinction in metabolic demands,cancer cells metabolize glucose in a manner that is distinct from that of cells in normal tissues,called “Warburg effect”,or termed “aerobic glycolysis”.Some genes' structure and function alteration in tumors cells might finally leads to metabolic reprogramming,because of metabolic manner alteration based on “Warburg effect”.Tumor cells could change a variety of signal pathways through gene mutation,which might regulate their metabolism and mainly shows in enhanced glycolysis,increased glucose uptake and consumption,and strengthened lactate production.Metabolic reprogramming of tumor cells could lead to metabolic abnormalities,which might be an active process so as to meet their demands for proliferation,and alter the distribution of nutrients in metabolic networks simultaneously.Recent years,this view of metabolic reprogramming in cancer seems more convincing and reasonable,but there are few evidences shows that whether cells metabolism would be reprogrammed in a long-term process with chronic inflammation during normal intestinal tissue cells developing for cancer cells.Therefore,we proposed that standing with a long time recurrent inflammation,namely the process of normal epithelium – inflamed epithelium(colitis)– dysplasia – cancer,cell metabolic manner might alter in responding to the complicated and changing inflammatory microenvironment induced by various inflammatory signal to meet the needs of themselves to develop into cancer cells.Herein,considering the above hypothesis,we have examined the alteration of a series of metabolic indexes during the process of simulating inflammation both in vivo and in vitro,for the purpose of exploring whether cells have metabolic reprogramming and its possible mechanism in inflammatory state.?Objectives? 1.Establish both acute and chronic colitis mouse models with DSS treatment 2.Detect the expression of glycolytic enzymes in vivo 3.Observe metabolic alteration stimulated by inflammatory signal in cell level4.Explore the related mechanism of metabolic reprogramming under inflammatory microenvironment?Methods and Results?1.Glycolytic enzymes were upregulated in DSS-induced acute colitis modelIn order to uncover the possible alterations of the key metabolic enzymes under inflammation microenvironment,we established both acute and chronic colitis mouse models with DSS treatment.In the acute DSS-induced colitis model,mice were administered 2.5% or 4% DSS respectively by adding it in drinking water.Body weight loss,colon length and histological analysis were observed after 7 days from first administration.Following DSS treatment,both the body weight and colon length of colitis mice were dramatically decreased,though there is only slight difference between 2.5% and 4% groups.In addition,we evaluated the inflammation status of colonic mucosa by HE staining and the colon histological score.A large number of lobular necroinflammatory cells were observed in the DSS group.Considering the dramatic loss of body weight,we propose that metabolic hallmarks might be affected following this acute inflammation.Expectedly,both m RNA and protein levels of hexokinase-2(HK2)and PKM2 were increased in colon epithelial cells following 7-day DSS treatment,accompanying with the induction of pho-STAT3 and c-Myc.2.Chronic inflammation conferred to a constant induction of the metabolic hallmarks associated with cancerBesides the acute inflammation model,we further analyzed the metabolic hallmarks alteration in chronic colitis model.For induction of chronic colitis,mice received three cycles of treatment with DSS.Each cycle consisted of 2.5% DSS in drinking water for 5 days,followed by a 10-day interval with normal drinking water.Histological evaluation of intestinal mucosa samples did not reveal any histopathological changes in the control group,but there are obvious abnormalities like distortion or inflammatory infiltration in the DSS group.Immunohistochemical staining showed that c-Myc,HK2,PFK,PKM2 and pho-STAT3 expression increased significantly under chronic inflammation condition.Interestingly,the key metabolic enzymes like LDH-A and PKM2,which playing critical roles in the later stage of aerobic glycolysis pathway,increased dramatically in chronic DSS-induced colitis mice,but failed be observed in acute colitis model.These results suggest that body itself has a capability to resist and adapt the inflammatory microenvironment in short time of acute inflammation,but this might be attenuated while inflammation prolonged and exacerbated.Therefore,normal cells with chronic inflammation might finally turn to metabolic reprogramming and early-stage tumor development.3.IL-6 increased glycolytic enzyme expressions and lactate productionRecent studies suggest that pro-inflammatory cytokine IL-6 is an important mediator within the cancer microenvironment to promote cancer cells proliferation.Thus,we propose that IL-6 might be able to alter the cell metabolic program and behaviors.Herein,we used human intestinal epithelial cell lines(HIEC)and colorectal cancer cells to determine the biological role of IL-6.We found that IL-6 treatment increased cell viability in each cell lines,especially in HIEC cells.Accordingly,flow cytometry analysis and colony formation assay further confirmed that IL-6 had a direct growth stimulatory impact on colon cells,including HIEC or CRC cells.Moreover,IL-6 inhibited the late apoptosis of HIEC cells induced by 5-Fu.Notably,IL-6 treatment significantly increased both m RNA and protein levels of the key glycolytic enzymes in HIEC/HT-29 cells.Further,the enzyme activity of LDH-A and PKM2 showed comparatively high levels under the stimulation of IL-6.Expectedly,IL-6 treatment caused a relatively higher enzyme activity in SW620 than HIEC cells,indicating that malignant and wide-spread metastasis cancer cells seem to be more competent to finish glycolysis process compared to the normal cells.In addition,IL-6 also increased the lactate production ability of each cells.Therefore,IL-6 induced inflammatory environment has a direct stimulatory effect on aerobic glycolysis in both human normal and colon cancer cells.4.IL-6 induces cell metabolic reprogramming via STAT3/c-Myc axisAll detection and results mentioned above were just functional information related to aerobic glycolysis,but underlying mechanism of inflammatory microenvironment reprogrammed the metabolic process of normal tissue cells toward fast-proliferating cells or cancer cells have remained elusive.Recent studies demonstrating the significance of IL-6/STAT3 signaling in promoting colitis-associated cancer,namely IL-6 signal may activate STAT3 and has direct stimulatory impact on CRC cell proliferation.Accordingly,we treated cells with S3I-201,a small-molecule inhibitor of STAT3 activation.As expected,the STAT3 phosphorylation was suppressed after cells treated with S3I-201 accompanying with the reduction of c-Myc and HK2 expression.Additionally,S3I-201 could further block IL-6 induced c-Myc and glycolytic enzyme expression.Owing to the m RNA and protein levels of c-Myc were both increased once stimulated by inflammatory signal,we then inhibited c-Myc expression via treating cells with Myc inhibitor JQ1.Interestingly,JQ1 could also interrupt the upregulation of glycolytic enzyme expression induced by IL-6 without altering phos-stat3 level.Our data demonstrated that STAT3/c-Myc axis played a critical role in inflammation induced metabolic reprogramming.?Conclusion? In our studies,we found that inflammatory signal might induce metabolic reprogramming,but there are some distinctions between DSS-induced acute colitis model and chronic inflammation.Interestingly,During the early stage of acute colitis,expressions of the glycolytic enzymes increased much higher in the early steps than that in the late steps,however,in chronic colitis model,more key glycolytic enzymes were shown highly expressed constantly,indicating the metabolic reprogramming was induced by long-term inflammatory signaling.In addition,we also confirmed the functional indexes in vitro following the treatment with IL-6,indicating that IL-6 induced inflammatory environment has a direct stimulatory effect on aerobic glycolysis.In the end,we had explored the mechanism of metabolic reprogramming induced by long-standing chronic inflammation,and found that STAT3/c-Myc axis might play a key role in this process.From the above,our study showed that cancer associated metabolic reprogramming in intestinal epithelial cell could be induced by a long-term chronic inflammation.These metabolism alterations are associated with the character of “Warburg effect” or aerobic glycolysis,such as enhanced glycolytic capability and lactate production.Perhaps,this metabolic characteristic,differing from cell that in normal tissues,might be novel early diagnosis biomarkers of chronic colitis associated colorectal cancer,or even as novel potential therapeutic targets for chronic inflammation.