Effect Of ART1 On The Growth Of Colorectal Cancer In Diabetic With High NE Status Mice And Its Mechanism

Background and aim: Colorectal cancer is one of the most common malignant tumors in recent 20 years.Its incidence rate showed a rapid growth trend in our country,and the mortality rate has slip to the third rank in all of malignant tumors.The incidence of type 2 diabetes,which also is increasing in the world,is expected will rise to 4.4%(about 439 million people)in 2030.Epidemiological and clinical studies have shown that there are many common risk factors between diabetes and colorectal cancer,and both diseases could promote each other.The incidence and mortality risk of colorectal cancer increase in the people with diabetes,and the prognosis of colorectal cancer is poor.Previous studies have shown that high insulin,increased reactive oxygen species and so on are associated with the occurrence and development of colorectal cancer.At the same time,the increase of norepinephrine in diabetes is also an important cause of increased risk of colorectal cancer.ART1(arginine specific single ADP ribosyltransferase 1),a kind of important mono-ADP ribosylation transferase,is reported that have a close relationship with a variety of cell biological behavior.Previous studies havesuggested that the expression of ART1 can affect the proliferation,invasion,metastasis,differentiation,angiogenesis,autophagy and apoptosis of colon cancer cell to involve in regulating the occurrence and development of colon cancer.ART1 is associate with the expression of phosphorylated GSK-3beta protein,which is a negative regulator of hepatic glycogen regulation in insulin signaling pathway,suggesting that ART1 may play a role in glucose metabolism.However,the effect of ART1 on the growth of colorectal cancer and its mechanism under high NE status has not been reported.This paper aims to based on the earlier research and study from three aspects.Firstly,the change of ART1 expression was detected in diabetic patients with colorectal cancer.Secondly,balb/C diabetic mice model was established,and then choose the preliminary constructed CT26 cells with different expression level of ART1 to establish transplantation tumor mice.In addition,CT26 cells with different expression level of ART1 induced by norepinephrine.The effect of ART1 on growth of colorectal cancer in diabetic with high NE and its possible molecular mechanism was investigated in vivo and in vitro.Further to provide experimental basis for ART1 as a candidate target for the treatment of diabetes mellitus complicated with colorectal cancer.Methods: This study is divided into three parts.1.Expression of ART1 in colorectal cancer patients with diabetes mellitus.The expression of ART1 in colorectal cancer was detected by immunohistochemistry.Clinical pathological features and ART1 expression intensity changes of CRCD group(n=37)and CRCO group(n=23)was compared.The relationship between ART1 expression intensity anddiabetes was analyzed.2.The effect of ART1 on the growth of colorectal cancer in diabetic with high NE state(1)in vitro GFP-ART1 CT26 cells,GFP-Vector CT26 cells,GFP-Sh ART1 CT26 cells,Un-transfection CT26 cells were been chosen.1)CCK8 method was used to evaluate the influence of ART1 on the CT26 cell proliferation in different concentrations or processing time of NE.2)The flow cytometry assessment was used to evaluated the cell cycle distribution of each CT26 group with NE treatment.(2)in vivo 1)Establishment of diabetic mouse model A diabetic Balb/c mouse model was established by feeding with high fat diet and 1% streptozotocin(STZ)intraperitoneal injection.Balb/c mice were injected intraperitoneally with saline as control.Blood samples which were collected from the tail vein were detected the level of blood glucose,norepinephrine(NE)and insulin for identifying the successful construction of the diabetic mouse model.2)The detection of size ?weight of transplanted tumor and survival time of tumor bearing with different ART1 levels in diabetic mice Diabetic Balb/c mice was experimental group,and non diabetic Balb/c mice was control group.Balb/c mice were inoculated with four groups of CT26 cells(GFP-ART1group?GFP-sh ART1 group?Un-transfection group and GFP-Vector group)to establish the transplantation tumor model.The changes of body weight,tumor volume and weight and survival time of tumor-bearing mice in each group were observed.3)Effects of ART1 on the growth of colorectal cancer transplanted tumor in mice with different NE levels of diabetic Balb/c mice The model of Balb/c mice with low NE level of diabetes mellitus was established by left renal denervation operation.Blood glucose,norepinephrine(NE)and insulin levels in collected tail vein blood were detected to identify the successful construction of the low NE level of diabetes mouse model.After the diabetic Balb/c mice were inoculated with GFP-ART1 CT26 cells,the Balb/c mice undertook operation(LRD group)was set as the experimental group,the Balb/c mice without operation(GFP-ART1 group)and undertook sham operation(LSO group)were set as control group.The tumor volume and weight were observed in each group.3.The mechanism of ART1 affects the growth of colorectal cancer in diabetic with high NE state.(1)in vitro Effect of ART1 on ART1?P-AKT?AKT?m TOR?STAT3 expression in colon cancer CT26 cells induce by high NE.GFP-ART1 CT26 cells,GFP-Vector CT26 cells,GFP-Sh ART1 CT26 cells,Un-transfection CT26 cells were been chosen.Each group was induced by NE(the final concentration is 1? mmol/l).The changes of ART1,p-AKT,m TOR,STAT3,cyclin D1 and c-myc protein expressions in each group were detected by western blot.(2)in vivo 1)The expression of ART1,P-AKT,m TOR and STAT3 in colorectal cancer transplanted tumor of Balb/C mice with diabetic.Western blot was used to the expression of ART1,p-AKT,AKT,m TOR,STAT3 protein expression in tumor tissue of diabetic mice andnon-diabetic mice.2)The effect of ART1 on expressions of ART1,P-AKT,m TOR and STAT3 in colorectal cancer transplanted tumor of diabetic Balb/C mice with different NE levels.Western blot was used to detect the expression of ART1,p-AKT,AKT,m TOR,STAT3 in tumor tissue of the non operation group(GFP-ART1 group),sham operation group(LSO group)and operation group(LRD group).Results: 1.Expression of ART1 in colorectal cancer patients with diabetes mellitus.(1)ART1 expression in human colorectal carcinoma detected by immunohistochemical staining.ART1 mainly located in membrane and cytoplasm of colorectal carcinoma cells.The expression of ART1 in CRCD group was significantly higher than that in CRCO group(p<0.05).Even after age,sex,tumor location,depth of invasion and other factors distortion,diabetes was still an independent related factor of ART1 expression intensity 2.The effect of ART1 on the growth of colorectal cancer in diabetic with high NE state(2)in vitro 1)Effects of ART1 on proliferation of CT26 cells induced by different NE concentrations CCK8 assay was used to detect the proliferation activity of the cells.The results showed that proliferation activity of GFP-ART1,Un-transfection,GFP-Vector group cell increased with the the concentration of NE.However,the proliferation activity of GFP-Sh ART1 group had no significant change.Compared with the other groups,cell proliferation activity of GFP-ART1 group was the highest,and cell proliferation activity of GFP-Sh ART1 group was the lowest(p<0.05).There was no significant difference between the Un-transfection group and GFP-Vector group(p>0.05).2)Effect of ART1 on proliferation of CT26 cells induced by NE with different processing time GFP-ART1 CT26 cells,GFP-Vector CT26 cells,GFP-Sh ART1 CT26 cells,Un-transfection CT26 cells were induced by NE(the final concentration is 1? mmol/l).The result of CCK8 showed that,in different induction treatment time,GFP-ART1 cell proliferation activity was the highest;GFP-Sh ART1 cell proliferation activity was the lowest(p<0.05).The proliferation activity of GFP-ART1,Un-transfection and GFP-Vector cells increased as the prolongation of treatment time(p<0.05),but the proliferation activity of GFP-Sh ART1 cell appeared to have no obvious change(P>0.05).There was no significant difference between the Un-transfection group and GFP-Vector group(p>0.05)3)The effect of ART1 on the cell cycle distribution in CT26 cells induced by NE CT26 cells induced by NE,not all the cells in the control group:(1)In GFP-ART1 group,non transfection group and empty vector group,the percentage of G1 phase decreased,the percentage of S phase increased and the cell proliferation index of PI increased significantly(p<0.05);(2)In GFP-sh ART1 group,the cell cycle distribution and PI had no obvious change(P>0.05).Whether CT26 cells treat with NE or not,the G1 ratio of GFP-ART1 group decreased,the S ratio and PI of it increased(p<0.05).There was no significant difference between the Un-transfection group and GFP-Vector group(p>0.05).(2)in vivo 1)Establishment of diabetic mouse model.The results showed that the body weight,blood glucose,insulin and the NE level in diabetic mice(n=48)were significantly higher than those in the control group(P<0.01),which prompted the diabetic Balb/c mice model was successfully established.2)Change of size ?weight of transplanted tumor and survival time of tumor bearing with different ART1 levels in diabetic mice After CT26 cells with the same expression of ART1 were injecting into the mice,compared with CRCO group,CRCD group had larger volume,heavier weight transplanted tumor and shorter survival time(p<0.05).Compared with non transfection group and the empty group,the largest and the heaviest of transplanted tumor and shortest survival time were in CRCD and CRCO mice were inoculated with GFP-ART1 CT26 cells(p<0.05);the transplanted tumor of CRCD and CRCO mice inoculated with GFP-sh ART1 CT26 cells was the smallest and lightest;the survival time of CRCD and CRCO mice is longest(p<0.05);there was no significant difference between the non transfection group and the empty group(p>0.05).3)Effects of ART1 on the growth of colorectal cancer transplanted tumor in mice with different NE levels of diabetic Balb/c mice Compared with no operation group(GFP-ART1 group)and sham operation group(LSO group),the levels of NE,insulin and blood glucosein LRD group were significantly decreased(p<0..01),the weight and volume of transplanted tumor in LRD group was decreased(P<0.01).There was no significant difference between the the no operation group(GFP-ART1 group)and sham operation group(LSO group)(p>0.05).3.The mechanism of ART1 affects the growth of colorectal cancer in diabetic with high NE state.(1)in vitro Effect of ART1 on ART1?P-AKT?AKT?m TOR?STAT3 expression in colon cancer CT26 cells induce by high NE.1)Compared with un-transfection group,expressions of ART1,p-AKT,m TOR,STAT3,Cyclin D1,c-myc protein were significantly increased in GFP-ART1 group(p<0.05)2)Compared with GFP-Vector group,expressions of ART1,p-AKT,m TOR,STAT3,Cyclin D1,c-myc protein were significantly reduced in the GFP-sh ART1 group(p<0.05)3)There was no significant difference between un-transfection group and GFP-Vector group.(2)in vivo 1)The expression of ART1,P-AKT,m TOR and STAT3 in colorectal cancer transplanted tumor of diabetic Balb/C mice with high NE state Western blot showed,compared with CRCO group,the expression of ART1,m TOR,STAT3,P-AKT protein in tumor tissues of CRCD group increased(p<0.01),and expression of AKT was no different between the two groups(P>0.05).2)The effect of ART1 on expressions of ART1,P-AKT,m TOR and STAT3 in colorectal cancer transplanted tumor of diabetic Balb/C mice withdifferent NE levels.Western blot showed that expressions of ART1 and AKT were no significant difference in the tumor tissue of no operation group(GFP-ART1 group),sham operation group(LSO group)and LRD group tumor tissue(P>0.05).The expression of P-AKT,m TOR and STAT3 in GFP-ART1 group and LSO group were significantly higher than those in GFP-ART1 group and LSD group(p<0.05).Conclusion: 1.ART1 in colorectal cancer patients complicated with diabetes was significantly increased.The expression of ART1 is higher in colorectal cancer with lymph node metastasis than that in colorectal cancer without metastasis.The expression of ART1 was irrelevant to gender,degree of differentiation and tumor site.However,after the age,gender,tumor location,invasion depth and other factors distortion,diabetes is still an independent relative factor associated with ART1 expression intensity.It indicated that ART1 may play a certain role in the growth of colorectal tumor with diabetic.2.The colon cancer transplanted tumor in diabetic mice grew faster,larger and heavier,and its survival times was shorter.There are growing trend in to accelerate these phenomenon in high expression of ART1.The silence ART1 has an inhibitory effect on the proliferation of colorectal cancer CT26 cell induced by NE,and cells were mainly arrested in G1 phase.These results suggest that ART1 can promote the growth of colorectal cancer transplanted tumor in diabetic mice.3 ? Under the condition of high NE,ART1 can modulates theAKT/m TOR/STAT3 signaling pathway through the regulation of AKT,and then interferes with the expression of downstream gene cyclin D1 and c-myc to affect the proliferation of colorectal cancer cells.ART1 is expected to be a candidate target for the treatment of colorectal cancer with diabetic accompanies high NE level.