High Norepinephrine Status Induced Growth of Colorectal Cancer With Type 2 Diabetes Mellitus Bene�ted From ADP-Ribosyltransferase 1

a hypothesis that high-norepinephrine-induced proliferation of colorectal cancer required expression of ADP-Ribosyltransferase 1, and raise ADP-Ribosyltransferase 1 might be a candidate target for treatment of diabetes-associated colorectal cancer


Introduction
At present, many studies have shown that type 2 diabetes mellitus (T2DM) is closely related to the occurrence and development of colorectal cancer (CRC).Compared with non-diabetic patients, T2DM patients have an increased risk of colorectal cancer and death, with a poor prognosis, but its speci c mechanism is not very clear [1,2,3].
Insulin resistance (IR) is the pathophysiological basis of T2DM.IR can activate the sympathetic nervous system (SNS), resulting in increased level of circulating norepinephrine (NE) [4,5,6].The long-term activation of SNS leads to the onset of metabolic syndrome and increases the risk of T2DM, which can further aggravate IR, causing a vicious cycle [7,8,9,10].A large number of studies indicate that NE can activate adrenergic receptors, in cAMP-PKA, AKT-mTOR, ERK-Mnk1 and other signaling pathways, thereby affecting the downstream signaling molecules STAT3, c-myc, MMP-2 expression and activity and thus affect the biological behavior of tumor cells.The role of NE may be inhibited when the level of NE changes or use the alpha and beta-receptor blocker to affect the activity of beta-adrenergic receptors on the surface of tumor cells [11,12,13,14,15,16,17].Therefore, T2DM with high level of NE induced activation of signaling pathways such as AKT/mTOR/STAT3, which might be a potential mechanism of patient suffering T2DM with higher risk of CRC.
ADP-ribosyltransferase-1 (ART1), an important single ADP ribose transferase, catalyzes the posttranslational modi cation of proteins by transferring a single ADP ribose group to the arginine residue of the protein, is believed to have a closely related to a variety of cellular biological behaviors [18,19].In the early stage, we reported that ART1 expression changes altered the phosphorylation level of AKT, as well as the activity and expression of mTOR, GSK-3, c-myc, then affected the proliferation, invasion, metastasis, differentiation, angiogenesis and apoptosis of colon cancer CT26 cells [20,21,22].AKT signaling pathway is one of the important pathways of cell survival, but also the important insulin signaling pathway.Activation of AKT can promote mTOR, STAT3 and other downstream substrate phosphorylation and exert extensive biological effects, suggesting that ART1 is associated with glucose metabolic diseases.However, the effects and mechanisms of ART1 on the growth of T2DM with high NE status in colorectal cancer have not been reported.
Here we illuminate whether ART1 affect AKT/mTOR/STAT3 signaling pathway and intervene its downstream genes expression such as CyclinD1 and c-myc through the regulation of AKT, subsequently in uencing tumor growth and proliferation in T2DM and CRC double attacked patients accompany with high level of NE, and what is the crosstalk between ART1 and NE.

Materials And Methods
Cell lines and animals CT26 cell line was obtained from Professor Yu-Quan Wei (Sichuan University, Chengdu, Sichuan, China), having successfully constructed ART1-short hairpin RNA (GFP-shRNA), ART1-over-expression (GFP-ART1) and vector-control (GFP-Vector) CT26 cells [23,24].All cell groups were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Hyclone) at 37°C in a 5% CO 2 incubator.BALB/c mice (6-8 weeks old,18-22 g) were obtained from the animal experimental center of Chongqing Medical University (Chongqing, China) and placed in the speci pathogen-free feeding room (20 CT26 cell survival assays and Flow cytometry analysis CCK8(CCK8 kit, Key Gen Biotechnology, Nanjing P R China) method was used to evaluate the in uence of ART1 on the CT26 cell proliferation in different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0µM) of NE for 24 or 1.0µM of NE for 12h, 24 h, 36 h or 48h.The absorbances (optical densities) were recorded with a universal microplate reader (Bio-Tek) at 450 nm.The assays were repeated at least three times.
The ow cytometry (Becton Dickison) assessment was used to evaluate the cell cycle distribution of each CT26 group with 1.0µM NE treatment for 48h.All experiments were conducted at least three times.

Establishment of diabetic mouse model
A diabetic Balb/c mouse model was established by feeding with high fat diet for six weeks and 1% streptozotocin (STZ, Sigma Chemical Co, St Louis, USA) 50mg/kg intraperitoneal injection, while Balb/c mice with normal diet were injected intraperitoneally with saline as control.Diabetes was de ned as a random glucose ≥11.1 mmol/L in tail vein blood (test strips, Advantage, Bayer, Contour TS), one week after STZ injection [25,26,27].
CT26 cell suspension (2x10 6 /ml x 200µl) was subcutaneously injected into the lateral skin of the right armpit of each mouse [30].After 14 days, six mice were randomly selected from each group for sacri ce, and the weight and volume of the subcutaneous tumor was recorded.The survival time of the rest of the mice in each group was recorded.Tumor volume was calculated according to the formula: Volume=the maximum diameter x the most trails2 x ½ [31] Renal Denervation (RD) and sham operation Another 12 diabetic Balb/c mice inoculated with GFP-ART1 CT26 cells, were further divided into three groups: (1) left RD (LRD, n=4) (2) left sham operation (LSO, n=4) (3) without operation (GFP-ART1 group, n=4).RDs or sham surgeries were performed as described previously [28,29].In brief, mice were anaesthetized with an intraperitoneal ketamine injection (87mg/kg).Following paravertebral line incision, kidneys were exposed and the renal arteries and veins were isolated from connective tissue.After stripping the visible nerves, the vessels were painted for 2 min with a solution of 10% phenol in absolute ethanol.The muscular layers of the abdominal wall were sutured with absorptive material and the skin was closed by non-absorptive lament.Animals recovered from anesthesia 10-20 min after the end of surgery.In sham operation, animals the renal nerves were isolated but preserved.As described earlier [29], correct denervation was assessed by measuring the renal tissue content of catecholamines using a ELISA KIT (Cloud-Clone, USA).Completeness of denervation was assumed if the norepinephrine tissue content was <10% of the mean value in the sham-operated groups [28].
Second days after the operation, the mice recovered, were inoculated with GFP-ART1CT26 cells on the right axillary fossa.After 2 weeks, the mice were killed.Kidneys and xenografted tumor were removed, weighing and measuring the tumor volume, they were frozen together in the liquid nitrogen to reserve.Besides, blood was obtained to measure.

Western blot analyses
The total proteins of cells and Balb/c mice transplantation tumors of the colorectal cancers were lysed with lysis buffer (Beyotime, Shanghai, China).

Other analytical Procedures
Insulin and NE levels of serum were measured by commercial enzyme-linked immunosorbent assay kit (Cloud-Clone, USA) according to the manual.

Statistical analysis
Statistical data was analyzed by SPSS 19.0 software (SPSS, Chicago, IL, USA).Enumeration data were checked by chi square test.Binary logistic regression analyzed the correction of T2DM and ART1 expression in CRC patients.All values are presented as means±SE.Unpaired Student t-test was used for two-group comparisons.A one-way ANOVA was using to compare among groups, followed by a least signi cant difference post hoc test to compare between groups.Differences were considered statistically signi cant at P <0.05.

High concentration of NE boosted proliferation of CT26 cells requiring of ART1 expression
Each group of CT26 cells with various expression level of ART1 (GFP-ART1, Un-transfection, GFP-Vector and GFP-ShART1 group CT26 cells) was induced for 24 hours at the end concentration of 0.2,0.4,0.6,0.8 and1 µM NE respectively.The results showed that with the increase of the concentration of NE, proliferation was raised gradually in groups of CT26 cells expressing ATR1 (GFP-ART1, Un-transfection and GFP-Vector groups), but not in ATR1 silenced CT26 cells (Figure 1A).We further studied the time-depend of NE on proliferation of GFP-ART1, Un-transfection, GFP-Vector and GFP-ShART1 group.Each group was treated with 12,24,36,48h at the end concentration of 1 µM NE, respectively.The data showed that cell proliferation activity of GFP-ART1, Un-transfection and GFP-Vector groups were all increased depend on prolongation of treatment time (from 12h to 36h), while treated for 48h, proliferation regress of these group was detected.But no conspicuous difference was measured in GFP-ShART1 group with prolongation of treatment time (Figure 1B).On the other hand, ow cytometry result showed that comparing with Un-transfection group and GFP-Vector group, ratio of G1 phase was decreased, while S phase and PI were increased in GFP-ART1 group, on the contrary, increased ratio of G1 phase and decreased of S phase and PI were detected in GFP-shART1 group.After NE induction, the result showed decreased percentage of G1 phase as well as increased percentage of S phase and the cell proliferation index of PI in groups of CT26 cells expressing ATR1 (GFP-ART1, Un-transfection and GFP-Vector groups).Still no obvious difference in cell cycle distribution and PI for ATR1 silenced CT26 cells (Figure 2, Table 1).These evidences suggested that environment of high concentration NE or appropriate treatment time of NE induced proliferation of CT26 cells requiring of ART1.GFP-ART1 vs un-transfection, GFP-vector vs GFP-shART1: * p<0.05 GFP-ART1 vs un-transfection, GFP-vector vs GFP-shART1:* p<0.05,** p<0.01

Establishment of Animal model of diabetes
A diabetic Balb/c mouse model was established by feeding with high fat diet and 1% STZ intraperitoneal injection.Blood test showed signi cantly higher glucose concentration, insulin levels and NE level of diabetic mice than normal fed mice, besides, higher weight was detected in diabetic mice (Figure 3, Table 4), indicating diabetic model of Balb/c mice was successfully established.Result showed that GFP-ART1 CT26 cells presented highest expression level of ART1, p-AKT, mTOR, STAT3, CyclinD1 and c-myc (P<0.01), while GFP-shART1 CT26 cells presented lowest expression level of these protein (P<0.01),suggesting ART1 contributed to up-regulate p-AKT, mTOR, STAT3, CyclinD1 and c-myc with high NE (Figure 8).
Expression pattern of ART1, p-AKT, mTOR, STAT3 in transplanted tumor of group of CRCD and CRCO mice and alteration after renal denervation performed on CRCD group Transplanted tumors of CRCD mice and CRCO mice were digested and detected by western blot.The expression of ART1, mTOR and STAT3 in CRCD group was signi cantly higher than that in CRCO group (p<0.01), and there was no signi cant difference in the expression of AKT (p>0.05), but the expression of P-AKT was signi cantly higher for CRCD group than CRCO group (p<0.01)(Figure 9).In vivo data also illustrated that high-NE environment boosted expression of ATR1, as well as P-AKT, mTOR and STAT3.
To discuss effect of NE on ART1-depended proliferation of CT26 cells, GFP-ART1 CT26 cells were inoculated in non-surgery group, LSO group (sham operation) and LRD group (renal denervation), transplanted tumors were digested and detected by western blot.The result showed that no signi cant difference of ART1 and AKT protein expression in these three groups (P>0.05),but expression of P-AKT (p<0.01),mTOR p<0.05 and STAT3 p<0.05 protein decreased obviously in group LRD (Figure 10).This result demonstrated after LRD performed, expression of P-AKT, mTOR and STAT3 was decreased consistently, however, interestingly, ART1 and AKT was not decreased, suggesting that expression of ART1 was not depended on NE, however, NE indeed impacted expression of proliferation-relative proteins.

Discussion
T2DM is a common comorbidity in colorectal cancer patients, besides, it is considered as a risk factor for this cancer and a prognostic factor for adverse survival outcomes as well [32].However, the molecular mechanism of this connection remains elusive.T2DM is a metabolic disease characterized by insulin resistance, hyperinsulinemia, chronic activation of the sympathetic nervous system (SNS), increased circulating NE levels, and a high NE status [7,8,9,10].It was also reported that increase of NE level, possibly through beta-adrenergic receptor signaling promoting colon cancer liver metastasis [33].Besides, large case-control studies had also found that long-term use of norepinephrine antagonists or beta-receptor blocking agent drug could reduce the risk of malignant tumors including colorectal cancer [16,17,34].
Therefore, high level of NE seemed like not only related with T2DM but also with CRC development.
In this study, we found that ART1 was required in proliferation of colorectal cancer cell in high-NE environment accompanied with diabetes.Our further research revealed that ART1 activated the AKT-mTOR pathway, resulting in increased STAT3 phosphorylation and ultimately promoting Cyclin D1 and c-myc expression and cell proliferation.
Our previous studies [20,21,22,23] showed high expression of ART1 boosted malignant biological behavior of CRC, besides, ADP-ribosylation was proved to impact glucose metabolism in several pathway [35].However, there is no research of effect of ART1 on colorectal cancer associated with diabetes mellitus.Therefore, ART1 expression changes in colorectal cancer with type T2DM were discussed in this study.Consistent with our previous study, in spite of normal or high fatty diet, higher expression of ART1 was observed prefer in mice with lymph node metastasis than mice without metastasis, hinting ART1 related to malignant behavior of CRC.More importantly, result showed higher expression of ART1 after fed with high fatty diet rather than fed with normal diet.These evidences suggested that ART1 related to CRC-associated glucose metabolism disorders, but the mechanism of the effect remains to be further studied.
On the other hand, in vivo experimentation showed that diabetes accompany was a positive factor for growth of xenograft when ART1 at same level, while ART1 accelerated growth of xenograft and shortened survival time of diabetic and non-diabetic mouse models.Namely, both diabetes and ART1 encouraged growth of xenograft, however, when ART1 was silenced, difference was still detected between diabetic and non-diabetic mouse models, suggested that diabetes-accelerated growth of xenograft was not totally depend on ART1.
Furthermore, in vitro study showed that with increase of NE concentration and induction time, ART1-high CT26 cells presented consistent increase of proliferation, but no obvious change was detected in ART1silenced CT26 cells with increase of NE concentration and induction time.These illuminated that NE promoted proliferation of colorectal cancer CT26 cells depending on ART1.Based on what we found, it was concluded that, due to complicate factors in vivo co-effecting proliferation of cancer, although diabetes-accelerated growth of xenograft was not totally depend on ART1, NE promoting proliferation of colorectal cancer cells depended on ART1.Phosphorylated STAT3 is linked to the activation of Akt/mTOR, and this pathway is involved in cell proliferation and cell growth.Result of both in vitro and in vivo experiments showed that expression of ART1 was positive associated with expression of p-AKT, mTOR and STAT3, rather than AKT, with high NE environment.After LRD reducing blood level of NE, expression of p-AKT, mTOR and STAT3 protein was decreased obviously, but the expression of ART1 and AKT protein in transplanted tumor had no obvious change, besides, the volume and weight of the transplanted tumor decreased signi cantly, clari ed that reduce of NE level was an effective strategy for inhibition of xenograft growth.Consistent with our result, Lima-Seolin et al. found an increase in the activity of AKT/mTOR/STAT3 signaling pathway in the NE condition as well [36], our results suggested that NE was not required for expression of ART1, but was required for ART1-induced activation of AKT/mTOR/STAT3 signaling pathway.
Since STAT3 could increase transcription of Cyclin, D1 and c-myc, and promoted the proliferation of cells inde nitely, we further investigated the expression of Cyclin, D1, and c-myc proteins.The results indicated that expression of ART1 up-regulated the expression of CyclinD1 and c-myc proteins in the condition of high NE.Thus, combining previous results, we conjectured that ART1 could up-regulate expression of CyclinD1 and c-myc proteins by activating the AKT/mTOR/STAT3 signal pathway with high NE, and promote the proliferation and growth of CT26 cells.

Conclusion
In summary, this study proposed a hypothesis that ART1-induced activation of AKT/mTOR/STAT3 pathway and promoted the proliferation of colorectal cancer by up-regulating expression of Cyclin D1, c-myc protein subsequently, requiring high-NE environment.Although, this study has certain limitation, the further molecular mechanism needs to be proved, might provide an explanation for T2DM (high NE status) with poorer prognosis of CRC, moreover, give a rise to ART1 could be a therapy target for T2DM-associated colorectal cancer patients.

Proliferation
of variety ATR1 level of CT26 cells in environment with NE.A showed the proliferation of variety ATR1 level of CT26 cells treated with different concentration of NE; B showed the proliferation of variety ATR1 level of CT26 cells treated same concentration of NE with different time.

Figure 2 Cell
Figure 2

Figure 9 Effect
Figure 9 -26˚C,12 h:12 h light-dark cycle) of the animal experimental center at Chongqing Medical University.Mouse were then randomly divided into two groups and fed either a normal-chow diet (NCD) or a high-fat diet (HFD).The NCD (catalog no.5001, Research Diets Inc., New Brunswick, NJ, USA) provided 59% calories from carbohydrates, 20% from protein, and 19% from fat, 14.61KJ/g.The HFD (catalog no.9398, Research Diets Inc) contained 30% of calories from carbohydrates, 18% from protein, and 50% from fat, 19.75KJ/g.All experimental procedures were approved by the Animal Experimentation Ethics Committee (Chongqing Medical University) and in accordance with the National Health and Medical Research Council of China Guidelines on Animal Experimentation.

Table 1
Cell cycle distribution and proliferative indices (PI) of CT26 cells in eight groups were determined by ow cytometry χ̅ ±SD

Table 2
In uence of ART1 on the cell proliferation activity of mouse colon carcinoma CT26 cells treated by NE with difference levels χ̅ ±SD

Table 3
In uence of ART1 on the cell proliferation activity of mouse colon carcinoma CT26 cells treated by NE with difference times χ̅ ±SD

Table 4
The changes of body weight in mice were established after transplantation of tumor model Diabetic mice were inoculated subcutaneously with mice colon cancer CT26 cells into right axillary fossa.The results showed that, after inoculated with the same ART1 expression level CT26 cells, diabetic mice lost more weight than non-diabetic mice (p<0.01),although absolute value of body weight was still higher

Table 5
Effect of ART1 expression on weight situation of transplanted tumors of Balb/C mouse χ̅ ±SD

Table 6
Effect of ART1 expression on weight situation of transplanted tumors of Balb/C mouse χ̅ ±SD high ART1 expressing CT26 cells and longest in mice injected with ATR1 silenced CT26 cells p<0.01 (Table.7).
We still investigated the impact of ART1 and diabetes on survival time of mice models of transplant tumor.The data demonstrated that after inoculated with the same ART1 expression level CT26 cells, the survival time of in CRCD group were shorter than CRCO group P<0.05 (Figure6).Furthermore, with different ART1 expression levels CT26 cells injection, survival time of both CRCD and CRCO groups was the shortest in mice injected with

Table 7
The survial time of mice for eight groups χ̅ ±SD [28,29]oup: GFP-ART19)s Un-transfection, GFP-shART1 vs GFP-Vector, ## p<0.01;GFP-ART1 vs GFP-shART1: ▲▲ p<0.01;Renal denervation reduced level of blood glucose and growth of xenograft with high ART1 in CRCD miceWe established animal models of renal denervation successfully since NE level of kidney and plasma in operated group (LRD) were decreased over 10%[28,29](p<0.01).Then, we measured blood glucose and plasma insulin levels of each group, also volume and weight of transplanted tumor.The result showed lower value of blood glucose and plasma insulin as well as smaller and lighter tumor of operated group, compared with the sham-operated group (LSO) or non-operated group (p<0.01),whilenosigni cant change was observed between non-operated group and LSO group P>0.05(Figure 7, Table.8,9).

Table 9
Effect of NE level on weight and volum situation of transplanted χ̅ ±SD Western blot was used to detect protein expression level in different ART1 level CT26 cells with high NE.