Effects Of AGEs On Proliferation Of Human Colon Carcinoma Cell Line SW-480 And Intervention Of Metformin

【BACKGROUND】As the improvement of people's living standard, the aging of populaion and change of life style, the prevalance of diabetes mellitus grows fastly.Diabetes mellitus can cause damage to organs and tissues all over the body,such as:eyes,kidney,nerves,heart,blood vessels and so on,which seriously affects diabetic patients'quality of life and survival rate.Recent studies found that the risk of malignant tumors of diabetic patients significantly increased,such as liver cancer,colorectal carcinoma, pancreatic carcinoma,gastric cancer and so on, It was reported that malignant tumors ranked the second in the total mortality of diabetic patients,became an important cause of deaths of diabetic patients.Colorectal cancer is one of the most common malignant tumors of digestive system in our country, its prevalance ranked the fourth in all the malignant tumors in our country. Recently, the prevalance of colorectal cancer grows fastly as the development of economy and improvement of people's living standard in our country. A great quantity of epidemilogical evidences showed that the morbility and mortility of colorectal cancer of diabetic patients significantly increased compared with non-diabetic patients. Thus, the association between diabetes and colorectal cancer and its underlying mechanism are receiving more and more attention.Now, the molecular mechanism underlying the association of diabetes mellitus and colorectal cancer is still unclear, it was considered to be associated with hyperglycemia, insulin resistance and so on.Recent studies found that advanced glycation end products (AGEs) is closely associated with carcinogenesis,so it may be also involved in the formation and development of colorectal cancer in diabetic patients. In 1993,IKI found that AGEs can induce production of growth factor IL-6 and promote the growth of renal cell carcinoma.He also held that AGEs may account for the high incidence of renal cell carcinoma in diabetic patients. Abe also found that AGEs can promote of the growth and metastasis of human melanoma cell lines G361 and A375, and he found that the level of AGEs of nude mice was significantly higher in beds of implanted human melanoma tumor than in normal skin, and using anti-RAGE neutralizing antibodies can prolong survival of nude mice by inhibiting the growth of human melanoma and blocking its invasion to the lung.Advanced glycation end products are a heterogeneous population of protein and lipid adducts that are formed through a posttranslational, non-enzymatic glycoxication reaction. The level of AGEs in the body of healthy people is less, because the glycoxication reaction processes very slowly, while the reaction and formation of AGEs accelerates in the body of diabetic patients due to hyperglycemia. RAGE is one of the most characteristic and important receptors of AGEs.The expression of RAGE increased in the environment of hyperglycemia and oxidative stress. Recently, it was found that experession of RAGE also increased in malignat tumors such as gastric cancer, colorectal cancer and so on. It was proved that AGEs promotes the initiation and development of chronical complications of diabetes through the interaction with RAGE. Recent studies showed that AGEs-RAGE was not only closely associated with complications of diabetes mellitus, but also closely related with carcinogenesis.Abe held that AGEs-RAGE system may be associated with carcinogenesis, AGEs-RAGE promotes oxidative stress which causes DNA injury, while oxidative stress promotes the formation of AGEs and the upregulation of RAGE, the positive feedback loop leads to the formation and development of malignant tumors.Besides, the ROS mediated via AGEs-RAGE also cause a lot of signal transduction that are associated with proliferation and apoptosis, may also promote the procession of tumors. Sebekova also held that AGEs-RAGE may lead to the formation and development of malignant tumors of patients of advanced kidney disease.Now, the role of AGEs/AGEs-RAGE in chronic complications of diabetes is clearly elucidated, but its role in the formation and development of tumors has not been fully understood, and further study is necessary. And exploration of the role of AGEs in tumor cells is significant for the prevention and treatment of tumors in patients with diabetes.Recent years, the relationship between therapies of diabetes and malignant tumors has been receiving more and more attention. Recent epidemiological studies have shown that, The insulin sensitizer metformin not only lower blood glucose, but also has potential anti-tumor effects, can reduce the cancer risk in diabetic patients, so receives more and more attention.Metformin is the first-line drug for type 2 diabetes, it regulates glucose homeostasis through inhibition of liver glucose production, an increase of glucose uptake in peripheral organs, supression of lipoclasis and improvement of insulin resistance.by activiation of AMP-activiated protein kinase. AMPK is one of the most important signaling pathways of energy metabolism, It plays an important role in the maintenance of AMP/ATP ratio, synthesis and degradation of protein and fat, stress reaction.It has been proved that, on one hand, activiation of AMPK by phosphorylation can block the cell cycle through activation of p53-p21 pathway, on the other hand, activated AMPK can inhibit growth of tumor cells by inhibiting the mTOR signaling pathway. Therefore, in addition to lowering blood glucose, metformin may act as an anti-tumor agent through activation of AMPK.Until now, the mechanism of anti-tumor effects of metformin has not yet been fully elucidated, further study is needed.It was reported that, in addition to the activation of AMPK signaling pathway, metormin can also inhibit the formation of AGEs, block a series of signaling pathways mediated by the interaction of AGEs with its receptors. Schurman found that intervention of metformin removed the detrimental effects of osteoblast cell induced by AGEs in vitro. So, does intervention of metformin can reverse the AGEs-induced tumor cells'growth?Currently, studies of AGEs and metformin in colon cancer cells are less. Therefore, we will culture human colon carcinoma cell line SW-480 as a model, observe the effects of AGEs and metformin on the proliferation of SW-480 cells and explore the underlying mechanism, then further study the effects of intervention of metformin in the proliferation of SW-480 cells induced by AGEs and the corresponding mechanism.Chapter 1 Effects of AGEs on proliferation of human colon carcinoma cell line SW-480 and study of the mechanism[Objective]To observe effects of AGEs on proliferation of SW-480 cells and explore the underlying mechanism.【Methods】1,The experiment object was human colon carcinoma cell line SW-480.SW-480 cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS),100ug/ml penicillin and 100ug/ml streptomycin at 37℃,the cells were passaged every other day.2,Preparation of AGE-BSA and BSA:AGE-BSA and BSA were prepared by incubation of BSA in the presence or absence of the glucose at 37℃for 2 months. And then they were dialyzed against 1×PBS for 24h to remove unbound sugars,at last they were identified by spectrofluorometer. The specimen of AGEs was shown 102.67U/mg protein, while that of BSA was shown as 12.84U/mg protein. The level of endotoxin was less than 0.25EU/ml.3,Cell proliferation assay Cells were seeded in 96-well plates, after the cells' adhesion to wall, they were incuated at RPMI1640 midium for 24 hours, then they were classed into five groups:blank group, control group, experment groups(50ug/ml,100ug/ml,500ug/ml AGE-BSA), every group contains four wells and corresponding medium, the medium was changed every day. After 72 hours, the cells of 96-well plates were added MTT, after incubating for four hours, removed the medium and added DMSO, then measure the OD after ten minute's vibration.4,Cell cycle analyzed by FCM cells were seeded at six-well plates, after the cells' adhesion to wall, they were incuated at RPMI1640 midium for 24 hours,then the cells were incubated at midum with 100ug/ml AGE-BSA and midum with 100 ug/ml BSA for 72h, after the incubation,the cells were digested into monoplasts, after being washed twice with PBS,fixed the cells in cold 70% alchhol overnight, then removed the alchhol and washed the cells with PBS twice, added the PI staining solution(the final concentration of PI and RNAase A were both 50ug/ml), after 30 minutes away from the light, analyzed the results with FCM.5,Western blot analysis for CyclinD1 expression:After collecting the cells of control group and experiment group(100ug/ml AGE-BSA 72h), the cell extracts were prepared using lysis buffer, then added the samples and protein marker in the gel, after SDS-PAGE electro-ionization, transferred the protein of the gel to the PVDF membrane, put the PVDF membrane into 50g/L milk for 2 hours, then put it into the fluid containing Cyclin D1 antibody overnight at 4℃,washed the PVDF membrane for three times, incubated the PVDF membrane in the fluid containing horseradish peroxidase-conjugated anti-rabbit antibody for 1h at room temperature,washed it for three times,then exposed the straps using the ECL coloring reagent, scanned the straps and analyze them with quantity one software.6,Telomerase activity explored by TRAP siliver staining:Extracted the telomerase of the control group and experiment group(100ug/ml AGE-BSA 72h) according to the tolomerase activity kit,mixed 5μL 10×TRAP buffer,1μL dNTPs,1μL Taq-DNA polymerase,1μL TS primer,2μL telomerase extracts, with 39μL water, then keeped the mixture at 23℃for 30min, added 1μL CX primer to the mixture,amplificated 36 cycles,the parameter of circulation is 4℃,30s; 50℃,30s; 72℃,90s,and then extended at 72℃for 10 minutes. Mixed the 9ul PCR products with lul loading buffer, electro-ionize in the gel, then siliver stained the gel, took a picture of the gel, and analyzed the gel with quantity one software.7,All values were represented by means±standard deviation. Statistics analysis was carried out with SPSS 13.0. The differences among groups were analyzed by One-way ANOVA or t-test.The received level of significance is P<0.05.[Results]1,The cell viability of the four groups were:BSA (0.729±0.083),50ug/ml AGE (1.042±0.178),1 OOug/ml AGE (1.440±0.242),500ug/mlAGE (1.845±0.489), there were significant differences among the four groups (F= 50.280, P=0.000) AGEs increased the proliferation of SW-480 cells in a dose dependent mode.2,The proportion of the cells at G0/G1 stage of control group and expriment group (100ug/ml AGE-BSA,72h) were 56.02±0.58,51.93±1.01 respectively,there were significant difference between the two groups in G0/G1 (t=6.109,P=0.004); While the cells at S stage were 31.86±3.27,34.32±4.06 respctively and at G2/M stage were 12.13±2.83,13.76±4.07 respectively. There were no significant difference between the two groups in S and G2/M (t=-0.818,P=0.459) (t=-0.568,P=0.600).3,The expression of CyclinD1 of the experiment group (1.27±0.04) treated with 100ug/ml AGE-BSA increased significantlly compared with the control group (0.74±0.04) (t=-17.436,P=0.000)4,Telomerase activity of the experiment group(100ug/ml AGE-BSA 72h) (35.51±0.57) increased significantlly compared with the control group (28.91±0.34) (t=-17.364,P=0.000)[Conclusion]1. AGEs can increase proliferation of SW-480 cells in a dose dependent mode.2. AGEs increased the proliferation of SW-480 cells mainly by upregulating the expression of Cyclin Dl to shorten G0/G1 and increasing the telomerase activity.Chapter 2 Effects of metformin on proliferation of human colon carcinoma cell line SW-480 and study of the mechanism[Objective]To observe the effects of metformin on proliferation of SW-480 cells and explore the underlying mechanism.[Methods]1,The experiment object was SW-480 cells, they were cultured like the first chapter;2,Metformin (MET) was dissolved with PBS, the control group was added with the same amount of PBS.3,The SW-480 cells were grouped into four groups:PBS group, 1mmol/L MET, 5mmol/L MET, 10mmol/L MET group.in MTT assay, the other experiments of this part were grouped into PBS group and 5mmol/L MET group.4,The mtt assay, cell cycle, expression of Cyclin D1, telomerase activity was detected like the first chapter.5,All values were represented by means±standard deviation. Statistics analysis was carried out with SPSS 13.0. The differences among groups were analyzed by One-way ANOVA or t-test.The received level of significance is P<0.05.【Results】1,Metformin decreased the proliferation of SW-480 cells in a dose and time dependent mode. The group treated with lmmol/L metformin grew slower than the control group after the third day (P＜0.05 versus the control group).the group treated with 5mmol/L and 10mmol/L metformin grew slower than the control group after the first day (P＜0.05 versus the control group).2,The proportion of the cells at G0/G1 stage of control group and expriment group (5mmol/L metformin,72h) were 55.81±0.63,63.38±0.99 respectively, the G0/G1 of experiment group increased significantly compared with the control group(P=0.000); while the cells at S stage were 31.11±3.05,25.29±1.64 respctively,the S stage of the experiment group was significantly lower than the control group(P=0.023); G2/M stage were 13.09±3.00,11.33±2.60 respectively,there were no singnificant difference betwwen the two groups(P=0.412).3,The expression of CyclinD1 of the experiment group(0.68±0.06) treated with metformin attenuated significantlly comared with the control group(0.89±0.04) (P=0.001) 4,Telomerase activity of the experiment group(47.52±0.06)decreased significantlly compared with the control group(52.48±0.06) (P=0.000)【Conclusion】1. Metformin decreased the proliferation of SW-480 cells in a dose and time dependent mode,2. Metformin decreased the proliferation of SW -480 cells mainly by downregulating the experession of CyclinDl to block the cell cycle at G0/G1 and decreasing the telomerase activity.Chapter 3 Effects of metformin on proliferation of SW-480 cells induced by AGEs and study of the mechanism【Objective】To observe effects of intervention of metformin on proliferation of SW-480 cells induced by AGEs and explore the underlying mechanism.【Methods】1,The experiment object was SW-480 cells, they were cultured like the first chapter;2,SW-480 cells were treated with 100ug/ml BSA,100ug/ml AGE and 100ug/ml AGE+5mmol/L MET (A+M) respectively for 72 hours.3,AGE-BSA, BSA and metformin were prepared ike the first chapter.4,The mtt assay, cell cycle, expression of Cyclin D1, telomerase activity was detected like the first chapter.5,All values were represented by means±standard deviation. Statistics analysis was carried out with SPSS 13.0. The differences among groups were analyzed by One-way ANOVA, LSD was used for the differences of the two groups. The received level of significance is P＜0.05.【Results】1,The cell viability of the three groups were:BSA (0.726±0.089),100ug/mlAGE (1.148±0.187), AGE+MET (0.565±0.047),there were significant difference among the three groups (F=46.012 P=0.000) The viability of 100ug/ml AGE group were higher than BSA group (P=0.000),The viability of AGE+MET group was lower than the AGE group (P=0.000)2,The proportion of cells at G0/G1 stage were:BSA(33.39±0.62),100 ug/mlAGE (35.61±0.51), AGE+MET (31.01±1.12) respectively, there were significant difference among the three groups (F=36.581 P=0.000),the G0/G1 of AGE group was lower compared with BSA group(P=0.000),and the AGE+MET group was higher than AGE group(P=0.000); while the cells of the three groups at S stage were 31.22±2.92,33.91±3.41,31.51±4.34 respctively,there were no significant differences among the three groups(F=0.671 P=0.535); and at G2/M stage were 13.09±3.00,14.81±3.33,11.42±4.12 respectively and there were no significant differences among the three groups (F=0.488 P=0.629)3,The expression of CyclinDl were:The groups of BSA(1.12±0.05),100 ug/mlAGE(1.58±0.04),AGE+MET(1.23±0.06),there were significant difference among the three groups (F=72.875 P=0.000).AGEs increased the expression of CyclinDl significantly compared with the BSA group (P=0.000), The CyclinD1 expression of AGE+MET group was significantly lower compared with AGE group (P=0.000)4,The telomerase activity were:BSA(33.39±0.62),100ug/ml AGE(35.61±0.51), AGE+MET(31.01±1.12),there were significant difference among the three groups(F=25.028 P=0.001.AGE-BSA increased the telomerase activety compared with the BSA group (P=0.014).The telomerase activity of AGE+MET group was lower compared with AGE group (P=0.000).【Conclusion】1. Metformin can reverse the proliferation of SW-480 cells induced by AGEs.2. The mechanism of metformin reversed the proliferation of SW-480 cells induced by AGEs may be associated with regulation of cell cycle by CyclinD1 and inhibition of telomerase activity.