Investigation Of Warburg Effect In The Colorectal Tumorigenesis Of Diabetes And The Inhibition Effect Of Metformin

BackgroundColorectal cancer is one of the common gastrointestinal malignant tumors. The morbidity and mortality of colorectal cancer in malignant tumor rank the second place in western developed countries. Epidemiologic findings to date have shown that type 2 diabetes mellitus is closely related to the increased risk of colorectal cancer. Several similar risk factors have been found between diabetes mellitus and colorectal cancer. Those include a "Western Lifestyle", with diets low in fruits and vegetables or fiber and high in fat and cooked meat, as well as restricted physical activity. There are no clinical data to date can confirm the relationship between them, and the mechanism during the tumorigenesis of diabetes combined colorectal cancer is unclear;Metformin is an oral antidiabetic agent in the biguanide class. It is the first-line drug of choice for the treatment of type 2 diabetes. Epidemiological studies have found that taking metformin can obviously reduce the tumorigenesis of diabetes combined colorectal cancer in patients with diabetes. But the mechanism of the metformin inhibition effect is not clear.Glycolysis and oxidative phosphorylation are the two common pathway of glucose metabolism in body. In the presence of oxygen, most cells primarily metabolize glucose to pyruvate via glycolysis and then completely oxidize pyruvate to carbon dioxide through the citric acid cycle. However, tumor cells generate as much as 60% of their ATP through glycolysis, and the glycolytic rate of tumor cells is up to 200 times higher than that of normal cells, regardless of the presence or absence of oxygen. This phenomenon is known as the Warburg effect.In the present study, we will research the influence of warburg effect during the tumorigenesis of diabetes combined colorectal cancer and the inhibition effect of metformin through animal model.Objective1. Induce colorectal cancer in diabetic rats by DMH. Detect the influence of DMH in the tumorigenesis of colorectal cancer and the enzyme activity in diabetic rats.2. Treat the DMH induced rats with metformin. Detect the influence of metformin on the tumorigenesis of colorectal cancer and the expression of enzyme in colon tissues.Methods Part â… All rats were randomly divided into four groups:(A) Control group; (B) STZ group; (C) DMH group; (D) STZ+DMH group. The type 2 diabetes animal model was induced using high-fat feeding in combination with a low dose of STZ. Then induce colorectal cancer using DMH. Detect the formation of ACF, the number, the volum and the incidence of tumor. Detect the proliferative index (PI) of tissue cells and the enzyme activity that involved in glucose metabolism.Part â…¡In vivo experiments:All rats were randomly divided into five groups, (A) Control group; (B) STZ group; (C) STZ+DMH group; (D) STZ+MET group and (E) STZ+DMH+MET group. The type 2 diabetes animal model was induced using high-fat feeding in combination with a low dose of STZ. Then induce colorectal cancer using DMH. At the same time treated with metformin. Detect the formation of ACF, the number, the volum and the incidence of tumor and the inhibition effect of metformin. Detect the proliferative index of tissue cells, the expression and the activity of enzymes that involved in glucose metabolism.In vitro experiments:Treat the human colon cancer cells LoVo and HT-29 with metformin for different time at different concentration. Detect the influence of metformin on the cell growth using MTT. Western Blot was used to detect the expression of enzymes that involved in glucose metabolism.ResultsPart â… 1. Serological analysis indicate that the rats were hyperglycemia and insulin resistance. These mean the type 2 diabetes animal model was induced successfully.2. ACF were detected in DMH group and STZ+DMH group. But there was no ACF in the other groups. The number of ACF in STZ+DMH group was significant higher than that in DMH group.3. Tumors were detected in DMH group and STZ+DMH group. The number, the volum and the incidence of tumors in STZ+DMH group were higher than that in DMH group. No tumors were detected in the Control group and STZ group.4. Proliferative index were significantly increased after the induce of DMH. The PI in STZ+DMH group was significant higher than that in DMH group.5. The activities of HK and PK were significantly increased after the induce of DMH and STZ+DMH group was higher than DMH group. But the activity of PDH was decreased. When compared with Control group, the activity of HK and PK in STZ group were all increased but PDH was decreased.Part â…¡1. ACF were detected in STZ+DMH group and STZ+DMH+MET group. MET treatment decreased the formation of ACF. No ACF were detected in Control group, STZ group and STZ+MET group.2. Tumors were detected in STZ+DMH group and STZ+DMH+MET group. The number, the volum and the incidence of tumors in STZ+DMH+MET group were all decreased than the DMH group. No tumor was formed in the Control group, STZ group and STZ+MET group.3. Proliferative index were significantly increased in STZ+DMH group and STZ+DMH+MET group and the treatment of metformin inhibited the Proliferative index.4. In STZ+DMH group and STZ+DMH+MET group, the expression of PKM2 in colon tissues were significantly increased, but the increase was inhibited by metformin. The expression of IDH1 in STZ+DMH+MET group was increased after the treatment of metformin but it was still lower than the other three groups. Western blot were in good agreement with the immunohistochemical results.5. The activity of HK in STZ+DMH group and STZ+DMH+MET group were all increased but the activity of PDH was decreased. The treatment of metformin inhibited the alteration.6. The treatment of metformin inhibited the cell growth. The expression of PKM2 was increased but the expression of IDH1 was decreased after the metformin treatment. With the increasing MET concentration and treatment times, the activity of HK gradually decreased. In contrast, the activity of PDH gradually increased.ConclusionDue to the increased glycolysis rate, the risk of colorectal cancer in diabetic rats was increased. Diabetes promotes DMH-induced colorectal cancer by increasing the activity of glycolytic enzymes in rats. As a drug for diabetes treatament, metformin could inhibited the tumorigenesis of colorectal cancer in diabetic rats. Reversed Warburg effect involved in this process may be the mechanism.