The Underlying Mechanism Of AMPK Phosphorylation Induced By Metformin As An Inhibition Of Gastrointestinal Cancer Proliferation, Invasion And Metastasis

The incidence, diagnosis and mortality rate of gastrointestinal cancer are increasing continuously in recent years worldwide, especially for gastric cancer, liver cancer, colon cancer and pancreatic cancer. The incidence of gastric cancer, colon cancer and liver cancer ranks 2nd to 4th within all kinds of cancers in China and pancreatic cancer also arouses our attention because of the fact that most patients have locally advanced or distant metastatic disease at the time of diagnosis, precluding radical resection because of the insidious, aggressive natural history of the disease. These gastrointestinal cancers will lead to nutritional insufficiency, electrolyte imbalance, impaired glycemic control and frequent hospitalizations, affecting the quality of patients’lives adversely or even causing death. Even though outcomes in patients with these four gastrointestinal cancers have been improved by the use of potentially curative resection and adjuvant treatments such as chemotherapy and biological therapies, early recurrence and metastasis still occur in a substantial proportion of them after these treatments. Thus, to investigate the underlying mechanism that how these gastrointestinal cancers proliferate, invade and metastasize will help improve the therapeutic effect and prognosis.Metformin (1,1-dimethylbiguanide hydrochloride) is a well-known and first-line treatment for 2DM patients, which is a biguanide derived from the French lilac (Galega officinalis). The hypoglycemic mechanism of metformin can be concluded into three ways: reducing hepatic glucose production, increasing insulin-stimulated glucose uptake in skeletal muscle and decreasing gastrointestinal glucose absorption. Metformin is low-cost and approximately 120 million people are using metformin worldwide. Besides the treatment of type 2 diabetes, it is also used for polycystic ovarian syndrome, metabolic syndrome, and diabetes prevention. The use of metformin in diabetic patients has been associated with significantly lower risks of cancer incidence and mortality. In recent years, several clinical research found that this drug could improve the overall survival among diabetic patients with breast, prostate, colorectal or head and neck cancers. Recent retrospective analyses indicate that metformin inhibits cell proliferation in several human malignancies, including gastric carcinoma, pancreatic cancer, medullary thyroid cancer and endometrial carcinoma. Although the anti-carcinoma effect of metformin is being discovered by more and more clinical research, the underlying mechanism is still unclarified.AMPK(Adenosine monophosphate (AMP)-activated protein kinase) is a heterotrimeric serine/threonine kinase composed of a catalytic (a) subunit and two regulatory (β and γ) subunits. It is a known cellular metabolic sensor and plays an important role in the control of energy homeostasis in response to external stresses. By activating the LKB1-AMPK-ACC signaling pathway, metformin suppresses hepatic gluconeogenesis and induces fatty acid oxidation, thus lowering glucose levels and increasing insulin sensitivity. Recent studies have documented that metformin, the pharmacological activation of AMPK, is able to induce AMPK phosphorylation and impair mechanistic downstream targets, thereby inhibiting protein synthesis and cell growth. However, the molecular mechanisms for the anti-tumor effects of metformin have not yet been clearly elucidated.To investigate the molecular mechanisms for the anti-tumor effects of metformin, in the current study, we are going to demonstrate:1. Metformin can induce AMPK phosphorylation.2. Metformin can inhibit the proliferation and invasion of gastric cancer and pancreatic cancer.3. Metformin inhibits the proliferation of gastric cancer cells and pancreatic cancer cells through activating AMPK/mTOR signaling pathway.4. Metformin inhibits the proliferation, invasion and metastasis of pancreatic cancer cells, hepatocellular carcinoma cells and colorectal cancer cells through activating AMPK/Akt/FoxMl signaling pathway.1. Metformin could induce AMPK phosphorylationObjective:To test the activity of AMPK in human cancer tissue and to verify the regulation of AMPK by metforminMethods:Collecting human gastric cancer and adjacent tissue pathological slices and using immunohistochemistry to test the expression level of p-AMPK in both tissue. The nude mice xenograft model was applied to establish mouse models with subcutaneou transplantation tumor with gastric cancer cells. Metformin was used to treat these mice and immunohistochemistry was performed in the subcutaneou transplantation tumors collected from them. p-AMPK expression level was tested between metformin-treated group and metformin-untreated group.Result:The expression level of p-AMPK in human gastric cancer was decreased compared with adjacent tissue. The expression level of p-AMPK in the subcutaneou transplantation tumors of metformin-treated nude mice was much higher than the control group did.Conclusion:The low activity of AMPK in human gastric cancer tissue suggested a cancer suppressor function. Metformin could induce AMPK phosphorylation and increase the activity of AMPK in cancer tissues.2. Metformin could inhibit the proliferation and invasion of gastric cancer and pancreatic cancerObjective:To test the suppressive effects of metformin treatment on gastric cancer and pancreatic cancerMethods:Gastric cancer cells and pancreatic cancer cells were cultivated in cell culture medium with different concentration of metformin. The cancer cell proliferation and colony formation were observed. Wound healing assay was performed to evaluate the invasion ability of pancreatic cancer cells between metformin-treated group and metformin-untreated group. Flow cytometry was used to investigate the change of gastric cancer cell cycle after a 48h-treatment of metformin in different concentrations. The nude mice xenograft model was applied to establish mouse models with subcutaneou transplantation tumor with gastric cancer cells and pancreatic cancer cells. Metformin was used to treat these mice and the observation of subcutaneou transplantation tumors was carried out. Collecting the case data of patients with both 2DM and gastric cancer and treated with metformin. Analyzing the median survival time and making comparison with metformin-untreated group.Result:After the treatment of metformin, the cell proliferation and colony formation ability of gastric cancer cells and pancreatic cancer cells decreased, correlating with the degree of metformin concentration. Of metformin treatment group and the group of pancreatic cancer cells to scratch, visible after 24 h of the scratch width of treatment group was obviously less than metformin treatment group. The proportion of cells in the S cell cycle phase was remarkedly decreased in metformin-treated cancer cells compared with untreated cells. The gastric cancer cells and pancreatic cancer cells xenograft tumor growth was significantly reduced with metformin treatment compared with untreated mice. Metformin users had a longer median survival time than that in non-metformin users.Conclusion:Metformin is a protective factor for gastric cancer patients with 2DM. Metformin can inhibit gastric cancer cells and pancreatic cancer cells proliferation and colony formation in vitro and growth in nude mice. Metformin can also affect the cell cycle of gastric cancer cells.3. Metformin inhibited the ability of gastric cancer cells proliferation via activating AMPK/mTOR signaling pathwayObjective:To study the underlying mechanism of the inhibition function to gastric cancer cells proliferation of metformin.Methods:Gastric cancer cells were cultivated in cell culture medium with different concentration of metformin for 24h and 48h. The cancer cells were then collected and cell lysis was performed to get the protein inside of the cells. By using Western blot, the expression level of AMPK and mTOR and its downstream targets were tested between each cell groups. The nude mice xenograft model was applied to establish mouse models with subcutaneou transplantation tumor with gastric cancer cells. Metformin was used to treat these mice and the observation of subcutaneou transplantation tumors was carried out to test the expression level of AMPK and mTOR and its downstream targets.Results:We found that the expression level of p-AMPK increased in a metformin treatment dose-dependent manner, while the expression level of p-mTOR decreased in a metformin treatment dose-dependent manner as well as the expression level of p-70S6K, pS6 and p4E-BP1.Conclusion:Metformin reduced the expression level of some proteins associating with cell proliferation via AMPK/mTOR signaling pathway, thus inhibiting the proliferation ability of gastric cancer cells.4. Metformin inhibited the ability of pancreatic cancer cells, hepatocellular carcinoma cells and colorectal cancer cells proliferation, invasion and metastasis via activating AMPK/Akt/FoxMl signaling pathwayObjective:To study the underlying mechanism of the inhibition function to pancreatic cancer cells, hepatocellular carcinoma cells and colorectal cancer cells proliferation, invasion and metastasis by metformin.Methods:Pancreatic cancer cells, hepatocellular carcinoma cells and colorectal cancer cells were cultivated in cell culture medium with different concentration of metformin for 24h and 48h. The cancer cells were then collected and cell lysis was performed to get the protein inside of the cells. By using Western blot, the expression level of AMPK, Akt, FoxM1 and other kinds of proteins were tested between each cell groups.Results:We found that the expression level of p-AMPK and E-cadherin increased in a metformin treatment dose-dependent and duration-dependent manner, while the expression level of p-Akt, FoxMl, (3-catenin, cyclin B1, cyclin D1, vimentin and N-cadherin decreased in a metformin treatment dose-dependent and duration-dependent manner.Conclusion:Metformin increased and reduced the expression level of some proteins associating with cancer cell proliferation, invasion and metastasis via AMPK/Akt/FoxMl signaling pathway, thus inhibiting the proliferation, invasion and metastasis ability of pancreatic cancer cells, hepatocellular carcinoma cells and colorectal cancer cells.In conclusion:The low activity of AMPK in human gastric cancer tissue suggested a cancer suppressor function. Metformin could induce AMPK phosphorylation and increase the activity of AMPK in cancer tissues.1. Metformin is a protective factor for gastric cancer patients with 2DM. Metformin can inhibit gastric cancer cells and pancreatic cancer cells proliferation and colony formation in vitro and growth in nude mice. Metformin can also affect the cell cycle of gastric cancer cells.2. Metformin reduced the expression level of some proteins associating with cell proliferation via AMPK/mTOR signaling pathway, thus inhibiting the proliferation ability of gastric cancer cells.3. Metformin increased and reduced the expression level of some proteins associating with cancer cell proliferation, invasion and metastasis via AMPK/Akt/FoxMl signaling pathway, thus inhibiting the proliferation, invasion and metastasis ability of pancreatic cancer cells, hepatocellular carcinoma cells and colorectal cancer cells.