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Esophageal squamous cell carcinomas (ESCC) have become a severe threat to health and the current treatments for ESCC are frequently not effective. Recent epidemiological studies suggest that the anti-hyperglycemic agent metformin may reduce the risk of developing cancer, including ESCC, among diabetic patients. However, the antitumor effects of metformin on ESCC and the mechanisms underlying its cell cycle regulation remain elusive. The findings reported herein show that the anti-proliferative action of metformin on ESCC cell lines is partially mediated by AMPK. Moreover, we observed that metformin induced G0/G1 phase arrest accompanied by the up-regulation of p21CIP1 and p27KIP1. In vivo experiments further showed that metformin inhibited tumor growth in a ESCC xenograft model. Most importantly, the up-regulation of AMPK, p53, p21CIP1, p27KIP1 and the down-regulation of cyclinD1 are involved in the anti-tumor action of metformin in vivo. In conclusion, metformin inhibits the growth of ESCC cells both in cell cultures and in an animal model. AMPK, p53, p21CIP1, p27KIP1 and cyclinD1 are involved in the inhibition of tumor growth that is induced by metformin and cell cycle arrest in ESCC. These findings indicate that metformin has the potential for use in the treatment of ESCC.

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Metformin is used as a first-line therapy for type 2 diabetes, with reports of its usefulness for the prevention and control of several types of cancers. This study investigated the effects of metformin on hepatocellular carcinoma (HCC). The human HCC cell lines HepG2 and PLC/PRF/5 were cultured and treated with metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of adenosine monophosphate (AMP)-activated protein kinase. Changes in cell viability and cell cycle distribution were evaluated by MTT and flow cytometry, respectively. Apoptosis was assessed by fluorescent-dye staining. An HCC model was established in 6- to 8-week-old BALB/c-nu mice by subcutaneous injection of PLC/PRF/5 cells. After 1 week, mice were treated intragastrically with metformin or vehicle. Tumor xenograft tissues were examined using immunohistochemistry for evaluation of the the expression of cyclin D1, p21CIP and p27KIP. HCC cells and tissues from the in vitro and in vivo experiments, respectively, were subjected to protein extraction and western blotting. We found that metformin treatment reduced HCC cell viability in a dose-dependent manner similar to AICAR treatment. In addition, metformin treatment induced HCC cell cycle arrest at G1/G0 phase and apoptosis. Intragastric treatment of the mouse PLC/PRF/5 cell xenograft model with metformin showed that metformin not only blocked tumor progression, but also reduced tumor morbidity. Treatment with metformin upregulated the expression of p21CIP and p27KIP, but downregulated cyclin D1 levels, both in vitro and in vivo. Metformin treatment also upregulated the expression of phosphorylated AMPK protein in xenograft tissues. These findings indicate that metformin warrants further evaluation as a novel therapeutic and preventive strategy against HCC.

AMP-activated protein kinase (AMPK) is a central metabolic sensor and plays an important role in regulating glucose, lipid and cholesterol metabolism. Therefore, AMPK is a key therapeutic target in diabetes. Recent pilot studies have suggested that diabetes drugs may reduce the risk of cancer by affecting the AMPK pathway. However, the association between AMPK and the proliferation of hepatocellular carcinoma (HCC) is unknown. In this study, we investigated the relationship between AMPK activity and the proliferation of HCC in cell lines, nude mice and human clinic samples. We first investigated the relationship between AMPK activity and cell proliferation in two HCC cell lines, PLC/PRF/5 and HepG2, by two AMPK activators, 5-aminoimidazole-4-carboxamide-1-h-D-ribofuranoside (AICAR) and metformain. AICAR and metformin treatment significantly inhibited the proliferation of HCC cells and induced cell cycle arrest at G1-S checkpoint. We then observed that metformin abrogated the growth of HCC xenografts in nude mice. The clinical pathology of AMPK activity in HCC, including cell proliferation, differential grade, tumor size and microvessel density, was studied by using 30 clinical tissue samples. In HCC tissue samples, phosphorylated AMPK was expressed mainly in cytoplasm. AMPK activity decreased significantly in HCC in comparison with paracancerous liver tissues (P<0.05). AMPK activity was negatively correlated with the level of Ki-67 (a marker of cell proliferation), differential degradation and tumor size (P<0.05), but not with microvessel density, hemorrhage or necrosis in HCC. Our findings suggest that AMPK activity inhibits the proliferation of HCC and AMPK might be an effective target for prevention and treatment of HCC.