Metformin Induces Autophagy via the AMPK-mTOR Signaling Pathway in Human Hepatocellular Carcinoma Cells.

BACKGROUND: Metformin may exert the anticancer effect on multiple types of cancers and some potential mechanisms have been suggested. Our study was designed to determine the effect of metformin on the cell autophagy and autophagic flux via the AMPK-mTOR signaling pathway in human hepatocellular carcinoma (HCC) cells. METHODS: MHCC97H and HepG2 cell lines were cultured and treated without and with metformin at various concentrations (2, 5, 10 and 20 mM) for 48 h. Then, 10 mM was determined as the optimal concentration and the HCC cells were treated with metformin for 12, 24, 48, and 72 h. MTT assay was used to assess the cell viability and Western blotting was used to determine the expression of proteins (LC3-II, p62, phospho-AMPKα, phospho-mTOR, mTOR, phospho-p70 S6 Kinase, p70 S6 Kinase, PARP1, Caspase-9 and Caspase-3). Autophagy inhibitor 3-methyladenine, EGFP-LC3 and mCherry-GFP-LC3B plasmid transfection were used for further study. RESULTS: Metformin inhibited significantly the viability of MHCC97H and HepG2 cells in a dose- and time-dependent manner. For the apoptotic properties, activation of Caspase-9 and Caspase-3 and PARP cleavage were not observed after treatment with metformin. MHCC97H cells were transfected with a EGFP-LC3 plasmid and treatment with metformin could lead to the increased level of LC3-II and decreased level of p62. In metformin-induced autophagy, AMPK expression was activated, and the phosphorylation levels of mTOR and p70 S6 Kinase were inhibited. Metformin treatment and mCherry-GFP-LC3B plasmid transfection showed that metformin could induce the autophagic flux. 3-Methyladenine (3-MA) partly abolished this effect. CONCLUSION: Metformin could induce the autophagy, autophagic flux, and activate the AMPK-mTOR signaling pathway in human HCC cells.

Expression and Significance of Insulin Receptor Substrate 1 in Human Hepatocellular Carcinoma.

BACKGROUND: Insulin receptor substrate 1 (IRS-1) is an important molecule of the insulin signal transduction pathway and has been associated with the occurrence and development of many tumors, including hepatocellular carcinoma (HCC). Our study was designed to determine the expression and significance of IRS-1 in human HCC. METHODS: Two hundred and forty specimens were drawn from 140 patients, including 100 HCC tissues and 100 paracancerous (PC) tissues from 100 HCC patients, 20 liver cirrhosis (LC) tissues from 20 LC patients, and 20 chronic hepatitis (CH) tissues from 20 CH patients. Baseline and pathological characteristics were included, and the expression of IRS-1 was examined by immunohistochemical (IHC) staining. Binary logistic regression model calculation was used for multivariate analysis. RESULTS: The total positive rates of IRS-1 expression were 41.0%, 17.0%, 15.0%, and 10.0% in HCC, PC, LC and CH tissues, respectively. IRS-1-positive signals were brown in color and located in the nucleus and cytoplasm. Compared with PC, LC, and CH tissues, a significantly increased expression was observed in human HCC tissues (P < 0.001, P = 0.028, and P = 0.008). Eight of the total 240 specimens had the strong immunostaining of IRS-1 expression, and all of them were HCC tissues. After control of the age, gender, and HBV and HCV infection, IRS-1 expression was independently associated with the diagnosis of HCC (OR 6.60, 95% CI 2.243-19.425, P = 0.001). CONCLUSIONS: Positive expression of IRS-1 in HCC was increased significantly and may play an important role in the occurrence and development of human HCC.

Increased Level of Systolic Blood Pressure in Hepatocellular Carcinoma Patients with Diabetes Mellitus.

BACKGROUND: More than 50% of patients with type 2 diabetes mellitus (DM) also have hypertension. Moreover, hypertension has been regarded as one paraneoplastic phenomenon of hepatocellular carcinoma (HCC). Our study was designed to determine the relationship between blood pressure and DM in HCC patients. PATIENTS AND METHODS: A total of 879 HCC patients were included and 151 (17.2%) were diagnosed with DM. Multivariable logistic regression analysis was used to determine the relationship and the results were expressed as adjusted odds ratios (AORs) and their 95% confidence intervals (CIs). Considering the effect of potential confounders, sub-group analysis was performed. We would further study the association of systolic blood pressure (SBP) with fasting glucose, and the association between DM duration/treatment and SBP level. RESULTS: Compared with non-diabetic patients, the diabetic patients had increased levels of SBP (133.7±18.5 mmHg vs 128.3±15.2 mmHg, P=0.001) and fasting blood glucose (9.13±3.04 mmol/L vs 5.18±1.08 mmol/L, P<0.001), an elder age (58.5±10.2 years vs 55.3±11.2 years, P=0.001), a higher percentage of cirrhosis diagnosis (60.9% vs 48.2%, P=0.004), lower percentages of drinking (18.5% vs 30.8%, P=0.002) and smoking (30.5% vs 43.7%, P=0.003), and decreased levels of GGT (median/interquartile-range 88/53-177 U/L vs 117/58-248 U/L, P=0.037), platelet count (121.4±76.6 ×109/L vs 151.2±82.8 ×109/L, P<0.001) and hemoglobin (124.3±25.5 g/L vs 133.6±24.2 g/L, P<0.001). Multivariable analysis showed that, statistically significant differences were found for SBP ≥140 mmHg (AOR=2.101; 95% CI, 1.424-3.100; P<0.001), smoking (AOR=0.637; 95% CI, 0.415-0.979; P=0.040), hemoglobin (AOR=0.990; 95% CI, 0.983-0.998; P=0.010) and platelet count (AOR=0.996; 95% CI, 0.994-0.999; P=0.009). For the relationship between SBP and DM, the positive result was supported by most (10/14) of the subgroup analyses. CONCLUSION: SBP level was increased in HCC patients with diabetes mellitus.

[Effect of baicalin on liver fatty acid binding protein in oxidative stress model in vitro].

OBJECTIVE: To investigate the effect of baicalin on liver fatty acid binding protein in oxidative stress model in vitro. METHODS: (1) Cellular oxidative stress in vitro was induced by incubating cells with 400µmol/L hydrogen peroxide (H2O2) for 20 minutes at 37 degrees C in the dark. After Chang liver cell line was treated with different dose of baicalin for 24, 48 and 72 hours. MTT assay was employed to detect cell viability, and then the hydrogen peroxide (TC50) of the different dose of baicalin was calculated. (2) Based on MTT assay, cells were treated with three different doses of baicalin (25, 50, 100 µmol/L) for 24 and 48 hours before being exposed to 400 µmol/L H2O2 for 20 minutes at 37 degrees C. Then, reactive oxygen species (ROS) assay and activity assays of superoxide dismutase (SOD) and reduced glutathione hormone (GSH) were evaluated. (3) Realtime PCR and Western blotting were applied to explore the influence of baicalin on the expression level of L-FABP. (4) One-way ANOVA was used for results statistical analysis. RESULT: (1) MTT assay showed baicalin treatment at 25, 50, 100 µmol/L for 24 and 48 hours was feasible (83.60% ± 3.47%, 72.36% ± 2.18%, 70.16% ± 2.04% for 24 hours; 84.93% ± 3.11%, 76.16% ± 2.45%, 72.72% ± 2.31% for 48 hours, P > 0.05, F = 386.24, 475.92 respectively). Meanwhile, we found by the linear regression model that the median toxic concentration of baicalin for 48 hours was 170.6 µmol/L, and the median toxic concentration of baicalin for 24 hours was 153.2 µmol/L. (2) ROS assay showed dichlorofluorescin in all baicalin-treated cells after stress was significantly reduced (37.0 ± 3.30, 22.90 ± 3.84, 29.60 ± 2.52 for 24 hours respectively, P < 0.05, F = 70.06; 35.77 ± 2.35, 21.80 ± 3.10, 23.87 ± 1.98 for 48 hours respectively, P < 0.05, F = 110.92) as compared with the H2O2-treated cells. Moreover, 50 µmol/L baicalin treatment for 48 hours was the optimal condition against ROS generation (21.80 ± 3.10, P < 0.01, F = 110.92). Furthermore, the activities of intracellular SOD and GSH was increased significantly (51.53 ± 1.91 µg/mg for SOD, P < 0.05, F = 93.81; 49.85 ± 1.45 U/mg for GSH, P < 0.05, F = 92.51). (3) Although realtime PCR analysis indicated 50 µmol/L baicalin treatment for 48 hours could have no changes of the level of L-FABP expression under the oxidative stress condition, western blotting analysis indicated 50 µmol/L baicalin treatment for 48 hours could increase up to about 80% for the level of L-FABP expression. CONCLUSION: Baicalin was suggested to be able to enhance both L-FABP expression and activity of intracellular SOD and GSH, and therefore protected hepatocytes from oxidative stress.