OBJECTIVE: To investigate effects of berberine (BBR) on cholesterol synthesis in HepG2 cells with free fatty acid (FFA)-induced steatosis and to explore the underlying mechanisms. METHODS: A steatosis cell model was induced in HepG2 cell line fed with FFA (0.5 mmol/L, oleic acid:palmitic acid = 2:1), and then treated with three concentrations of BBR; cell viability was assessed with cell counting kit-8 assays. Lipid accumulation in cells was observed through oil red O staining and total cholesterol (TC) content was detected by TC assay. The effects of BBR on cholesterol synthesis mediators were assessed by Western blotting and quantitative polymerase chain reaction. In addition, both silent information regulator 1 (SIRT1) and forkhead box transcription factor O1 (FoxO1) inhibitors were employed for validation. RESULTS: FFA-induced steatosis was successfully established in HepG2 cells. Lipid accumulation and TC content in BBR groups were significantly lower (P < 0.05, P < 0.01), associated with significantly higher mRNA and protein levels of SIRT1(P < 0.05, P < 0.01), significantly lower sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy 3-methylglutaryl-CoA reductase levels (P < 0.05, P < 0.01), as well as higher Acetyl-FoxO1 protein level (P < 0.05, P < 0.01) compared to the FFA only group. Both SIRT1 inhibitor SIRT1-IN-1 and FoxO1 inhibitor AS1842856 blocked the BBR-mediated therapeutic effects. Immunofluorescence showed that the increased SIRT1 expression increased FoxO1 deacetylation, and promoted its nuclear translocation. CONCLUSION BBR can mitigate FFA-induced steatosis in HepG2 cells by activating SIRT1-FoxO1-SREBP2 signal pathway. BBR may emerge as a potential drug candidate for treating nonalcoholic hepatic steatosis.
Berberine/pharmacology* ; Cholesterol ; Forkhead Box Protein O1/genetics* ; Humans ; Non-alcoholic Fatty Liver Disease/drug therapy* ; Sirtuin 1/genetics* ; Sterol Regulatory Element Binding Proteins

OBJECTIVETo investigate the effect of Metformin on the proliferation and collagen synthesis of the human keloids fibroblasts as well as the effect on phosphorylation of Akt/FoxO1 signal transduction pathway.

METHODSFibroblasts of keloid were divided into control group treated with medium solution and experimental groups treated with different concentrations of Metformin. 48 h later CCK-8 assay was adopted to evaluate cell survival; Western blot was performed to detect the Akt and FoxO1 phosphorylation; and Hydroxyproline reagent kit was used to detect the collagen synthesis.

RESULTSWith different concentrations (30, 60, 90, 120 mmol/L) of Metformin, the absorbance of cultured keloid fibroblasts detected by CCK8 assay decreased by (13.30 ± 2.04)%, (22.64 ± 4.70)%, (54.00 ± 5.34)% and (63.12 ± 3.48)%. The growth of fibroblasts was suppressed by Metformin in a dose-dependent manner. It showed that the level of phoshpo-akt and phoshpo-foxOl in keloids fibroblasts in experimental groups was lower than that in the control group and the collagen synthesis were also decreased in experimental groups, all in a dose-dependent manner (P < 0.05, P < 0.01).

CONCLUSIONSMetformin can effectively inhibit the proliferation and collagen synthesis of the human keloids fibroblasts in vitro, which may be associated with the suppression of phosphorylation of Akt/FoxO1 signaling pathway

Cell Proliferation ; drug effects ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; metabolism ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; metabolism ; Humans ; Keloid ; pathology ; Metformin ; pharmacology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects

BACKGROUNDPancreatic β cells are susceptible to fatty acid-induced apoptosis. The 17β-estradiol (E2) protects pancreatic β cells from apoptosis, mediated by the estrogen receptor-α (ERα). The mRNA level and promoter activity of leukemia-related protein (LRP) 16 were significantly increased by E2 in ER-α and LRP16 was a co-activator of ER-α. The aim of the study was to assess the effects of LRP16 on fatty acid-induced apoptosis in MIN6 cells.

METHODSCells with over-expressing LRP16 were obtained by lipidosome transfection. Insulin content and glucose-stimulated insulin secretion (GSIS) were examined by radioimmunoassay. Western blotting was applied to detect protein expression. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and flow cytometry. The forkhead boxO1 (FoxO1) subcellular localization was determined by immunocytochemical analysis.

RESULTSMIN6-LRP16 cells with overexpression of LRP16 were successfully established, and protein expression of LRP16 was 2.29-fold of that of control cells (MIN6-3.1, P < 0.05). Insulin content and GSIS in MIN6-LRP16 were substantially increased compared with those in control cells. When cells were stimulated with glucose, increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and serine-threonine kinase (Akt) were observed in MIN6-LRP16. When cells were under palmitate pressure, the TUNEL-positive rate in MIN6-LRP16 was (17.0 ± 0.5)%, while it in MIN6-3.1 was (22.0 ± 0.4)%. In palmitate-treated cells, attenuated Akt phosphorylation was observed, but the attenuation in Akt activity was partially restored in MIN6-LRP16 cells. Meanwhile, nuclear localization of FoxO1 in MIN6-LRP16 was apparently reduced compared with that in control cells.

CONCLUSIONSLRP16 regulated insulin content and GSIS in MIN6 cells by ERK1/2 and Akt activated way. Meanwhile, LRP16 overexpression protected MIN6 cells from fatty acid-induced apoptosis by partially restoring Akt phosphorylation and inhibiting FoxO1 nuclear redistribution. Therefore, LRP16 played important roles not only in insulin content and GSIS but also in the antilipotoxic effect mediated by Akt/FoxO1 signaling.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Cell Line, Tumor ; Fatty Acids ; pharmacology ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; genetics ; metabolism ; Mice ; Neoplasm Proteins ; genetics ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; genetics

Diabetes-associated liver injury becomes a dominant hepatopathy, leading to hepatic failure worldwide. The current study was designed to evaluate the ameliorative effects of ginsenoside Rh1 (G-Rh1) on liver injury induced by T2DM. A T2DM model was established using C57BL/6 mice through feeding with HFD followed by injection with streptozotocin at 100 mg·kg^-1.. Then the mice were continuously administered with G-Rh1 (5 and 10 mg·kg^-1), to explore the protective effects of G-Rh1 against liver injury. Results showed that G-Rh1 exerted significant effects on maintaining the levels of FBG and insulin, and ameliorated the increased levels of TG, TC and LDL-C induced by T2DM. Moreover, apoptosis in liver tissue was relieved by G-Rh1, according to histological analysis. Particularly, in diabetic mice, it was observed that not only the increased secretion of G6Pase and PEPCK in the gluconeogenesis pathway, but also inflammatory factors including NF-κB and NLRP3 were suppressed by G-Rh1 treatment. Furthermore, the underlying mechanisms by which G-Rh1 exhibited ameliorative effects was associated with its capacity to inhibit the activation of the Akt/FoxO1 signaling pathway induced by T2DM. Taken together, our preliminary study demonstrated the potential mechnism of G-Rh1 in protecting the liver against T2DM-induced damage.
Animals ; Chemical and Drug Induced Liver Injury, Chronic ; Cholesterol, LDL/pharmacology* ; Diabetes Mellitus, Experimental/metabolism* ; Diabetes Mellitus, Type 2/metabolism* ; Forkhead Box Protein O1/pharmacology* ; Ginsenosides ; Insulin/metabolism* ; Liver ; Mice ; Mice, Inbred C57BL ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Streptozocin

Objective: To explore the apoptosis-inducing effect of the Chinese medicinal compound CFF-1 on prostate cancer cells and its related molecular mechanisms. METHODS: Normal prostate WPMY-1 cells and prostate cancer LNCaP, CWR22Rv1, PC3 and DU145 cells were treated in dehydrated alcohol with CFF-1 at 0, 2, 5, or 10 mg/ml for 24 hours. Then the viability of the prostate cells was detected by morphological observation, MTT and CCK-8 assay, nuclear condensation and disruption measured by DAPI staining, the cell cycle and apoptosis calculated by flow cytometry, the activity of the PI3K/AKT/FOXO1 signaling pathway and the expressions of its downstream apoptosis- and cycle-related proteins determined by Western blot. RESULTS: CFF-1 significantly arrested the cell cycle in the G1 phase, decreased the cell viability and increased the nuclear condensation and disruption in a dose-dependent manner, and elevated the apoptosis rate of prostate cancer cells. At the molecular level, CFF-1 dose-dependently reduced the activity of the PI3K/AKT signaling pathway and phosphorylation of the FOXO1 protein, increased the transcription activity of FOXO1, and eventually regulated the expressions of cell apoptosis- and cycle-related genes. CONCLUSIONS The Chinese medicinal compound CFF-1 can significantly inhibit the growth, arrest the cycle, and induce the apoptosis of prostate cancer cells by decreasing the activity of the PI3K/AKT/FOXO1 signaling pathway, which suggests its potential clinical application value in the treatment of prostate cancer.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Drugs, Chinese Herbal ; pharmacology ; Forkhead Box Protein O1 ; metabolism ; Humans ; Male ; Neoplasm Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum (ER) stress.

METHODSA model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxo1 at the protein level. The expression of Bim, CHOP and Foxo1 at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim.

RESULTSTreatment of the melanoma cells with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin (3 µmol/L) for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37 ± 0.05, 0.13 ± 0.02 and 0.02 ± 0.01 in Mel-RM cells, while 0.41 ± 0.06, 0.16 ± 0.04 and 0.21 ± 0.03 in MM200 cells, respectively. The expression of Bim mRNA was significantly reduced compared with that in the control groups of the two cell lines (P < 0.01). siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was (5.69 ± 0.38)%, significantly lower than that of the siRNA control group (40.32 ± 1.64)% and blank control group (35.46 ± 2.01)% (P < 0.01). In the melanoma cells after exposure to tunicamycin (3 µmol/L) for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxo1 at mRNA level significantly decreased and the expressions at protein level were down-regulated, too.

CONCLUSIONSThe lack of Bim up-regulation contributes to the resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions. Transcription factors CHOP and Foxo1 may be responsible for the dysregulation of Bim in melanoma cells upon ER stress.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Bcl-2-Like Protein 11 ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Endoplasmic Reticulum Stress ; drug effects ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; genetics ; metabolism ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Melanoma ; genetics ; metabolism ; pathology ; Membrane Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transcription Factor CHOP ; genetics ; metabolism ; Tunicamycin ; pharmacology