Journal: Molecular Medicine Reports

Article Title: Metformin inhibits growth of human non-small cell lung cancer cells via liver kinase B-1-independent activation of adenosine monophosphate-activated protein kinase

doi: 10.3892/mmr.2016.4830

Figure Lengend Snippet: Metformin activated AMPK and inhibited the mTOR signaling pathway in H460 and H1299 cells. The H460 and H1299 cell lines were treated with 10 mM metformin for the indicated times or treated with the indicated concentrations of metformin for 6 h. Following treatment, protein extracts were examined by western blot for p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin protein expression levels. Data are representative of a minimum of three independent experiments. p, phosphorylated; AMPK, adenosine monophosphate-activated protein kinase; mTOR, mammalian target of rapamycin; p70S6K, 70-kDa ribosomal protein S6 kinase.

Article Snippet: Primary antibodies against p-mTOR, mTOR, p-70-kDa ribosomal protein S6 kinase (p70S6K) and p-p70S6K were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA).

Techniques: Western Blot, Expressing

Journal: Molecular Medicine Reports

Article Title: Metformin inhibits growth of human non-small cell lung cancer cells via liver kinase B-1-independent activation of adenosine monophosphate-activated protein kinase

doi: 10.3892/mmr.2016.4830

Figure Lengend Snippet: Knockdown of AMPK with siRNA reversed the effects of metformin on non-small cell lung cancer cells. (A) Cells were treated with 10 mM metformin for 6 h after transfection with si-AMPK, and examined by western blot for p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin protein expression levels. Data are representative of a minimum of three independent experiments. (B) Cells were transfected with si-AMPK or si-NC. Following transfection, at 24 h, cells were treated with 0, 5, 10 or 20 mM metformin for 48 h, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess cell viability. Data from three independent experiments are presented as the mean ± standard error of the mean. * P<0.05 vs. the si-NC group. p, phosphorylated; AMPK, adenosine monophosphate-activated protein kinase; met, metformin; mTOR, mammalian target of rapamycin; p70S6K, 70-kDa ribosomal protein S6 kinase; si, small interfering; NC, negative control.

Article Snippet: Primary antibodies against p-mTOR, mTOR, p-70-kDa ribosomal protein S6 kinase (p70S6K) and p-p70S6K were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA).

Techniques: Transfection, Western Blot, Expressing, Negative Control

Journal: BioMed Research International

Article Title: δ -Tocotrienol Oxazine Derivative Antagonizes Mammary Tumor Cell Compensatory Response to CoCl 2 -Induced Hypoxia

doi: 10.1155/2014/285752

Figure Lengend Snippet: Effects of 150 µ M CoCl 2 (hypoxic, but not cytotoxic dose) alone and in combination with compound 44 ( 44 ), on mitogenic/survival signaling and hypoxic response marker protein levels in +SA mammary tumor cells. +SA cells were seeded at concentration of 1.5 × 10 6 in 100 mm culture dishes and allowed to attach overnight. The following day, cells were divided into groups and exposed to their respective treatments for a 24 hr incubation period. Afterwards, whole cell lysates were prepared for Western blot analysis for Akt, PI3K, phospho-Akt (p-Akt, Ser473), mTOR, phospho-mTOR (p-mTOR, Ser2448), HIF-1 α , phospho-p70S6K (p-p70S6K, Ser424), phospho-eIF-4E1 (p-eIF-4E1, Ser209), and phospho-4E-BP1 (p-4E-BP1, Thr37). Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding α -tubulin, as shown in the bar graphs below their respective Western blot image. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane ± SEM. * P < 0.05 as compared to the hypoxic CoCl 2 -treated controls.

Article Snippet: Antibodies for p-p70S6K (#GTX530304, Ser424), p-eIF-4E (#GTX50268, Ser209), and p-4E-BP1 (#GTX61987, Thr37) were purchased from Gene Tex Inc. (Irvine, CA, USA).

Techniques: Marker, Concentration Assay, Incubation, Western Blot

Journal: BioMed Research International

Article Title: δ -Tocotrienol Oxazine Derivative Antagonizes Mammary Tumor Cell Compensatory Response to CoCl 2 -Induced Hypoxia

doi: 10.1155/2014/285752

Figure Lengend Snippet: (a) Average +SA tumor volume in the treatment groups at the start (Day 0) and end (Day 11) of the treatment period. +SA mammary cells (1 × 10 6 ) suspended in 100 μ L of 0.05 M PBS were injected into the number 4 abdominal mammary fat pad of syngeneic female BALB/c mice. Once tumors reached 4-5 mm in diameter, mice were divided into different treatment groups (8 mice/group) and treated with intralesional injections of lipid nanoemulsion formulations of α -tocopherol ( α T), δ -tocotrienol ( δ T 3 ), or δ -tocotrienol oxazine derivative, compound 44 ( 44 ), at a dose of 0–120 μ g/20 μ L every other day throughout the experimental period. The untreated control group (C) was added to ensure that intralesional injection of the α -tocopherol nanoemulsion did not influence tumor growth in a nonspecific manner. Data points indicate the average tumor volume (cm 3 ± SEM) for 8 mice/group ± SEM in each treatment group. * P < 0.05, as compared with the α -tocopherol-treated negative control group. (b) Western blot analysis of HIF-1 α , phospho-Akt (p-Akt, Ser473), phospho-mTOR (p-mTOR, Ser2448), phospho-p70S6K (p-p70S6K, Ser424), phospho-eIF-4E1 (p-eIF-4E1, Ser209), phosphor-4E-BP1 (p-4E-BP1, Thr37), and phospho-ERK1/2 (p-ERK1/2, Thr202/Tyr204) in +SA mammary tumors grown in syngeneic BALB/c mice exposed to the various treatments. Lysates prepared from each tumor were separated by polyacrylamide gel electrophoresis (40 μ g/lane) followed by Western blot analysis. α -Tubulin was visualized to ensure equal sample loading in each lane. Each Western blot is a representative image of data obtained for experiments that were repeated at least three times. Scanning densitometric analysis was performed on all blots in triplicate and the optical density of each band was normalized with that of the corresponding α -tubulin, as shown in bar graphs. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane ± SEM. * P < 0.05, as compared with the α -tocopherol-treated negative control group. C: Untreated control; α -T: α -tocopherol-treated negative control; δ T 3 : δ -tocotrienol; 44 : δ -tocotrienol oxazine derivative, compound 44 .

Article Snippet: Antibodies for p-p70S6K (#GTX530304, Ser424), p-eIF-4E (#GTX50268, Ser209), and p-4E-BP1 (#GTX61987, Thr37) were purchased from Gene Tex Inc. (Irvine, CA, USA).

Techniques: Injection, Control, Negative Control, Western Blot, Polyacrylamide Gel Electrophoresis