m aicar  (Toronto Research Chemicals)

Journal: Molecular Oncology

Article Title: Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer

doi: 10.1002/1878-0261.12370

Figure Lengend Snippet: Significant increase in number of apoptotic cells is dependent on drug concentration and time exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100 n m ), (B) AICAR (0–10 m m ), and (C) drug combinations (0/0–2/20, 4/40 and 10/100, m m /n m ) show that increase in number of apoptotic cells is dose‐dependent using annexin V‐ FITC conjugated to PI by flow cytometry. In addition, treatment of the cells with drug combinations for 24, 48, and 72 h (D) shows that increase in number of apoptotic cells is time‐dependent. Apoptotic data were confirmed in cells by measuring apoptotic protein expression. Lysates from cells treated with rapamycin (20 n m ), AICAR (2 m m ) or rapamycin+ AICAR (20 n m /2 m m ) for 72 h were subjected to Western blot analysis to measure PARP and caspase 3 cleavages. (E) Significant increase was detected in cleavage of PARP at 85 kD a and caspase 3 at 22 and 17 kDa in 786‐O cells treated with drug combinations compared with cells treated with each drug alone for 72 h. GAPDH was used as a loading control. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P < 0.01.

Article Snippet: Cells were treated with serial concentrations of AICAR (0, 2, 4, 10 m m ), rapamycin (0, 20, 40, 100 n m ) or drug combinations (0/0, 2/20, 4/40, 10/100, m m /n m , AICAR/rapamycin) for 72 h. Cells were treated with drug combinations (2 m m /20 n m , AICAR/rapamycin) for 24, 48, and 72 h. AICAR was obtained from Cayman Chemical (Ann Arbor, MI, USA) and rapamycin from Sigma‐Aldrich (St. Louis, MO, USA).

Techniques: Concentration Assay, Flow Cytometry, Expressing, Western Blot

Journal: Molecular Oncology

Article Title: Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer

doi: 10.1002/1878-0261.12370

Figure Lengend Snippet: Decrease in cell proliferation by drug combinations is dependent on the drug concentration and duration of exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100 n m ), (B) AICAR (0–10 m m ), and (C) drug combinations (0/0–2/20, 4/40 and 10/100, m m /n m ) show that the decrease in cell proliferation is dose‐dependent using the 3 H‐thymidine incorporation assay. In addition, treatment of the cells with drug combinations for 24, 48, and 72 h (D) show that the significant decrease in cell proliferation is time‐dependent. Cell proliferation data were confirmed in cells by measuring proliferative protein expression. Lysates from cells treated with rapamycin (20 n m ), AICAR (2 m m ) or rapamycin+ AICAR (20 n m /2 m m ) for 72 h were subjected to Western blot analysis to measure PCNA and cyclin D1. (E) A significant decrease in expression of PCNA and cyclin D1 was detected in cells treated with a single drug, whereas drug combinations abolished the expression of both proteins detected in cells, providing evidence of the additive effect of drug combinations in reducing cell proliferation. A combination of drugs abolished Akt survival kinase and blocked HIF ‐2α and VEGF in 786‐O cells. Cells were treated with rapamycin (20 n m ), AICAR (2 m m ) or drug combination (20 n m rapamycin/2 m m AICAR ) for 72 h. Cell lysates were subjected to Western blot analysis to measure p‐Akt, HIF ‐2α, and VEGF expression. (F, G) A significant decrease in p‐Akt, HIF ‐2α, and VEGF expression was seen in cells treated with single drug; however, the additive effect of drug combinations showed complete abolishment in HIF ‐2α and VEGF as well as in phosphorylation of Akt at Ser 473 expression compared with cells treated with single drug and control cells. GAPDH was used as a loading control. Further evidence of the effect of drug combinations on HIF ‐2α function was measured by luciferase assay. (H) The additive effect of drug combinations caused a more than 90% decrease in the promoter activity of HIF ‐2α compared with control cells using the Luciferase Reporter Assay System normalized to Renilla activity and measured by a luminometer. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P < 0.01.

Article Snippet: Cells were treated with serial concentrations of AICAR (0, 2, 4, 10 m m ), rapamycin (0, 20, 40, 100 n m ) or drug combinations (0/0, 2/20, 4/40, 10/100, m m /n m , AICAR/rapamycin) for 72 h. Cells were treated with drug combinations (2 m m /20 n m , AICAR/rapamycin) for 24, 48, and 72 h. AICAR was obtained from Cayman Chemical (Ann Arbor, MI, USA) and rapamycin from Sigma‐Aldrich (St. Louis, MO, USA).

Techniques: Concentration Assay, Thymidine Incorporation Assay, Expressing, Western Blot, Luciferase, Activity Assay, Reporter Assay

Journal: Molecular Oncology

Article Title: Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer

doi: 10.1002/1878-0261.12370

Figure Lengend Snippet: Drug combinations significantly decreased cell migration and cell invasion of kidney cancer cells. 786‐O cells treated with rapamycin (20 n m ), AICAR (2 m m ) or a drug combination (20 n m rapamycin/2 m m AICAR ) for 72 h were added into the upper well chamber of a 96‐well plate. (A, B) Cells treated with drug combination have a significantly low number of migrated cells and (C, D) invaded cells compared with cells treated with a single drug or control cells. The total number of migrated and invaded cells was counted using counting software and the images of migrated or invaded cells were taken using a Nikon light inverted microscope. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P < 0.01.

Article Snippet: Cells were treated with serial concentrations of AICAR (0, 2, 4, 10 m m ), rapamycin (0, 20, 40, 100 n m ) or drug combinations (0/0, 2/20, 4/40, 10/100, m m /n m , AICAR/rapamycin) for 72 h. Cells were treated with drug combinations (2 m m /20 n m , AICAR/rapamycin) for 24, 48, and 72 h. AICAR was obtained from Cayman Chemical (Ann Arbor, MI, USA) and rapamycin from Sigma‐Aldrich (St. Louis, MO, USA).

Techniques: Migration, Software, Inverted Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation

doi: 10.1074/jbc.RA117.001254

Figure Lengend Snippet: Mitochondria-targeted paraquat induces insulin resistance downstream of Akt. A and B , 3T3-L1 adipocytes ( A ) or L6 myotubes ( B ) overexpressing HA-GLUT4 and PDGFR were serum-starved, treated with DMSO or the indicated doses of MitoPQ ( mPQ ) for 2 h, and assessed for plasma membrane–localized HA-GLUT4 following incubation without ( basal ) or with 100 n m insulin, 20 ng/ml PDGF, or 2 m m AICAR and 100 μ m A-769662 ( A769 ) for 20 min where indicated ( n = 4, mean ± S.E. ( error bars ), two-sample t test corrected for multiple comparisons; *, p < 0.05; comparisons as indicated). C , 3T3-L1 adipocytes ( left ) and L6 myotubes ( right ) were serum-starved and treated with DMSO or the indicated doses of MitoPQ for 2 h and insulin where indicated. Phosphorylation status of indicated sites was assessed by immunoblot. Immunoblots were quantified by densitometry and normalized to insulin-treated control cells (indicated by the dotted line ) ( n = 4, mean ± S.E., one-sample t test; *, p < 0.05). D , insulin resistance was induced in 3T3-L1 adipocytes by treatment with 10 n m insulin ( CI ) for 24 h or 2 ng/ml TNFα ( TNF ) for 96 h and in adipose tissue by 14-day HFHSD feeding. 3T3-L1 adipocytes or adipose tissue explants were serum-starved for 2 h and incubated without or with 100 n m insulin for 20 min. Phosphorylation status of the indicated sites was assessed by immunoblotting. Immunoblots were quantified by densitometry and normalized to insulin-treated control cells (indicated by the dotted line ) (insulin and TNFα, n = 3; HFHSD, n = 6; mean ± S.E., one-sample t test; *, p < 0.05). E and F , 3T3-L1 adipocytes were serum-starved and treated with DMSO or the indicated doses of MitoPQ for 2 h, and protein synthesis was assessed by incorporation of [ 3 H]leucine into protein in the presence and absence of 100 n m insulin ( n = 4, mean ± S.E., two-sample t test corrected for multiple comparisons) ( E ), or lipolysis was assessed via release of glycerol into culture medium after stimulation with 1 n m isoproterenol with and without incubation with 100 n m insulin ( n = 4, mean ± S.E., two-sample t test corrected for multiple comparisons) ( F ).

Article Snippet: Glucose uptake was assessed following the addition of [ 3 H]2DOG (final concentration 0.375 μCi/ml) and [ 14 C]mannitol (0.05 μCi/ml) with or without 10 n m insulin or 2 m m AICAR (Toronto Research Chemicals).

Techniques: Incubation, Western Blot