Journal: International Journal of Molecular Sciences
Article Title: ABT-751 Induces Multiple Anticancer Effects in Urinary Bladder Urothelial Carcinoma-Derived Cells: Highlighting the Induction of Cytostasis through the Inhibition of SKP2 at Both Transcriptional and Post-Translational Levels
Figure Lengend Snippet: ABT-751 inhibits SKP2 transcription and subsequent translation in UBUC cells through dysregulation of the AKT–CHUK–NFKBIA–NFKB signaling pathway. ( A ) BFTC905 cells were treated with ABT-751 (0.6 μM) for 24 h and the levels of the components of SKP2 E3 ligase (CDC20 and FZR1 proteins) were determined by immunoblots. ( B ) The cells were pretreated with 4 μM of MG132, a proteasome inhibitor, for 6 h, then treated with a specified concentration of ABT-751 (BFTC905, 0.6 μM and J82, 0.7 μM) for another 18 h. Next, nuclear/cytosol fractionation along with immunoblot analysis were performed. ( C ) The mRNA levels of SKP2 , TP 53, CDKN 1 A , CDKN 1 B , RB 1, E 2 F 1 and TFDP 1 were analyzed by quantitative RT-PCR. ( D ) The expression levels of the SKP2 gene regulator-associated proteins were determined by immunoblots in BFTC905 and J82 cells. Cells were treated with a specified concentration of ABT-751 (BFTC905, 0.6 μM and J82, 0.7 μM) for 24 h, ( E ) AKT1 kinase activity via a co-immunoprecipitation assay and ( F ) nuclear/cytosol fractionation along with immunoblot analysis were performed. ( G ) The cells were treated with a specified concentration of ABT-751 (BFTC905, 0.6 μM and J82, 0.7 μM) for 24 h, cell lysates were subjected to the co-immunoprecipitation assay with anti-RELA antibody. ( H ) Immunoblot analysis was used to compare the alterations of SKP2 -regulatory protein levels after treatment with a PI3K-AKT inhibitor, LY294002 (10 μM) for 2 h and ABT-751 (BFTC905, 0.6 μM and J82, 0.7 μM) for 24 h. ( I ) BFTC905 cells were treated with ABT-751 (0.6 μM) or LY294002 (10 μM) and SKP2 mRNA levels were analyzed by quantitative RT-PCR. ( J ) Transfection of a constitutive active AKT1 plasmid, pHRIG-AKT1, into BFTC905 cells for 24 h, suppressed AKT1-induced SKP2 mRNA levels. Relative protein levels were quantified using Image J software. The results are expressed as the means ± SD. Pan-actin, GAPDH and PARP1 served as loading, cytosolic and nuclear controls, respectively, in each gel. Statistical significance: * p
Article Snippet: Quantitative Reverse Transcription-Polymerase Chain Reaction Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was applied to quantify the mRNA expression levels of several genes using predesigned TaqMan® reagents from ThermoFisher (SKP2 : Hs01021864_m1 (59 bp); TP 53: Hs01034249_m1 (108 bp); CDKN 1A : Hs00355782_m1 (66 bp); CDKN 1B : Hs01597588_m1 (151 bp); RB 1: Hs01078066_m1 (72 bp); E 2F 1: Hs00153451_m1 (84 bp); TFDP 1: Hs00955488_g1 (102 bp); glyceraldehyde- 3-phosphate dehydrogenase (GAPDH ): Hs02758991_g1 (93 bp); CD 44 molecule, Indian blood group (CD 44): Hs01075861_m1 (70 bp); cadherin 1 (CDH 1): Hs01023895_m1 (80 bp); vimentin (VIM ): Hs00185584_m1 (73 bp); MTOR : Hs00234508_m1 (103 bp)) along with LightCycler® 96 System (Roche, Basel, Switzerland) and ΔΔCT calculation.
Techniques: Western Blot, Concentration Assay, Fractionation, Quantitative RT-PCR, Expressing, Activity Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Software