RLIP76 Regulates PI3K/Akt Signaling and Chemo-Radiotherapy Resistance in Pancreatic Cancer
Figure 1
Comparison of RLIP76 levels in pancreatic cancer cells vs non-malignant cells.
Aliquots of crude membrane fractions of pancreatic cancer cells (BxPC-3 and Panc-1) and normal control cells (HUVEC) containing 100 µg protein were used for SDS-PAGE and Western blotting. Intensity of the full-length RLIP76 protein (∼95 kDa) band was quantified by scanning densitometry using Innotech Alpha Imager HP. β-actin was used as an internal control (panel A). Impact of anti-RLIP76 IgG, RLIP76 siRNA and RLIP76 antisense on normal and pancreatic cancer cells: Effect of anti-RLIP76 IgG (40 µg/ml final concentration) on the cell survival was determined by MTT assay [29], [30]. Depletion of RLIP76 expression by RLIP76 siRNA and RLIP76 antisense (each 10 µg/ml final concentration) was done, using Transmessenger Transfection-Reagent-kit (Qiagen), and Maxfect Transfection-Reagent (Molecula, Inc.), respectively, according to the manufacturer's instructions. Cell survival was measured by MTT cytotoxicity assay 48 h after treatment. The values are presented as mean ± SD from two separate determinations with eight-replicates each (n = 16), black bars, normal HUVEC cells; gray bars, BxPC-3 pancreatic cancer cells (panel B) * p<0.05, ** p<0.01 compared to respective controls. Effect of RLIP76 antisense on PI3K and Akt signaling in pancreatic cancer cells: RLIP76 antisense caused inhibition of PI3K/Akt pathway in BxPC-3 and Panc-1 cells. Cells were treated with 10 µg/ml of RLIP76 antisense for 24 h and immune-blotted for pPI3K, PI3K, pAkt, Akt, Bim, Bcl2, cyclin B1 and CDK4. The same blot was stripped and reprobed for GAPDH to ensure equal protein loading (panel C). Bar diagram shows the quantitation of respective Western blots. Dotted line represents no significant change as observed with scrambled antisense (panel D).