RRM2 Regulates Bcl-2 in Head and Neck and Lung Cancers: A Potential Target for Cancer Therapy

Knockdown of RRM2 induces apoptosis via the mitochondria-mediated intrinsic pathway. A, Tu212 (left) and A549 (right) cells were transfected with siC or siR2. After 72 hours, cell lysates were analyzed by Western blotting with the indicated antibodies. A single set of blots is pictured from three independent experiments. B, Tu212 (top) and A549 (bottom) cells were transfected with siC or different siRNAs against RRM2, siR2, siR2-1, and siR2-2. After 72 hours, cell lysates were analyzed by Western blotting with the indicated antibodies. A single set of blots is pictured from three independent experiments. C, mitochondrial fraction (MF) and cytosolic fraction (CF) of Tu212 and A549 cells were isolated 72 hours after transfection with 5 nmol/L siR2, 5 nmol/L siC, or no treatment (NT). Western blotting was conducted to detect Cytochrome C and Cox-4.

Apoptosis induction by knockdown of RRM2 is Bcl-2 dependent. A, cell lysates of Tu212 and A549 were collected 72 hours after transfection with 5 nmol/L siC or siR2. Western blotting was conducted to detect antiapoptotic Bcl-2 family proteins. B, cell lysates of Tu212 and A549 were collected 72 hours after transfection with 5 nmol/L siC or different siRNAs against RRM2, siR2, siR2-1, and siR2-2. Western blotting was conducted to detect Bcl-2 protein. C, apoptosis analysis (error bars, mean ± SD from three independent experiments). D, Western blotting was conducted with specific antibodies 72 hours after transfection with 5 nmol/L siC or siR2 in Tu212 and Bcl-2 overexpressing Tu212 (Tu212/Bcl-2) cell lines.

Apoptosis induction by knockdown of RRM2 is p53, p73, and Akt independent. A, Western blotting for p53 and p73 expression in Tu212 and A549 cells 72 hours after transfection with siC or siR2. B, Western blotting for the indicated proteins in A549 and p53-knockdown A549 (A549/sh p53) cell lines after transfection with 5 nmol/L siC or siR2. A representative blot of three independent experiments is presented. C, apoptosis analysis (error bars, mean ± SD from three independent experiments). D, Western blot analysis 72 hours after transfection with 5 nmol/L of siC or siR2 in H1299 and two clones of dominant-negative p73-expressing H1299 (H1299/dN p73 Cl-7, and Cl-10) cell lines. E, Western blotting for p-Akt and Akt in Tu212 and A549 cells 72 hours after transfection with siC or different concentrations of siR2. F, apoptosis analysis. G, Western blot of whole-cell lysates from A549 and Akt-overexpressing A549 (A549/Akt) cell lines 72 hours after siRNA transfection.

RRM2 regulates Bcl-2 through direct protein–protein interaction and partially influences Bcl-2 mRNA. A, RT-PCR for Bcl-2 and GAPDH mRNA levels 72 hours after transfection of Tu212 and A549 cells with 5 nmol/L siC or siR2. RRM2 was normalized to GAPDH levels within the same sample. B, Tu212 cells were stained with anti-RRM2 (green) and anti-Bcl-2 (red), and nuclei were counterstained with DAPI (blue). A z-stack of optical sections was created at 0.59-μm intervals using a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.). Inset, top shows magnification of white box in merged figure. Inset, bottom shows z-axis reconstructions along the bars indicated in inset top. Bar, 50 μm. (C) Tu212 and (D) A549 cells were stained with anti-RRM2 (green), anti-Bcl-2 (red), and nuclei (blue) 48 hours after transfection with 5 nmol/L siC or siR2. Bar, 50 μm. E, Co-immunoprecipitation (IP) with anti-RRM2 (left) or anti-Bcl-2 (right) and immunoblotting with anti-Bcl-2 or anti-RRM2 in Tu212 and A549 cells. IM, immunoblotting.

Depletion of RRM2 reduces Bcl-2 protein expression and regulates protein stability. A and B, Tu212 cells were transfected with 5 nmol/L siC or siR2, reseeded after 24 hours into 60-mm dishes, and 24 hours later treated with cyclohexamide (100 μg/mL) for 0, 1, 3, 6, 9, and 12 hours. Cell lysates were collected at indicated time points, and Western blotting was conducted with specific antibodies. C, Tu212 and A549 cells were transfected with 5 nmol/L siC or siR2 and 24 hours later treated with 10 μmol/L MG132 for 2 hours before analyzing cell lysates by Western blotting. D, Bcl-2 expression was detected in xenograft tumor tissue by IHC analysis. The animal study was conducted previously by treating with 4 doses of siCON1 (control nanoparticle) or CALAA-01 (RRM2 siRNA-nanoparticle). Representative images shown from siCON1 and CALAA-01 10 mg/kg groups (brown stain for Bcl-2 and nuclei were counterstained by hematoxylin, blue; magnification ×200). IB, immunoblot.