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Supplementary Figures 1 - 6 from RRM2 Regulates Bcl-2 in Head and Neck and Lung Cancers: A Potential Target for Cancer Therapy

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posted on 2023-03-31, 17:51 authored by Mohammad Aminur Rahman, A.R.M. Ruhul Amin, Dongsheng Wang, Lydia Koenig, Sreenivas Nannapaneni, Zhengjia Chen, Zhibo Wang, Gabriel Sica, Xingming Deng, Zhuo (Georgia) Chen, Dong M. Shin

PDF file - 427K, Figure S1. (A) Western blotting was performed for the indicated proteins in Tu212 and Bcl-2 overexpressing Tu212 (Tu212/Bcl-2) cell lines (upper panel) and in Tu686 and Bcl-2 overexpressing Tu686 (Tu686/Bcl-2) cell lines (lower panel) (B) Apoptosis analysis (error bars are mean � SD from 3 independent experiments) of indicated cell lines upon treatment with siC or siR2. Figure S2. (A) Western blotting was performed for the indicated proteins in H1299 and two clones of dominant negative p73 overexpressing H1299 (H1299/dN p73 cl-7 and cl-10) cell lines (upper panel) and in H358 dominant negative p73 overexpressing H358 (H358/dN) cell lines (lower panel). (B) Apoptosis analysis (error bars are mean � SD from 3 independent experiments) of indicated cell lines upon treatment with siC or siR2. Figure S3. (A) Western blotting was performed for the indicated proteins in A549 and Akt overexpressing A549 (A549/Akt) cell lines (upper panel) and in Tu686 and Akt overexpressing Tu686 (Tu686/Akt) cell lines (lower panel) (B) Apoptosis analysis (error bars are mean � SD from 3 independent experiments) of indicated cell lines upon treatment with siC or siR2. Figure S4. Gallery images of a z-stack. Tu212 cells were stained with anti-RRM2 (green), anti-Bcl-2 (red) and 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 ) collected Inc.). A total of 32 images were collected, shown in a gallery of merged images collected in the z-stack in a single display. Figure S5. The relationship between RRM2 (IHC) and RRM2 (QD-IHF) in HNSCC. Spearman's correlation coefficient was estimated between RRM2 (IHC) and RRM2 (QD-IHF) expression level in tumor tissues from HNSCC patients. Figure S6. The relationship between RRM2 (IHC) and RRM2 (QD-IHF) in NSCLC. Spearman's correlation coefficient was estimated between RRM2 (IHC) and RRM2 (QD-IHF) expression levels in tumor tissues from NSCLC patients.

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ARTICLE ABSTRACT

Purpose: Ribonucleotide reductase subunit M2 (RRM2) plays an active role in tumor progression. Recently, we reported that depletion of RRM2 by systemic delivery of a nanoparticle carrying RRM2-specific siRNA suppresses head and neck tumor growth. The aim of this study is to clarify the underlying mechanism by which RRM2 depletion inhibits tumor growth.Experimental Design: siRNA-mediated gene silencing was carried out to downregulate RRM2. Immunoblotting, reverse-transcriptase PCR, confocal microscopy, tissue fractionation, gene overexpression and knockdown were employed to analyze critical apoptosis signaling. Conventional immunohistochemistry and quantum dot-based immunofluorescence were applied to detect RRM2 and Bcl2 expression and localization in tissue samples from patients and mice.Results: Knockdown of RRM2 led to apoptosis through the intrinsic pathway in head and neck squamous cell carcinoma (HNSCC) and non–small cell lung cancer (NSCLC) cell lines. We showed that Bcl-2 is a key determinant controlling apoptosis, both in vitro and in vivo, and that RRM2 depletion significantly reduces Bcl-2 protein expression. We observed that RRM2 regulates Bcl-2 protein stability, with RRM2 suppression leading to increased Bcl-2 degradation, and identified their colocalization in HNSCC and NSCLC cells. In a total of 50 specimens each from patients with HNSCC and NSCLC, we identified the colocalization of Bcl-2 and RRM2 and found a significant positive correlation between their expression in HNSCC (R = 0.98; P < 0.0001) and NSCLC (R = 0.92; P < 0.0001) tumor tissues.Conclusions: Our novel findings add to the knowledge of RRM2 in regulating expression of the antiapoptotic protein Bcl-2 and reveal a critical link between RRM2 and Bcl-2 in apoptosis signaling. Clin Cancer Res; 19(13); 3416–28. ©2013 AACR.

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    Clinical Cancer Research

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    Keywords

    Cell Death And Senescencebcl-2 pathwaysGene RegulationAntisense control of gene expressionHead, Neck & Oral CancersLung Cancer

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