Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan 8;11(1):e0145780.
doi: 10.1371/journal.pone.0145780. eCollection 2016.

BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

Samra Hafeez  1 , Mahwish Urooj  1 , Shamiala Saleem  1 , Zeeshan Gillani  1 , Sumaira Shaheen  1 , Mahmood Husain Qazi  1 , Muhammad Imran Naseer  2 , Zafar Iqbal  3 , Shakeel Ahmed Ansari  2 , Absarul Haque  4 , Muhammad Asif  5 , Manzoor Ahmad Mir  6 , Ashraf Ali  4 , Peter Natesan Pushparaj  2 , Mohammad Sarwar Jamal  4 , Mahmood Rasool  2
Affiliations

BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

Samra Hafeez et al. PLoS One. .

Abstract

This study has been undertaken to explore the therapeutic effects of deguelin and specific siRNAs in HeLa cells. The data provided clearly show the silencing of ERK 1/2 with siRNAs and inhibition of ERK1/2 with deguelin treatment in HeLa cells. Additionally, we are providing information that deguelin binds directly to anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 in the hydrophobic grooves, thereby releasing BAD and BAX from dimerization with these proteins. This results in increased apoptotic activity through the intrinsic pathway involved in rupture of mitochondrial membrane and release of cytochrome C. Evidence for inhibition of ERK1/2 by deguelin and escape of BAD phosphorylation at serine 112 through ERK/RSK pathway has been further fortified by obtaining similar results by silencing ERK 1/2 each with specific siRNAs. Increase in BAD after treatment with deguelin or siRNAs has been interpreted to mean that deguelin acts through several alternative pathways and therefore can be used as effective therapeutic agent.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared no competing interests exist.

Figures

Fig 1
Fig 1. MTT Assay in HeLa cells.
The MTT assay was carried out after plating HeLa cells at a density of 5000 cells/well in 96 well plates. The cells were treated with different doses of deguelin. It shows a regression curve of cell viability at the indicated doses.
Fig 2
Fig 2. Docking of deguelin on anti-apoptotic proteins.
(A) Structure of deguelin arrow indicates the binding of deguelin on Bcl-2 (B) The docking of deguelin on BH3 domain of hydrophobic group of anti-apoptotic protein Bcl-xl (C) Binding of deguelin on MCL-1 (D) Binding of deguelin molecule on ERK-1 near the ATP binding site. Docking was carried out using bio-informative tools as described in materials and methods.
Fig 3
Fig 3. The binding of deguelin molecule on ERK-1 and ERK-2 near the ATP binding site.
Docking was carried out using bio-informative tools as described in materials and methods. It may be observed that the molecule binds to ERK1/2 in the ATP pocket, thereby, preventing the phosphorylation of ERK.
Fig 4
Fig 4. The figure shows the blotting of various proteins isolated from normal cells and those treated with various doses of either deguelin or transfected with siRNAs specific for silencing of ERK1/2.
(A) Cells transfected with various doses of ERK-1 specific siRNA. (B) Cells transfected with various doses of ERK-2 specific siRNA. (C) BAD expression in cells transfected with either ERK-1 or ERK-2 siRNAs. (D) ERK-1 and ERK-2, (E) BAD, (F) Bcl-xl, (G) BAX and (H) Cytochrome-C levels in cells treated with various doses of deguelin. (I) The house-keeping control (β-actin).
See this image and copyright information in PMC

References

    1. Chun K-H, Kosmeder JW, Sun S, Pezzuto JM, Lotan R, et al. (2003) Effects of deguelin on the phosphatidylinositol 3-kinase/Akt pathway and apoptosis in premalignant human bronchial epithelial cells. Journal of the National Cancer Institute 95: 291–302. - PubMed
    1. Yang Y, Ji C, Bi Z-g, Lu C-c, Wang R, et al. (2013) Deguelin induces both apoptosis and autophagy in cultured head and neck squamous cell carcinoma cells. PloS one 8: e54736 10.1371/journal.pone.0054736 - DOI - PMC - PubMed
    1. Wang Y, Ma W, Zheng W (2013) Deguelin, a novel anti-tumorigenic agent targeting apoptosis, cell cycle arrest and anti-angiogenesis for cancer chemoprevention (Review). Molecular and clinical oncology 1: 215–219. - PMC - PubMed
    1. Thamilselvan V, Menon M, Thamilselvan S (2011) Anticancer efficacy of deguelin in human prostate cancer cells targeting glycogen synthase kinase‐3 β/β‐catenin pathway. International Journal of Cancer 129: 2916–2927. - PubMed
    1. Kang HW, Kim JM, Cha MY, Jung HC, Song IS, et al. (2012) Deguelin, an Akt inhibitor, down-regulates NF-κB signaling and induces apoptosis in colon cancer cells and inhibits tumor growth in mice. Digestive diseases and sciences 57: 2873–2882. 10.1007/s10620-012-2237-x - DOI - PubMed

Publication types

MeSH terms