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NF-κB acts downstream of EGFR in regulating low dose cadmium induced primary lung cell proliferation

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Abstract

Apart from cytotoxicity cadmium has no special attributes towards cell’s physiological function. The role of cadmium with respect to cell growth is still under debate. Mitogen activated protein kinase and Ca2+/calmodulin dependent protein kinase dependent pathways are the two elaborately studied concerning cadmium induced cell proliferation. Low concentration of cadmium chloride (2.5 μM) was applied to mice primary lung epithelial cells and cell proliferation was measured both by cell cycle analysis and Brdu incorporation assay. Effects of differential dose of cadmium chloride on lung epithelial cells were evaluated morphologically by atomic force microscopy. RT-PCR and western blot altogether corroborated the specific signalling pathways concerning cadmium induced lung cell proliferation. Cadmium induced lung epithelial cells which over-expressed EGFR, were transfected with siEGFR, revealed downstream molecules and RNAi induced EGFR silencing. Use of siEGFR effectively prevents expression of proinflammatory and cell proliferative markers. Moreover N-acetyl cysteine and ascorbic acid mediated inhibition of EGFR and downstream signalling molecules indicate the involvement of reactive oxygen species. Exposure to low concentration of cadmium promotes the growth of primary mice lung epithelial cell by EGFR signalling. We have also transfected the primary lung epithelial cell with siRNA against the regulatory subunit of nuclear factor-κB (NF-κB) and the data shows that cadmium induced lung cell proliferation is the effect of EGFR mediated NF-κB activation.

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Abbreviations

CaMKII:

Ca2+/calmodulin-dependent protein kinase-II

c-Myc:

Myelocytomatosis viral oncogene homolog

Cox-2:

Cyclooxygenase-2

EGFR:

Epidermal growth factor receptor

Erk:

Extracellular signal-regulated kinases

IL-1β:

Interleukin-1β

IL-6:

Interleukin-6

Jnk:

c-Jun N-terminal kinase

MAPK:

Mitogen activated protein kinase

MMP:

Matrix metalloproteinases

NAC:

N-Acetyl cysteine

NF-κB:

Nuclear factor-κB

STAT3:

Signal transducer and activator of transcription-3

TNF-α:

Tumor necrosis factor-α

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Acknowledgments

Authors want to thank Mr. Soham Mitra for his help in immunofluorescence and western blot study. Authors are very much grateful to Mr.Tarun Keswani who helped a lot in cell cycle data analysis and Mr. Samir Jana and Mr.Subir Biswas, for their continuous help during cell maintenance and siRNA experiments. Authors would like to thank Dr. Tarak Das Basu and Lab members (University of Kalyani) for their immense help in providing AFM. Mr Santanu Paul helped us during statistical data analysis. Mr. Saiful Alam Mir provided some valuable protease inhibitors. Authors heartily thank Miss Suranjana Goswami for her valuable contribution in rewriting the manuscript and language correction. This work is fully supported by the grant of Indian Council of Medical Research (ICMR), Govt. of India, and sanctioned project 3/2/2/166/2008/NCD-III. This work was partly supported by grants from Department of Science and Technology, Govt. of India (SR/FT/L-42/2006). Also we like to thank Department of Biotechnology, Govt.of India (BT/PR9779/GBD 27/67/2007).

Conflict of interest

The authors have no competing interests.

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Authors and Affiliations

  1. Department of Zoology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India

    Subhadip Kundu, Suman Sengupta & Arindam Bhattacharyya

  2. Department of Environmental Science, University of Kalyani, Kalyani, 241235, West Bengal, India

    Subhadip Kundu, Suman Sengupta & Arindam Bhattacharyya

Authors
  1. Subhadip Kundu
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  2. Suman Sengupta
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  3. Arindam Bhattacharyya
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Correspondence to Arindam Bhattacharyya.

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Supplementary Fig. 1

Application of natural antioxidant AA. a Ascorbic acid down regulated the expression of EGFR and its downstream molecules indicated cadmium induced ROS generation and up regulation of these molecules are shown by western blot result. Bar graphs indicated the values of above figures represented by relative density of the bands normalized to β-actin. Data represent three independent experiments (*p < 0.05). b ELISA of specific cytokines depicted that ROS production controlled their regulations in cadmium-induced lung EC. Bar graphs indicated the values of above figures represented by relative density of the bands normalized to β-actin. Data represent three independent experiments with (*p < 0.05; **p < 0.01) (JPEG 40 kb)

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Kundu, S., Sengupta, S. & Bhattacharyya, A. NF-κB acts downstream of EGFR in regulating low dose cadmium induced primary lung cell proliferation. Biometals 26, 897–911 (2013). https://doi.org/10.1007/s10534-013-9666-7

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  • DOI: https://doi.org/10.1007/s10534-013-9666-7

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