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Antagonizing canonical Wnt signaling pathway by recombinant human sFRP4 purified from E. coli and its implications in cancer therapy

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Abstract

The Wnt signaling pathway plays a predominant role in aberrant proliferation in myriad of cancers. In non-cancerous cells, Wnts are blocked by the secreted frizzled-related proteins (sFRPs) that are generally downregulated in cancer cells. We have purified and characterized bacterially expressed glutathione S-transferase-tagged SFRP4 from a novel clone generated from human cell origin. Cervical cancer (HeLa) and lung cancer (A549) cells, in which Wnt and associated genes were found to be expressed, were treated with the purified recombinant sFRP4, which revealed a significant dose-dependent cell growth inhibition up to 40 %. The current investigation on functionality of this bacterially produced recombinant sFRP4 in arresting cancer cell proliferation is the first of its kind, where G2/M phase arrest and early apoptosis were evident. Increase in phosphorylated β-catenin in sFRP4 treatment indicated inhibition of Wnt pathway, which was further confirmed by downregulation of pro-proliferative genes, namely cyclin D1, c-myc, and survivin. Functional activity of recombinant sFRP4 was further exploited in co-therapy module with chemotherapeutic drugs to decipher molecular events. Collectively, our study on purified recombinant sFRP4 from bacterial host holds great promise in targeting Wnt signaling for exploring new strategies to combat cancer.

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Abbreviations

sFRP:

Secreted frizzled-related protein

GST:

Glutathione S-transferase

CRD:

Cysteine-rich domain

NLD:

Netrin-like domain

MALDI TOF:

Matrix-assisted laser desorption/ionization time of flight

DMEM:

Dulbecco’s modified Eagle’s medium

cDNA:

Complementary deoxyribonucleic acid

RNA:

Ribonucleic acid

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

PBS:

Phosphate-buffered saline

PMSF:

Phenylmethylsulfonyl fluoride

EDTA:

Ethylenediaminetetraacetic acid

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

HRP:

Horseradish peroxidase

HCl:

Hydrochloric acid

PI:

Propidium iodide

FACS:

Fluorescence-activated cell sorting

IAP:

Inhibitor of apoptosis

FITC:

Fluorescein isothiocyanate

AO:

Acridine orange

EB:

Ethidium bromide

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Acknowledgments

Authors acknowledge financial supports from the Department of Biotechnology Programme Support (BT/PR13560/COE/34/44/2015) and the Department of Electronics and Information Technology [No. 5(9)/2012-NANO (Vol. II)]. Authors also acknowledge the support of the Centre for Nanotechnology and the Central Instruments Facility, IIT Guwahati.

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Authors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 39, India

    Archita Ghoshal & Siddhartha Sankar Ghosh

  2. Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, 39, India

    Siddhartha Sankar Ghosh

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  1. Archita Ghoshal
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Ghoshal, A., Ghosh, S.S. Antagonizing canonical Wnt signaling pathway by recombinant human sFRP4 purified from E. coli and its implications in cancer therapy. Mol Cell Biochem 418, 119–135 (2016). https://doi.org/10.1007/s11010-016-2738-6

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