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Evaluation of Anticancer and Anti-hemolytic Activity of Azurin, a Novel Bacterial Protein from Pseudomonas aeruginosa SSj

  • M. C. Sereena
  • Denoj SebastianEmail author
Article
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Abstract

Cancer is the second leading cause of death in the world. About 18 milllion people were affected with cancer in 2018. Research in novel anti-cancer agents of bacterial origin thus gain importance. Azurin is a novel bacterial protein that has unique ability to specifically target cancer cells without any harmful effect to normal cells. In this study, cytotoxic effect of azurin from Pseudomonas aeruginosa SSj expressed in pET22b (+) vector and standard azurin (Sigma) were studied on (MCF7), (HCT15), and (VERO) cell lines by MTT assay. Apoptosis assay was performed using Hoechst staining and confirmed by FRET based assay on colon cancer cell lines (SW480). In vivo ascites antitumor model assay was also done to detect the in vivo cytotoxicity of azurin. Anti-hemolytic potential of purified azurin on human erythrocytes was also evaluated. Azurin induced cytotoxicity in both HCT15 and MCF7 cell lines, without having any adverse effect on the normal cells. Cell cycle analysis by Hoechst staining and FRET analysis, both showed evidence of apoptosis induced by azurin. In vivo ascites antitumor model proved that azurin treatment can increase the lifespan of tumor-bearing mice. Anti-hemolytic assay showed a significant increase of inhibition of hemolysis.

Keywords

Azurin MTT assay FRET Hemolytic assay SW 480 

Abbreviations

MCF7

Michigan Cancer Foundation-7

HCT 15

Human colorectal carcinoma

SW480

Spectral karyotyping of the human colon cancer cell line

MTT

3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyl tetrazolium bromide

DMEM

Dulbecco’s modified Eagle medium

FBS

Fetal bovine serum

PBS

Phosphate-buffered saline

DLA

Daltons lymphoma ascites

EYFP

Enhanced yellow fluorescent protein

ECFP

Enhanced cyan fluorescent protein

DAPI

4′,6-Diamidino-2-phenylindole

FSC

Forward scatter

SSC

Side scatter

FRET

Fluorescence resonance energy transfer

DEVD

Aspartic acid–glutamic acid–valine–aspartic acid

Notes

Compliance with Ethical Standards

Conflict of interest

Authors have no conflict of interest regarding the publication of paper.

Research Involving Animal Rights

All applicable institutional guidelines for the care and use of animals were followed.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Life SciencesUniversity of CalicutThenjipalamIndia

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