Azurin Synthesis from Pseudomonas Aeruginosa MTCC 2453, Properties, Induction of Reactive Oxygen Species, and p53 Stimulated Apoptosis in Breast Carcinoma Cells

Sankar Ramachandran,Abhijit Mazumadar,Mahitosh Mandal,Siddik Sarkar

doi:10.4172/1948-5956.1000069

Abstract

Breast cancers are usually treated with surgery and radiation excretes adverse effects. Azurin, a potent anticancer redox protein secreted by Pseudomonas aeruginosa (P. aeruginosa) species has been reported to have activity against breast cancer cell lines; this had prompted researchers to search for novel methods to enhance this protein’s production. Researchers previously have reported on the synthesis of blue copper protein azurin from different microbial sources specifically from P. aeruginosa. Our investigation used customized methods to focus on synthesizing azurin from different strains of P. aeruginosa with apparent homogeneity. We screened the growth of different P. aeruginosa strains (1934, 741, 2453, and 1942) for the synthesis of azurin and for enhanced azurin production. We exposed azurin properties using matrix-assisted laser desorption/ionization, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Fourier transform infrared spectroscopy. Additional studies of possible molecular mechanisms and reactive oxygen species (ROS) generation of P. aeruginosa 2453 secreted azurin are needed. We examined which strain among P. aeruginosa strains 1934, 741, 2453, and 1942 best enhanced azurin production. Our current study also revealed which strain of the four had the strongest antiproliferative effect of azurin. P. aeruginosa MTCC (Microbial Type culture collection) 2453 was the strain that secreted the most azurin and showed remarkable apoptosis in breast carcinoma cells like T- 47D and ZR-75-1. This study demonstrates customized methods to synthesize azurin from different strains of P. aeruginosa with apparent homogeneity and their apoptotic effects on breast carcinoma cells with possible molecular mechanisms and ROS.

Highlights

Among cancer incidence breast cancer comprises 10.4% among women than men alarmingly high
Cell cycle analysis of the azurin treated breast cancer cells was performed using fluorescence activated cell sorting (FACS) scan (Becton Dickinson Immunocytometery Systems California, USA ) we evaluated the intensity of cytotoxicity because of induction of apoptosis
The dialysate was blended with diethyl amino ethyl cellulose (DEAE) cellulose beads to remove the flavor proteins and unwanted proteins

Summary

Introduction

Among cancer incidence breast cancer comprises 10.4% among women than men alarmingly high. A redox protein, recently captured the interest of biomedical researchers as an anticancer therapeutic agent that can enter human breast cancer cells and induce apoptosis mediated by the tumor suppressor P53 protein [3]. Since azurin has been reported to be a potential anticancer protein against breast cancer cell lines [7], researchers are searching for novel methods to enhance its production of azurin. Synthesis of a pure microbial metabolite like azurin from P. aeruginosa strain 2453 reduces toxic effects in regression of cancer treatment. Our prospective study is focused on enhanced azurin synthesis from four different strains of P. aeruginosa (1934, 741, 2453, and 1942) with apparent homogeneity and stability by adding both CuSo4 and KNO3 in the culture medium. Our interest extended to analyze the differences in secondary structure of purified azurin from all strains and its impact on apoptosis with possible molecular mechanisms and ROS generation in breast carcinoma cells

Materials and Methods

Results

Discussion