Molecular screening and genetic diversity analysis of anticancer Azurin-encoding and Azurin-like genes in human gut microbiome deduced through cultivation-dependent and cultivation-independent studies
Azurin, a bacteriocin produced by a human gut bacterium Pseudomonas aeruginosa, can reveal selectively cytotoxic and induce apoptosis in cancer cells. After overcoming two phase I trials, a functional region of Azurin called p28 has been approved as a drug for the treatment of brain tumor glioma by FDA. The present study aims to improve a screening procedure and assess genetic diversity of Azurin genes in P. aeruginosa and Azurin-like genes in the gut microbiome of a specific population in Vietnam and global populations. Firstly, both cultivation-dependent and cultivation-independent techniques based on genomic and metagenomic DNAs extracted from fecal samples of the healthy specific population were performed and optimized to detect Azurin genes. Secondly, the Azurin gene sequences were analyzed and compared with global populations by using bioinformatics tools. Finally, the screening procedure improved from the first step was applied for screening Azurin-like genes, followed by the protein synthesis and NCI in vitro screening for anticancer activity. As a result, this study has successfully optimized the annealing temperatures to amplify DNAs for screening Azurin genes and applying to Azurin-like genes from human gut microbiota. The novelty of this study is the first of its kind to classify Azurin genes into five different genotypes at a global scale and confirm the potential anticancer activity of three Azurin-like synthetic proteins (Cnazu1, Dlazu11, and Ruazu12). The results contribute to the procedure development applied for screening anticancer proteins from human microbiome and a comprehensive understanding of their therapeutic response at a genetic level.
KeywordsAnticancer proteins Azurin Cancer therapy Genetic diversity Microbiome NCI in vitro screening
Authors would like to acknowledge the British Council under the frame of HAPIE and TEAM-SIE projects (www.vuheie.org) to CHL, MP, and VDN for enhancing the partnership among higher education institutions, companies, and hospitals in Vietnam and the UK. We would also like to give special thanks to Professor Arsénio M. Fialho at the Instituto Superior Técnico, University of Lisbon, Portugal, for his valuable comments and contribution to the manuscript, and Professor Simeon Keates at the Faculty of Engineering and Science, University of Greenwich, UK, for the research administration support.
This work was supported by a grant no. 106-YS.04-2014.40 from the Vietnam National Foundation for Science and Technology Development (NAFOSTED) to VDN.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Research and Education Committee of Nha Trang University (REC-NTU) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants or their parents as minors (less than 16 years old) included in the study.
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