Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

DNA damage and necroptosis induced by peroxidase from proso millet in human colorectal cancer cells

Xiaodong Cui¹ , Zhongqi Cao¹, Zhuanhua Wang^1,2

¹Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology; ²School of Life Science, Shanxi University, Taiyuan 030006, China.

For correspondence:- Xiaodong Cui Email: cuixiaodong@sxu.edu.cn Tel:+865317011499

Accepted: 30 April 2019 Published: 31 May 2019

Citation: Cui X, Cao Z, Wang Z. DNA damage and necroptosis induced by peroxidase from proso millet in human colorectal cancer cells. Trop J Pharm Res 2019; 18(5):975-983 doi: 10.4314/tjpr.v18i5.10

© 2019 The authors.
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited..

Abstract

Purpose: To investigate the effects of cationic peroxidase from proso millet (PmPOD) on DNA damage and necroptosis in human colon cancer HCT116 and HT29 cells.

Methods: Cell necroptosis and cell cycle was stained using Annexin V-FITC and cell cycle kits, respectively, and evaluated by flow cytometry. Lipid raft on the membrane was disrupted by cholesterol depletion and the location of PmPOD observed by confocal microscopy. Comet assays were used to detect DNA damage, and different inhibitors were also used. Knockdown of p53 or ectopic p53 expression in HCT116 cells were transfected p53 siRNA and pCMV3-TP53-myc plasmid, and p53 expression analyzed by western blotting.

Results: Pre-treatment of HCT116 and HT29 cell lines with the specific necroptosis inhibitor Nec-1 prevented PmPOD-induced necroptosis, whereas the apoptosis inhibitor, z-VAD-fmk, had no effect. The entry of PmPOD is necessary for induction of DNA damage and necroptosis. Furthermore, PmPOD induced cell cycle arrest at S phase, as well as DNA DSBs in vivo, as reflected by numerous γ-H2AX foci in CRC cells. However, the tumor suppressor protein, p53, alleviated PmPOD-induced DNA damage and necroptosis.

Conclusions: These results demonstrate that PmPOD-induced DNA DSBs in CRC cells is the main cause of necroptosis, and that the tumor suppressor protein, p53, alleviates PmPOD-induced necroptosis by promoting p53-mediated repair pathways.

Keywords: Cationic peroxidase, Necroptosis, DNA double strand breaks, Tumor suppressor protein p53, ^7;-H2AX