Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 21, 2017; 23(19): 3427-3439
Published online May 21, 2017. doi: 10.3748/wjg.v23.i19.3427
Oxidative stress-induced mitochondrial dysfunction in a normal colon epithelial cell line
Nandakumar Packiriswamy, Kari F Coulson, Susan J Holcombe, Lorraine M Sordillo
Nandakumar Packiriswamy, Kari F Coulson, Susan J Holcombe, Lorraine M Sordillo, Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, United States
Author contributions: Packiriswamy N, Coulson KF, Holcombe SJ and Sordillo LM designed the study; Packiriswamy N, Coulson KF and Holcombe SJ performed the experiments; Packiriswamy N, Coulson KF, Holcombe SJ, and Sordillo LM interpreted the data, prepared the manuscript, and approved the final version of the article to be published.
Supported by an endowment from the Matilda R. Wilson Fund in Detroit, Michigan.
Institutional animal care and use committee statement: The Animal Use and Care Committee at Michigan State University approved the protocols used in this study.
Conflict-of-interest statement: None of the authors have conflicts of interest to report.
Data sharing statement: No additional data are available for sharing.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Lorraine M Sordillo, Professor, Meadow Brook Chair, Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, 784 Wilson Road, East Lansing, MI 48824, United States. sordillo@msu.edu
Telephone: +1-517-4328821 Fax: +1-517-4328822
Received: December 12, 2016
Peer-review started: December 14, 2016
First decision: December 29, 2016
Revised: January 13, 2017
Accepted: March 15, 2017
Article in press: March 15, 2017
Published online: May 21, 2017
Processing time: 158 Days and 12.7 Hours
Abstract
AIM

To determine how a normal human colon cell line reacts to microbial challenge as a way to study oxidative stress-induced responses associated with inflammatory bowel disease.

METHODS

Normal human colon epithelial cells (ATCC® CRL.1790™) were stimulated with either heat killed E. coli or heat killed murine cecal contents (HKC) and examined for several relevant biomarkers associated with inflammation and oxidative stress including cytokine production, mitochondrial autophagy and oxidant status. TNFα, IL-1β and IL-8 protein concentrations were measured within the supernatants. Fluorescent microscopy was performed to quantify the production of reactive oxygen species (ROS) using an oxidation responsive fluorogenic probe. Mitochondrial morphology and mitochondrial membrane potential was assessed by dual staining using COXIV antibody and a dye concentrating in active mitochondria. Mitochondrial ROS scavenger was used to determine the source of ROS in stimulated cells. Autophagy was detected by staining for the presence of autophagic vesicles. Positive controls for autophagy and ROS/RNS experiments were treated with rapamycin and chloroquine. Mitochondrial morphology, ROS production and autophagy microscopy experiments were analyzed using a custom acquisition and analysis microscopy software (ImageJ).

RESULTS

Exposing CRL.1790 cells to microbial challenge stimulated cells to produce several relevant biomarkers associated with inflammation and oxidative stress. Heat killed cecal contents treatment induced a 10-12 fold increase in IL-8 production by CRL.1790 cells compared to unstimulated controls at 6 and 12 h (P < 0.001). Heat killed E. coli stimulation resulted in a 4-5 fold increase in IL-8 compared to the unstimulated control cells at each time point (P < 0.001). Both heat killed E. coli and HKC stimulated robust ROS production at 6 (P < 0.001), and 12 h (P < 0.01). Mitochondrial morphologic abnormalities were detected at 6 and 12 h based on reduced mitochondrial circularity and decreased mitochondrial membrane potential, P < 0.01. Microbial stimulation also induced significant autophagy at 6 and 12 h, P < 0.01. Lastly, blocking mitochondrial ROS generation using mitochondrial specific ROS scavenger reversed microbial challenge induced mitochondrial morphologic abnormalities and autophagy.

CONCLUSION

The findings from this study suggest that CRL.1790 cells may be a useful alternative to other colon cancer cell lines in studying the mechanisms of oxidative stress events associated with intestinal inflammatory disorders.

Keywords: Colon cancer cell line; CRL.1790 cells; Inflammation; Mitochondria; Microbial stimulation; Interleukin-8; Autophagy

Core tip: The normal human colon cell line, CRL.1970, can recapitulate oxidative stress-induced responses associated with inflammatory bowel disease following microbial challenge including enhanced production of reactive oxygen species (ROS), inflammatory cytokines, and enhanced mitochondrial autophagic responses. Scavenging mitochondrial ROS inhibited mitochondrial morphologic changes and autophagy suggesting that CRL.1790 cells can be used to study oxidative events associated with intestinal inflammatory disorders.