Changes in mitochondrial stability during the progression of the Barrett's esophagus disease sequence

Abstract

Background: Barrett's esophagus follows the classic step-wise progression of metaplasia-dysplasia-adenocarcinoma. While Barrett's esophagus is a leading known risk factor for esophageal adenocarcinoma, the pathogenesis of this disease sequence is poorly understood. Mitochondria are highly susceptible to mutations due to high levels of reactive oxygen species (ROS) coupled with low levels of DNA repair. The timing and levels of mitochondria instability and dysfunction across the Barrett's disease progression is under studied.

Methods: Using an in-vitro model representing the Barrett's esophagus disease sequence of normal squamous epithelium (HET1A), metaplasia (QH), dysplasia (Go), and esophageal adenocarcinoma (OE33), random mitochondrial mutations, deletions and surrogate markers of mitochondrial function were assessed. In-vivo and ex-vivo tissues were also assessed for instability profiles.

Results: Barrett's metaplastic cells demonstrated increased levels of ROS (p < 0.005) and increased levels of random mitochondrial mutations (p < 0.05) compared with all other stages of the Barrett's disease sequence in-vitro. Using patient in-vivo samples, Barrett's metaplasia tissue demonstrated significantly increased levels of random mitochondrial deletions (p = 0.043) compared with esophageal adenocarcinoma tissue, along with increased expression of cytoglobin (CYGB) (p < 0.05), a gene linked to oxidative stress, compared with all other points across the disease sequence. Using ex-vivo Barrett's metaplastic and matched normal patient tissue explants, higher levels of cytochrome c (p = 0.003), SMAC/Diablo (p = 0.008) and four inflammatory cytokines (all p values <0.05) were secreted from Barrett's metaplastic tissue compared with matched normal squamous epithelium.

Conclusions: We have demonstrated that increased mitochondrial instability and markers of cellular and mitochondrial stress are early events in the Barrett's disease sequence.

Keywords: Barrett’s esophagus; Mitochondrial instability; Oxidative stress.

Fig. 1

Random mitochondrial point mutations in-vitro. There was a significantly increased frequency of random mitochondrial DNA mutations in the QH cells (mean 7.710 × 10⁻⁵, SD 2.770 × 10⁻⁵) (n = 5) compared to HET1A (mean 2.560 × 10⁻⁵, SD 1.015 × 10⁻⁵) (n = 3), Go (mean 2.730 × 10⁻⁵, SD 2.440 × 10⁻⁵) (n = 5) and OE33 (mean 2.500 × 10⁻⁵, SD 1.430 × 10⁻⁵) (n = 5) cells. This demonstrated that random mutations were an early event in this in-vitro model of Barrett’s progression. *p ≤ 0.05

Fig. 2

Random mitochondrial point deletions in-vivo. Wilcoxon matched-paired signed rank tests demonstrated a significantly increased level of deletions in the SIM matched normal tissue compared with SIM (p = 0.031) and a trend towards increased deletions in HGD/EAC-matched normal tissue compared with areas of HGD/EAC (p = 0.063). Mann Whitney-U test demonstrated significantly increased frequencies of deletions in SIM compared to HGD/EAC tissue (p=0.043). *p ≤ 0.05

Fig. 3

Mitochondrial function, ROS levels, across the Barrett’s disease sequence (n = 5). ROS was significantly lowest in the HET1A cells and highest in the QH cells. ROS levels were significantly increased in the QH cells compared with Go (p = 0.003) and OE33 (p < 0.0001) cell lines. ROS levels were 1.3 times higher in the Go cell line compared with the OE33s (p = 0.020). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005

Fig. 4

Expression of CYGB along the Barrett’s disease sequence. The expression of CYGB is demonstrated in normal (mean 0.425, standard error of mean [SEM] 0.231), SIM (mean 11.013, SEM 8.493), LGD (mean 6.03, SEM 1.555), HGD (mean 3.580, SEM 1.580) and EAC biopsies (mean 4.581, SEM 0.991). SIM over-expressed CYGB relative to LGD and EAC. There was a significant increase in CYGB in SIM, LGD, HGD and EAC samples when compared with normal squamous epithelium. *p < 0.05, **p < 0.005

Fig. 5

a-f Mitochondrial proteins and inflammatory cytokines levels in explant cultured media in SIM tissue and surrounding matched-normal tissue. Wilcoxon matched-pairs signed rank tests demonstrated significantly increased levels of a cytochrome c (n=12), b SMAC/Diablo (n=8), c IL-1beta (n=12), d IL-6 (n=12), e IL-8 (n=12) and f TNF-alpha (n=12) in SIM tissue compared to surrounding normal epithelium. *p ≤ 0.05, **p ≤ 0.005 and ***p ≤ 0.0005