Gastroesophageal Reflux Induces Protein Adducts in the Esophagus.

Epidemiologic studies have identified gastroesophageal reflux disease (GERD) as the strongest risk factor for esophageal adenocarcinoma. However, it remains not well understood how gastroesophageal reflux facilitates tumor development. In this study, we investigated, for the first time, the role of isolevuglandins (isoLGs), which are formed through free radical and enzymatic cyclooxygenation of polyunsaturated fatty acids. Structurally, isoLGs are categorized as lipid-derived γ-ketoaldehydes that are highly reactive with free amines on lysine residues forming LG-lysine lactam protein adducts, protein-protein, and protein-DNA crosslinks.2, 3

To investigate the reflux-induced cellular alterations, we exposed esophageal cells derived from the normal esophagus (EPC2), Barrett’s esophagus (CP-A), and cancer (TE-7) to acidic growth medium (pH 4.0), supplemented with 100 μM bile salts cocktail (BA/A). The composition, total bile salts concentration and pH, were selected based on previous measurements conducted in patients with GERD. IsoLGs were analyzed using D11 single chain antibody. This antibody was generated by screenings of phage-display libraries and tested to specifically recognize isoLG protein adducts independently of protein amino acid sequences.5, 6 We found that treatment of esophageal cells with acidic bile salts led to significant accumulation of isoLG protein adducts compared with untreated control (Figure 1A, Supplementary Figure 1A and B). Notably, multiple proteins were adducted after treatment with BA/A, which was indicated by a strong increase in the intensities of multiple protein bands. Induction of isoLG protein adducts was also observed using immunofluorescence with D11 scFv (Supplementary Figure 2).

Figure 1

Reflux induces the formation of isoLG protein adducts. (A) Treatment with BA/A leads to significant accumulation of isoLG adducts in TE-7 (n = 3) cells. D11 positivity was arbitrarily set at 1 in control samples. (B) Representative image (×20) of immunohistochemical staining for isoLG protein adducts in the mouse esophagus after induction of reflux by surgery (n = 7). Mice with sham surgery were used as a control (n = 5). Mice with reflux showed significantly higher levels of isoLGs compared with control sham mice (P < .05). Inset shows cells at a ×40 magnification. (C) Representative images (×20) of immunohistochemical staining for isoLG in esophageal biopsies collected from patients with GERD (n = 10) and healthy individuals (n = 9). A trend was found toward increasing formation of isoLG protein adducts in patients with GERD (P = .07). Inset shows cells at a ×40 magnification. Experiments conducted in vitro were evaluated using the Student t test. The Mann-Whitney test was used for data sets generated in vivo. *P < .05, **P < .01. Data are represented as mean ± standard deviation. Ctrl, control; H&E, hematoxylin-eosin; IHC, immunohistochemical; ns = not significant.

Supplementary Figure 1

Formation of isoLG protein adducts (A) EPC-2 cells were treated with BA/A for 15 minutes and analyzed for isoLG protein adducts with D11 antibody. Treatment with BA/A led to significant accumulation of isoLG adducts in EPC-2 cells. D11 positivity was arbitrarily set at 1 in control samples. (B) The same as A, but CP-A cells are shown. (C) Representative mass spectrometry chromatograms show levels of isoLG-lysine lactam adducts in cellular extracts collected from control (untreated) and BA/A-treated TE-7 cells in the presence or absence of 2-HOBA. Statistical analyses were performed with the Student t test. *P < .05, **P < .01. Data are represented as mean ± standard deviation.

Supplementary Figure 2

Representative images of isoLG-positive cells. Analyses were performed using immunofluorescence with D11 antibody after treatment with BA/A for 18 hours. Cell nuclei were stained with DAPI. Significant increase in isoLG positivity was found in treated EPC-2 (A) and TE-7 (B) cells. ***P < .001. At least 150 cells were assessed in each experiment. Statistical analyses were performed with Student t test. Data are represented as mean ± standard deviation.

The formation of LG-lysine lactam adducts was further verified by liquid chromatography-electrospray-ionization-tandem mass spectrometry in TE-7 cells. Our quantification analyses were based on specific transitions from the molecular ion at m/z = 479.2 to the specific fragment at m/z = 332.1. The elution of the 2 fragment ions found in BA/A-treated samples was similar to the [13C6] lysine–lactam internal standard. As an additional control we analyzed TE-7 cells treated with BA/A in the presence of the specific isoLG scavenger 2-hydroxybenzylamine (2-HOBA; 50 μM). Mass spectrometry analyses found an upregulation of LG-lysine lactam adducts after treatment of esophageal cells with BA/A. Treatment with 2-HOBA inhibited the effect of BA/A, providing further support to our findings (Supplementary Figure 1C).

To investigate the formation of isoLG protein adducts in vivo, we used a mouse model of esophageal reflux injury. A section of the mouse jejunum was transected and then anastomosed to the esophagus resulting in increased reflux. Using immunohistochemistry with D11 antibody, the levels of isoLG protein adducts were compared in esophageal specimens collected from animals with reflux and control animals with sham surgery (n = 12). We found significant accumulation of isoLG protein adducts in the esophagus of animals affected by reflux (Figure 1B).

To further investigate isoLG adducts in vivo, we conducted a small-scale study in patients with GERD. Esophageal biopsies collected from 10 GERD patients and 9 healthy individuals were immunostained with D11 antibody and analyzed for isoLG protein adducts. We found a trend toward increasing formation of isoLG protein adducts in patients with GERD (P = .07). An increased staining for the isoLG adducts was observed in 50% (5 out of 10) of patients with GERD (Figure 1C). Normal subjects primarily showed low or undetectable levels of isoLGs. Interestingly, some patients with GERD showed a strong nuclear staining showing that adducts form on nuclear proteins (Figure 1C, inset).

To investigate how induction of isoLGs by reflux affects proteins, we focused our studies on the regulation of p53 protein because it plays a key tumor suppressor role in the esophagus. Given that isoLGs have strong hydrophobic properties and may cause protein aggregation, we separately analyzed soluble and insoluble cellular fractions, which were prepared as described in the Supplementary Methods section. Surprisingly, we found that the solubility of the p53 protein significantly decreased after treatment with BA/A, whereas the insoluble cellular fraction was enriched with p53 protein aggregates in all tested cell lines (Figure 2A, Supplementary Figure 3A and B). p53 precipitation was prevented by 2-HOBA, suggesting that isoLGs are responsible for the precipitation of p53 protein (Figure 2A, Supplementary Figure 3A and B).

Figure 2

Acidic bile salts cause the formation of adducts on p53 protein and its precipitation. (A) p53 protein is precipitated after treatment of CP-A cells with BA/A. 2-HOBA prevents precipitation of p53 protein. (B) Analysis of isoLG protein adducts using D11 scFv after immunoprecipitation of p53 protein with p53-specific antibody (DO1) in CP-A cells. Exposure of esophageal cells to BA/A significantly increases levels of p53 protein adducts. Each analysis is representative of 3 independent experiments and values are expressed as mean ± standard deviation. D11 positivity was arbitrarily set at 1 in control samples. Statistical analyses were performed with the Student t test. *P < .05, **P < .01. Ctrl, control; ns = not significant.

Supplementary Figure 3

Acidic bile salts cause the formation of adducts on p53 protein and its precipitation. (A) p53 protein is precipitated after treatment of EPC-2 cells with BA/A. 2-HOBA prevents precipitation of p53 protein. (B) The same as A, but HET-1A cells are shown. (C) Analysis of isoLG protein adducts using D11 scFv after immunoprecipitation of p53 protein with p53-specific antibody (DO1) in EPC-2 cells. Exposure of esophageal cells to BA/A significantly increases levels of p53 protein adducts. Each analysis is representative of 3 independent experiments and values are expressed as mean ± standard deviation. D11 positivity was arbitrarily set at 1 in control samples. Statistical analyses were performed with the Student t test. *P < .05, **P < .01.

To directly analyze p53 protein adducts, cellular lysates were immunoprecipitated with p53-specific antibody (DO1). The immunoprecipitated p53 protein was then analyzed for the isoLG adducts by Western blotting using D11 scFv. We found that BA/A led to the formation of p53 isoLG protein adducts. Notably, 2-HOBA inhibited the formation of adducts on p53 protein further supporting our findings (Figure 2B, Supplementary Figure 3C). Then, the effect of 2-HOBA was compared with antioxidants (tempol and N-acetylcysteine), which were shown to inhibit the production of reactive oxygen species by acidic bile salts at tested concentrations. Although both tested antioxidants had some inhibitory effect, suppression of isoLG protein adducts was significantly stronger by 2-HOBA (Supplementary Figure 4).

Supplementary Figure 4

Effect of 2-HOBA, Tempol, and NAC on the formation of isoLG protein adducts. EPC-2 and CP-A cells were treated with BA/A alone or in combination with either 2-HOBA or 20 μM of Tempol or 20 μM of NAC. Treated cells were collected and analyzed for the formation of isoLG protein adducts. D11 positivity was arbitrarily set at 1 in control samples. Results from 3 independent experiments are shown. Statistical analyses were performed with the Student t test. *P < .05, **P < .01, ***P < .001. Data are represented as mean ± standard deviation. NAC, N-acetylcysteine.

In summary, this study revealed, for the first time, that gastroesophageal reflux leads to the formation of isoLG and accumulation of isoLG protein adducts causing precipitation and inactivation of p53 tumor suppressor. We also found that isoLG scavenger 2-HOBA efficiently suppresses accumulation of isoLG adducts in the esophagus.