S-equol shifts afatinib-altered mucosal immune-epithelial defense indices toward baseline and attenuates increases in DEFA5 and SOX9 promoter methylation indices.

BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) frequently cause gastrointestinal adverse events, including diarrhea, and are associated with alterations in intestinal mucosal defense. Previous studies have shown that afatinib alters Paneth cell-associated α-defensin 5 (DEFA5)-related antimicrobial defense and epithelial transport-related molecular readouts, including cystic fibrosis transmembrane conductance regulator (CFTR) expression. OBJECTIVE: We investigated whether S-equol, a gut microbiota-derived isoflavone metabolite, shifts selected afatinib-associated mucosal immune-epithelial and promoter methylation readouts toward their corresponding control values under defined post-exposure and post-dose intervention schedules. METHODS: Mature Caco-2 monolayers were exposed to 20 µM afatinib for 24 h, washed, and subsequently treated with either 600 µM S-equol or 600 µM R-equol for 24 h. Rats received afatinib at 5.2 mg/kg once daily for 14 consecutive days, followed 2 h after the final afatinib dose by a single oral dose of S-equol at 7.8 mg/kg. Tissues were collected 24 h after the final afatinib dose, corresponding to 22 h after S-equol administration. In a separate differentiation-stage experiment, Caco-2 cells were exposed to 1000 nM afatinib for 24 h starting Day 1, treated with 60 µM S-equol for 24 h starting Day 7, and harvested on Day 14. RESULTS: In mature Caco-2 monolayers, afatinib reduced DEFA5, WNT3, and LGR5 protein abundance relative to that under the vehicle-control condition. Subsequent S-equol treatment increased the abundance of all three proteins relative to that under the afatinib-only condition and shifted these readouts toward their corresponding control values. R-equol produced similar directional shifts in WNT3 and LGR5 protein abundance but not in DEFA5 protein abundance. In rats, post-dose S-equol shifted afatinib-reduced intestinal Defa5 and Pigr mRNA expression and lipopolysaccharide-stimulated Peyer's patch IgA output toward their corresponding control values, while attenuating afatinib-associated increases in CFTR protein abundance, Peyer's patch IgA protein abundance, and salivary IgA concentration. Salivary IgA concentration was positively associated with IgA protein abundance in Peyer's patch across treatment groups. In the differentiation-stage experiment, S-equol attenuated afatinib-associated increases in the DEFA5 and SOX9 promoter methylation indices and shifted afatinib-reduced DEFA5 protein abundance toward the control value. CONCLUSIONS: Across complementary epithelial and animal models, S-equol shifted selected afatinib-associated mucosal immune-epithelial and promoter methylation readouts toward their corresponding control values. These findings identify molecular responses that warrant further investigation using direct intestinal functional measurements and models that evaluate compatibility with afatinib-mediated tumor control.