Establishment of a multicomponent dietary bioactive human equivalent dose to delete damaged Lgr5+ stem cells using a mouse colon tumor initiation model

Multicomponent therapy has gained interest for its potential to synergize and subsequently lower the effective dose of each constituent required to reduce colon cancer risk. We have previously showed that rapidly cycling Lgr5(+) stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk. In the present study, we quantified the dose-dependent synergistic properties of dietary n-3 polyunsaturated fatty acids (PUFA) and curcumin (Cur) to promote targeted apoptotic deletion of damaged colonic Lgr5(+) stem cells. For this purpose, both heterogeneous bulk colonocytes and Lgr5(+) stem cells were isolated from Lgr5-EGFP-IRES-CreER(T2) knock-in mice injected with azoxymethane (AOM). Isolated cells were analyzed for DNA damage (gamma H2AX), apoptosis (cleaved caspase-3), and targeted apoptosis (both gamma H2AX and cleaved caspase-3) at 12 h post-AOM injection. Comparison of the percentage of targeted apoptosis in Lgr5(+) stem cells (GFP(high)) across a broad bioactive dose-range revealed an ED50 of 16.0 mg/day n-3 PUFA + 15.9 mg/day Cur. This corresponded to a human equivalent dose of 3.0 g n-3 PUFA + 3.0 g Cur. In summary, our results provide evidence that a low dose (n-3 PUFA + Cur) combination diet reduces AOM-induced DNA damage in Lgr5(+) stem cells and enhances targeted apoptosis of DNA-damaged cells, implying that a lower human equivalent dose can be utilized in future human clinical trials.