To better understand how the stem cells respond to environmental factors such as diet and carcinogen, we investigated the chemo-protective effects of dietary agents (n-3 PUFA and curcumin) on DNA damage response in colonic stem cells isolated from Lgr5-EGFP-IRES-CreER^T2 knock in mice injected with AOM. We demonstrated that n-3 PUFA and curcumin synergize to promote targeted apoptosis of damaged Lgr5+ stem cells in part by enhancing p53 signaling in Lgr5+ stem cells at the tumor initiation stage. In addition, at the pre-tumor stage of tumorigenesis in colon cancer, we demonstrated n-3 PUFA and curcumin combination synergistically reduces nuclear ?-catenin levels in aberrant crypt foci, a surrogate marker of colon cancer. In order to assess the dose-dependency of n-3 PUFA and curcumin action, we also calculated the median effective concentration and the Human equivalent dose of n-3 PUFA + curcumin required to remove DNA damaged Lgr5+ stem cells by targeted apoptosis. In order to further elucidate the effects of oncogenesis on the biophysical properties of the colonocyte plasma membrane at the pre tumor stage, we generated CDX2P-CreER^T2 -Apc^580S/+; KrasLSL-^G12D/+ (ACK) transgenic mice. Our findings demonstrate for the first time that oncogenic Apc and Kras increase plasma membrane order by perturbing cholesterol homeostasis and promoting cell proliferation. Genes associated with cholesterol uptake and de novo synthesis of cholesterol are enhanced in ACK mice. This process is associated with the upregulation of Myc signaling, a well-known upstream mediator of cholesterol homeostasis. Our preliminary findings also indicate that the addition of exogenous cholesterol can dose-dependently promote cell proliferation in colonic cell lines and mouse colonic organoids. In complementary experiments, we also investigated the chemo-protective effects of dietary agents on cholesterol homeostasis and plasma membrane order in ACK mice. Our findings indicate that perturbed plasma membrane order and cholesterol homeostasis is ameliorated by n-3 PUFA + curcumin feeding. In summary, our results indicate for the first time that fish oil plus curcumin synergistically reduce colon cancer risk in part by modulating (i) p53 signaling in Lgr5+ stem cells, and (ii) plasma membrane properties implicated in the regulation of the colon cancer cells and tumor development.
