|Abstract:||Colorectal cancer is a relentless disease plaguing over one million people just in the United States. Survival rates are highly dependent on the stage of malignancy, with a low survival rate once the cancer has metastasized. Therefore, it is crucial to understand the molecular changes that occur during the progression of colorectal cancer tumorigenesis to determine effective treatments. Colorectal cancer risk in the United States is highly tied to lifestyle aspects: diet, body weight, and activity. Determining dietary changes to assist in preventing or slowing progression to metastasis could increase survival rates.
Anthocyanins, a subcategory of flavonoids, are natural red, purple, and blue pigments in a variety of plants consumed in a healthy diet. Many colorful fruits, vegetables, cereals, and legumes are rich in anthocyanins. The reported health benefits of anthocyanins and foods containing them include anti-carcinogenic, anti-diabetic, and anti-obesity effects. It is our long-term goal to understand the mechanisms of action that anthocyanins and anthocyanin-rich extracts can exert to inhibit colorectal tumorigenesis and progression towards metastasis. We hypothesize that anthocyanins and anthocyanin-rich extracts can hinder colorectal cancer progression by induction of apoptosis, suppression of angiogenesis, and stimulation of immune response. To test this hypothesis, we utilized several in vitro and in silico models to demonstrate potential mechanisms of action of the anthocyanins and extracts to inhibit colorectal cancer progression. Utilizing the knowledge we gained on anthocyanins and anthocyanin-rich extracts through these studies, we tested an anthocyanin-enriched and black lentil extract in an in vivo carcinogenesis model in the final aim.
We tested a variety of anthocyanin-rich extracts in vitro: red and purple corns, black lentil, red and purple grapes, sorghum, black rice, black and purple beans, purple sweet potato, blue wheat, purple carrot, and black peanut. Throughout, we used two colon cancer cell lines, HCT 116 and HT-29, which have different phenotypic and genotypic characteristics to give a more thorough analysis of the anthocyanins and anthocyanin-rich extract effects. The first aim utilized colored corn extracts and showed anti-proliferative effects on HCT 116 and HT-29 human colorectal cancer cells. The concentration of 50% inhibition (IC50) ranged from 1.1 to 6.3 mg/mL; the red corn had the highest potential to inhibit the cancer cell viability. All purple corn extracts increased apoptotic cells (19-53%) and impacted markers of apoptosis (BAX, BCL-2, and cytochrome C). Angiogenesis markers were also affected; colored corn extracts induced a decrease of the expression of vascular endothelial growth factor. In aim two, eleven phenolic-rich cereal, legume, vegetable, and fruit extracts were tested on HCT 116 and HT-29 cells to dig deeper into the effects of chemical composition on inhibition potential. Inhibition of HCT 116 and HT-29 cells significantly and strongly correlated with total phenolics (r = 0.87 and 0.77, respectively, p < 0.05) and delphinidin-3-O-glucoside (D3G) concentration correlated with HT-29 inhibition (r = 0.69, p < 0.05). Black lentil had the lowest IC50 for both cell lines (HCT 116: 0.9 mg/mL, HT-29: 1.4 mg/mL). Plant extracts induced apoptosis and arrested cells in G1. Anthocyanins exhibited tyrosine kinase inhibitory potential in silico and biochemically; cyanidin-3-O-glucoside (C3G) had one of the highest binding affinities with all of the tested pro-angiogenic kinases. Aim three utilized insight on chemical composition discovered in aim two to focus on the impact of pure phenolics on human colorectal cancer cells and determine the effects on immune-suppressing checkpoints, programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1). D3G and its metabolites, delphinidin and gallic acid, had IC50 values ranging from 81-600 µg/mL. C3G, D3G, and delphinidin decreased PD-L1 fluorescence intensity by 39%, 30%, and 27%, respectively. C3G reduced PD-L1 expression in HCT 116 cells by 40%. Anthocyanins decreased PD-1 expression in peripheral blood mononuclear cells in monoculture by 41% and 55%, and co-culture with HCT-116 and HT-29 cells by 39% and 26% (C3G) and 50% and 51% (D3G), respectively. In the final aim, a mouse model was used to determine the effect of a black lentil water extract and D3G-enriched extract on azoxymethane (AOM) and dextran sodium sulfate (DSS) induced colitis-associated carcinogenesis. The black lentil group had a lower disease activity index throughout and at the end of 2.4 compared to 6.3 of the AOM/DSS control (p<0.05). Mice in the black lentil group had an average of 7.8 ± 1.0 neoplasms while the AOM/DSS control had 12.8 ± 0.9 (p<0.05). Interleukin-6 protein expression was downregulated in colon mucosa (-44.5%), plasma (-72.6%) and gene expression was reduced in colon polyp tissue (fold change: -4.0) in the black lentil group compared to the AOM/DSS control. Although the D3G group did not have significant macroscopic changes compared to the AOM/DSS control, gene expression in the non-polyp tissue compared to the healthy control tissue was very similar with only four genes significantly modified (Secreted phosphoprotein 1 and CXCL2, CXCL5, and CXCL10). Black lentil and D3G-enriched extracts impacted interleukin-17 signaling, cytokine-cytokine receptor interaction, and intestinal immune network pathways. Black lentil treatment showed both anti-carcinogenic and anti-inflammatory properties while the D3G treatment demonstrated a decrease in the expression of pro-inflammatory genes.
Overall, anthocyanin-rich extracts showed potential to inhibit colorectal cancer progression. In successful completion of this project, we have contributed to the understanding of the anti-proliferative, pro-apoptotic, anti-angiogenic, and stimulatory immune response effects of anthocyanins and anthocyanin-rich extracts on colorectal cancer in silico, in vitro, and in vivo.