Combination of vitamins A, D2 and D3 reduce tumor load and alter the expression of miRNAs that regulate genes involved with apoptosis, tumor suppression, and the epithelial-mesenchymal transition in HCT-116 colon cancer cells

Abstract

Introduction: In previous work, we have shown the synergistic effects of combinations of vitamins A, D2, and D3 in reducing the proliferation of HCT-116 colon cancer cells. This combination also induced apoptosis and altered gene expression patterns as determined by transcriptomic profiling. Aims: The aims of this study were to investigate the effects of vitamins A and D in HCT-116 Crl:NU(NCr)-Foxn1nu mouse xenographs. Also, to determine potential mechanisms of action using miRNA-seq and correlated these data with results from RNA-seq.Methods: HCT-116 colon cancer cells were cultured and used for the xenograph study. Crl:NU(NCr)-Male Foxn1nu mice were injected subcutaneously with HCT-116-cells (1 X 106 in 100 µl 50% Matrigel and Sterile PBS), and after 7 days, were treated with vitamin A and D in feed for 21 days. The mice were imaged on day 28, sacrificed and the tumors were excised and measured. RNA was isolated from the HCT-116 cells and tumors, and RNA-seq and miRNA-seq were performed.Results: There was a concentration-dependent reduction in the HCT-116 colon cancer cell viability after treatment with vitamin A and D combinations. In Crl:NU(NCr)-Foxn1nu mice injected with HCT-116 colon tumor cells, treatment with vitamin A (25,000 IU) and vitamin D (4,000 IU) or vitamin A (35,000 IU) and vitamin D (5,000 IU) for 21 days significantly reduced tumor growth by ~38% and ~58%, respectively (p < 0.001). In the HCT-116 cells and excised tumors, treatment upregulated the expression of Bim, Bax, p53, and PTEN, and downregulated AXIN2, ID2 and DDX20 mRNAs, all well-known pro-apoptotic proteins, tumor suppressors, and molecules involved in the epithelial-mesenchymal transition. Ingenuity Pathway Analysis of miRNA-seq paired with RNA-seq showed correlations between miRNAs expression and expression of these genes. For example, miRNA-mRNA correlation pairing showed that treatment downregulated miR-30c-3p and miR-125b-3p which was associated with upregulated TP53. Upregulation of the tumor suppressor PTEN was associated with the downregulation of eleven miRNAs, including oncogenic miR17 and miR21.Conclusion: The results suggest that the combinations of vitamins A and D reduced the HCT-116 tumor burden in mice and altered the expression of miRNAs directly associated with genes in the apoptosis, tumor suppression, and epithelial-mesenchymal transition pathways. The data supports the hypothesis that vitamin A and D combinations impact multiple cancer signaling pathways and thus may be more effective in the treatment and prevention of cancer, as well as reducing cancer metastasis. Keywords: apoptosis, cholcalciferol, colon cancer, DDX20, epithelial-mesenchymal transition, ergocalciferol, p53, RNA-seq, miRNA-seq, PTEN, synergism, transcriptome

Combinations of vitamin A and D induced are synergistic in breast cancer cells and alter gene expression in the endoplasmic reticulum stress, unfolded protein and estrogen signaling canonical pathways

“…ATRA+D2+D3 combinations were added to the wells at the IC50 concentration for a final volume of 100 μl per well. The Caspase-Glo ® 3/7 and Caspase-Glo ® 8 assay kits (Promega Corporation, Madison, WI, USA) were used according to the manufacturer's protocols as we have described [33][34]. A Synergy HT Plate reader (Biotek, Winooski, VT, USA) and the Gen5 1.11 software analyzed and determined luminescence.…”

“…A Synergy HT Plate reader (Biotek, Winooski, VT, USA) and the Gen5 1.11 software analyzed and determined luminescence. The ApoToxGlo™ Triplex Assay kit was used according to the manufacturer's instructions and luminescence was measured using the Synergy HT Plate reader (Biotek, Winooski, VT) and Gen5 Table 1: Human primer sequences used for real-time PCR analysis are presented below and were obtained from previously published works [33][34][38][39][40].…”

“…Several studies have shown that vitamin A and D are more effective in combination for the treatment of colon, prostate, gastric epithelial cell cancers, and leukemia [12,[31][32][33][34]. This approach is rational because of the nature of the dimerization interaction between the Vitamin D Receptor (VDR) and the RAR/RXR.…”

“…To determine the background luminescence, control wells in the 96 well plates contained appropriate media with 10% FBS and 1% penicillin/streptomycin but no cells. Vitamins A, D2, D3, and ATRA were purchased from Sigma-Aldrich (St. Louis, MO) and analyzed by LC/MS as we have described [32][33]. Prior to adding the vitamins, A, D3, or D2 compounds at 1-100 µg/ml in 0.01% DMSO and combinations thereof new medium was added to the wells.…”

“…The 96-well plates were incubated at 37ºC with 5% CO2. After 72 hours, 100 μl of CellTiter-Glo 2.0 Reagent (Promega Corporation, Madison, WI, USA) was added to each well and the plate processed as we have described [32][33]. A Synergy HT Plate reader (Biotek, Winooski, VT) and Gen5 1.11 software were used to measure luminescence.…”