Transcriptomic analysis reveals that combinations of vitamins A, D2, and D3 have synergistic effects in HCT-116 colon cancer cells by altering the expression of genes involved in multiple canonical pathways including apoptosis, regulation of the...

Abstract

Integrated systems biology approaches suggest that combinations of nutrients may be more effective against cancer due to the large number of signaling pathways associated with cancer initiation and promotion. In a previous work, we have reported that combinations of vitamins A (as all trans-retinoic acid, ATRA), D2, and D3 act synergistically to induce apoptosis in colon and gastric cancer cells. In this work, we use whole-genome transcriptomic profiling to detect gene expression changes using RNA-seq to more comprehensively investigate the biological pathways affected by the combination of vitamin D2, D3 and ATRA. HCT-116 colon cancer cells were harvested, RNA was isolated and RNA-seq libraries were prepared using a Universal Plus mRNASeq kit. Sequencing was carried out on NovaSeq 6000. General quality-control metrics were obtained using FastQC and raw reads were aligned to human reference genome hg38 using STAR and BWA MEM. ENSEMBL genes were quantified using FeatureCounts, and differential expression statistics were computed using EdgeR. Specific gene expression was validated using qPCR. Transcriptomic analysis showed that of 26,313 genes analyzed, the expression of 8,402 genes was significantly altered (4030 up-regulated and 4373 down-regulated, FDR<0.05) in the treated cells, of which, 3,621 genes were differentially expressed (fold change <-1 or >+1 and an FDR <0.05). IngenuityÒ Pathway analysis revealed the involvement of 97 canonical pathways, with the top pathways including: mechanisms of cancer, apoptosis, myc-mediated apoptosis, regulation of the epithelial mesenchymal transition, and immunity. Keywords: apoptosis, cholcalciferol, colon cancer, caspase, CRMP1, ergocalciferol, IL-12, NOTCH1, RNA-seq, SMAD7, 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].…”

“…prepared the RNA-seq libraries as we have previously described [33][34]. Briefly, library construction was performed using 250 ng of total RNA/sample and 15 PCR cycles [33][34].…”

“…prepared the RNA-seq libraries as we have previously described [33][34]. Briefly, library construction was performed using 250 ng of total RNA/sample and 15 PCR cycles [33][34]. The final amplified libraries were purified, and the library fragment size distribution (range 264 and 294 nt) was determined by electrophoresis on a 2200…”

“…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.…”