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

Abstract

Introduction: Numerous studies over the past 30 years have shown that bioactive compounds present in functional foods, including vitamins, minerals and phytochemicals reduce cancer risk. For example, vitamins A and D derivatives found in fruits, vegetables, and dairy products, reduce the growth of breast, bladder, head, neck, lung, prostate, and skin cancers. However, the effects of these combined vitamins have not been previously reported for breast cancer.Aims: To investigate the activities of vitamin A (all-trans-retinoic acid; ATRA), as well as vitamins D2 and D3 in combination in the breast epithelial cancer cell lines T47D:A18, MCF-7, and SK-BR-3 and perform whole genome analysis in MCF-7 cells using RNA-seq.Methods: Breast cancer cells were cultured in appropriate media and treated with ATRA, D2 or D3 alone in concentrations from 1-10 μg/ml, or in combination at 1, 5, and 10 μg/ml. The CellTiter-Glo® 2.0 assay, Caspase-Glo®3/7, Caspase®8, and ApoTox-Glo™ Triplex assays measured cell viability and apoptosis. The effect of treatment on autophagy in MCF-7 cells was measured with a CYTO-ID® Autophagy Detection Kit 2.0. The whole transcriptome analysis was assessed using mRNA-seq and qPCR.Results: Separately, ATRA, D2, and D3 all reduced the viability of all breast cancer cell lines tested, with median inhibitory concentrations (IC50) between 2.1 to 31.7 μg/ml. However, when breast cancer cells were treated with combinations of ATRA+D2+D3, the IC50 was reduced indicating synergism. In MCF-7 cells, 5-flurouracil (5-FLU) had an IC50 of 1.37 µg/ml, while the vitamin A and D combination had an IC50 of 1.5 µg/ml, indicating the combination was ~90% as effective as 5-FLU. Treatment of MCF-7 cells with ATRA+D2+D3 enhanced caspase 3/7 activity, as well as the expression of Bax, BAD, PTEN and p53 (apoptosis canonical pathway), as well as induced autophagy. Whole genome analysis of treated MCF-7 cells showed a significant upregulation in gene expression in the autophagy, endoplasmic reticulum stress and the unfolded protein response apoptosis canonical pathways. Furthermore, MCF-7 cells treated with ATRA+D2+D3 demonstrated significant downregulation of gene expression in estrogen-mediated S phase entry and estrogen signaling canonical pathways suggesting antiestrogenic effects. Conclusions: Vitamins A and D combinations had synergistic effects in breast cancer cells and induced both apoptosis and autophagy. Transcriptional profiling showed significant alterations in gene expression patterns and upregulation of multiple cancer signaling pathways supporting the hypothesis that combining vitamins A and D is a more effective treatment than either vitamin alone. Keywords: all-trans-retinoic acid (ATRA), Vitamin D2, Vitamin D3, breast cancer cells, apoptosis, estrogen receptors

Peonidin-3-O-glucoside and Resveratrol Increase the Viability of Cultured Human hFOB Osteoblasts and Alter the Expression of Genes Associated with Apoptosis, Osteoblast Differentiation and Osteoclastogenesis

“…RNA was isolated from harvested osteoblasts using Trizol (ThermoFisher Scientific, Waltham, MA, USA) and used for RNA-Seq. Total RNA was quantified using a NanoDrop™ One Spectrophotometer, and the RNA quality was assessed on an Agilent 4200 TapeStation and RNA Screen Tape as we have previously described [ 14 , 15 ]. Remaining DNA concentrations were <10%.…”

“…A Universal Plus mRNASeq kit was used to generate the mRNA-seq library (Tecan, Männedorf, Switzerland, PN: 0520B-A01) with 250 ng of the purified RNA and 15 PCR cycles, as we have described [ 14 , 15 ]. An Agencourt RNAClean XP system was used to clean up the amplified libraries (Beckman Coulter, Brea, CA, USA).…”

“…The final library concentrations were determined by PCR analysis. A NovaSeq 6000 was used for sequencing using methods we have previously described [ 14 , 15 ]. For confirmation of gene expression, qPCR was performed as we have described using primers (Origene, Rockville, MD, USA) as listed in Supplemental Figure S3 .…”

“…Canonical signaling pathways and other biological networks that overlapped with differential gene expression were analyzed using the predicted protein function of ENSEMBL using the Ingenuity ® Pathway Analysis software 01-22-01 (Qiagen, Germantown, MD, USA), as we have described [ 14 , 15 , 23 ]. Alteration of gene expression was filtered by FC of ≤−1 and ≥1 and an FDR ≤ 0.05 to determine overlapping canonical pathways in IPA’s databases [ 23 ].…”