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

Like China, Mexico has a traditional system of medicine dating back almost 5000 years that incorporates the healing practices of pre-Columbian civilizations, including the Maya and Aztec. Mexican Traditional Medicine (MTM) women depend on MTM practices and herbal medicines for their primary healthcare needs as limited access and high costs of Western medicine is a significant problem. The aims of this work were to determine the medicinal plants more commonly used in MTM for women’s reproductive health issues and assess the clinical data supporting their use. Data from multiple sources was compiled and information on plants commonly used by women in Mexico MTM for the treatment of fertility and menstruation issues, pregnancy, and menopause was analyzed. Analysis of the data show that 185 species of plants representing > 60 families were used for a wide range of reproductive health issues. Some of these plants have been used in MTM for fertility regulation in women of which 35 species were used as emmenagogues and abortifacients. Approximate 40 species were used for the symptoms of premenstrual syndrome, heavy menstrual bleeding, and dysmenorrhea. In terms of pregnancy, 35 species were used for postpartum care and to facilitate breastfeeding, 16 species were used as oxytocic agents to induce labor and speed birth, and six plant species were used to prevent miscarriage. Fourteen plant species were reported to treat infertility or promote fertility, and seven species were used to treat uterine prolapse. Three plants species were reported to treat menopause and two plants were used for osteoporosis. Analysis of the clinical data for commonly used medicinal plants showed some clinical support for the use of these plants in MTM. In Mexico, women use medicinal plants for almost every aspect of reproductive health. While some plants have clinical data, most medicinal plants used in MTM have no safety or efficacy data available and could serve as the basis of future investigations.