Vitamin A and D3 combinations reduce breast cancer tumor load in a postmenopausal MCF-7 xenograft mouse model in a dose- and time- dependent manner

Introduction: Earlier, we documented that a combination of vitamins A and D3 synergistically inhibited the growth of MCF-7, T48:A18 and SKBR3 breast cancer cells with the best activity seen in the ER+ cell line MCF-7. Transcriptomic analysis of treated MCF-7 cells also showed that the combination significantly upregulated the apoptosis and unfolded protein response canonical pathways, and reduced estrogen signaling. Objective: This study aimed to explore the impact of increasing vitamin A and D3 dose combinations over time in a postmenopausal model of breast cancer using ovariectomized athymic female mice bearing MCF-7 xenografts and further analyze mechanisms of action in MCF-7 cells using RNA-seq analysis. Methods: MCF-7 breast cancer cells were grown in culture for the xenograft experiments. Athymic female mice were injected with MCF-7 cells (1 x 10^6 in 100 µl of 50% Matrigel mixed with sterile PBS) via subcutaneous injection. Once the tumors reached an average volume of 100 mm³, the mice were randomly divided into four groups and treated with different vitamin A and D3 combinations. Tumor sizes and mouse body weights were monitored on a biweekly basis. After the treatment period, the mice were euthanized, and the tumors were surgically removed and measured. RNA-seq data from the treated MCF-7 cells were then further evaluated using IPA. Results: As compared with controls, treatment with vitamin A (25,000 IU) and vitamin D (10,000 IU) led to a significant reduction in tumor volume >70%, (p < 0.05-0.01) in OVX athymic mice with MCF-7 xenografts as determined by a two-tailed Student T test. Over the treatment period, the tumor volume in mice treated with vitamin A (10,000 IU) and vitamin D (5,000 IU) or vitamin A (25,000 IU) and vitamin D (5,000 IU) also trended downward and was statistically significant using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (p<0.05 and p<0.0001, respectively) but was not significant using a two-tailed Student T test. In cultured MCF-7 cells, Ingenuity Pathway Analysis of mRNA-seq data showed that the vitamin A and D combination significantly altered the expression of 101 genes out of 864 in the molecular mechanisms in cancer canonical pathway, downregulating gene expression in the integrin/P13K/Akt/mTOR pathway. Conclusions: The findings showed that the combination of vitamins A and D3 effectively reduced tumor burden in a postmenopausal MCF-7 xenograft mouse model, with effects that were both dose-dependent and time-dependent. The combination also significantly altered the expression of genes in the molecular mechanisms of cancer canonical pathway in cultured MCF-7 cells. These preclinical data support the use of vitamins A and D3 in the management of estrogen-dependent breast cancers, with the caveat that higher doses and longer treatment periods may be needed to observe anti-tumor effects. Keywords: Apoptosis, autophagy, breast cancer, cell cycle, integrin, postmenopausal, P13K, tumor load, xenograft