Calcitriol represents the most active VD metabolite and hormonal form, which modulates calcium homeostasis through actions on the kidney, bone, and intestinal tract. However, calcitriol is also known for its potent anticancer effects. In particular, calcitriol inhibits breast cancer cells proliferation and tumorigenesis.
1. Calcitriol in Combination with Chemo/Radiotherapy in BC
Most cancer cells can respond to calcitriol by expressing the VDR. In particular, BC cells have shown higher VDR protein levels compared to benign breast tissue 
. Through this transcription factor, calcitriol exerts many anticancer effects, including growth inhibition, induction of cell differentiation, anti-inflammatory activity, cell cycle arrest, oncogenes downregulation, and many others that place calcitriol as a natural endogenous cancer-preventive antineoplastic factor 
. This has been the basis of the vast number of studies designed to study calcitriol and its analogs as pharmacological options in the oncological setting 
. Notably, calcitriol not only acts as an antineoplastic agent, but also can help to overcome drug resistance, increase the susceptibility to chemotherapy and even potentiate the effects of conventional chemotherapeutic agents and radiation therapy.
A vast number of preclinical studies has explored the potential enhancement of calcitriol anticancer effects by its combination with conventional chemotherapeutic regimens. This, of course, has the additional benefit of allowing for dose reduction of the chemotherapeutic drug, while at the same time minimizing unwanted side effects.
2. Calcitriol in Combination with Endocrine Therapy
Endocrine therapy has been used for the management of early and advanced hormone-positive BC. This therapy functions by blocking the estrogen signaling or by inhibiting estrogen synthesis. Among the most commonly used endocrine therapeutic factors are tamoxifen, raloxifene; fulvestrant; and AIs 
The antiestrogens tamoxifen and fulvestrant competitively inhibit the binding of estradiol to the ER. Tamoxifen induces changes in ER conformation resulting in the recruitment of coactivators or corepressors. Depending on the interaction of ER-tamoxifen complex, tamoxifen can act as either partial agonists or antagonists of ER function in a tissue-, cell-, and promoter-specific manner. Due to these selective activities, tamoxifen is classified as a selective estrogen receptor modulator (SERM). Based on its antagonist action, it is used for the treatment of women with metastatic BC, as adjuvant therapy of primary BC, as well as for the reduction of BC risk 
. Fulvestrant, on the other hand, has no agonistic effects, since it interrupts ER dimerization and nuclear localization, blocking ER-mediated transcriptional activity associated with tumor progression, invasion, metastasis, and angiogenesis. This antiestrogen also accelerates receptor degradation, and therefore is considered as a selective estrogen receptor down-regulator (SERD) 
. Fulvestrant is used to treat hormone receptor-positive advanced BC in postmenopausal women without previous endocrine therapy or with disease progression following endocrine therapy 
In vitro studies in MCF-7 and ZR-75-1 BC cell lines have shown that the combined treatment of calcitriol and tamoxifen inhibited, in a cytostatic way, cell proliferation to a greater extent than either compound alone 
, through inducing apoptosis 
. This combination allowed reducing the doses of calcitriol. Moreover, calcitriol diminished the estradiol-stimulated growth of the two ER-positive cell lines. The pharmacological effect of both compounds was classified as an additive interaction 
. Interestingly, tamoxifen treatment increased in a dose-dependent fashion the levels of VDR, thus favoring calcitriol biological effects 
Notably, theVD3 analog EB1089 is more potent to inhibit the proliferation of MCF-7 cells compared to calcitriol. In MCF-7 cells stimulated with estradiol, the co-treatment of EB1089 with fulvestrant suppressed the estradiol-stimulated growth of MCF-7 cells and produced a higher inhibitory effect than either compound alone 
. 22-oxa-l,25-dihydroxyvitamin D3, (22-oxa-calcitriol) is another synthetic analog of calcitriol that inhibits BC cell growth regardless of ER status without raising serum calcium concentrations 
. Combining this analog with tamoxifen enhanced the 22-oxa-calcitriol antitumor effect in an ER-positive BC model 
. In addition, Ro24-553, another calcitriol analog, inhibits mammary carcinogenesis by extending tumor latency and reducing tumor incidence. Its combination with tamoxifen in a murine model increased the anti-estrogenic actions of tamoxifen, resulting in the reduction of tumor burden and incidence 
On the other hand, within the pathophysiology of BC, it is known that it can metastasize to the bones. A risk factor related to this is the increase of bone resorption 
. In this sense, it was demonstrated that calcitriol and its analogs, EB1089 and KH1060, stimulated calcium release in a dose-dependent manner from long bones of fetal mice. Significantly, tamoxifen, or fulvestrant treatment inhibited the bone resorption promoted by calcitriol and its analogs 
. Hence, the potential side effect of treating BC patients with calcitriol or its analogs is the increased risk of skeletal metastases due to the stimulation of bone resorption, which could be reduced by combining them with antiestrogens, thus taking advantage of the antiproliferative proprieties of VD compounds.
An important antitumoral mechanism of tamoxifen is the reduction of glucose uptake. However, calcitriol co-treatment was found to significantly attenuate this effect in MCF-7 cells. In order to avoid this, combining calcitriol with Glucose-6-phosohate dehydrogenase-inhibiting regimens could improve substantial antitumor effects observed by the combination 
3. Calcitriol in Combination with Histone Modifiers
Histone modification can be defined as a post-translational alterations at the N-terminal histone tails; acetylation and methylation are the two most recognized mechanisms regulating the epigenetic effects of gene expression, genomic stability, DNA damage response, and cell cycle checkpoint integrity. Both mechanisms are importantly related to cancer development 
HDACs are enzymes associated with transcriptional repression. Histone deacetylase inhibitors (HDACI) are a class of compounds that interfere with the function of HDAC, inducing a hyperacetylation status of chromatin; the above disturbs the gene expression through modulating the chromatin structure 
. It has been widely reported that changes in cancer cells lead to hypomethylation and hypermethylation of specific DNA regions, mainly within the promoters of tumor suppressor genes 
. Trichostatin A (TSA or 7-(4-(dimethylamino)phenyl)-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide) is the most potent HDACI chemical agent that has been discovered and widely employed in BC models 
. In different BC cell lines, this compound increases CYP24A1 expression in concentrations between 3 to 400 nM 
. The latter points out that the use of HDACI can perturb the effects of VD-derived compounds by decreasing its bioavailability, and with this its antineoplastic effects.
4. Calcitriol in Combination with Kinase Inhibitors in BC
Different types of kinases are implicated in the growth of BC cells. MAPK, PI3K/AKT signaling pathway, Janus kinase (JAK)–signal transducer, and activator of transcription (STAT) pathway are mitogenic routes that have outstanding participation in cancer cell proliferation. These and other cellular signaling pathways are stimulated after the activation of various growth receptors 
. Specifically, in HER2-positive and TNBC cells, the overexpression and hyperactivation of different epidermal growth factor receptor family members such as EGFR and HER2 are common. The co-expression of these receptors confers poor outcomes and a high rate of metastasis. It is important to mention that these receptors are activated by a series of phosphorylations in their tyrosine kinase residues. Thus, drugs known as TKIs are generally employed to counter their activation. It has been reported that the combination of calcitriol or different analogs with tyrosine kinase inhibitors such as gefitinib, lapatinib, or neratinib resulted in a greater antiproliferative and apoptotic effect than either drug alone in TNBC and HER2-positive BC cells 
. Notably, the combination of calcitriol with different TKIs downregulated the MAPK and PI3K phosphorylation 
. The overall synergistic effect of the combined treatment of calcitriol with TKIs can be attributed to the presence of VDREs in EGFR promoter, which regulate the expression of growth factor receptors. Moreover, calcitriol can avoid the binding of different ligands to EGFR 
. Regarding this point, it has been described that calcitriol can modulate the activation of MAPK by non-genomic routes 
. The coadministration of calcitriol with gefitinib and with gefitinib plus dexamethasone has also been probed in clinical trials involving solid tumors such as BC 
. However, these studies were focused on evaluating the maximum tolerated dose (MTD) of calcitriol in a combined scheme administration. The authors reported no antitumor activity in patients with solid tumors when the drugs were administered together.
In combination with dovitinib, a multi-kinase inhibitor, calcitriol has also been demonstrated to have a synergistic antiproliferative effect in TNBC in in vitro and in vivo models. At the molecular level, the combination of these compounds induced cell death and inhibited tumor growth of BC cells to a greater extent than each compound alone 
. Of note, at clinically achievable and safe concentrations, the combination of calcitriol with dovitinib allowed reducing the dose of the kinase inhibitor while preserving its antiproliferative effect. The latter suggested that lower dovitinib dosing is feasible by the co-treatment, which may decrease its adverse effects and avoid the generation of resistance in therapeutic applications.
On the other hand, the synergistic effect on cell proliferation of calcitriol in combination with ruxolitinib, a JAK1 and JAK2 inhibitor, was also demonstrated in BC cells with or without the presence of ER 
. The combined treatment negatively modulated the protein levels of JAK2, phosphorylated JAK2, c-Myc protein, CCND1m and induced the apoptosis regulator Bcl-2, Bcl-2-like protein 1, and caspase-3 
. These findings indicate that the combination of TKIs with calcitriol can be favored in ER-negative BC cells, while its combination with other kinds of inhibitors such as ruxolitinib can be helpful to both panoramas. In fact, the combination of calcitriol with kinase inhibitors has shown promising results in different types of cancer 
In conclusion, the simultaneous treatment of calcitriol or its analogs with TKI’s in TNBC and HER2-positive BC cells is significantly better than monotherapy as antineoplastic treatment, resulting in a greater antiproliferative and pro-apoptotic effect. Part of the increased effect could be attributed to the regulation of growth factor receptors expression and activation by calcitriol. The overall preclinical evidence provides the basis for the potential use of this therapeutic combination in BC patients whose tumors overexpress TK receptors.
5. Calcitriol in Combination with Non-Steroidal Analgesic Drugs in BC
The sustained inflammatory environment in the cancer context is associated with enhanced cell proliferation and carcinogenesis promotion. In fact, the employment of different anti-inflammatory molecules, including nonsteroidal anti-inflammatory drugs (NSAIDs), for counteracting the inflammatory status has contributed to reducing the risk and incidence of several cancers and to inhibit cancer growth 
. The NSAIDs inhibit COX enzyme activity, which exists as two isoforms: COX-1 and COX-2. The first is expressed ubiquitously in many tissues and cell types, while the second one is induced by a variety of stimuli and is involved in inflammatory processes. COX-1 and COX-2 convert arachidonic acid to prostaglandins, which promote proliferation, inflammation and play an essential role in neoplasms development and progression, including BC 
. On the other hand, calcitriol exhibits significant anti-inflammatory actions that contribute to its antineoplastic effects 
. For many years, different kinds of pro-inflammatory molecules such as prostaglandins and thromboxanes have been associated with bad prognosis, recurrence of the disease, and poor survival rate in patients with BC 
. Different studies have reported that the combination of calcitriol with celecoxib, an inhibitor of COX-2, significantly reduced BC cell proliferation in a synergistic manner as compared to each single agent; an effect that was independent of ER presence 
. In addition, calcitriol can downregulate COX-2 protein and gene expression in BC cell lines with or without ER expression 
, an effect that was attributed to its immunomodulatory role and the link between VD3 and prostaglandin metabolism 
Related to the above, the antiangiogenic effects of calcitriol may be mediated by the inhibition of prostaglandins, which are important proangiogenic factors 
, in addition to the modulation of vascular endothelial growth factor (VEGF) 
In conclusion and considering that inflammation is considered one of the hallmarks of cancer, the anti-inflammatory compounds have been widely evaluated as antineoplastic agents alone or combined with calcitriol, resulting in synergistic antiproliferative effects independently of the BC phenotype. Therefore, further studies are necessary to determine the benefit of this therapeutic strategy.
6. Calcitriol in Combination with Immunomodulatory Agents in BC
There are few reports on the combination of calcitriol with immunomodulatory agents in BC. However, in order to counteract the hypercalcemic effect evoked by calcitriol, schemes based on glucocorticoids have emerged regarding this point 
. In addition, glucocorticoids enhance VDR transcription in many cell types 
. Thus, different combinations of calcitriol with these agents have been explored, specifically in prostate cancer. The results are controversial, some of them pointing out that the combination of dexamethasone is safe, feasible, and has antitumor activity 
, while others report a lack of significant antitumoral activity in prostate cancer 
. However, in BC cells, pre-clinical studies demonstrated that the combination of dexamethasone synergizes the antitumoral effects of calcitriol 
On the other hand, the role of calcitriol and its receptor has shown crucial activity in the proper activation of the immune system, particularly for T-cell development, differentiation, polarization, and function 
. In BC, tumor-infiltrating lymphocytes (TIL) play an important role against cancer cells in the tumor microenvironment; however, depending on the cellular signals, TILs can modify their phenotype and exhibit pro-tumoral actions 
. In this regard, in an orthotopic BC mouse model, it has been demonstrated that VD3 supplementation accompanied with a low-fat diet can avoid the progression of BC tumors. In contrast, in a regimen based on a high-fat diet also combined with VD3 supplementation, the growth of mammary tumors was evident. The above was correlated with changes in the activation status and infiltration of T CD8+ lymphocytes promoted by the inflammatory conditions associated with overweight. Importantly, VD supplementation also showed a reduction of both adipogenic markers and pro-inflammatory cytokines 
. These findings add different mechanisms of action of how VD3 supplementation can slow down the growth and development of tumors of mammary origin.
In addition, a vast number of immune cells express the CYP27B1 enzyme and the VDR, which favor both the conversion of circulating calcidiol into the active form and its intracellular signaling, respectively 
. In relation to this, it is important to remark that VD3 deficiency has correlated with a lack of successful response to immune checkpoint inhibitors (anti-PD-1, anti-PD-L1, or anti-CTLA4) in patients with metastatic renal carcinoma as compared with patients with high VD3 serum levels 
. The above prompts to consider that VD3 supplementation is an important adjuvant strategy to avoid the prevalence of cancer. In addition, VD3 supplementation could favor the therapeutic response in the onco-immuno-biological background. Of note, the relationship between hypovitaminosis of VD3 with immunotherapy in the BC context has been scarcely explored.
It has been demonstrated that CB1093 analog improves the responsiveness of BC cells to TNFα-induced cell death by promoting TNFα-induced cytosolic phospholipase A2 (PLA2) activation 
. Similarly, results from our laboratory demonstrated that the combination of calcitriol with TNFα resulted in a more significant antiproliferative effect than the drug alone in ER-positive and ER-negative BC cells 
Considering the magnitude of the problems generated by VD deficiency, and taking into account all the benefits of calcitriol as an antineoplastic and immunomodulatory agent, it is highly recommended to assess VD serum levels followed by its supplementation when necessary, in women with high risk of BC development or in-treatment for this pathology.
7. Calcitriol in Combination with Histamine Inhibitors in BC
Histamine is one of the first proinflammatory mediators to be described, and its primary sources are basophils and mast cells, which are distributed widely in the skin and mucosa. In response to allergic stimuli, a complex interaction between inflammatory cells is activated, and several inflammatory mediators are produced; among these, histamine, which regulates the maturation and activation of leukocytes and directs their migration to target sites where they cause chronic inflammation 
. Additionally, in vivo and in vitro studies have described that histamine is involved in cell proliferation, migration, and invasion of several cancers 
. Accordingly, the use of antihistamines has shown promising effects to fight this pathology, as in the case of astemizole, a non-sedating second-generation antihistamine, commonly prescribed for the treatment of allergies. This drug has been repurposed as an antineoplastic agent, since in addition to H1-histamine receptors blockade, it targets several other molecules involved in cancer development, such as P-glycoprotein and the voltage-gated potassium channels EAG1 and human EAG related genes (HERG) 
. Regarding BC, astemizole has shown to exert antiproliferative effects against both hormone-dependent and non-hormone-dependent BC cell lines, as well as in primary cell cultures derived from breast tumors 
. The antineoplastic effects of astemizole also have been evaluated in conjoint with other antineoplastic agents, including calcitriol 
. In BC cells, this antihistamine compound synergized the antiproliferative activity of calcitriol by downregulating CYP24A1, upregulating the VDR, and targeting EAG1 
. Moreover, in vivo studies showed that the co-administration of astemizole and calcitriol to mice xenografted with human BC cells inhibited tumor growth more efficiently than each drug alone 
. In summary, the therapeutic use of this antihistamine with calcitriol could be beneficial as adjuvant therapy for BC, independently of the tumor phenotype, since the molecular targets of these compounds are the VDR and EAG1 channel, both of them highly expressed in BC.