A High Dose of Calcitriol Inhibits Glycolysis and M2 Macrophage Polarization in the Tumor Microenvironment by Repressing mTOR Activation: in vitro and Molecular Docking Studies.

BACKGROUND/AIMS: Macrophages interact with tumor cells within the tumor microenvironment (TME), which plays a crucial role in tumor progression. Cancer cells also can instruct macrophages to facilitate the spread of cancer and the growth of tumors. Thus, modulating macrophages-cancer cells interaction in the TME may be therapeutically beneficial. Although calcitriol (an active form of vitamin D) has anticancer properties, its role in TME is unclear. This study examined the role of calcitriol in the regulation of macrophages and cancer cells in the TME and its influence on the proliferation of breast cancer cells. METHODS: We modeled the TME, in vitro, by collecting conditioned medium from cancer cells (CCM) and macrophages (MCM) and culturing each cell type separately with and without (control) a high-dose (0.5 µM) calcitriol (an active form of vitamin D). An MTT assay was used to examine cell viability. Apoptosis was detected using FITC (fluorescein isothiocyanate) annexin V apoptosis detection kit. Western blotting was used to separate and identify proteins. Quantitative real-time PCR was used to analyze gene expression. Molecular docking studies were performed to evaluate the binding type and interactions of calcitriol to the GLUT1 and mTORC1 ligand-binding sites. RESULTS: Calcitriol treatment suppressed the expression of genes and proteins implicated in glycolysis (GLUT1, HKII, LDHA), promoted cancer cell apoptosis, and reduced viability and Cyclin D1gene expression in MCM-induced breast cancer cells. Additionally, calcitriol treatment suppressed mTOR activation in MCM-induced breast cancer cells. Molecular docking studies further showed efficient binding of calcitriol with GLUT1 and mTORC1. Calcitriol also inhibited CCM-mediated induction of CD206 and increased TNFα gene expression in THP1-derived macrophages. CONCLUSION: The results suggest that calcitriol may impact breast cancer progression by inhibiting glycolysis and M2 macrophage polarization via regulating mTOR activation in the TME and warrants further investigation in vivo.

Protective effects of eicosapentaenoic acid in adipocyte-breast cancer cell cross talk.

Breast cancer is the leading cause of death in women among all cancer types. Obesity is one of the factors that promote progression of breast cancer, especially in post-menopausal women. Increasingly, adipose tissue is recognized for its active role in the tumor microenvironment. We hypothesized that adipocytes conditioned medium can impact breast cancer progression by increasing inflammatory cytokines production by cancer cells, and subsequently increasing their motility. By contrast, eicosapentaenoic acid (EPA), an anti-inflammatory n-3 polyunsaturated fatty acid, reduces adipocyte-secreted inflammatory factors, leading to reduced cancer cell motility. To test these hypotheses, we investigated the direct effects of EPA on MCF-7 and MDA-MB-231 breast cancer cells and the effects of conditioned medium from 3 T3-L1 or human mesenchymal stem cells (HMSC)-derived adipocytes treated with or without EPA supplementation on breast cancer cells. We observed that conditioned medium from HMSC-derived adipocytes significantly increased mRNA transcription levels of cancer-associated genes such as FASN, STAT3 and cIAP2, while EPA-treated HMSC-derived adipocytes significantly reduced mRNA levels of these genes. However, direct EPA treatment significantly reduced mRNA content of these tumor-associated markers (FASN, STAT3, cIAP-2) only in MDA-MB-231 cells not in MCF-7 cells. Conditioned medium from EPA-treated 3 T3-L1 adipocytes further decreased inflammation, cell motility and glycolysis in cancer cells. Our data confirms that adipocytes play a significant role in promoting breast cancer progression and demonstrates that EPA-treated adipocytes reduced the negative impact of adipocyte-secreted factors on breast cancer cell inflammation and migration.

Cytotoxicity of Selenium Immunoconjugates against Triple Negative Breast Cancer Cells.

Within the subtypes of breast cancer, those identified as triple negative for expression of estrogen receptor α (ESR1), progesterone receptor (PR) and human epidermal growth factor 2 (HER2), account for 10⁻20% of breast cancers, yet result in 30% of global breast cancer-associated deaths. Thus, it is critical to develop more targeted and efficacious therapies that also demonstrate less side effects. Selenium, an essential dietary supplement, is incorporated as selenocysteine (Sec) in vivo into human selenoproteins, some of which exist as anti-oxidant enzymes and are of importance to human health. Studies have also shown that selenium compounds hinder cancer cell growth and induce apoptosis in cancer cell culture models. The focus of this study was to investigate whether selenium-antibody conjugates could be effective against triple negative breast cancer cell lines using clinically relevant, antibody therapies targeted for high expressing breast cancers and whether selenium cytotoxicity was attenuated in normal breast epithelial cells. To that end, the humanized monoclonal IgG1 antibodies, Bevacizumab and Trastuzumab were conjugated with redox selenium to form Selenobevacizumab and Selenotrastuzumab and tested against the triple negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231 as well as a normal, immortalized, human mammary epithelial cell line, HME50-5E. VEGF and HER2 protein expression were assessed by Western. Although expression levels of HER2 were low or absent in all test cells, our results showed that Selenobevacizumab and Selenotrastuzumab produced superoxide (O2•-) anions in the presence of glutathione (GSH) and this was confirmed by a dihydroethidium (DHE) assay. Interestingly, superoxide was not elevated within HME50-5E cells assessed by DHE. The cytotoxicity of selenite and the selenium immunoconjugates towards triple negative cells compared to HME-50E cells was performed in a time and dose-dependent manner as measured by Trypan Blue exclusion, MTT assay and Annexin V assays. Selenobevacizumab and Selenotrastuzumab were shown to arrest the cancer cell growth but not the HME50-5E cells. These results suggest that selenium-induced toxicity may be effective in treating TNBC cells by exploiting different immunotherapeutic approaches potentially reducing the debilitating side effects associated with current TNBC anticancer drugs. Thus, clinically relevant, targeting antibody therapies may be repurposed for TNBC treatment by attachment of redox selenium.

Protective properties of n-3 fatty acids and implications in obesity-associated breast cancer.

Obesity is well documented as a risk factor for developing breast cancer, especially in postmenopausal women. Adipose tissue in the breast under obese conditions induces inflammation by increasing macrophage infiltration and pro-inflammatory cytokines that in turn up-regulates genes and signaling pathways, resulting in increased inflammation, cell proliferation and tumor growth in the breast. Due to their potent anti-inflammatory effects, n-3 polyunsaturated fatty acids (n-3 PUFA) are a promising and safe dietary intervention in reducing breast cancer risk. Here, we briefly review current status of breast cancer and its relationship with obesity. We then review in depth, current research and knowledge on the role of n-3 PUFA in reducing/preventing breast cancer cell growth in vitro, in vivo and in human studies, and how n-3 PUFA may modulate signaling pathways mitigating their effects on breast cancer development.

Paclitaxel induces apoptosis in breast cancer cells through different calcium--regulating mechanisms depending on external calcium conditions.

Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an "Enhanced Calcium Efflux" mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel's stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis.