PRIMA-1MET Does Not Restore Vitamin D Sensitivity to MDA-MB-231 and MDA-MB-468 Triple-Negative Breast Cancer Cells.

Vitamin D is a steroid hormone that causes growth suppression in cultured cells. We had previously discovered that the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 did not have growth suppression with vitamin D, while MCF-7 did. MCF-7 cells are not triple-negative and have wild-type p53. Both MDA-MB-231 and MDA-MB-468 have mutations in p53 and these mutations were a possible explanation for the lack of growth suppression with vitamin D. Our hypothesis was that reactivation of p53 in the triple-negative cell lines would cause them to become sensitive to vitamin D. We chose to use the small molecule PRIMA-1MET to reactivate p53 as it has been previously shown to restore function to the p53 mutants present in MB-231 and MB-468. We then measured the ability of vitamin D and its analogues calcipotriol and EB1089 to suppress growth in the presence of PRIMA-1MET. Here, we show that while PRIMA-1MET can kill the breast cancer cells investigated in this study, it does not restore their sensitivity to vitamin D or its analogues.

Effect of Vitamin D on Paclitaxel Efficacy in Triple-negative Breast Cancer Cell Lines.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer that disproportionately affects women with darker skin and is often treated with paclitaxel (PTX). Here, the effect of vitamin D on p53-positive DU4475 cells and its ability to decrease the IC50 of PTX in these cells were investigated. MATERIALS AND METHODS: The growth inhibitory effects of vitamin D on DU4475 cells and the effect of PTX plus vitamin D on overall TNBC cell viability was assessed using CellTiter-Glo®. RESULTS: Vitamin D increased proliferation of DU4475 cells at low concentrations and lowered the IC50 of PTX. However, it did not change the IC50 of PTX in MDA-MB-231 cells which remained largely viable. CONCLUSION: The effect of vitamin D on DU4475 cell viability was different than in other TNBC cells. The effect of PTX on DU4475 cells was enhanced with vitamin D. MDA-MB-231 cells were relatively resistant to the effects of PTX.

Altered cytotoxicity of ROS-inducing compounds by sodium pyruvate in cell culture medium depends on the location of ROS generation.

Induction of oxidative stress by drugs and other xenobiotics is an important mechanism of cytotoxicity. However, in vitro studies on the relationship between oxidative stress and cytotoxicity in cultured cells is frequently complicated by the fact that cell culture medium components affect reactive oxygen species (ROS) exposures in ways that vary with the mode of ROS production. The objectives of this study were to first determine the mode of ROS induction by certain model compounds when they are applied to cultured cells, and then to determine how ROS induction and cytotoxicity were affected by the ROS-quenching medium component pyruvate. Three compounds, eseroline, benserazide, and pyrogallol induced H2O2 in cell culture media independent of cells. However, another compound, menadione, induced H2O2 in a manner largely dependent on the MDA-MB-231 breast cancer cells used in this study, which is consistent with its known mechanism of inducing ROS through intracellular redox cycling. 1 mM pyruvate, as well as catalase, reduced the H2O2 in culture wells with each ROS inducer tested but it only reduced the cytotoxicity of cell-independent inducers. It reduced the cytotoxicity of benserazide and pyrogallol >10-fold and of eseroline about 2.5-fold, but had no effect on menadione cytotoxicity. From this data, it was concluded that depending on the mechanism of ROS induction, whether intra- or extracellular, a ROS-quenching medium component such as pyruvate will differentially affect the net ROS-induction and cytotoxicity of a test compound.

Vitamin D effect on growth and vitamin D metabolizing enzymes in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that disproportionately affects women with darker skin. Epidemiological studies indicate that higher vitamin D levels prevent incidence of TNBC and translate to higher survival rates in those that have TNBC. MATERIALS AND METHODS: The growth inhibition effects of two forms of vitamin D were assessed in MCF-7 and three TNBC lines using CellTiter-Glo. Expression of vitamin D-metabolizing enzymes was measured after vitamin D treatment by quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: MCF-7 was growth inhibited by vitamin D at high concentrations but the TNBC lines were not. All cell lines demonstrated large increases in CYP24A1 mRNA levels under vitamin D treatment but there was little change in CYP27B1 or VDR mRNA levels. CONCLUSION: These TNBC cell lines are resistant to growth inhibition by vitamin D. This could be due to large inactivation of vitamin D by CYP24A1 or by another mechanism.

Automation and miniaturization of the bioluminescent UGT-Glo assay for screening of UDP-glucuronosyltransferase inhibition by various compounds.

The uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes is involved in the metabolism of various compounds. These enzymes transfer a hydrophilic glucuronic acid moiety to their substrates, rendering them more water soluble and amenable to excretion. The UGTs act on various endogenous substrates, such as bilirubin, 17ß-estradiol, and testosterone, and drugs and other xenobiotics. The function of these enzymes is essential for the clearance of drugs and toxicants, and alteration of UGT activity is a potential cause of adverse drug-drug interactions in vivo. This has stimulated an increased interest in the study of UGT function and inhibition, and the desire to profile new drug entities against UGT enzymes, similar to CYP450 profiling. However, certain factors have hindered the development of a robust method for UGT profiling. Current methods for assessing UGT enzyme activity are laborious and involve protein precipitation and/or chromatographic separation steps, which are not amenable to rapid screening applications for UGT inhibitors or substrates. The approach presented here is a bioluminescent assay for measuring UGT enzyme activity and inhibition in vitro. Using flexible, robust instrumentation in a 384-well microplate format, this study highlights the quick and easy assay implementation for estimation of inhibition kinetics with a variety of known and suspected UGT substrates and inhibitors.