Synergistic in-vitro effects of combining an antiglycolytic, 3-bromopyruvate, and a bromodomain-4 inhibitor on U937 myeloid leukemia cells.

This project investigated the in-vitro effects of a glycolytic inhibitor, 3-bromopyruvate (3-BrP), in combination with and a new in silico-designed inhibitor of the bromodomain-4 (BRD-4) protein, ITH-47, on the U937 acute myeloid leukemia cell line. 3-BrP is an agent that targets the altered metabolism of cancer cells by interfering with glucose metabolism in the glycolytic pathway. ITH-47 is an acetyl-lysine inhibitor that displaces bromdomain 4 proteins from chromatin by competitively binding to the acetyl-lysine recognition pocket of this bromodomain and extraterminal (BET) BRD protein, thereby preventing transcription of cancer-associated genes and further cell growth. Cell growth studies determined the IC50 after 48 h exposure for 3-BrP and ITH-47 to be 6 and 2 µmol/l, respectively. When combined, 2.4 and 1 µmol/l of 3-BrP and ITH-47, respectively, inhibited 50% of the cell population, yielding a synergistic combination index of 0.9. Subsequent mechanistic studies showed that the IC50 concentrations of ITH-47 and 3-BrP and the combination increased observable apoptotic bodies and cell shrinkage in U937 cells treated for 48 h. Cell cycle analysis showed an increase in the sub-G1 fraction in all treated cells, suggesting that cell death was increased in the treated samples. Annexin-V-FITC apoptosis analysis showed a statistically significant increase in the number of cells in early and late apoptosis, indicating that cell death occurred through apoptosis and not necrosis. Only U937 cells exposed to ITH-47 showed a decrease in mitochondrial membrane potential compared with the vehicle control. Reactive oxygen species production was decreased in all treated samples. ITH-47-exposed cells showed a decrease in c-Myc, Bcl-2, and p53 gene expressions. 3-BrP-treated cells showed an increase in c-myc and p53 gene expressions. The combination of ITH-47 and 3-BrP lead to downregulation of c-myc and Bcl-2 genes. ITH-47 exposure conditions yielded a marked decrease in c-myc protein levels as well as a decrease in Ser70 phosphorylated Bcl-2. Analysis of 3-BrP and the combination of ITH-47 and 3-BrP test conditions indicated an increase in p53 protein levels. This novel study is the first to investigate the in-vitro synergistic therapeutic effect of ITH-47 and 3-BrP. The current study contributes toward unraveling the in-vitro molecular mechanisms and signal transduction associated with a novel combination of BRD inhibitors and antiglycolytic agents, providing a basis for further research on these combinations.

In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17-ß-estradiol analogue in breast adenocarcinoma cells.

2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-ß-estradiol analogue, was tested on the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 µM of C19 on MCF-7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.