Radiosensitization of Breast Cancer Cells with a 2-Methoxyestradiol Analogue Affects DNA Damage and Repair Signaling In Vitro.

Radiation resistance and radiation-related side effects warrant research into alternative strategies in the application of this modality to cancer treatment. Designed in silico to improve the pharmacokinetics and anti-cancer properties of 2-methoxyestradiol, 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) disrupts microtubule dynamics and induces apoptosis. Here, we investigated whether pre-exposure of breast cancer cells to low-dose ESE-16 would affect radiation-induced deoxyribonucleic acid (DNA) damage and the consequent repair pathways. MCF-7, MDA-MB-231, and BT-20 cells were exposed to sub-lethal doses of ESE-16 for 24 h before 8 Gy radiation. Flow cytometric quantification of Annexin V, clonogenic studies, micronuclei quantification, assessment of histone H2AX phosphorylation and Ku70 expression were performed to assess cell viability, DNA damage, and repair pathways, in both directly irradiated cells and cells treated with conditioned medium. A small increase in apoptosis was observed as an early consequence, with significant repercussions on long-term cell survival. Overall, a greater degree of DNA damage was detected. Moreover, initiation of the DNA-damage repair response was delayed, with a subsequent sustained elevation. Radiation-induced bystander effects induced similar pathways and were initiated via intercellular signaling. These results justify further investigation of ESE-16 as a radiation-sensitizing agent since pre-exposure appears to augment the response of tumor cells to radiation.

2-Methoxyestradiol and its derivatives inhibit store-operated Ca2+ entry in T cells: Identification of a new and potent inhibitor.

T cell activation starts with formation of second messengers that release Ca2+ from the endoplasmic reticulum (ER) and thereby activate store-operated Ca2+ entry (SOCE), one of the essential signals for T cell activation. Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca2+ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca2+ entry via SOCE without affecting other ion channels and pumps involved in Ca2+ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.

Design, Synthesis, and Evaluation of Novel 2-Methoxyestradiol Derivatives as Apoptotic Inducers Through an Intrinsic Apoptosis Pathway.

In order to discover novel derivatives in the anti-tumor field, reported anti-tumor pharmacophores (uridine, uracil, and thymine) were combined with 2-methoxyestradiol, which has been characterized as having excellent biological properties in terms of anti-tumor activity. Thus, 20 hybrids were synthesized through etherification at the 17ß-OH or 3-phenolic hydroxyl group of 2-methoxyestradiol, and evaluated for their biological activities against the human breast adenocarcinoma MCF-7 cell lines, human breast cancer MDA-MB-231 cell lines, and the normal human liver L-O2 cell lines. As a result, all the uridine derivatives and single-access derivatives of uracil/thymine possessed good anti-proliferative activity against tested tumor cells (half maximal inhibitory concentration values from 3.89 to 19.32 µM), while only one dual-access derivative (21b) of thymine possessed good anti-proliferative activity (half maximal inhibitory concentration ≈ 25 µM). Among them, the uridine derivative 11 and the single-access derivative of uracil 12a possessed good anti-proliferative selectivity against tested tumor cells. Furthermore, basic mechanism studies revealed that hybrids 11 and 12a could induce apoptosis in MCF-7 cells through mitochondrial pathway. These hybrids induced morphological changes in MCF-7 cells, causing mitochondrial depolarization. These two hybrids also had the following effects: arrest of the cell cycle at the G2 phase; upregulation of Apaf-1, Bax, and cytochrome c; downregulation of Bcl-2 and Bcl-xL for both mRNA and protein; and increase of the expression for caspase-8 and -9. Finally, apoptotic effector caspase-3 was increased, which eventually caused nuclear apoptosis at least through an intrinsic pathway in the mitochondria. Additionally, hybrids 11 and 12a could specifically bind to estradiol receptor alpha in a dose-dependent manner.

Discovery of chalcone-modified estradiol analogs as antitumour agents that Inhibit tumour angiogenesis and epithelial to mesenchymal transition.

Angiogenesis plays an essential role in tumourigenesis and tumour progression, and anti-angiogenesis therapies have shown promising antitumour effects in solid tumours. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, has been regarded as a potential antitumour agent mainly targeting angiogenesis. Here we synthesized a novel series of chalcones based on 2-methoxyestradiol and evaluated their potential activities against tumours. Compound 11e was demonstrated to have potent antiangiogenic activity. Further studies showed that 11e suppressed tumour growth in human breast cancer (MCF-7) xenograft models without obvious side effects. Evaluation of the mechanism revealed that 11e targeted the epithelial to mesenchymal transition (EMT) process in MCF-7 cells and inhibited HUVEC migration and then contributed to hindrance of angiogenesis. Thus, 11e may be a promising antitumour agent with excellent efficacy and low toxicity.