Victor Pribluda

2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF.

Inhibition of angiogenesis is an important new modality for cancer treatment. 2-methoxyestradiol (2ME2) is a novel antitumor and antiangiogenic agent, currently in clinical trials, whose molecular mechanism of action remains unclear. Herein, we report that 2ME2 inhibits tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo. Mechanistically, we found that 2ME2 downregulates hypoxia-inducible factor-1 (HIF) at the posttranscriptional level and inhibits HIF-1-induced transcriptional activation of VEGF expression. Inhibition of HIF-1 occurs downstream of the 2ME2/tubulin interaction, as disruption of interphase MTs is required for HIF-alpha downregulation. These data establish 2ME2 as a small molecule inhibitor of HIF-1 and provide a mechanistic link between the disruption of the MT cytoskeleton and inhibition of angiogenesis.

Withaferin A is a potent inhibitor of angiogenesis.

The medicinal plant Withania somnifera is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. In Ayurveda, the major Traditional Indian medicine system, extracts from W. somnifera are distinctively employed for the treatment of arthritis and menstrual disorders. Because these conditions involve angiogenic processes we hypothesized that the W. somnifera extracts might contain angiogenesis inhibitors. We employed an endothelial cell-sprouting assay to monitor the purification of substances from W. somnifera root extracts and isolated as the active principle the previously known natural product withaferin A. We show that withaferin A inhibits human umbilical vein endothelial cell (HUVEC) sprouting in three-dimensional collagen-I matrix at doses which are relevant to NF-kappa B-inhibitory activity. Withaferin A inhibits cell proliferation in HUVECs (IC50=12 nM) at doses that are significantly lower than those required for tumor cell lines through a process associated with inhibition of cyclin D1 expression. We propose that the inhibition of NF-kappa B by withaferin A in HUVECs occurs by interference with the ubiquitin-mediated proteasome pathway as suggested by the increased levels of poly-ubiquitinated proteins. Finally, withaferin A is shown to exert potent anti-angiogenic activity in vivo at doses that are 500-fold lower than those previously reported to exert anti-tumor activity in vivo. In conclusion, our findings identify a novel mode of action of withaferin A, which highlights the potential use of this natural product for cancer treatment or prevention.

2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate.

Abstract2-Methoxyestradiol, once considered an inacitve end-metabolite of estradiol, has recently emerged as a very promising agent for cancer treatment. It is orally active in a wide range of tumor models, and inhibits tumor growth at doses showing non clinical signs of toxicity. 2ME2 targets both the tumor cell and endothelial cell compartments by inducing apoptosis in rapidly proliferating cells and inhibiting blood vessel formation at several stages in the angiogenic cascade. Moreover, the ability of 2ME2 to inhibit metastic spread in several models adds to its therapeutic value for cancer treatment at various stages of the disease. Though the mechanism of action is still undefined, several potential molecular targets and pathways of activation have been suggested.

2-Methoxyestradiol Inhibits Hypoxia-Inducible Factor 1α, Tumor Growth, and Angiogenesis and Augments Paclitaxel Efficacy in Head and Neck Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinomas have been reported to overexpress hypoxia-inducible factor (HIF)-1α, a transcription factor that promotes expression of angiogenesis factors and resistance to programmed and therapy-induced cell death. 2-Methoxyestradiol (2ME2) is a natural compound with HIF-1α inhibitory activity that is currently being evaluated in phase 1 and 2 clinical trials for advanced solid tumors and multiple myeloma. To our knowledge, this is the first study to evaluate the effects of 2ME2 in head and neck squamous cell carcinoma. Experimental Design: In the present study, we investigated the effects of 2ME2 alone and in combination with paclitaxel, an active agent in recurrent or advanced head and neck squamous cell carcinoma. Results: 2ME2 exhibited antiproliferative and cytotoxic effects in a panel of five head and neck squamous cell carcinoma cell lines in the 0.5 to 10 μmol/L range, including induction of G2-M blockade, caspase-3/7 activation, and apoptosis at 48 hours. 2ME2 resulted in decreased nuclear HIF-1α–binding activity and affected the expression of downstream genes, such as bid, a proapoptotic bcl-2 family member, and vascular endothelial growth factor, a proangiogenic cytokine. The up-regulation of Bid (57.5% at 12 hours, P < 0.0006) and inhibition of vascular endothelial growth factor secretion (57.7% at 24 hours, P < 0.015; and 50.3% at 48 hours, P < 0.0006) could be partially attributed to the effects on HIF-1α, because HIF-1α small interfering RNAs produced similar effects. Finally, in vivo, in a xenograft model of head and neck squamous cell carcinoma using UM-SCC-11A cells, 2ME2 exhibited antitumor and antiangiogenic activity, as measured by CD31 immunostaining. Conclusions: These results provide support for the use of 2ME2 in combination with paclitaxel for the treatment of recurrent or advanced head and neck squamous cell carcinoma.

Synthesis of 2- and 17-substituted estrone analogs and their antiproliferative structure–activity relationships compared to 2-methoxyestradiol

A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2 and have diminished metabolic liability. The analogs were evaluated for antiproliferative activity against MDA-MB-231 breast tumor cells, antiangiogenic activity in HUVEC, and estrogenic activity on MCF-7 cell proliferation. Several analogs were evaluated for metabolic stability in human liver microsomes and in vivo in a rat cassette dosing model. This study lead to several 17-modified analogs of 2ME2 that have similar or improved antiproliferative and antiangiogenic activity, lack estrogenic properties and have improved metabolic stability compared to 2ME2.

Synthesis, antiproliferative, and pharmacokinetic properties of 3- and 17-double-modified analogs of 2-methoxyestradiol.

The syntheses of 21 analogs of 2-methoxyestradiol are presented, including ENMD-1198 which was selected for advancement into Phase 1 clinical trials in oncology. These analogs were evaluated for antiproliferative activity using breast tumor MDA-MB-231 cells, for antiangiogenic activity in HUVEC proliferation assays, and for estrogenic activity in MCF-7 cell proliferation. The most active analogs were evaluated for iv and oral pharmacokinetic properties via cassette dosing in rat and in mice pharmacokinetic models.