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Hepatocellular carcinoma (HCC) is associated with a poor prognosis. 2-methoxyestradiol (2-ME) is currently under preclinical evaluation as a treatment for many malignancies, but the utility of the drug in terms of HCC treatment remains unclear. Here, we explored the effect of 2-ME on human huh7 cell proliferation and apoptosis and discuss the possible molecular mechanisms involved. The MTT assay showed that proliferation was markedly inhibited by 2-ME (at 5, 10, 15, and 20 μmol/L) in a time- and dose-dependent manner. Moreover, flow cytometry indicated that 2-ME induced cell cycle arrest at the G2/M phase, and early apoptosis. We used Western blotting and PCR to detect the expression of vascular endothelial growth factor (VEGF) and Bcl-2; 2-ME decreased the mRNA/protein expression levels of both effectors. Furthermore, 2-ME remarkably suppressed xenograft tumor growth in nude mice, and no visible toxicity was observed in either the liver or kidneys. Immunohistochemically, the Bcl-2 and VEGF expression levels were significantly lower than those of controls. Thus, 2-ME inhibited huh7 cell proliferation, promoted apoptosis, and suppressed xenograft tumor growth in nude mice, perhaps reflecting the effects of the drug on VEGF and Bcl-2 expression., (Copyright © 2019. Published by Elsevier Inc.)

Although hypoxia-inducible factor-1α (HIF-1α) has been extensively studied in brain injury following hypoxia-ischemia, the role of HIF-1α in early brain injury (EBI) after subarachnoid hemorrhage (SAH) remains unclear. The present study was under taken to investigate a potential role of HIF-1α in EBI after SAH. Rats (n=60) were randomly divided into sham+vehicle, SAH+2-methoxyestradiol (2ME2), and SAH+vehicle groups. The SAH model was induced by endovascular perforation and all the rats were subsequently sacrificed at 24h after SAH. We found that treatment with 2ME2 suppressed the expression of HIF-1α, BNIP3 and VEGF and reduced cell apoptosis, blood-brain barrier (BBB) permeability, brain edema, and neurologic scores. Double fluorescence labeling revealed that HIF-1α was expressed predominantly in the nuclei of neurons and TUNEL-positive cells. Our work demonstrated that HIF-1α may play a role in EBI after SAH, causing cell apoptosis, BBB disruption, and brain edema by up-regulating its downstream targets, BNIP3 and VEGF. These effects were blocked by the HIF-1α inhibitor, 2ME2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: It is of clinical importance to find methods to overcome bortezomib resistance. In the current study, we clarified the relationship between resistance to bortezomib and the differentiation status of myeloma cells, and explored the feasibility of induction of differentiation in overcoming bortezomib resistance in myeloma., Methods: Cell morphology, immunoglobulin light-chain protein secretion levels, and XBP-1 expression were used to evaluate the differentiation status of myeloma cells. Low dose 2-ME2 alone or in combination with ATRA was used to induce differentiation in myeloma cells., Results: The differentiation status of myeloma cells was related to myeloma sensitivity to bortezomib. After successful induction of differentiation, the myeloma cells were more sensitive to bortezomib with decreased growth and an increased rate of apoptosis. Induction of differentiation increased the proteasome workload in myeloma cells by increasing immunoglobulin secretion, while reducing proteasome capacity by decreasing proteasome activity. The imbalance between increased proteasome workload and decreased proteasome capacity is a possible mechanism by which induction of differentiation overcomes myeloma resistance to bortezomib., Conclusion: The current study demonstrated, for the first time, that myeloma differentiation status is associated with myeloma sensitivity to bortezomib and that induction of differentiation can overcome myeloma resistance to bortezomib., (Copyright © 2012 Elsevier Inc. All rights reserved.)

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF1alpha) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1alpha in HEK293 cells was found not to be due to an effect on HIF1alpha synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1alpha in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho(-) cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1alpha stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect.

Few therapeutic treatment options are available for patients suffering from metastatic androgen-independent prostate cancer. We investigated the ability of the estrogen metabolite 2-methoxyestradiol to inhibit the proliferation of a variety of human prostate cancer cell lines in vitro and to inhibit the growth of androgen-independent prostate cancer in a transgenic mouse model in vivo. Our results showed that 2-methoxyestradiol is a powerful growth inhibitor of LNCaP, DU 145, PC-3, and ALVA-31 prostate cancer cells. Cell flow cytometry of 2-methoxyestradiol-treated DU 145 cells showed a marked accumulation of cells in the G2/M phase of the cell cycle and an increase in the sub-G1 fraction (apoptotic). In addition, staining for annexin V, changes in nuclear morphology, and inhibition of caspase activity support a role for apoptosis. More importantly, we showed that 2-methoxyestradiol inhibits prostate tumor progression in the Ggamma/T-15 transgenic mouse model of androgen-independent prostate cancer without toxic side effects. These results in cell culture and an animal model support investigations into the clinical use of 2-methoxyestradiol in patients with androgen-independent prostate cancer., (Copyright 2001 Academic Press.)

Estrogen receptors (ERs) are widely held to mediate the ability of 17 beta-estradiol (estradiol) to attenuate injury-induced proliferation of vascular smooth muscle cells (VSMCs) leading to vascular lesions. However, recent findings that estradiol prevents injury-induced vascular lesion formation in knock-out mice lacking either ER alpha or ER beta seriously challenge this concept. Here we report that the local metabolism of estradiol to methoxyestradiols, endogenous metabolites of estradiol with no affinity for ERs, is responsible for the ER-independent inhibitory effects of locally applied estradiol on rat VSMC growth. These finding imply that local vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of circulating estradiol. Thus, interindividual differences, either genetic or acquired, in the vascular metabolism of estradiol may define a given female's risk of cardiovascular disease and influence the cardiovascular benefit she receives from estradiol replacement therapy in the postmenopausal state. These findings also imply that nonfeminizing estradiol metabolites may confer cardiovascular protection in both women and men., (Copyright 2000 Academic Press.)

The aromatase enzyme, which converts androstenedione to oestrone, regulates the availability of oestrogen to support the growth of hormone-dependent breast tumours. Cytokines, such as interleukin 6 (IL-6) and tumour necrosis factor alpha (TNFalpha) or prostaglandin E(2) (PGE(2)), can stimulate aromatase activity. These factors may originate from cells of the immune system that infiltrate breast tumours. Paclitaxel, which is used in the treatment of breast cancer, stabilizes microtubules and has previously been shown to rapidly down-regulate TNF-receptors on human macrophages. The endogenous oestrogen metabolite, 2-methoxyestradiol (2-meOE2), also acts to stabilize microtubules. In this study, we have examined the ability of paclitaxel or 2-meOE2 to antagonise TNFalpha-stimulated aromatase activity in stromal fibroblasts derived from normal or malignant breast tissues. Paclitaxel inhibited basal and TNFalpha-stimulated aromatase activities by 88% and 91% respectively. 2-MeOE2 also reduced basal and TNFalpha-stimulated aromatase activities by 46% and 56% respectively. Both paclitaxel and 2-meOE2 also inhibited stimulation of aromatase activity by IL-6 plus its soluble receptor and PGE(2). The 16alpha-hydroxylated derivative of 2-meoE2 and 2-meOE3, which does not bind to microtubules, was less effective at inhibiting TNFalpha-stimulated aromatase activity. Increased 2-hydroxylation of oestrogens, and subsequent formation of their 2-methoxy derivatives, may be associated with a reduced risk of breast cancer. It is possible that the pathway of oestrogen metabolism may influence the ability of stromal cells to respond to cytokine stimulation., (Copyright 1999 Academic Press.)

2-Methoxyestradiol (2ME) is an endogenous metabolite of estradiol (E2) and is known to inhibit tumor angiogenesis. In the present study, the direct effects of 2ME on the vascular endothelial cells were examined. 2ME enhanced apoptosis and beta-galactosidase expression in bovine vascular endothelial cells. A nitric oxide (NO) donor S-nitroso-N-acetyl penicillamin (SNAP) also enhanced beta-galactosidase expression, suggesting a possible role of NO in mediating the action of 2ME. 2ME increased the cellular content of nitric oxide synthase (NOS) and the production of NO. In addition, 2ME altered the membrane localization pattern of NOS. These suggest that the effects of 2ME on apoptosis and senescence of vascular endothelial cells were mediated, at least partly, by NOS and NO.

The natural estrogen metabolite 2-methoxyestradiol (2ME) is anti-angiogenic in vivo and a strong growth inhibitor in vitro. The growth inhibition is due to mitotic arrest and apoptosis. These effects are reminiscent of those induced by taxol, and appear to be mediated by inhibition of microtubule dynamics. Here we have studied the cellular response to 2ME in regard to potential mediators of the observed cellular changes. 2ME treatment increases the insoluble polymerized fraction of cellular tubulin similar to taxol, and in contrast to the microtubule depolymerizing drugs such as colcemid and vincristine. This stabilization following 2ME treatment is accompanied by phosphorylation and inactivation of Bcl-2 increasing gradually from 2-24 hours. To study the pathway leading to Bcl-2 phosphorylation we analyzed Raf-1 and JNK/SAPK kinases, both of which have been reported to be involved in Bcl-2 inactivation. Our results indicate that Raf-1 is phosphorylated in response to 2ME, but this occurs later than Bcl-2 phosphorylation suggesting that Raf-1 is not directly phosphorylating Bcl-2. JNK/SAPK was activated rapidly after 2ME treatment. However, this activation was transient and returned to undetectable levels by 2 hours of treatment, demonstrating that JNK/SAPK is not directly phosphorylating Bcl-2. Taken together with previous results indicating that overexpression of JNK/SAPK leads to Bcl-2 phosphorylation, our results would support a model where JNK/SAPK is indirectly phosphorylating Bcl-2.

The endogenous estrogen metabolite 2-methoxyestradiol (2-MeOE2) suppresses experimental tumor growth in vivo and inhibits angiogenesis activity in vitro. Moreover, 2-MeOE2 has been observed to block mitosis in cell cultures. As high concentrations of 2-MeOE2 prevent microtubule assembly in vitro, the mitotic arrest has been attributed to inhibition of tubulin polymerization. Here we report that concentrations of 2-MeOE2 that cause complete metaphasal arrest do not inhibit the assembly of mitotic spindles. In contrast to the chromosomal dispersal seen in cells arrested by the tubulin depolymerizing drug colcemid, the chromosomes of cells treated with 2-MeOE2 remained in the metaphasal plate indicating a functional defect of the mitotic spindle. The 2-MeOE2 arrest resembles those induced by compounds affecting microtubule dynamics such as taxol and vinblastine. The 2-MeOE2 block is also similar to that induced by several anti-calmodulin agents. Given that metaphase to anaphase transition is a calmodulin-dependent step and our observation that 2-MeOE2 inhibits calmodulin activity in vitro, we suggest that the 2-MeOE2 metaphasal arrest may occur via inhibition of calmodulin.

The effect of endogenous estrogens on sex-hormone-binding globulin (SHBG) production was studied in HepG2 cells. 17 beta-estradiol, estrone, and estrogens from both 2- and 16 alpha-hydroxylative pathways stimulated SHBG production, but not in parallel with their binding affinities for the estrogen receptor. Thus, the underlying mechanism may be other than a pure interaction with the estrogen receptor.