Growth inhibition and induction of apoptosis by 2-methoxyestradiol in rat osteosarcoma and malignant fibrous histiocytoma cell lines (original) (raw)
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Oncology Reports, 2004
2-Methoxyestradiol (2-ME) has been found to possess antitumor activity in vivo and in vitro. It has been suggested that 2-ME induces apoptosis resulting in G 2 /M arrest of tumor cells. In this study, the effect of 2-ME was evaluated in rat osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines. 2-ME was used at final concentrations of 100 nM to 2 ÌM. The effect of 2-ME on cell growth was measured by the MTS assay. Induction of apoptosis and activation of caspase-3 were investigated along with apoptosis-related gene expression. The data showed that 2-ME significantly inhibited cell growth, inducing apoptosis. The activity of caspase-3 was increased at 20 h and 40 h in both cell lines. 2-ME induced p16 expression, which was possibly involved in the apoptotic process. These results suggested that the 2-ME-induced apoptosis of rat osteosarcoma and rat MFH cells was accompanied by caspase-3 activation through p16 induction.
Reproductive Sciences, 2008
Objective: The estrogen metabolite 2-methoxyestradiol has shown antitumorigenic action in some epithelial tumors. In the present work we investigate its effects in ovarian cancer used alone or in combination with other apoptotic-inducing reagents such as tumor necrosis factor-related apoptosis-inducing ligand. Methods: To assess the effect of 2-methoxyestradiol, dose response and time courses in ovarian cancer and normal cells were conducted. Apoptosis was confirmed through DNA laddering, by flow cytometry, and Western blotting of proteins involved in the apoptotic cascade. Results: 2-Methoxyestradiol induced apoptosis in ovarian cancer cells but not in normal counterparts. 2-Methoxyestradiol activates both the intrinsic and extrinsic apoptotic pathways. 2-Methoxyestradiol-mediated apoptosis involves reactive oxygen species generation and caspasedependent and caspase-independent mechanisms. We also demonstrate that 2-methoxyestradiol selectively induces an additive/synergistic apoptotic response in ovarian cancer cells when used in combination with tumor necrosis factor-related apoptosis-inducing ligand. Conclusions: 2-Methoxyestradiol, alone or in combination with tumor necrosis factor-related apoptosis-inducing ligand, should be considered as a potential treatment for ovarian cancer.
Induction of apoptosis and G2/M arrest by 2-methoxyestradiol in human cervical cancer HeLaS3 cells
Anticancer research
It has been demonstrated that 2-Methoxyestradiol (2-ME), one of the estrogen metabolites, induces apoptosis in many different tumor cell lines. In the present study, the effects of 2-ME on human cervical cancer HeLaS3 cells and on normal cervical epithelial cells were evaluated. Acridine orange staining, DNA fragmentation arrays and flow cytometry were used to measure the apoptosis and cell cycle progression. In addition, the effect of 2-ME on expression of iNOS was measured by Western blot. 2-ME inhibited the growth of HeLaS3 cells. This growth inhibition was accompanied by apoptosis and G2/M cell cycle arrest. 2-ME increased the expression of iNOS in parallel with apoptosis. Moreover, apoptosis was prevented by the iNOS inhibitor 1400W. 2-ME treatment resulted in a slight increase of the G2/M population, but no apoptosis, in normal cervical epithelial cells. There was no synergetic effect between E2 and 2-ME. 2-ME induced apoptosis via the iNOS pathway and caused G2/M cell cycle a...
The Journal of Steroid Biochemistry and Molecular Biology, 2010
2-Methoxyestradiol (2ME) is an endogenous metabolite of 17β-estradiol exerting both antiangiogenic and antimitogenic effects in vitro and in vivo . 2ME is a target for 17β-hydroxysteroid dehydrogenase-mediated metabolism that explains its low bioavailability due to rapid metabolic breakdown . Results from preclinical tumor models in animals suggest that maintaining a plasma concentration of 2ME in the range of 3-17ng/ml (10-56nM) is needed for efficient anti-tumor activity . 2ME is registered as Panzem R by Entremed, Inc. (Rockville, MD) and is currently being used in clinical trials by making use of a novel nanocrystal dispersion (NCD) drug delivery system . The NCD formulation of 2ME was shown to have improved bioavailability resulting in plasma concentration levels within the range needed for anti-tumor activity . 2ME plays as role in the abrogation of microtubule dynamics and the inhibition of protein translation and activity of the anaphasepromoting complex (APC/C) . 2ME inhibits angiogenesis by interacting with endothelial tubulin dynamics which in turn negatively affects the expression and activity of hypoxia-inducible factor 1-alpha (HIF-1a) and vascular endothelial growth factor (VEGF) . 2ME can also induce antiangiogenic effects through apoptosis as a result of increased phosphorylation of protein kinase B (Akt), activation of c-jun N-terminal-(JNK), extracellular signal-regulated-(ERK) and p38-kinases, activation of the intrinsic apoptotic pathway through inactivation of B-cell lymphoma 2 (Bcl-2) and Bcl-xL proteins as well as the upregulating the extrinsic pathway by increasing the expression of death receptor 5 (DR5) leading to activation of caspase-8 . Fukui and Zhu (2008) demonstrated that 2ME induced the phosphorylation of Bcl-2 proteins, but did not significantly alter the levels of Bcl-2associated X protein (Bax) and Bcl-2 expression in MDA-MB-435 M14 melanoma derived metastatic cells . This is in contrast to the results found by wherein 2ME altered the ratio of Bax/Bcl-2 expression levels in esophageal 2 cancer cells and cervical carcinoma cells . In addition, 2ME induces apoptosis through the p38 pathway and not the JNK pathway in ovarian carcinoma cells, but activates and induces apoptosis through both the JNK and p38 pathways in prostate cancer cells . MCF-7 cells are known to be caspase-3 deficient, since they do not express the CASP-3 gene as a result of a 47-base pair deletion within exon 3 of the gene, thus causing abrogated translation of CASP-3 mRNA . These observations, among others, indicate that the mechanism of action of 2ME-induced growth inhibition is cell line specific.
2-methoxyestradiol induces caspase-independent, mitochondria-centered apoptosis in DS-sarcoma cells
International Journal of Cancer, 2004
The anti-cancer potential of the natural estrogen metabolite 2-methoxyestradiol is associated with microtubuli interaction, anti-angiogenetic effects and inhibition of superoxide dismutase leading to apoptosis. The effectors of apoptotic signaling through 2-methoxyestradiol, however, are cell type-dependent. We investigated the effect of 2-methoxyestradiol on several events associated with apoptosis in rat DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to mitochondria was identified as an initial apoptotic event that was accompanied by a decrease in mitochondrial transmembrane potential and the formation of reactive oxygen species (ROS) followed by mitochondrial release of apoptosis inducing factor and endonuclease G. In addition, 2-methoxyestradiol treatment caused upregulation of death receptor ligands FasL and TNF␣ and induced caspase-8 activation. The pan caspase inhibitor Z-VAD-FMK did not suppress apoptotic cell death, however, indicating that the major pro-apoptotic effect of 2-methoxyestradiol is mediated by a caspase-independent mechanism. Furthermore, ROS do not seem to play a pivotal role in the toxic/apoptotic effect of 2-methoxyestradiol in DS-sarcoma cells because supplementation with various antioxidants provided only limit protection. Colony formation was not affected by antioxidants. Therefore, in DS-sarcoma cells, the breakdown of mitochondrial integrity with the subsequent release of mitochondrial nucleases is the main factor in 2-methoxyestradiol mediated cell death.
2-Methoxyestradiol Induces Apoptosis in Cultured Human Anaplastic Thyroid Carcinoma Cells
Thyroid, 2006
Anaplastic thyroid carcinoma (ATC) is one of the most malignant tumors in humans, and currently there is no effective treatment. In the present study we investigated the effect of an endogenous estrogen metabolite, 2methoxyestradiol (2-ME), on the growth of human ATC cells. 2-ME treatment had a strong growth inhibitory effect on five human ATC cell lines (HTh7, HTh 74, HTh83, C643, and SW1736), but showed no effect on one cell line (KAT-4). Cell cycle analysis of the growth-inhibited cells showed that 2-ME induced a G 2 /M-arrest, followed by an increased fraction of cells in sub-G 1. Analysis of internucleosomal DNA laddering as well as DNA fragmentation in a terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay demonstrated a high number of cells undergoing apoptosis after 2-ME treatment. An increased activation of caspase-3 and caspase-8 by 2-ME was observed, and inhibition of caspase-3 decreased the apoptotic effect. Addition of 2-ME increased activity of p38 mitogen-activated protein kinase (MAPK) in the sensitive HTh7 as well as the refractory KAT-4 cells, however, activation of stress-activated protein kinase/c-jun aminoterminal kinase (SAPK/JNK) was seen only in the HTh7 cells. Inhibitors of p38 MAPK and SAPK/JNK significantly attenuated the 2-ME effect. Taken together, our data demonstrate an antiproliferative and apoptotic effect of 2-ME on ATC cells involving activation of MAPKs.
2-METHOXYESTRADIOL Induces Interferon Gene Expression and Apoptosis in Osteosarcoma Cells
Bone, 2002
2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17-estradiol, has been implicated as a physiological inhibitor of tumor cell proliferation. In this study, the effects of 2-ME on cultured osteosarcomatous cells were investigated. Dose-dependent growth inhibition was observed in MG63 and TE85 human osteosarcoma cells exposed to 2-ME. The cell killing by 2-ME was ligandspecific; the immediate precursor (2-hydroxyestradiol), the parent compound (17-estradiol), and the equivalent metabolite of estrone (2-methoxyestrone) exhibited less potency and efficacy. Furthermore, 2-ME was similarly effective at killing immortalized human fetal osteoblastic cells (hFOB) with and without estrogen receptor-␣ and- and rat osteosarcoma cells (ROS17/2.8). The cytotoxicity of 2-ME was selective to transformed and immortalized osteoblastic cells; 2-ME (2 m) had no effect on the proliferation of primary cultures of human osteoblasts. Co-treatment with the potent estrogen receptor ligand, ICI-182,780, did not reduce 2-ME-induced osteosarcoma cell death, implying that this action is not mediated by conventional estrogen receptors. The expression levels of bone matrix protein genes, type 1 collagen and osteonectin, were transiently reduced after 2-ME treatment, suggesting that the surviving cells are capable of producing bone matrix. The 2-ME-mediated killing of osteosarcoma cells was due to the induction of apoptosis; treatment induced expression of interferon genes within 12 h and histological evidence of apoptosis within 48 h of 2-ME treatment. Thus, our results demonstrate that 2-ME is highly cytotoxic to osteosarcoma cells but not normal osteoblasts. These findings suggest that further study of 2-ME as a potential intervention for treatment of osteosarcoma is warranted.
Sulphamoylated 2-Methoxyestradiol Analogues Induce Apoptosis in Adenocarcinoma Cell Lines
PLoS ONE, 2013
2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1-25 mM) was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 mM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues.
Cancer research, 2003
2-Methoxyestradiol (2ME2), a natural metabolite of estradiol, is a potent antitumor and antiangiogenic agent. In vitro, 2ME2 inhibits the proliferation of a wide variety of cell lines and primary cultures, and in numerous models in vivo, it has been shown to be an effective inhibitor of tumor growth and angiogenesis. 2ME2 is currently in several Phase I and Phase II clinical trials under the name Panzem. Although various molecular targets have been proposed for this compound, the mechanism by which 2ME2 exerts its effects is still uncertain. This study shows that 2ME2 uses the extrinsic pathway for induction of apoptosis. 2ME2 treatment results in up-regulation of death receptor 5 (DR5) protein expression in vitro and in vivo and renders cells more sensitive to the cytotoxic activities of the DR5 ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). 2ME2-induced apoptosis requires caspase activation and kinetic studies show the sequential activation of caspase-8, c...
A 2-methoxyestradiol bis-sulphamoylated derivative induces apoptosis in breast cell lines
Cell & Bioscience, 2015
Introduction: Research involving antimitotic compounds identified 2-methoxyestradiol (2ME2), as a promising anticancer endogenous metabolite. Owing to its low bioavailability, several in silico-designed 2ME2 analogues were synthesized. Structure-activity relationship studies indicated that an already existing 17-β-estradiol analogue, namely (8R,13S,14S,17S)-2-ethyl- 13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) to exert potential in vitro anticancer activity. Methods: This study investigated the in vitro apoptotic influence of EMBS in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7); an estrogen receptor-negative breast epithelial cell line (MDA-MB-231) and a non-tumorigenic breast cell line (MCF-12A). Cell cycle progression, a phosphatidylserine flip, caspase 6-, 7-and 8 enzyme activity levels, Bcl-2 phosphorylation status at serine 70 and Bcl-2-and p53 protein levels were investigated to identify a possible action mechanism for apoptotic induction. Results: The xCELLigence real-time label-independent approach revealed that EMBS exerted antiproliferative activity in all three cell lines after 24 h of exposure. A G 2 M block was observed and apoptosis induction was verified by means of flow cytometry using propidium iodide and Annexin V-FITC respectively. EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. EMBS exposure resulted in a statistically significant increase in p53 protein expression, decreased Bcl-2 protein expression and a decrease in pBcl-2(s70) phosphorylation status in all three cell lines. Results support the notion that EMBS induces apoptosis in all three cell lines. Conclusion: This study includes investigation into the apoptotic hallmarks exerted by EMBS after exposure of three cell lines namely MCF-7-, MDA-MDA-231-and MCF-12A cells. Increased caspase 6-, caspase 7-and caspase 8 activities, upregulation of p53 protein expression and a decrease in phosphorylation status of Bcl-2 at serine 70 in tumorigenic and non-tumorigenic lines were demonstrated.