Elsevier

Biochemical Pharmacology

Volume 94, Issue 4, 15 April 2015, Pages 257-269
Biochemical Pharmacology

Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells

https://doi.org/10.1016/j.bcp.2015.02.011Get rights and content

Abstract

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E2)-induced cell cycle arrest and 2-MeO-E2-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E2 (0.5–1.0 μM) caused G2/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G2/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E2 and the G1/S blocking agent aphidicolin resulted in G1/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E2-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10–20 μM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0 μM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5–1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.

Graphical abstract

The apoptogenic activity of 2-MeO-E2 was attributable to mitotic spindle damage, prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.

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Introduction

2-Methoxyestradiol (2-MeO-E2), an endogenous metabolite of 17β-E2, has been examined as a promising anticancer drug candidate [1]. Recently, 2-MeO-E2 has received great attention due to its anticancer activity along with its few undesirable side effects. The majority of tumor cell lines appear to be sensitive to the in vitro anti-proliferative properties of 2-MeO-E2 at concentrations ranging from 0.08 μM to 5.0 μM [2], [3]. Numerous studies have reported that the anticancer effects of 2-MeO-E2 at pharmacological concentrations are exerted by inducing apoptosis, arresting the cell cycle at the G1/S boundary and/or the G2/M boundary, and potentially inhibiting angiogenesis [4], [5], [6], [7], [8], [9].

The 2-MeO-E2-induced apoptosis of tumor cells appears to be mediated by several different mechanisms, including up-regulation of the death receptor (DR5), p53, and p21, down-regulation of Bcl-2, phosphorylation of Bcl-2 and Bcl-xL, generation of reactive oxygen species (ROS), activation of c-Jun N-terminal kinase (JNK), and mitochondrial cytochrome c release in an estrogen receptor (ER)-independent manner [3], [10], [11], [12], [13]. With respect to 2-MeO-E2-induced cell cycle arrest, its interference in cellular microtubule formation, which occurs via reducing the tubulin polymerization rate, has been implicated [14]. Additionally, the G2/M-promoting doses of 2-MeO-E2 and paclitaxel (a microtubule-polymerizing drug) have been shown to exert similar effects on the cell cycle and apoptosis in human prostate cancer cells [15]. These previous studies suggest that the mechanism underlying 2-MeO-E2-induced cell cycle arrest may be similar to those of microtubule-targeting drugs, which commonly induce the disruption of mitotic spindles and loss of microtubule function, leading to arrest at the M phase due to activation of the mitotic spindle assembly checkpoint [16], [17]. However, several studies have reported that following treatment with 2-MeO-E2, tumor cells undergo cell cycle arrest at the G1/S phase or late G2 phase rather than the M phase, along with apoptosis [4], [5], [6], [9]. In addition, 2-MeO-E2 appears to inhibit tubulin polymerization by interacting with its colchicine-binding site, and the Ki value of 2-MeO-E2 for inhibition of colchicine binding appears to be 22 μM, which is a much higher concentration than that which is required to induce apoptosis [14]. Furthermore, it has been reported that Bcl-2 overexpression in Jurkat T cells via retroviral transduction can prevent 2-MeO-E2 (0.5–1.0 μM)-induced apoptosis via p27Kip1-mediated G1/S arrest and NF-κB activation, suggesting that Jurkat T cells might arrest at the G1/S phase prior to undergoing apoptosis in the presence of 2-MeO-E2 (0.5–1.0 μM) [9]. Although these previous studies raised the possibility that 2-MeO-E2 at low doses (0.5–1.0 μM) could induce apoptosis independently of microtubule damage and subsequent mitotic arrest, the correlation between cell cycle arrest and apoptosis in tumor cells following 2-MeO-E2 treatment requires further investigation in order to clarify the anticancer activity of 2-MeO-E2.

Recently, to obtain direct evidence for a causal link between 2-MeO-E2-induced cell cycle arrest and 2-MeO-E2-induced apoptosis, we also decided to take advantage of Bcl-2 overexpression, which can inhibit 2-MeO-E2-induced apoptosis. Bcl-2 overexpression has previously been utilized to determine the correlation between p53-mediated G1 arrest and p53-mediated apoptosis in murine M1 myeloid leukemia cells, in which p53-mediated G1 arrest was not detectable unless the simultaneous induction of p53-mediated apoptosis was delayed by Bcl-2 overexpression [18]. In this study, to examine whether 2-MeO-E2 arrests cell cycle progression at the G1/S phase and/or the G2M phase and the mechanism by which 2-MeO-E2-induced cell cycle arrest activates the apoptotic death pathway, we investigated the apoptogenic mechanism of 2-MeO-E2 (0.1–1.0 μM) using Jurkat T cell clone stably transfected with an empty vector (JT/Neo) or a Bcl-2 expression vector (JT/Bcl-2). To further examine the dependency of 2-MeO-E2-induced apoptotic events on G1/S arrest and/or G2/M arrest, we investigated the effect of aphidicolin (APC), which is known to arrest cell cycle progression at the G1/S border [19], [20], on 2-MeO-E2-induced apoptosis.

Section snippets

Reagents, antibodies, and cells

2-MeO-E2, APC, 3,3′dihexyloxacarbocyanine iodide (DiOC6), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Chemical (St. Louis, MO, USA). An ECL western blot kit was purchased from Amersham (Arlington Heights, IL, USA), and the Immobilon-P membrane was obtained from Millipore Corporation (Bedford, MA, USA). The anti-caspase-3 antibody was purchased from Pharmingen (San Diego, CA), and the anti-poly (ADP-ribose) polymerase (PARP), anti-Bax, anti-Bim, anti-Bcl-2, anti-Bcl-xL,

Apoptogenic effect of 2-MeO-E2 on human Jurkat T cell clones JT/Neo and JT/Bcl-2

To examine whether the Bcl-2-sensitive mitochondrial apoptotic pathway is a key mediator of the cytotoxicity of 2-MeO-E2 (0.1–1.0 μM), the cytotoxic effect of 2-MeO-E2 was compared using JT/Neo and JT/Bcl-2 cells. As determined via the MTT assay, the viabilities of JT/Neo cells exposed to 0.1 μM, 0.5 μM, or 1.0 μM 2-MeO-E2 for 20 h were 98.8%, 52.3%, and 35.6%, respectively, whereas those of JT/Bcl-2 cells were 98.1%, 77.3%, and 72.1%, respectively (Fig. 1A). When 2-MeO-E2-induced apoptotic DNA

Discussion

In this study, we demonstrate that 2-MeO-E2 (0.5–1.0 μM) induces, prior to provoking apoptosis, an aberrant bipolar microtubule network along with nuclear envelope breakdown and failure of the chromosomes to congress at the metaphase plate, all of which represent prometaphase arrest. This prometaphase arrest caused by 2-MeO-E2 treatment was more apparently observed in JT/Bcl-2 cells overexpressing Bcl-2 because the overexpression of Bcl-2 did not influence the prometaphase arrest but could

Acknowledgements

This study was supported by a grant from the National Research Foundation of Korea funded by the Korean government (NRF-353-2009-2-F00021).

References (56)

  • A. Aihara et al.

    The selective Aurora B kinase inhibitor AZD1152 as a novel treatment for hepatocellular carcinoma

    J. Hepatol.

    (2010)
  • R.M. Angell et al.

    N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl) amides as potent, selective, inhibitors of JNK2 and JNK3

    Bioorg. Med. Chem. Lett.

    (2007)
  • H.S. Park et al.

    Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56lck in human Jurkat T cells

    Biochem. Pharmacol.

    (2011)
  • C.R. Han et al.

    Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells

    Biochim. Biophys. Acta

    (2013)
  • R.D. Van Horn et al.

    Cdk1 activity is required for mitotic activation of aurora A during G2/M transition of human cells

    J. Biol. Chem.

    (2010)
  • L. Danel et al.

    Distribution of androgen and estrogen receptors among lymphoid and haemopoietic cell lines

    Leuk. Res.

    (1985)
  • R. Ohi et al.

    Regulating the onset of mitosis

    Curr. Opin. Cell Biol.

    (1999)
  • D.J. Burke et al.

    Linking kinetochore-microtubule binding to the spindle checkpoint

    Dev. Cell

    (2008)
  • J.E. Chipuk et al.

    The BCL-2 family reunion

    Mol. Cell

    (2010)
  • G. De Chiara et al.

    Bcl-2 phosphorylation by p38 MAPK: identification of target sites and biologic consequences

    J. Biol. Chem.

    (2006)
  • V.S. Pribluda et al.

    2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate

    Cancer Metastasis Rev.

    (2000)
  • S. Verenich et al.

    Therapeutic promises of 2-methoxyestradiol and its drug disposition challenges

    Mol. Pharm.

    (2010)
  • A. Maran et al.

    2-Methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest

    J. Cell. Biochem.

    (2008)
  • Y. Gui et al.

    2-Methoxyestradiol induces cell cycle arrest and mitotic cell apoptosis in human vascular smooth muscle cells

    Hypertension

    (2006)
  • G. Ray et al.

    Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways

    Biochemistry

    (2006)
  • S.L. Mooberry

    New insights into 2-methoxyestradiol, a promising antiangiogenic and antitumor agent

    Curr. Opin. Oncol.

    (2003)
  • N. Gao et al.

    2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Akt-dependent process

    Oncogene

    (2005)
  • R.J. D’Amato et al.

    2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site

    Proc. Natl. Acad. Sci. U. S. A.

    (1994)
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