Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells
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.
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)
- et al.
2-Methoxyestradiol-biology and mechanism of action
Steroids
(2010) - et al.
Mechanisms of 2-methoxyestradiol-induced apoptosis and G2/M cell-cycle arrest of nasopharyngeal carcinoma cells
Cancer Lett.
(2008) - et al.
Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27Kip1-dependent G1/S cell cycle arrest in conjunction with NF-kB activation
Biochem. Pharmacol.
(2009) - et al.
2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells
Cancer Lett.
(2002) - et al.
Mechanisms for 2-methoxyestradiol-induced apoptosis of prostate cancer cells
FEBS Lett.
(2002) - et al.
Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol
Biochim. Biophys. Acta
(2012) 2-Methoxyestradiol and paclitaxel have similar effects on the cell cycle and induction of apoptosis in prostate cancer cells
Cancer Lett.
(2006)- et al.
Dissection of the genetic programs of p53-mediated G1 growth arrest and apoptosis: blocking p53-induced apoptosis unmasks G1 arrest
Blood
(1995) - et al.
Synchronized generation of reactive oxygen species with the cell cycle
Life Sci.
(2004) - et al.
A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
Blood
(2010)