Biochemical Pharmacology 94 (2015) 257–269
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Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells Seung Tae Lee a, Ji Young Lee a, Cho Rong Han a, Yoon Hee Kim a,b, Do Youn Jun c, Dennis Taub d, Young Ho Kim a,* a
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea b Daegu Science High School, Daegu 706-852, Republic of Korea c Institute of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea d Center for Translational Studies, Medical Services, Veterans Administration Medical Center, Department of Veteran Affairs, Washington, DC 20422, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 December 2014 Received in revised form 18 February 2015 Accepted 18 February 2015 Available online 27 February 2015
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 2MeO-E2 (0.5–1.0 mM) caused G2/M arrest, Bak activation, Dcm 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 mM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0 mM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5–1.0 mM) 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. ß 2015 Elsevier Inc. All rights reserved.
Keywords: Mitotic spindle damage Prometaphase arrest Cdk1 activation Phosphorylation of Bcl-2 family proteins Mitochondrial apoptosis
1. Introduction 2-Methoxyestradiol (2-MeO-E2), an endogenous metabolite of 17b-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 mM to 5.0 mM [2,3]. Numerous studies have
* Corresponding author. Tel.: +82 53 950 5378; fax: +82 53 955 5522. E-mail address: [email protected] (Y.H. Kim). http://dx.doi.org/10.1016/j.bcp.2015.02.011 0006-2952/ß 2015 Elsevier Inc. All rights reserved.
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 [4–9]. 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 [3,10–13]. With respect to 2-MeO-E2induced cell cycle arrest, its interference in cellular microtubule formation, which occurs via reducing the tubulin polymerization
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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 [4–6,9]. 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 mM, 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 mM)induced apoptosis via p27Kip1-mediated G1/S arrest and NF-kB 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 mM) [9]. Although these previous studies raised the possibility that 2-MeO-E2 at low doses (0.5–1.0 mM) 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 2MeO-E2. Recently, to obtain direct evidence for a causal link between 2MeO-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 mM) 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. 2. Materials and methods
The anti-caspase-9, anti-p-Cdk1 (Tyr-15), anti-p-Cdk1 (Thr-161), anti-p-Cdc25C (Thr-48), anti-Cdc25C, anti-p-Bcl-2 (Thr-56), antip-Bcl-2 (Ser-70), anti-p-Mcl-1 (Ser-159/Thr-163), anti-p-AURKA (Thr-288), anti-p-c-Jun (Ser-63), and anti-a-tubulin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), and the anti-p-histone H1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The anti-Bak (Ab-1) and anti-Bax (6A7) antibodies were obtained from Calbiochem (San Diego, CA, USA). The anti-p-Bcl-xL (Ser-62) antibody was obtained from Invitrogen (Carlsbad, CA, USA). The Cdk1 inhibitor RO3306 was purchased from Tocris Bioscience (Ellisville, MO, USA), and the AURKA inhibitor MLN8237 and Aurora B kinase (AURKB) inhibitor AZD1152-HQPA were obtained from Selleck (Huston, TX, USA). The JNK inhibitor SP600125 and the p38 MAPK inhibitor SB202190 were purchased from Biomol (Plymouth Meeting, PA, USA). The JNK inhibitor IX and the ROS sensitive probe dihydroethidium (DHE) were obtained from Santa Cruz Biotechnology. Human acute leukemia Jurkat T cell clone, stably transfected with a Bcl-2 expression vector (JT/Bcl-2) or with an empty vector (JT/Neo) was kindly provided by Dr. Dennis Taub (Gerontology Research Center, NIA/NIH, Baltimore, MD, USA). Both JT/Neo cells and JT/Bcl-2 cells were maintained in RPMI 1640 medium containing 10% FBS, 20 mM HEPES (pH 7.0), 5 105 M b-mercaptoethanol, 100 mg/ml gentamicin, and 400 mg/ml G418 (A.G. Scientific Inc., San Diego, CA, USA). JT/ Bcl-2 cells overexpressing Bcl-2 and JT/Neo cells were identified by western blot analysis. These stable clones were kept in culture for no more than 3 months before the studies, and used in numerous our previous investigations including a recent study [21]. To investigate the effect of the Cdk1 inhibitor (RO3306) [22], the AURKA inhibitor (MLN8237) [23], the AURKB inhibitor (AZD1152-HQPA) [24], the JNK inhibitors (SP600125 or JNK inhibitor IX) [25,26], and the p38 MAPK inhibitor (SB202190) [27] on 2-MeO-E2-induced apoptotic events, JT/Neo cells were pretreated with the individual inhibitors for 1 h prior to 2MeO-E2 treatment for 20 h. 2.2. Flow cytometric analysis Flow cytometric analysis to measure cell cycle state of Jurkat T cells exposed to 2-MeO-E2 was performed on a FACS Calibur (BD Sciences, San Jose, CA, USA) as described elsewhere [28]. The extent of necrosis was detected using an Annexin V-FITC apoptosis kit as previously described [28]. The changes in the mitochondrial membrane potential (Dcm) following 2-MeO-E2 treatment were measured after staining with DiOC6 [29,30]. Activation of Bak and Bax in Jurkat T cells following 2-MeO-E2 treatment was measured as previously described [31]. To measure intracellular ROS accumulation, the cells were treated with DHE at 37 8C for 30 min, and the fluorescence intensity was analyzed by flow cytometry (FACS Aria III system, BD Sciences) with an excitation wavelength of 488 nm [32].
2.1. Reagents, antibodies, and cells 2.3. Immunofluorescence microscopy 2-MeO-E2, APC, 3,30 dihexyloxacarbocyanine iodide (DiOC6), and 40 ,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, anti-Mcl-1, anti-Cdk1, anti-cyclin B1, anti-Aurora A kinase (AURKA), anti-histone H1, anti-p-histone H3 (Ser-10), anti-histone H3, anti-lamin B, and anti-b-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Immunostaining of Jurkat T cells treated with 2-MeO-E2 was performed as previously described [33]. 2.4. Preparation of cell lysates and western blot analysis Cell lysates were prepared by suspending 5 106 Jurkat T cells in 300 ml of lysis buffer as described elsewhere [28]. An equivalent amount of protein lysate (20 mg) was electrophoresed on a 4–12% NuPAGE gradient gel and then electrotransferred to an ImmobilonP membrane. Protein detection was performed using an ECL western blot kit according to the manufacturer’s instructions.
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Densitometry was performed using ImageQuant TL software (Amersham, Arlington Heights, IL). The arbitrary densitometric units for each protein of interest were normalized to the densitometric units for b-actin. 2.5. Extraction of monomeric and polymeric tubulin The monomeric fraction was prepared by extracting cells in monomeric extraction buffer (20 mM PIPES, 0.14 M NaCl, 1 mM MgCl2, 1 mM EGTA, 0.5% NP-40, and 0.5 mM PMSF, pH 6.8) as previously described [34] with some modifications. After centrifugation at 13,000 g for 10 min at room temperature, the NP-40soluble fraction containing monomeric tubulin was collected. The polymeric tubulin fraction was prepared by disrupting the remaining insoluble material in RIPA buffer (0.15 M NaCl, 1% deoxycholate, 1% NP-40, 0.1% SDS, and 10 mM Tris, pH 7.4) followed by centrifugation. An equivalent amount of each fraction sample was electrophoresed on a 4–12% NuPAGE gradient gel. Western blot analysis for a-tubulin was performed as described in Section 2. 2.6. Statistical analysis Unless otherwise indicated, each result in this article is representative of at least three separate experiments. The values are expressed as the means standard deviation of these experiments. Statistical significance was calculated using Student’s t-test. P values 2.5 mM SP600125, >10 nM the JNK inhibitor IX, or >10 mM SB202190 alone for 20 h (data not shown). When JT/Neo cells were treated with concomitantly treated with 1 mM 2-MeO-E2 and 2.5 mM Sp600125, approximately 48% of the 2MeO-E2-induced c-Jun phosphorylation at Ser-63 was prevented; however, the 2MeO-E2-induced Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation was not influenced, excluding the involvement of MAPKs in the 2-MeO-E2-induced phosphorylation of Bcl-2 family proteins.
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Immunofluorescence microscopic analysis revealed that prometaphase arrest was induced in Jurkat T cells treated with 2MeO-E2, based on the breakdown of the nuclear envelope and the failure of the chromosomes to congress at the metaphase plate due to an aberrant bipolar network of microtubules. This result is consistent with several previous studies demonstrating that tumor cells treated with microtubule-damaging drugs typically display impaired microtubule assembly, resulting in mitotic arrest and apoptosis [16,17,33]. Because the 2-MeO-E2-induced prometaphase arrest was rapidly coupled to apoptotic morphological changes, including membrane blebbing and apoptotic body formation, we decided to examine the effect of 2-MeO-E2 on cellular microtubule polymerization by utilizing JT/Bcl-2 cells that are resistant to undergoing apoptosis due to Bcl-2 overexpression. In accordance with a previous report [14], treatment with 2-MeOE2 (10–20 mM) reduced the proportion of intracellular polymeric tubulin. However, 2-MeO-E2 (0.5–5.0 mM) appeared to increase the proportion of intracellular polymeric tubulin, as did treatment with paclitaxel (0.01–0.1 mM). The present results indicate that the induction of mitotic arrest in Jurkat T cells by treatment with 2MeO-E2 (0.5–1.0 mM) was initiated by an enhancement in intracellular tubulin polymerization. In conclusion, our results demonstrate that the 2-MeO-E2 (0.5– 1.0 mM)-induced apoptotic signaling pathway in Jurkat T cells was proceeded by enhanced microtubule polymerization, causing impairment of the mitotic spindle network, prometaphase arrest, Cdk1 activation, and phosphorylation of Bcl-2, Mcl-1, and Bim, which render the cells susceptible to the onset of mitochondriadependent apoptosis by triggering Bak activation, loss of Dcm, and subsequent caspase cascade activation. These results also clarify the molecular and cellular mechanism underlying the anticancer effect of 2-MeO-E2. Acknowledgements This study was supported by a grant from the National Research Foundation of Korea funded by the Korean government (NRF-3532009-2-F00021). References [1] A.O. Mueck, H. Seeger, 2-Methoxyestradiol-biology and mechanism of action, Steroids 75 (2010) 625–631. [2] V.S. Pribluda, E.R. Gubish, T.M. LaVallee, A. Treston, G.M. Swartz, S.J. Green, 2Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate, Cancer Metastasis Rev. 19 (2000) 173–179. [3] S. Verenich, P.M. Gerk, Therapeutic promises of 2-methoxyestradiol and its drug disposition challenges, Mol. Pharm. 7 (2010) 2030–2039. [4] A. Maran, K.L. Shogren, M. Benedikt, G. Sarkar, R.T. Turner, M.J. Yaszemski, 2Methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest, J. Cell. Biochem. 104 (2008) 1937–1945. [5] Y. Gui, X.L. Zheng, 2-Methoxyestradiol induces cell cycle arrest and mitotic cell apoptosis in human vascular smooth muscle cells, Hypertension 47 (2006) 271–280. [6] G. Ray, G. Dhar, P.J. Van Veldhuizen, S. Banerjee, N.K. Saxena, K. Sengupta, S.K. Banerjee, Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways, Biochemistry 45 (2006) 3703–3713. [7] Y.M. Lee, C.M. Ting, Y.K. Cheng, T.P. Fan, R.N. Wong, M.L. Lung, N.K. Mak, Mechanisms of 2-methoxyestradiol-induced apoptosis and G2/M cell-cycle arrest of nasopharyngeal carcinoma cells, Cancer Lett. 268 (2008) 295–307. [8] S.L. Mooberry, New insights into 2-methoxyestradiol, a promising antiangiogenic and antitumor agent, Curr. Opin. Oncol. 15 (2003) 425–430. [9] C. Batsi, S. Markopoulou, E. Kontargiris, C. Charalambous, C. Thomas, S. Christoforidis, P. Kanavaros, A.I. Constantinou, K.B. Marcu, E. Kolettas, Bcl-2 blocks 2methoxyestradiol induced leukemia cell apoptosis by a p27Kip1-dependent G1/S cell cycle arrest in conjunction with NF-kB activation, Biochem. Pharmacol. 78 (2009) 33–44. [10] A.M. Carothers, S.A. Hughes, D. Ortega, M.M. Bertagnolli, 2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells, Cancer Lett. 187 (2002) 77–86. [11] N. Gao, M. Rahmani, P. Dent, S. Grant, 2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Aktdependent process, Oncogene 24 (2005) 3797–3809.
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Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells Seung Tae Lee a, Ji Young Lee a, Cho Rong Han a, Yoon Hee Kim a,b, Do Youn Jun c, Dennis Taub d, Young Ho Kim a,* a
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea b Daegu Science High School, Daegu 706-852, Republic of Korea c Institute of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea d Center for Translational Studies, Medical Services, Veterans Administration Medical Center, Department of Veteran Affairs, Washington, DC 20422, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 December 2014 Received in revised form 18 February 2015 Accepted 18 February 2015 Available online 27 February 2015
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 2MeO-E2 (0.5–1.0 mM) caused G2/M arrest, Bak activation, Dcm 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 mM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0 mM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5–1.0 mM) 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. ß 2015 Elsevier Inc. All rights reserved.
Keywords: Mitotic spindle damage Prometaphase arrest Cdk1 activation Phosphorylation of Bcl-2 family proteins Mitochondrial apoptosis
1. Introduction 2-Methoxyestradiol (2-MeO-E2), an endogenous metabolite of 17b-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 mM to 5.0 mM [2,3]. Numerous studies have
* Corresponding author. Tel.: +82 53 950 5378; fax: +82 53 955 5522. E-mail address: [email protected] (Y.H. Kim). http://dx.doi.org/10.1016/j.bcp.2015.02.011 0006-2952/ß 2015 Elsevier Inc. All rights reserved.
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 [4–9]. 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 [3,10–13]. With respect to 2-MeO-E2induced cell cycle arrest, its interference in cellular microtubule formation, which occurs via reducing the tubulin polymerization
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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 [4–6,9]. 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 mM, 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 mM)induced apoptosis via p27Kip1-mediated G1/S arrest and NF-kB 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 mM) [9]. Although these previous studies raised the possibility that 2-MeO-E2 at low doses (0.5–1.0 mM) 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 2MeO-E2. Recently, to obtain direct evidence for a causal link between 2MeO-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 mM) 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. 2. Materials and methods
The anti-caspase-9, anti-p-Cdk1 (Tyr-15), anti-p-Cdk1 (Thr-161), anti-p-Cdc25C (Thr-48), anti-Cdc25C, anti-p-Bcl-2 (Thr-56), antip-Bcl-2 (Ser-70), anti-p-Mcl-1 (Ser-159/Thr-163), anti-p-AURKA (Thr-288), anti-p-c-Jun (Ser-63), and anti-a-tubulin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), and the anti-p-histone H1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The anti-Bak (Ab-1) and anti-Bax (6A7) antibodies were obtained from Calbiochem (San Diego, CA, USA). The anti-p-Bcl-xL (Ser-62) antibody was obtained from Invitrogen (Carlsbad, CA, USA). The Cdk1 inhibitor RO3306 was purchased from Tocris Bioscience (Ellisville, MO, USA), and the AURKA inhibitor MLN8237 and Aurora B kinase (AURKB) inhibitor AZD1152-HQPA were obtained from Selleck (Huston, TX, USA). The JNK inhibitor SP600125 and the p38 MAPK inhibitor SB202190 were purchased from Biomol (Plymouth Meeting, PA, USA). The JNK inhibitor IX and the ROS sensitive probe dihydroethidium (DHE) were obtained from Santa Cruz Biotechnology. Human acute leukemia Jurkat T cell clone, stably transfected with a Bcl-2 expression vector (JT/Bcl-2) or with an empty vector (JT/Neo) was kindly provided by Dr. Dennis Taub (Gerontology Research Center, NIA/NIH, Baltimore, MD, USA). Both JT/Neo cells and JT/Bcl-2 cells were maintained in RPMI 1640 medium containing 10% FBS, 20 mM HEPES (pH 7.0), 5 105 M b-mercaptoethanol, 100 mg/ml gentamicin, and 400 mg/ml G418 (A.G. Scientific Inc., San Diego, CA, USA). JT/ Bcl-2 cells overexpressing Bcl-2 and JT/Neo cells were identified by western blot analysis. These stable clones were kept in culture for no more than 3 months before the studies, and used in numerous our previous investigations including a recent study [21]. To investigate the effect of the Cdk1 inhibitor (RO3306) [22], the AURKA inhibitor (MLN8237) [23], the AURKB inhibitor (AZD1152-HQPA) [24], the JNK inhibitors (SP600125 or JNK inhibitor IX) [25,26], and the p38 MAPK inhibitor (SB202190) [27] on 2-MeO-E2-induced apoptotic events, JT/Neo cells were pretreated with the individual inhibitors for 1 h prior to 2MeO-E2 treatment for 20 h. 2.2. Flow cytometric analysis Flow cytometric analysis to measure cell cycle state of Jurkat T cells exposed to 2-MeO-E2 was performed on a FACS Calibur (BD Sciences, San Jose, CA, USA) as described elsewhere [28]. The extent of necrosis was detected using an Annexin V-FITC apoptosis kit as previously described [28]. The changes in the mitochondrial membrane potential (Dcm) following 2-MeO-E2 treatment were measured after staining with DiOC6 [29,30]. Activation of Bak and Bax in Jurkat T cells following 2-MeO-E2 treatment was measured as previously described [31]. To measure intracellular ROS accumulation, the cells were treated with DHE at 37 8C for 30 min, and the fluorescence intensity was analyzed by flow cytometry (FACS Aria III system, BD Sciences) with an excitation wavelength of 488 nm [32].
2.1. Reagents, antibodies, and cells 2.3. Immunofluorescence microscopy 2-MeO-E2, APC, 3,30 dihexyloxacarbocyanine iodide (DiOC6), and 40 ,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, anti-Mcl-1, anti-Cdk1, anti-cyclin B1, anti-Aurora A kinase (AURKA), anti-histone H1, anti-p-histone H3 (Ser-10), anti-histone H3, anti-lamin B, and anti-b-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Immunostaining of Jurkat T cells treated with 2-MeO-E2 was performed as previously described [33]. 2.4. Preparation of cell lysates and western blot analysis Cell lysates were prepared by suspending 5 106 Jurkat T cells in 300 ml of lysis buffer as described elsewhere [28]. An equivalent amount of protein lysate (20 mg) was electrophoresed on a 4–12% NuPAGE gradient gel and then electrotransferred to an ImmobilonP membrane. Protein detection was performed using an ECL western blot kit according to the manufacturer’s instructions.
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Densitometry was performed using ImageQuant TL software (Amersham, Arlington Heights, IL). The arbitrary densitometric units for each protein of interest were normalized to the densitometric units for b-actin. 2.5. Extraction of monomeric and polymeric tubulin The monomeric fraction was prepared by extracting cells in monomeric extraction buffer (20 mM PIPES, 0.14 M NaCl, 1 mM MgCl2, 1 mM EGTA, 0.5% NP-40, and 0.5 mM PMSF, pH 6.8) as previously described [34] with some modifications. After centrifugation at 13,000 g for 10 min at room temperature, the NP-40soluble fraction containing monomeric tubulin was collected. The polymeric tubulin fraction was prepared by disrupting the remaining insoluble material in RIPA buffer (0.15 M NaCl, 1% deoxycholate, 1% NP-40, 0.1% SDS, and 10 mM Tris, pH 7.4) followed by centrifugation. An equivalent amount of each fraction sample was electrophoresed on a 4–12% NuPAGE gradient gel. Western blot analysis for a-tubulin was performed as described in Section 2. 2.6. Statistical analysis Unless otherwise indicated, each result in this article is representative of at least three separate experiments. The values are expressed as the means standard deviation of these experiments. Statistical significance was calculated using Student’s t-test. P values 2.5 mM SP600125, >10 nM the JNK inhibitor IX, or >10 mM SB202190 alone for 20 h (data not shown). When JT/Neo cells were treated with concomitantly treated with 1 mM 2-MeO-E2 and 2.5 mM Sp600125, approximately 48% of the 2MeO-E2-induced c-Jun phosphorylation at Ser-63 was prevented; however, the 2MeO-E2-induced Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation was not influenced, excluding the involvement of MAPKs in the 2-MeO-E2-induced phosphorylation of Bcl-2 family proteins.
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Immunofluorescence microscopic analysis revealed that prometaphase arrest was induced in Jurkat T cells treated with 2MeO-E2, based on the breakdown of the nuclear envelope and the failure of the chromosomes to congress at the metaphase plate due to an aberrant bipolar network of microtubules. This result is consistent with several previous studies demonstrating that tumor cells treated with microtubule-damaging drugs typically display impaired microtubule assembly, resulting in mitotic arrest and apoptosis [16,17,33]. Because the 2-MeO-E2-induced prometaphase arrest was rapidly coupled to apoptotic morphological changes, including membrane blebbing and apoptotic body formation, we decided to examine the effect of 2-MeO-E2 on cellular microtubule polymerization by utilizing JT/Bcl-2 cells that are resistant to undergoing apoptosis due to Bcl-2 overexpression. In accordance with a previous report [14], treatment with 2-MeOE2 (10–20 mM) reduced the proportion of intracellular polymeric tubulin. However, 2-MeO-E2 (0.5–5.0 mM) appeared to increase the proportion of intracellular polymeric tubulin, as did treatment with paclitaxel (0.01–0.1 mM). The present results indicate that the induction of mitotic arrest in Jurkat T cells by treatment with 2MeO-E2 (0.5–1.0 mM) was initiated by an enhancement in intracellular tubulin polymerization. In conclusion, our results demonstrate that the 2-MeO-E2 (0.5– 1.0 mM)-induced apoptotic signaling pathway in Jurkat T cells was proceeded by enhanced microtubule polymerization, causing impairment of the mitotic spindle network, prometaphase arrest, Cdk1 activation, and phosphorylation of Bcl-2, Mcl-1, and Bim, which render the cells susceptible to the onset of mitochondriadependent apoptosis by triggering Bak activation, loss of Dcm, and subsequent caspase cascade activation. These results also clarify the molecular and cellular mechanism underlying the anticancer effect of 2-MeO-E2. Acknowledgements This study was supported by a grant from the National Research Foundation of Korea funded by the Korean government (NRF-3532009-2-F00021). References [1] A.O. Mueck, H. Seeger, 2-Methoxyestradiol-biology and mechanism of action, Steroids 75 (2010) 625–631. [2] V.S. Pribluda, E.R. Gubish, T.M. LaVallee, A. Treston, G.M. Swartz, S.J. Green, 2Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate, Cancer Metastasis Rev. 19 (2000) 173–179. [3] S. Verenich, P.M. Gerk, Therapeutic promises of 2-methoxyestradiol and its drug disposition challenges, Mol. Pharm. 7 (2010) 2030–2039. [4] A. Maran, K.L. Shogren, M. Benedikt, G. Sarkar, R.T. Turner, M.J. Yaszemski, 2Methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest, J. Cell. Biochem. 104 (2008) 1937–1945. [5] Y. Gui, X.L. Zheng, 2-Methoxyestradiol induces cell cycle arrest and mitotic cell apoptosis in human vascular smooth muscle cells, Hypertension 47 (2006) 271–280. [6] G. Ray, G. Dhar, P.J. Van Veldhuizen, S. Banerjee, N.K. Saxena, K. Sengupta, S.K. Banerjee, Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways, Biochemistry 45 (2006) 3703–3713. [7] Y.M. Lee, C.M. Ting, Y.K. Cheng, T.P. Fan, R.N. Wong, M.L. Lung, N.K. Mak, Mechanisms of 2-methoxyestradiol-induced apoptosis and G2/M cell-cycle arrest of nasopharyngeal carcinoma cells, Cancer Lett. 268 (2008) 295–307. [8] S.L. Mooberry, New insights into 2-methoxyestradiol, a promising antiangiogenic and antitumor agent, Curr. Opin. Oncol. 15 (2003) 425–430. [9] C. Batsi, S. Markopoulou, E. Kontargiris, C. Charalambous, C. Thomas, S. Christoforidis, P. Kanavaros, A.I. Constantinou, K.B. Marcu, E. Kolettas, Bcl-2 blocks 2methoxyestradiol induced leukemia cell apoptosis by a p27Kip1-dependent G1/S cell cycle arrest in conjunction with NF-kB activation, Biochem. Pharmacol. 78 (2009) 33–44. [10] A.M. Carothers, S.A. Hughes, D. Ortega, M.M. Bertagnolli, 2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells, Cancer Lett. 187 (2002) 77–86. [11] N. Gao, M. Rahmani, P. Dent, S. Grant, 2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Aktdependent process, Oncogene 24 (2005) 3797–3809.
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