Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27^Kip1-dependent G1/S cell cycle arrest in conjunction with NF-κB activation

Abstract

2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21^Cip1/Waf1 and up-regulating p16^INK4A. Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27^Kip1. Bcl-2 and p27^Kip1 were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-κB activity and p50 levels were increased by 2-ME2 and suppression of NF-κB signaling reduced p27^Kip1 expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27^Kip1 in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27^Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.

Introduction

2-Methoxyestradiol (2-ME2), a catechol estrogen, is a natural metabolic by-product of 17β-estradiol that acts independently of estrogen receptors to inhibit angiogenesis and tumor cell proliferation and to induce apoptosis in vitro and in vivo[1], [2], [3], [4], [5], [6], [7], [8].

Multiple discrete mechanisms are involved in the specific anti-proliferative action of 2-ME2 in tumor cells including both G1/S and G2/M cell cycle phase arrest. 2-ME2 was shown to arrest the growth of many human cancer cell lines in vitro, including, Jurkat cells [9], multiple myeloma [10], epithelial [11], [12], [13], [14], [15], [16], melanoma [17] and medulloblastoma cancer cells [18] and transformed fibroblasts [19] in G2/M phase. G2/M cell cycle arrest was characterized by the induction of cyclin B and Cdc2 kinase activity [9], [11], [13], [17]. Others, however, showed that 2-ME2 inhibited the growth of pancreatic cancer cells by prolonging S-phase [20] or by inducing both G1/S and G2/M arrest of human osteosarcoma cells [21] or of pancreatic cell lines [22]. In contrast, 2-ME2 had no effect on the growth of normal cells [13], [15], [17], [19], [23] including lymphocytes [24]. The induction of apoptosis by 2-ME2 in tumor cells involves different molecular mechanisms. While several studies suggested that 2-ME2 can induce apoptosis both by p53-dependent and p53-independent mechanisms in various tumor cell types [8], [13], [15], [17], [18], [19], [23], [25], [26], scant evidence exists implicating NF-κB in 2-ME2-induced apoptosis [18], [27]. While p38/JNK-dependent NF-κB activation was required for 2-ME2-induced apoptosis in prostate cancer cells [27], in contrast a reduction in NF-κB transcriptional and DNA binding activity was observed in 2-ME2-induced apoptosis of medulloblastoma cells [18].

Further studies have implicated the anti-apoptotic members of the Bcl-2 family in 2-ME2-induced apoptosis [15], [27], [28], [29], [30]. The Bcl-2 family comprises two mutually opposing groups of proteins including: anti-apoptotic Bcl-2 and Bcl-X_L and pro-apoptotic Bak and Bax. While several models have been proposed to explain the mechanism by which Bcl-2 family members regulate apoptosis, the ratio of anti-apoptotic:pro-apoptotic Bcl-2 family members is one key factor dictating the relative sensitivity or resistance of cells to a wide variety of apoptotic stimuli [31], [32], [33]. In addition, Bcl-2 and Bcl-X_L are regulated by phosphorylation in their flexible loop between the BH4 and BH3 domains, which determines their cytoprotective function in response to cellular stresses as well as growth and survival factors [34]. Bcl-2 phosphorylation by ERK1/2 and PKCα kinases, either at the unique Ser70 residue or at multiple Thr69, Ser70, and Ser89 sites, positively regulates Bcl-2 anti-apoptotic function [35]. However, c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK)-mediated phosphorylation of Bcl-2 at multiple-sites hinders Bcl-2 survival function in paclitaxel-induced apoptosis [36], [37]. Thus, the type of stimulus, the regulatory pathways involved, and the degree and duration of phosphorylation at specific Bcl-2 residues produce different outcomes.

In response to 2-ME2, both Bcl-2 [15], [27], [28], [29] and Bcl-X_L[22], [30] are inactivated by phosphorylation at Ser70 and Ser62, respectively, mediated by JNK but not ERK1/2 [27], [29], [30], [37]. Whether Bcl-2 phosphorylation induced by microtubule destabilizing agents such as taxol [37], [38], [39] or 2-ME2 [15], [27], [28], [29] interferes with the heterodimerization of Bcl-2 to Bax remains elusive [37], [38], [39]. However, JNK-mediated phosphorylation of Bcl-2 leading to its inactivation in response to 2-ME2 will allow the pro-apoptotic members of the Bcl-2 family, to drive the cell towards death [31], [32], [33].

2-ME2-induced phosphorylation of Bcl-2, mediated by JNK/SAPK, has been correlated with apoptosis of prostate [15], [27] and leukemia cells [29]. Activation of JNK by 2-ME2 appears to be due to its ability to potently inhibit superoxide dismutase [40] resulting in enhanced formation of ROS [7], [24], [25], [26], [40] and Akt inhibition [26] to selectively kill tumor cells [24], [25], [26], [40].

Although available data point to Bcl-2 phosphorylation as a key executing signal for 2-ME2-induced apoptosis, Bcl-2's mechanisms of action in this context and its ability to protect cells from 2-ME2-induced apoptosis both remain undefined. We show here that 2-ME2 treatment of leukemia cells promoted a p53-independent apoptotic response characterized by Bcl-2 down-regulation and phosphorylation mediated by JNK/SAPK, Bak up-regulation, proteolytic cleavages of caspases-9, -3 and PARP-1. Moreover, ectopic over-expression of Bcl-2 in leukemia cells prevented all of these aspects of the 2-ME2-induced apoptotic response by orchestrating a p27^Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB.