Anaplastic large-cell lymphoma (ALCL) is a heterogeneous lymphoma category in which a subset of cases carry the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. In this study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in ALCL cell lines. LY293111 inhibited proliferation of both ALK(+) and ALK(−) ALCL cell in a dose-dependent fashion and induced complete G1–S cell cycle arrest, which was accompanied by upregulation of p27 and downregulation of cyclin E. Pretreatment with LY293111 for 4 h resulted in profound inhibition of serum-induced phosphorylation of extracellular-regulated kinases-1 and 2 and Akt and a concomitant increase in the phosphorylation of the stress-activated kinase c-jun N-terminal kinases (JNK). Simultaneously, LY293111 induced caspase-dependent apoptosis via activation of the intrinsic pathway, including early loss of mitochondrial inner transmembrane potential and the production of reactive oxygen species (ROS), cleavage of caspases-9, -3, poly ADP-ribose polymerase (PARP) and X-linked inhibitor of apoptosis. The phospho-JNK inhibitor SP600125 partially protected Sup-M2 cells from LY293111-induced apoptosis, PARP cleavage and ROS generation, suggesting a role for JNK in LY293111-induced cell death. These results warrant further studies of LY293111 in ALCL.
Anaplastic large-cell lymphoma (ALCL) was first identified by Stein et al1 as a new lymphoma category and featured as large lymphoid cells non-Hodgkin's lymphoma with strong expression of the cytokine receptor CD30 and a characteristic growth pattern. Most ALCLs express a constitutively active receptor tyrosine kinase, anaplastic lymphoma kinase (ALK), which represents 40–80% of ALCLs.2, 3, 4 ALCLs lacking ALK are clinically heterogeneous and have a very poor prognosis. ALK(+) ALCL predominantly affects young male patients and, if treated with chemotherapy, was considered to have a favorable prognosis. However, majority of the patients who relapse have a very poor prognosis, and almost all succumb to the disease within 2 years of diagnosis.5 There is thus a great need for the development of novel therapies for these malignancies.
Leukotriene B4 (LTB4) is known as one of the most potent chemoattractants and activators of leukocytes synthesized in myeloid cells from arachidonic acid. It also plays a crucial role in regulating apoptosis and promoting tumor growth.6, 7 It reportedly enhances cell proliferation in a variety of tumors, including lymphoma, melanoma, oral cancer, breast cancer, pancreatic cancer and colon cancer.8, 9, 10, 11, 12, 13, 14, 15 Several studies have shown that LTB4 augments DNA synthesis and enhances the replication of lymphocytes and malignant lymphoma cells.8, 16 Lindsay et al17 reported that LTB4 could induce a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). In addition, Loren18 reported that Drosophila melanogaster homologue of ALK DAlk was necessary for the ERK activation in the developing mesoderm. On the other hand, LTB4 may interact with receptors and activate G protein-regulated phospholipase C (PLC).19 PLC-γ may play a pivotal role in mitogenesis induced by ALK signaling, which is via the specific binding to Tyr664 site of NPM-ALK protein.4, 20
Since the constitutive activation of mitogen-activated protein kinase (MAPK) signaling has been reported to play crucial roles in the oncogenesis of lymphoma,21, 22 targeting the LTB4 receptor may represent a potential strategy for therapy of ALCLs. The 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxy-phenoxy]-propoxy]-phenoxy] benzoic acid sodium salt (LY293111) is a second-generation LTB4 receptor antagonist,23 that has demonstrated significant antitumor activity in Phase I–II clinical trials.24 It has been further shown that LY293111 inhibits the proliferation and induces the apoptosis of pancreatic cancer cells by inhibiting LTB4-induced ERK-1/2 phosphorylation,13 and reduced the growth of colon tumors in an in vivo animal model.7 However, the effects of LY293111 on hematological malignancies have not been investigated.
In this study, we examined therapeutic implications of LY293111 for the therapy of ALCL and elucidated the mechanisms of LY293111's growth-inhibitory effects. Our results demonstrate that LY293111 induced cell cycle arrest and apoptosis in both ALK(+) (Sup-M2 and Su-DHL-1) and ALK(−) (Mac-2A) ALCL cell lines. Mechanistic studies implicated engagement of the intrinsic apoptotic pathway via stress-activated JNK signaling.
Materials and methods
Chemicals and cell lines
LY293111 was provided by Eli Lilly and Co. (Indianapolis, IN, USA). The Sup-M2 and Su-DHL-1 cell lines were derived from CD30+, ALK(+) ALCLs and both carry the t(2;5) chromosomal translocation,25 and Mac-2A cells (a kind gift from Dr Kojo Elenitoba-Johnson, Salt Lake, UT, USA) were derived from a CD30+, ALK(−) ALCL.26
Cell viability, apoptosis assays and cell cycle analysis
The cells were cultured with the indicated concentrations of LY293111. Cell viability was assessed using the trypan blue dye exclusion method, and apoptosis was measured by flow cytometry using Annexin V staining as described previously.27 Sup-M2 cells were pulsed with 5-bromo-2-deoxyuridine (BrdU) for 45 min to 2 h and incubated with FITC-conjugated antibody against BrdU for 1 h. BrdU intensity and distribution were analyzed by flow cytometry.
Phosphorylation assays of JNK, Erk, Akt and Bcl-2 proteins
To determine the phosphorylation of the JNK, Erk, Akt and Bcl-2 proteins, the cells were pretreated with LY293111 for 3–4 h after 20 h low-serum (1% FCS) starvation, followed by addition of 15% FCS to stimulate protein phosphorylation. The phosphorylation levels were detected with antiphosphorylated antibodies by Western blot analysis.
Measurement of generation of reactive oxygen species and mitochondrial membrane potential
Hydroperoxide and superoxide production was determined using CM-H2DCFDA as described by Hail et al,28 with some modifications. The mitochondrial membrane potential (MMP; ΔΨm) was evaluated by loading with CMXRos for 1 h29 and measured by flow cytometry.
Results are expressed as means±s.e.m. Levels of significance were evaluated by a two-tailed paired Student's t-test, and P<0.05 was considered significant.
The details of the Materials and methods including primers and probes, antibodies, BrdU incorporation, RT-PCR, Real-time PCR and Western blot are available online as Supplementary Information.
LY293111 inhibits cell proliferation by promoting G1 cell cycle arrest
Cell viability was diminished by LY293111 in a dose-dependent manner in three ALCL cell lines (Figure 1a), and IC50 of ALK(−) cell Mac-2A was lower than IC50 of ALK(+) cell lines after 72 h treatment (Figure 1b). BrdU labeling showed that LY293111 inhibited growth of exponentially growing Sup-M2 cells by inducing G1 arrest in a dose-dependent fashion, with 50% reduction of cells in S phase at 5 μ M (Figure 1c). Following low-serum (1%) starvation and release with 15% serum, almost all Sup-M2 cells were arrested in the G1 phase even at a 2.5 μ M LY293111 (Figure 1d).
We next performed Western blot analysis to determine which proteins were involved in G1 phase arrest of the cells. The results demonstrated that LY293111 induced p27KIP1 and downregulated cyclin E proteins levels in all the ALCL cells; these effects were more apparent in ALK(−) Mac-2A compared to ALK(+) cells after 24-h LY293111 treatment (Figure 2a). Furthermore, LY293111 reduced cyclin E mRNA levels (four-fold decrease at 5 μ M, Sup-M2 cells) (Figure 2b). However, there was no significant change in the level of cyclin B1 protein, which is an important checkpoint protein for the passage of the cell from G2 to M phase in ALK(+) ALCL cell lines (Figure 2a). Only a minimal decrease in cyclin B1 levels was observed in Mac-2A cells (Figure 2a).
LY293111 induces apoptosis through a caspase-dependent mechanism
The translocation of membrane phospholipid phosphatidylserine and activation of caspase-3 with poly ADP-ribose polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP) cleavage have been well established as important indices of cell apoptosis. Apoptosis induction was observed in all these ALCL cell lines in a dose-dependent manner after 72-h LY293111 treatment, although ALK(+) cell lines were more sensitive at 10 μ M of LY293111 (Figure 3a). Conversely, pretreatment with the pan-caspase inhibitor z-VAD-fmk protected cells from the apoptosis induced by LY293111 (Figure 3a). Western blot analysis showed that LY293111 cleaved caspase-3 in a dose-dependent manner in ALCL cells. The cleavage of caspase-3 substrates PARP and XIAP were also seen especially in ALK(+) cells (Figure 3b). These findings indicate that LY293111-activated caspase-3 is functionally active in the cells.
Caspase-8 pathway is not essential for LY293111-induced cell apoptosis
We performed Western blot analysis to identify the upstream apoptotic pathways responsible for activating caspase-3 in LY293111-induced cell apoptosis. After 24 h of LY293111 treatment cleavage of caspase-9 was observed in all three ALCL cell lines, while caspase-8 levels did not change significantly (Figure 3c). Additionally, apoptosis induced by LY293111 in caspase-8 mutant Jurkat cells was not diminished compared to apoptosis induced in wild-type cells, and Western blot showed cleavage of caspase-9 and PARP in the caspase-8–mutant Jurkat cells (Figure 1, Supplementary Information). These results therefore imply that caspase-8 is not essential for LY293111-induced cell apoptosis.
LY293111 induces reactive oxygen species production and loss of the MMP
Mitochondrial damage is important to the apoptosis affected by the caspase-9 pathway and even in some models of extrinsic apoptosis.30 The resulting elevated intracellular amounts of reactive oxygen species (ROS) are sufficient to trigger cell death, and it has thus been suggested that ROS are biochemical mediators of apoptosis.31 To determine whether ROS are involved in the regulation of apoptosis induced by LY293111, the fluorescent CM-H2DCFDA product was assessed using flow cytometry. As shown in Figure 4a, after 1 h treatment, LY293111 induced production of ROS in a dose-dependent manner. In addition, 5 mM of ROS scavenger GSH almost completely protected cells from LY293111 generated ROS.
We further studied whether the LY293111-induced production of ROS was coincidental with changes in the MMP that occurred during apoptosis in the cells. As shown in Figure 4b, LY293111 led to a drop in mitochondrial potential, as reflected by a decrease in CMXRos fluorescence from a baseline value of 90–10% after 48 h of treatment at a concentration of 10 μ M. These data show that LY293111-induced apoptosis might be closely linked to mitochondrial function and membrane permeability. Further, ROS generation preceded loss of MMP, suggesting a likely causative role in mitochondrial damage.
LY293111 affected Bcl-2 family proteins and decreased Bcl-2 phosphorylation in ALK(+) ALCL cells
Since our data showed that LY293111-induced apoptosis of ALCL cells is mediated by the intrinsic pathway and is associated with loss of MMP, we investigated the effects of LY293111 on Bcl-2 and Bax proteins. The results demonstrate that LY293111 treatment downregulated Bcl-2 protein and induced p18 cleaved-Bax in ALK(+) ALCL cells, but showed no significant changes in ALK(−) Mac-2A cell (Figure 5a). In addition, Bcl-2 mRNA level decreased three-fold as determined by TaqMan Real Time PCR (Figure 5b) and serum-induced Bcl-2 phosphorylation was rapidly inhibited in a dose-dependent manner (inset in Figure 5b) in ALK(+) cells. No baseline Bcl-2 phosphorylation was detected in ALK(−) Mac-2A cells.
LY293111 affects apoptosis and ROS production via JNK phosphorylation
To investigate the possible involvement of several MAPKs in association with LY293111-induced apoptosis, we examined phosphorylation levels of SAPK/JNK, Erk and Akt by Western blot analysis. The results showed that LY293111 induced phosphorylation of the JNK protein. It also simultaneously produced a rapid decrease in the activity of Erk and Akt by inhibiting the phosphorylation of these proteins. These effects were more pronounced in ALK(+) Sup-M2 and Su-DHL-1 cells than in ALK(−) Mac-2A cells (Figure 6a). Since JNK functions as stress-activated kinase, while MEK/Erk and Akt phosphorylation contribute to cytoprotection, these results indicate that the regulation of phosphorylation of these proteins might contribute to LY293111-mediated inhibition of proliferation and cell death. To determine the functional significance of LY293111-induced JNK phosphorylation, we employed the specific phospho-JNK inhibitor SP600125 that fully blocked LY293111-induced JNK activation at a concentration of 2 μ M. However, it had no obvious effects on the level of Erk and Akt (Ser473) phosphorylation (Figure 6b). Furthermore, pretreatment with SP600125 decreased the number of apoptotic cells induced by LY293111 in a dose-dependent manner (Figure 6c). Western blot analysis showed that SP600125 abrogated LY293111-induced PARP cleavage, and LY293111-induced ROS production was reduced to an equivalent extent (Figure 6d, e). These findings show that JNK phosphorylation plays a crucial role in LY293111-induced apoptosis.
LY293111 is known to be a LTB4 antagonist, 5-lipoxygenase inhibitor and a peroxisome proliferator-activated receptor (PPAR)-γ agonist.32, 33 In this study, we demonstrate that LY293111 inhibited the growth of ALCL cells. These growth-inhibitory effects of LY293111 were accompanied by decreased G1–S phase cell cycle progression, with complete G1/S cell cycle block at low-serum conditions. The orderly progression of cells through the cell cycle depends on the coordinated interaction of key cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (Cdk) and Cdk inhibitory proteins p27Kip1.34 Inhibition of phospho-Akt activity in ALCL may decrease p27 phosphorylation and degradation, resulting in increased p27 level, which may benefit to cell cycle arrest in the G1 phase.35 Furthermore, RAS-activated RAF → MEK → ERK pathway plays an important role in the regulation of cyclin/cdk2 activity, at least in part, through the regulation of p27Kip1 expression.36 Our studies showed the decreased levels of phosphorylated Erk and Akt and induction p27Kip1, accompanied by downregulation of cyclin E following LY293111 treatment in all ALCL cell lines. All of these changes may contribute to the observed inhibition of cell growth. Of note, LY293111 diminished cyclin E levels at lower (2.5 μ M) concentrations in Mac-2A cells, which translated into lower IC50 values (2.306 μ M) for the inhibition of cell growth. This implies that cyclin E likely plays an important role as a mediator of growth arrest in ALK(−) ALCL cells.
The underlining mechanisms of LY293111 as a potent inducer of apoptosis remain unclear. Our studies demonstrated that LY293111-induced apoptosis in ALCL cell lines via intrinsic apoptotic pathway, which manifested by loss of the MMP, ROS production, activation of caspase-9 and downstream effector caspase-3, which in turn cleaved PARP and XIAP. Caspase-8 cleavage did not occur after 24 h LY293111 treatment in either ALK(+) or ALK(−) ALCL cell lines. Members of the Bcl-2 family of proteins are central regulators of mitochondrial-mediated apoptosis and cell death. Pretreatment of cells with radical scavenger GSH only partially reversed ROS generation suggesting that there is another mechanism for LY293111-induced ROS production. Since Bcl-2 has been shown to employ a radical-scavenging mechanism to block the onset of apoptosis,37 LY293111-reduced Bcl-2 phosphorylation might also play a role in ROS generation.38 Although Rassidakis et al39 showed differential expression of Bcl-2 and Bax in ALK(+) and ALK(−) ALCL patient samples (Bcl-2: 0 vs 57.8%, Bax: 72.2 vs 41.7%, respectively), Bcl-2 and Bax proteins were equally expressed in both ALK(+) and ALK(−) ALCL cell lines by Western blot analysis that may be attributable to biological differences between primary and cultured cells. In addition, previous studies have shown that treatment of ALK(+) ALCL cells with standard chemotherapeutic regimens results in apoptotic cell death through caspase-3 cleavage and activation of the intrinsic apoptotic pathway (see Drakos et al,40 and unpublished data). LY293111 reduced mRNA and protein level of antiapoptotic Bcl-2 and initiated Bax activation via increase in p18 cleaved-Bax in ALK(+) ALCL cell lines, shifting a balance towards lower Bcl-2/Bax ratio and favoring mitochondrial apoptosis. In contrast, no significant changes in Bcl-2/Bax levels were observed in Mac-2A cells. This finding likely explains more efficient apoptosis induction by LY293111 in ALK(+) cells compared to ALK(−) cells.
The phosphorylation of stress protein kinase JNK was strongly activated in ALK(+) ALCL cells after LY293111 treatment. Activated JNK has been shown to mediate the dephosphorylation of Bcl-241 and may be considered as a contributor to the apoptosis of ALK(+) ALCL cells. On the other hand, the phospho-JNK inhibitor SP600125 reduced LY293111-induced apoptosis and prevented PARP cleavage. Since SP600125 partially diminished LY293111's induction of ROS production, JNK phosphorylation may also participate in the regulation of ROS production, ultimately promoting apoptosis. Our findings indeed suggest that LY293111 triggers apoptosis by eliciting ROS production, which is partially mediated by JNK activation, followed by reductions in the Bcl-2 mRNA and protein levels as well as its phosphorylation, loss of MMP and caspase-9 activation. However, LY293111 did not significantly induce JNK activation or modulated Bcl-2/Bax proteins in the ALK(−) ALCL cells, implying alternative mechanisms involved in LY293111-mediated ALK(−) ALCL cell death.
In summary, we showed for the first time that LY293111 inhibits ALCL cells proliferation by arresting cells in the G1 phase of the cell cycle and induces the apoptosis via the mitochondrial pathway. While microarray studies have shown an abundant expression of 5-lipoxygenase in certain B-cell malignancies42 and recent report demonstrated the pivotal role of LTB4 in CLL,43 the contribution of this pathway in LY293111-mediated growth inhibition of ALCL remains to be investigated. LY293111 is orally available with reproducible pharmacodynamic effects in man44, 45 and is currently in Phase II trials in cancer patients. Our findings suggest potential therapeutic utility of LY293111 in the management of ALCL.
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We thank Drs Numsen Hail, Jr, Ismael J Samudio and Betty L Notzon for critical review of the manuscript. Grant support: Supported in part by grants from the National Institutes of Health (PO1 CA55164 and CA16672) and the Paul and Mary Haas Chair in Genetics (to MA) and a grant from Eli Lilly and Company (to MK).
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).
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