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- Materials and methods
- Conflict of interest
Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.
Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the western world, with an incidence of around 3·5 cases per 100 000 people per year (Dighiero & Hamblin, 2008). While the outcome of patients with CLL vary by many factors, such as age, disease status, associated genetic abnormalities and other co-morbid illnesses, the available therapies including alkylating agents, purine analogues, bendamustine, alemtuzumab, rituximab and, more recently, combination therapy with chemoimmunotherapy have not been curative (Montserrat & Moreno, 2008). Similar to chemotherapy combinations in other malignancies, drug resistance often ensues when treatment has been initiated. In particular, patients relapsing after chemoimmunotherapy have a poor outcome with a survival <2 years, warranting alternative therapies in CLL.
One alternative to circumvent drug resistance is to utilize agents that act through mechanisms that are different from that of the currently used therapies. The p38 mitogen-activated protein kinase (MAPK) pathway, initially identified for its role in stress and inflammatory response, was found to have a tumour suppressor function (Nebreda & Porras, 2000; Ono & Han, 2000; Dolado et al, 2007; Han & Sun, 2007; Hui et al, 2007a,b; Kennedy et al, 2007). The p38 MAPK pathway has been implicated in negative control of tumourigenesis through cell cycle control (Ambrosino & Nebreda, 2001), cell differentiation (Puri et al, 2000), cell proliferation, oncogene-induced senescence, replicative senescence, contact inhibition, DNA-damage response and induction of apoptosis (Nebreda & Porras, 2000; Bulavin & Fornace, 2004; Wada & Penninger, 2004; Han & Sun, 2007). Some chemotherapeutic drugs have been reported to induce cell apoptosis via p38 MAPK activation, including all-trans retinoic acid in medulloblastoma cells, vinka alkaloids in HeLa cells, taxol and cisplatin in several non-haematological cell lines (Deacon et al, 2003; Hallahan et al, 2003; Losa et al, 2003; Olson & Hallahan, 2004). Although the mechanism(s) by which p38 MAPK induces cell apoptosis is not fully understood, studies in myeloma and rat pheochromocytoma cells suggest a link between p38 MAPK and the Bcl-2 family protein and mitochondrial pathway (Seo et al, 2007; Cai & Xia, 2008). Together, these data suggest a potential use for p38 MAPK targeted therapeutic agents in cancer treatment.
In an attempt to develop a new class of agents targeting p38 MAPK activation, we used the immunosuppressive agent FTY720 as a lead compound to conduct structural optimization, which has been reported to mediate apoptosis in human Jurkat T lymphocytes, in part, via a p38-dependent mechanism (Matsuda et al, 1999). Among various derivatives examined, OSU-DY-7 [(R)-2-amino-3-(4-heptyloxy-phenyl)-propan-1-ol] represented the optimal agent, which exhibits unique ability to activate p38 MAPK in a variety of lymphoid malignancies including primary CLL cells. Here, we demonstrate that OSU-DY7 mediates cytotoxicity in lymphocytic cell lines and primary B-CLL cells, in part, via p38 MAPK-dependent down-regulation of BIRC5 expression.
- Top of page
- Materials and methods
- Conflict of interest
We have described here development of a novel D-tyrosinol derivative, OSU-DY7, which mediates cytotoxicity in primary CLL B cells and B cell lines representing CLL (MEC-1), ALL (697), and Burkitt lymphoma (Raji and Ramos) cell lines. The cytotoxic effect of OSU-DY7 is dependent on the activation of caspase and downstream PARP cleavage. Z-VAD-FMK, at concentrations that inhibited the activation of caspases, prevented OSU-DY7-mediated apoptosis. The partial inhibitory effect of Z-VAD-FMK suggested a potential additional mechanism in OSY-DY7-mediated cytotoxicity. Consistent with this hypothesis, OSU-DY7 induced activation of p38 MAPK in B-CLL cells and B cells lines.
Three lines of evidence suggested a potential role for p38 MAPK phosphorylation in OSU-DY7-mediated apoptosis. First, concentrations of OSU-DY7 that induced apoptosis resulted in time-dependent phosphorylation of p38 MAPK on Thr180 and Tyr182 residues, which has been shown to promote apoptosis (Wada & Penninger, 2004; Seo et al, 2007). Second, SB202190, which resulted in inhibition of p38 MAPK activity, also resulted in inhibition of OSU-DY7-induced apoptosis. Third, the OSU-DY7 resulted in phosphorylation of MAPKAPK2, a downstream target of p38 MAPK that is implicated in apoptosis (Ono & Han, 2000; Dolado et al, 2007). Concentrations of SB202190 that inhibited MAPKAP2 phosphorylation prevented OSU-DY7-mediated apoptosis in B cell lines and B cells from CLL patients.
Our finding, indicating that OSU-DY7 induced activation of p38 MAPK leading to cytotoxicity of CLL cells, is consistent with the potential role for p38 MAPK in a tumour suppressive effect (Nebreda & Porras, 2000; Ono & Han, 2000; Dolado et al, 2007; Han & Sun, 2007; Hui et al, 2007a,b; Kennedy et al, 2007). Interestingly, a negative role for p38 MAPK in cell survival has been documented, suggesting a complex role for p38 MAPK activation in cell growth and apoptosis (Juretic et al, 2001; Park et al, 2002; Wada & Penninger, 2004). Multiple key cell cycle controls are known to be targets of p38 MAPK. Lavoie et al (1996) demonstrated that p38 MAPK could inhibit cyclin D1 expression and was reversed by p38 MAPK inhibitor. Bulavin et al (2002) found that, in vivo, inactivated p38 MAPK expedited tumour formation by suppressing p53 activation. The same group further found p38 MAPK activation could suppress tumour appearance by modulating the CDKN2A tumour-suppressor gene (Bulavin et al, 2004). Importantly, a downstream signal of the p38 MAPK pathway, MAPKAPK-2, has been found to be a cell cycle checkpoint kinase, exhibiting its activity via phophorylation of CDC25 protein phosphatase (Manke et al, 2005). Several evidences suggest a correlation between p38 MAPK activation and apoptosis induction. For example, withdrawal of nerve growth factor from Rat PC-12 pheochromocytoma cell was found to cause sustained activation of p38 MAPK and JNK, as well as induction of apoptosis (Xia et al, 1995). Knockout studies further demonstrated decreased cell survival in cells lacking MKK6, p38αMAPK and MAPKAPK2 (Nebreda & Porras, 2000).
Despite the extensive description, the precise molecular mechanisms by which p38 MAPK causes cell apoptosis are not completely understood. A potential role for the regulation of Bcl-2 family and mitochondrial pathway has been implicated (Kennedy et al, 2007; Seo et al, 2007; Cai & Xia, 2008). Down-regulation of cIAP-1/2, XIAP and BIRC5, as well as accumulation of p53, Bax and Bak in mitochondria were noted in sulindac-induced p38 MAPK activation and cell apoptosis (Seo et al, 2007). OSU-DY7-induced downregulation of BIRC5 is consistent with the observations in human lung carcinoma cells where down-regulation of BIRC5 protein expression was caused by activation of the p38 MAPK pathway (Chao et al, 2004). Similar down modulation of BIRC5 by activation of p38 MAPK and JNK pathways was also observed in response to arsenic trioxide in lung adenocarcinoma cells (Cheng et al, 2006), and vitamin D3-mediated cell growth inhibition and apoptosis (Li et al, 2005). Together, these data suggest that p38 MAPK activation plays a role in tumour suppression. Although untreated primary CLL cells have low expression of BIRC5 compared with ALL or follicular lymphoma cells (de Graaf et al, 2005; Hui et al, 2006), up-regulation of BIRC5 is found in post-chemotherapy CLL cells, which may be one of the mechanisms by which CLL becomes chemoresistant (Hui et al, 2006).
The pro-apoptotic effect of p38 MAPK pathway has also been revealed in CLL. Pedersen et al (2002) demonstrated that rituximab, besides its activity to induce antibody-dependent cellular toxicity, could induce apoptosis in B-CLL through a p38 MAPK activation-dependent pathway, and inhibition of p38 MAPK reduced the degree of anti-CD20-induce apoptosis. In another study, a vitamin D3 analogue, EB1089, was reported to induce apoptosis in B-CLL cells via a mechanism involving p38 MAPK activation and ERK suppression (Pepper et al, 2003a). Interestingly OSU-DY7 also induced activation of p38 MAPK with concomitant down regulation of ERK1/2 phosphorylation (data not shown), suggesting a potential reciprocal regulation of these two signalling pathways. Pepper et al (2003b) also showed that flavopiridol-induced apoptosis in B-CLL was associated with activation of p38 MAPK and suppression of ERK activity. Altering the balance between these two pathways could provide a rationale for the p53-independent nature of flavopiridol-induced apoptosis.
In conclusion, these studies describe a newly synthesized D-tyrosinol derivative, OSU-DY7, which is active for B lymphocytic cell lines representing ALL, Burkitt lymphoma and CLL and primary B cells from CLL patients. p38 MAPK activation-dependent mechanisms involving down modulation of BIRC5 protein and BIRC5 mRNA and caspase-dependent apoptosis reveal OSU-DY7 as an attractive alternative therapy targeting the p38 MAPK pathway for CLL and other lymphocytic malignancies. Further studies are warranted to validate OSU-DY7 for clinical development for CLL and other B cell malignancies.