OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway
Li-Yuan Bai,1,2 Yihui Ma,3 Samuel K. Kulp,3 Shu-Huei Wang,4 Chang-Fang Chiu,1,2 Frank Frissora,4 Rajeswaran Mani,4 Xiaokui Mo,5 David Jarjoura,5 John C. Byrd,4 Ching-Shih Chen3 and Natarajan Muthusamy4
1Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan; 2Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan; 3Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, USA; 4Division of Hematology and Oncology, Department of Internal Medicine, The Ohio State University, USA; 5Center for Biostatistics, The Ohio State University, USA
Running Title:
OSU-DY7 activates p38 MAPK in B-CLL and Burkitt lymphoma Corresponding author: Natarajan Muthusamy, DVM., Ph.D. 455E, OSUCCC, 410, West 12th Avenue, Columbus, OH 43210 Tel: (614) 292-8135 Fax: (614) 292-3312 E-mail: [email protected]
Summary
Drug resistance and associated immune deregulation limit use of current therapies in
chronic lymphocytic leukemia (CLL), thus warranting alternative therapy
development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative
targeting p38 MAPK, mediates cytotoxicity in lymphocytic cell lines representing CLL
(MEC-1), acute lymphoblastic leukemia (697 cells), Burkitt lymphoma (Raji and
Ramos) and primary B cells from CLL patients in dose and time dependent manner.
The OSU-DY7-induced cytotoxicity is dependent on caspase activation as evidenced
by induction of caspase-3 activation and 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 an evidence for a role of OSU-DY7 in p38 MAPK activation and
BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus
Keywords: D-tyrosinol, chronic lymphocytic leukemia, p38 MAPK, apoptosis,
Introduction
Chronic lymphocytic leukemia (CLL) is the most common form of leukemia 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 has shown to be not 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 less than
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
MAPK pathway, initially identified for its role in stress and inflammatory response,
was found to have a tumor suppressor function (Nebreda & Porras, 2000; Ono & Han,
2000; Dolado et al, 2007; Han & Sun, 2007; Hui et al, 2007b; Hui et al, 2007a;
Kennedy et al, 2007). The p38 MAPK pathway has been implicated in negative
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
mdulloblastoma cells, vinka alkaloids in HeLa cells, taxol and cisplatin in several
non-hematological cell lines (Deacon et al, 2003; Hallahan et al, 2003; Losa et al,
2003; Olson & Hallahan, 2004). Although how 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,
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
Materials and methods
Cells and culture conditions
Blood from patients with CLL was obtained under a protocol approved by The Ohio
State University hospital internal review board. All patients had understood and
signed the informed consent in accordance with the Declaration of Helsinki. B-CLL
cells were isolated from freshly collected whole blood using Rosette-Sep kit
(STEMCELL Technologies, Vancouver, BC, Canada) according to the
manufacturer’s instructions. Human B-lymphocyte cell lines MEC-1, Raji, Ramos,
697 and isolated primary B-CLL cells were incubated using procedures previously
described (Liu et al, 2008). MEC-1 cell line was obtained from the German cell line
bank (Braunschweig, Germany) and Raji, 697 and Ramos cell lines were from
American Type Culture Collection (ATCC, Manassas, VA).
Reagents
OSU-DY7 was prepared from D-tyrosine, of which the synthetic procedure will be
published elsewhere. The identity and purity were confirmed by nuclear magnetic
resonance and mass spectrometry. The chemical structure of OSU-DY7 is shown in
Fig 1A. The pharmacological agents were purchased from the respective vendors:
MAP kinase kinase (MEK) inhibitor PD98059 (Calbiochem, Gibbstown, NJ);
Z-VAD-FMK (BIOMOL, Plymouth, PA); caspase-3 substrate (Ac-DMQD)2-Rh110
(AnaSpec, San Jose, CA); TRIzol reagent (Invitrogen, Carlsbad, CA); MG132
(Cayman Chemical, Ann Arbor, MI).
Cell viability and apoptosis assay
The cell viability was assessed by dual staining with annexin V conjugated to
flourescein isothiocyanate (FITC) and propidium iodide (PI). Cells (1× 106) were
stained by annextin V-FITC (BD Pharmingen, San Diego, CA) and PI (BD
Pharmingen) using a procedure previously published (Liu et al, 2008). Cells were
analyzed by a Beckman-Coulter EPICS XL cytometer (Beckman-Coulter, Miami, FL).
Annexin V-FITC and/or PI positive cells were identified as apoptotic cells. Viable cells
were those with both annexin V-FITC negative and PI negative staining. The viable
cells in each sample were expressed as % by normalizing annexin V-/PI- cells to
untreated control.
Analysis of caspase-3 activity
Caspase-3 activity was determined by using (Ac-DMQD)2-Rh110 (AnaSpec) as the
al, 2008).
MTS assay
Measurement of cell growth was performed using CellTiter 96 Aqueous
Non-radioactive Cell Proliferation Assay kit purchased from Promega (Madison, WI).
Cells (0.25× 106/mL for cell lines and 1× 106/mL for primary B-CLL) were placed in
200 µL volume in 96-well microtiter plates with indicated reagent and incubated in
37oC. MTS solution [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)- 2H-tetrazolium] and PMS (phenazine methoxulfate) solution were
mixed in 20:1 in volume. The colorimetric measurements were performed 4 hours
later at 490-nm wavelength by a VersaMax tunable microplate reader (Molecular
Devices, Sunnyvale, CA). The cell viability was expressed as a percentage of
absorbance value in treated sample compared to that observed in control vehicle
treated sample.
Western blotting
Cell lysates were prepared using RIPA buffer (150 mM NaCl, 50 mM Tris PH 8.0, 1%
NP40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) supplemented
Antibodies against various proteins were obtained from the following sources:
poly-ADP-ribose polymerase (PARP), Akt, p-Akt (Ser473), ERK1/2, p-ERK1/2
(pThr202Tyr204), JNK, p-JNK (Thr183Tyr185), p38 mitogen-activated protein kinase
(p38 MAPK), p-p38 MAPK (Thr180Tyr182), MAPKAPK2, p-MAPKAPK2 (Thr334)
(Cell Signaling, Danvers, MA); BIRC5 (R&D, Minneapolis, MN), tubulin (Santa Cruz
Biotechnology, Santa Cruz, CA); actin (MP Biomedicals, Solon, OH). The goat
anti-rabbit IgG-horseradish peroxidase (HRP) conjugates and goat anti-mouse
IgG-HRP conjugates were purchased from Jackson ImmunoResearch Laboratories,
Inc (West Grove, PA).
Reverse transcription and polymerase chain reaction
Cells (5×106) were washed with PBS twice and mRNA was extracted by TRIzol
reagent (Invitrogen, Carlsbad, CA). Reverse transcription to cDNA was performed
using BioRad iScript cDNA Synthesis Kit (BIO-RAD Laboratories, Hercules, CA).
Briefly, 1 µg of mRNA, 4 µL of 5 X iScript-Reaction Mix and 1 µL of iScript Reverse
Transcriptase were mixed in nuclease-free water with final volume of 20 µL. The
condition for reverse transcription was: 25oC for 5 min, 42oC for 30 min and 85oC for
5 min. The real-time PCR for BIRC5 was performed using Taqman Gene Expression
Briefly, 1 µL of cDNA, 12.5 µL of Taqman Universal PCR Master Mix (Applied
Biosystems, Foster City, CA) and 1.25 µL BIRC5 probe (Hs 00977611_g1, Applied
Biosystems) or RN18S1 control (Hs 03003631_g1, Applied Biosystems) were mixed
to DNAse-free water to final volume of 25 µL. The real-time PCR reaction was carried
on 7900HT Real-Time PCR System (Applied Biosystems).
Statistical analysis
All statistical analyses were performed by biostatisticians in the Center for
Biostatistics at the Ohio State University. Nonlinear mixed models were used to
obtain IC50. For comparisons, linear mixed models were used for modeling treatment
effect and patient random effect. Holm’s method was applied to adjust for multiplicity
and control the overall Type I error rate at α=0.05. SAS software (version 9.1, SAS
Institute, Inc., Cary, NC) was used for all statistical analyses.
Results
OSU-DY7 mediates cytotoxicity of B-lymphocytic cell lines and primary B cells from CLL patients
Lymphoid cell lines representative of CLL (MEC-1), ALL (697), Burkitt lymphoma
of OSU-DY7. The OSU-DY7 induced dose and time-dependent decrease in cell
survival (Fig 1B). The IC50 value for each of the cell lines is shown in Table 1.
In order to determine the cytotoxic efficacy of OSY-DY7 in B-CLL cells, freshly
isolated CD19+ B-CLL cells were treated with OSU-DY7 (ranging from 0, 1, 2, 4, 6, 8,
and 10 µM) and the cell viability was evaluated by annexin-V/PI staining analysis at
24 or 48 hours. The IC50 for 16 patients were 3.58 µM (95% CI: 2.60∼4.57) and 3.26
µM (95% CI: 2.20∼4.32) for 24 hours and 48 hours, respectively (Fig 1C, Table 1)
demonstrating maximal apoptosis was observed at 24 hours with no advantage for
extended exposure beyond this time period.
OSU-DY7 induces caspase-3 activation and PARP cleavage in B-lymphocytic cell lines and primary B-CLL cells
To investigate the relationship of OSU-DY7-mediated cytotoxicity and activation of
caspase 3, cells were treated with OSU-DY7 0 µM, 4 µM and 8 µM for 24 hours.
There was a significant linear increase in caspase-3 activity as concentration of
OSU-DY7 increased (Fig 2A, *p<0.0001 in Raji cells and *p=0.0048 in primary B-CLL
cells).
In order to determine if caspase activation results in PARP cleavage, Raji cells
incubated with OSU-DY7 at 0, 0.5, 1, and 2 µM for 24 hours. Lower concentrations of
OSU-DY7 were chosen for MEC-1 cells due to their relatively higher sensitivity
compared to Raji cells (see Table 1). The results showed OSU-DY7 induced PARP
cleavage in Raji and MEC-1 cell lines in a dose-dependent manner as evidenced by
appearance of the cleaved 89 Kd band (Fig 2B, left panels). Similar to the results
obtained in cell lines, OSU-DY7 lead to PARP cleavage in primary B-CLL cells in 6
independent patient samples. Representative results from two patients are shown in
Fig 2B.
In order to determine the relevance of caspase activation in OSU-DY7 mediated
cytotoxicity, we tested the effect of OSU-DY7 on cells pretreated with pancaspase
inhibitor Z-VAD-FMK. As shown in Fig 2C, OSU-DY7-mediated cytotoxicity was
partially yet significantly rescued by Z-VAD-FMK, in both cell lines and primary B-CLL
cells. In MEC-1 cells, 100 µM Z-VAD-FMK pretreatment rescued the
OSU-DY7-mediated cytotoxicity as evidenced by the significant increase in viable
CLL cells from 21.3% to 44.5%. Thus, Z-VAD-FMK significantly decreased
OSU-DY7-mediated MEC-1 cell killing by 52% (95% CI: 48.3% ∼ 55.7%, n=4,
*p<0.0001). Similar rescue effect of Z-VAD-FMK was also observed in Raji cells
where OSU-DY7-mediated cytotoxicity was partially rescued with the increased
OSU-DY7-mediated Raji cell killing by 23% (95% CI: 21.0∼25.6%, n=4, *p<0.0001)
(Fig 2C). Consistent with the cell line data, Z-VAD-FMK also rescued
OSU-DY7-mediated cytotoxicity in primary B-CLL cells (Fig 2D). Without
Z-VAD-FMK, the ratio in survival between OSU-DY7 and DMSO was 62.1% (95% CI:
55.3%∼69.7%), with the Z-VAD-FMK, the ratio was 74.3% (95% CI: 60.6%∼91.0%).
The Z-VAD-FMK significantly decreased the cell killing by OSU-DY7 by 16.5% (95%
CI: 3.9%∼27.4%, n=5, *p=0.0298).
OSU-DY7 induces phosphorylation of p38 MAPK in lymphoid cell lines and primary CLL cells
To investigate the possible mechanisms involved in OSU-DY7-mediated cytotoxicity,
pathways of Akt, ERK, p38 MAPK, JNK, NF-κB were examined. No differences in
levels of p-JNK (Thr183Tyr185), JNK and NF-κB between vehicle and OSU-DY7
treated groups were noted (data not shown). However, OSU-DY7 induced significant
increase of phosphorylation of p38 MAPK in a dose-dependent manner in Raji (n=4,
*p<0.0001) and MEC-1 (n=4, *p=0.0325) when comparing OSU-DY7-treated group
with DMSO (Fig 3A). In Raji cells, the ratio of p-p38 MAPK (Thr180Tyr182) versus
p38 MAPK after OSU-DY7 at 2, 4 and 8 µM concentrations increased 2.2 fold (1∼
increase in p-p38 MAPK was also observed in MEC-1 cells. The ratio after treatment
with OSU-DY7 0.5, 1 and 2 µM for 24 hours increased 1.1 fold (1∼1.3), 1.9 fold (1.2
∼2.8) and 2.9 fold (1.4 ∼ 5.5) respectively compared with control group. The
phosphorylated MAPKAPK2 (Thr334), a downstream target of p38 MAPK, was also
increased after OSU-DY7 treatment.
Consistent with the findings in cell lines, OSU-DY7 induced phosphorylation of
p38 MAPK and MAPKAPK2 in primary B-CLL cells (Fig 3B). The ratio of p-p38
MAPK and p38 MAPK increased 1.3 fold (1∼1.8), 2.3 fold (1.2∼3.4) and 90 fold (1
∼412) in cells from six CLL patients after treatment with OSU-DY7 2, 4 and 8 µM for
24 hours (n=6, *p=0.0004 when comparing OSU-DY7-treated group with DMSO
vehicle control).
The time course analysis of OSU-DY7-induced phosphorylation of p38 MAPK in
Raji cells revealed increased phosphorylation of p38 MAPK as early as 2 hours post
treatment with the progressive increase leading to ~ 44 fold increased
phosphorylation by 24 hours. The difference in phosphorylation of p38 MAPK
between the trends of OSU-DY7 and the negative control was significant (p=0.0022).
(Fig 3C).
To further examine the role of p38 MAPK pathway in OSU-DY7-mediated cytotoxicity,
p38 MAPK specific inhibitor SB202190 was used. OSU-DY7 induced increased
phosphorylation of MAPKAPK2 and PARP cleavage in Raji cells. SB202190
reversed the phosphorylated MAPKAPK2 and reduced the degree of PARP cleavage
(Fig 4A). Meanwhile, SB202190 rescued OSU-DY7-mediated cytotoxicity in Raji
cells partially either at 24 or 48 hours (Fig 4B). At 24 hours, without SB202190, the
ratio in survival between OSU-DY7 and DMSO was 34.1% (95% CI: 32.7%∼35.6%,
n=4); with SB202190, the ratio was 53.9% (95% CI: 51.7%∼56.2%, n=4). The
SB202109 significantly decreased OSU-DY7-mediated Raji cell killing by 36.7 %
(95% CI: 33.9%∼39.4%, *p<0.0001 when comparing SB202190 and OSU-DY7
interaction). Similarly, at 48 hours treatment, there was significant interaction
between SB202190 and OSU-DY7 (Fig 4B). Without SB202190, the ratio in survival
between OSU-DY7 and DMSO was 24.3% (95% CI: 22.7%∼26.1%, n=4), with the
SB202190, the ratio was 48.2% (95% CI: 51.7%∼56.2%, n=4). The SB202109
significantly decreased OSU-DY7-mediated Raji cell killing by 49.5 % (95% CI:
45.0%∼53.6%, *p<0.0001 when comparing SB202190 and OSU-DY7 interaction).
We then checked the salvage effect of SB202190 for OSU-DY7 in primary CLL
cells (Fig 4C). Briefly, without SB202190, the ratio in survival between OSU-DY7 and
ratio was 77.2% (95% CI: 68.5% ∼ 81.7%, n=4). The SB202109 significantly
decreased OSU-DY7-mediated cell killing by 24.3 % (95% CI: 14.1%∼33.3%,
*p=0.002 when comparing SB202190 and OSU-DY7 interaction).
OSU-DY7 down regulates BIRC5 via p38 MAPK activation
The underlying molecular mechanism by which p38 MAPK causes cell apoptosis is
not completely understood. We investigated pro-apoptotic and anti-apoptotic
proteins in response to OSU-DY7 treatment in Raji cells. Compared with DMSO
group, there was no significant change in expression of Bcl-2, Bcl-xl and cIAP2
proteins as detected by immunoblot analysis following OSU-DY7 treatment (data not
shown). However, significant reduction in expression of BIRC5 protein was observed
in OSU-DY7-treated group. The down-regulation of BIRC5 level could be partially
reversed by SB202190 (Fig 5A, left panel). Without P38 MAPK inhibitor SB202190,
OSU-DY7 significantly decreased BIRC5 expression (24% of DMSO, 95%CI: 21.5%
∼25.7%, p<0.0001, n=3). The pretreatment with 10 µM of SB202190 partially
rescued BIRC5 expression level by 1.9 fold (1.5∼2.1, *p<0.0001, n=3) (Fig 5A, right
panel). To further delineate the cause of down-regulation of BIRC5 protein, RT-PCR
analysis of BIRC5 transcripts was analyzed. As shown in Fig 5B, OSU-DY7
of SB202190 significantly prevented OSU-DY7 induced BIRC5 mRNA expression by
2.4 fold (Fig 5B, 95% CI: 1.56∼3.84 fold, *p=0.0026 when comparing SB202190 and
OSU-DY7 interaction, n=3). This implied that OSU-DY7 activated p38 MAPK that
suppressed BIRC5 expression by transcriptional inhibition. To investigate if the
down-regulation of BIRC5 protein level was related to increased proteasome activity,
Raji cells were treated with DMSO or 4 µM OSU-DY7 for 12 hours, followed by
DMSO or 10 µM of MG132, a proteasome inhibitor, for 12 hours. In contrast to
OSU-DY7 induced Mcl-1 protein that was partially rescued, MG132, failed to
modulate the BIRC5 protein level (Fig 5C). Collectively these results suggest that
OSU-DY7 induces activation of p38 MAPK that leads to down regulation of BIRC5
Discussion
We have described here development of a novel D-tyrosinol derivative,
OSU-DY7 that 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 activation of caspase and
downstream PARP cleavage. Z-VAD-FMK at concentration that inhibited the
activation of caspases prevented OSU-DY7 mediated apoptosis. The partial
inhibitory effect of Z-VAD-FMK suggested potential additional mechanism in
OSY-DY7 mediated cytotoxicity. Consistent with this hypothesis, the OSU-DY7
induced activation of p38 MAPK in B-CLL cells and B cells lines.
Three lines of evidences 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 that has been shown to promote apoptosis (Wada &
Penninger, 2004; Seo et al, 2007). Second, SB201190 that 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).
OSU-DY7 mediated apoptosis in B cell lines and B cells from CLL patients.
Our finding indicating OSU-DY7 induced activation of p38 MAPK leading to
cytotoxicity of CLL cells is consistent with the potential role for p38 MAPK in tumor
suppressive effect (Nebreda & Porras, 2000; Ono & Han, 2000; Dolado et al, 2007;
Han & Sun, 2007; Hui et al, 2007b; Hui et al, 2007a; Kennedy et al, 2007).
Interestingly a negative role for p38 MAPK in cell survival has been documented
suggesting the 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 and colleagues
demonstrated that p38 MAPK could inhibit cyclin D1 expression that was reversed by
p38 MAPK inhibitor (Lavoie et al, 1996). Bulavin DV found that inactivated p38 MAPK
in vivo would expedite tumor formation by suppressing p53 activation (Bulavin et al,
2002). The same group further found p38 MAPK activation could suppress tumor
appearance by modulating the CDKN2A tumor-suppressor gene (Bulavin et al, 2004).
Importantly, a downstream signal of p38 MAPK pathway, MAPKAPK-2, has been
found to be a member of the cell cycle checkpoint kinases, exhibiting it’s activity via
phophorylation of Cdc25 protein phosphatase (Manke et al, 2005). Several
evidences suggest a correlation between p38 MAPK activation and apoptosis
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 a 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
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 down regulation of BIRC5 is consistent with the observations in
human lung carcinoma cells where down-regulation of BIRC5 protein expression
was caused by activation of 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 tumor
suppression. Although untreated primary CLL cells have low expression of BIRC5
up-regulation of BIRC5 is found in post-chemotherapy CLL cells, which maybe 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 and colleagues 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 (Pedersen et al, 2002). In another study, a
vitamin D3 analog EB1089 was reported to induce apoptosis in B-CLL cells via a
mechanism involving p38 MAPK activation and ERK suppression (Pepper et al,
2003b). Interestingly OSU-DY7 also induces activation of p38 MAPK with
concomitant down regulation of ERK1/2 phosphorylation (data not shown)
suggesting a potential reciprocal regulation of these two signaling pathways. Pepper
C el al also showed that flavopiridol-induced apoptosis in B-CLL was associated with
activation of p38 MAPK and suppression of ERK activity (Pepper et al, 2003a).
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, that is active for B lymphocytic cell lines representing ALL, Burkitt
dependent mechanisms involving down modulation of BIRC5 protein and 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.
Acknowledgements
This work was supported by D Warren Brown Foundation, Specialized Center of
Research from the Leukemia and Lymphoma Society and Experimental
Therapeutics of Leukemia- SPORE grant (1 P50 CA 140158-01) from NCI.
Disclosure of Potential Conflicts of Interest
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Figure Legend
Fig 1. Chemical structure of OSU-DY7 and cytotoxicity study in B-lymphocytic cell
lines and primary B CLL cells. (A) Chemical structure of OSU-DY7 with a molecular
weight of 265.39. (B) MEC-1, 697, Raji, and Ramos cells (0.25×106 cells/mL) were
incubated with indicated concentrations of OSU-DY7 or DMSO vehicle for 24 hours
( ) or 48 hours (■). The cells were analysed with MTS assay, as described in “MTS
assay in Methods“ (n=3). (C) purified B-CLL cells (1×106 cells/mL) were incubated
with OSU-DY7 or DMSO for 24 hours ( ) or 48 hours (■). The cells were stained with
annexin V-FITC and PI for accessing cell viability (n=16).
Fig 2. OSU-DY7-mediated cytotoxicity is dependent on caspase activation and
apoptosis. (A) Raji cells (0.25×106 cells/mL) and primary B-CLL cells (1×106 cells/mL)
were incubated with OSU-DY7 or DMSO for 24 hours. Cells (1×106) were analyzed
for caspase-3 activity as described in “Analysis of caspase-3 activity in Methods”. The
numbers in each graph represent the percentage and the range of caspase-3 positive
cells (n=3). (B) OSU-DY7 induces PARP cleavage in Raji cell, MEC-1 cell and primary
B-cell CLL in 24 hours. Raji cells (0.25×106 cells/mL), MEC-1 cells (0.25×106 cells/mL)
Total cell lysates (10 µg) were used for western blot. The data represents results from
2 of 6 patient samples with similar observation. (C) OSU-DY7-induced cytotoxicity
can be rescued in part by Z-VAD-FMK in MEC-1 and Raji cells. In MEC-1 study, cells
(0.25×106 cells/mL) were pretreated with medium or 100 µM Z-VAD-FMK, followed by
incubation with DMSO or 2 µM OSU-DY7 for 24 hours. In Raji study, cells (0.25×106
cells/mL) were pretreated with medium or 50 µM Z-VAD-FMK, followed by incubation
with DMSO or 4 µM OSU-DY7 for 24 hours. The viability of cells was checked with
MTS assay (n=4). (D) OSU-DY7-induced cytotoxicity can be rescued in part by
Z-VAD-FMK in primary CLL cells. Primary B-CLL cells (1×106 cells/mL) were
pretreated with medium or 100 µM Z-VAD-FMK, followed by incubation with DMSO or
4 µM OSU-DY7 for 24 hours. At 24 hours, the cells were stained with annexin V-FITC
and PI for accessing cell viability (n=5).
Fig 3. OSU-DY7 induces phosphorylation of p38 MAPK. (A) Raji cells and MEC-1
cells (0.25×106 cells/mL) were incubated with OSU-DY7 or DMSO for 24 hours. Cell
lysates of 15 µg were used for western blot analysis. The ratio of p-p38 MAPK versus
p38 MAPK in indicated concentrations of OSU-DY7 was compared with that in DMSO.
▬ represents average of the values from 4 different experiments. ﹡p=0.0325 for Raji
DMSO control. (B) Primary B-CLL cells (1×106 cells/mL) were incubated with
OSU-DY7 or DMSO for 24 hours. Cell lysates of 15 µg at each point were used for
western blot analysis. The ratio of p-p38 MAPK versus p38 in indicated
concentrations of OSU-DY7 was compared with that in DMSO. ▬ represents average
of the values from 6 patients. ﹡p=0.0004 when comparing OSU-DY7-treated group
with DMSO control. (C) OSU-DY7 induces phosphorylation of p38 MAPK in Raji cells
in a time-dependent manner. Cell lysates of 15 µg at each point were used for western
blot analysis. The data shown here is a representative of two independent
experiments with similar results.
Fig 4. OSU-DY7-induced cytotoxicity is dependent on p38 MAPK activation. (A)
OSU-DY7 induces upregulation of phosphorylation of MAPKAPK2 that is reversed by
p38 MAPK inhibitor SB202190 in Raji cells. Cells (0.25×106 cells/mL) were pretreated
with medium or 10 µM SB202190 for 2 hours, followed by incubation with DMSO or 4
µM OSU-DY7 for 24 hours. Cell lysates of 15 µg at each point were used for western
blot analysis. (B) OSU-DY7-induced cytotoxicity is partially rescued by SB202190 in
Raji cells. Cells (0.25×106 cells/mL) were pretreated with medium or 10 µM
SB202190 for 2 hours, followed by incubation with DMSO or 4 µM OSU-DY7. The
cytotoxicity is partially rescued by SB202190 in primary CLL cells. B-CLL cells (1×106
cells/mL) were pretreated with medium or 10 µM SB202190 for 2 hours, followed by
incubation with DMSO or 4 µM OSU-DY7 for 24 hours. The cells were stained with
annexin V-FITC and PI to assess cell viability (n=4).
Fig 5. OSU-DY7-induced down regulation of BIRC5 protein and mRNA transcription
in p38 MAPK activation dependent manner. (A) Raji cells (0.25×106 cells/mL) were
pretreated with medium or 10 µM SB202190 for 2 hours, followed by DMSO or 4 µM
OSU-DY7 for 24 hours. Cell lysates of 15 µg at each point were used for western blot
(left panel). The ratio of BIRC5 versus actin protein was compared between OSU-DY7
alone and OSU-DY7 plus SB202190 (right panel, n=3). (B) Raji cells (0.25×106
cells/mL) were pretreated with DMSO or 10 µM SB202190 for 2 hours, followed by
DMSO or 4 µM OSU-DY7 for 24 hours. mRNA was reverse transcribed to cDNA
which was further compared for BIRC5 expression using real-time PCR. The data
shown here represents relative mRNA level of BIRC5 compared with DMSO-treated
cells. (C) Raji cells (0.25×106 cells/mL) were pretreated with DMSO or 4 µM
OSU-DY7 for 12 hours, followed by DMSO or 10 µM MG132 for 12 hours. Cell lysates
