p38 mapk is required for the antitumor activity of...
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p38 MAPK is required for the antitumor activity of the
vascular disrupting agent DMXAA
Qiong Wu, Haitian Quan, Yongping Xu, Yun Li, Youhong Hu, Liguang Lou
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
JPET Fast Forward. Published on March 13, 2012 as DOI:10.1124/jpet.112.191635
Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: p38 is required for the antitumor activity of DMXAA
Corresponding author: Liguang Lou, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. Phone:
86-21-5080 6056, Fax: 86-21-5080 7088. E-mail: [email protected]
Number of text pages: 33
Number of tables: 0
Number of figures: 9
Number of references: 39
Number of words in Abstract: 150
Number of words in Introduction: 745
Number of words in Discussion: 1219
Abbreviations: 5, 6-dimethylxantheonone-4-acetic acid, DMXAA; mitogen-activated
protein kinase, MAPK; tumor necrosis factor, TNF; vascular disrupting agents, VDAs;
extracellular signal-regulated kinase, ERK; c-Jun N-terminal kinase, JNK;
N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]benza
mide, PD0325901; 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-
imidazole, SB202190; 1,9-pyrazoloanthrone, SP600125; human umbilical vein
endothelial cells, HUVECs; enzyme-linked immunosorbent assay, ELISA;
SDS-polyacrylamide gel electrophoresis, SDS-PAGE; myosin light chain, MLC
Recommended section: Cellular and Molecular
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Abstract
5, 6-dimethylxanthenone-4-acetic acid (DMXAA), a potent vascular disrupting
agent, selectively destroys established tumor vasculature, causing a rapid collapse in
blood flow that ultimately leads to inhibition of tumor growth. Here, we demonstrate
that p38 MAPK is critically involved in DMXAA-induced cytoskeleton
reorganization in endothelial cells and TNF-α production in macrophages, both of
which were essential for DMXAA-induced vascular disruption. Inhibition of p38
MAPK significantly attenuated DMXAA-induced actin cytoskeleton reorganization in
human umbilical vein endothelial cells and TNF-α production in macrophages.
In vivo, p38 MAPK inhibition attenuated the immediate reduction in tumor blood
flow induced by DMXAA treatment (<30 min) by inhibiting actin cytoskeleton
reorganization in tumor vascular endothelial cells, and blunted the long-lasting (>4 h)
DMXAA-induced shutdown of the tumor vasculature by inhibiting intratumoral
TNF-α production. These results indicate that p38 MAPK plays a critical role in
DMXAA-induced endothelial cell cytoskeleton reorganization and TNF-α production,
thus regulating DMXAA-induced antitumor activity.
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Introduction
The tumor vasculature represents a promising target for cancer therapy. Vascular
disrupting agents (VDAs) are a new class of potential antitumor drugs that selectively
destroy the established tumor vasculature, shut down blood supply, deprive tumors of
oxygen and nutrition, and cause extensive tumor necrosis (Chaplin & Dougherty,
1999; Lippert, 2007; Philip E. Thorpe, 2003). There are two types of VDAs: small
molecules and ligand-directed agents. Currently, most small molecule VDAs are
tubulin inhibitors, such as combretastatin A-4P and ZD6126; the remainder are
synthetic flavonoids, including flavone acetic acid (FAA) and DMXAA (Cai, 2007).
5, 6-Dimethylxanthenone-4-acetic acid (DMXAA), also known as ASA404, is a
FAA derivative that acts as an effective VDA. DMXAA causes a rapid and
remarkable collapse in blood flow within 30 min after administration and leads to
complete vascular shutdown later (Tozer et al, 2005). Clinical studies have confirmed
the anti-vascular effect, demonstrating an increase in tumor vascular permeability
(Baguley et al, 2005), changes in tumor perfusion (Murata et al, 2001), and increased
plasma concentrations of the serotonin metabolite 5-hydroxyindole acetic acid (Zhao
L, 2002). DMXAA is thought to exert its antitumor effects through both induction of
apoptosis in tumor vascular endothelial cells (Ching et al, 2002a) and an indirect
vascular effect involving production of a spectrum of cytokines and chemokines,
including tumor necrosis factor-α (TNF-α) (Wang, 2004), interferon-β (Shirey et al,
2011),interferon-inducible protein 10 (Cao Z, 2001), and inducible nitric oxide
synthase (iNOS) (Peng et al, 2011). It has been reported that TNF-α is an important
cytokine involved in mediating the antitumor effects of DMXAA (Ching LM, 1999 ).
In situ hybridization studies of the murine Colon 38 tumor have shown that the extent
of DMXAA-induced TNF-α synthesis is greater in tumors than that in the spleen,
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liver, or serum (Joseph WR 1999). More importantly, previous results have
demonstrated that mice lacking genes for TNF or TNF receptor-1 (Ching LM, 1999 )
exhibit a partially diminished tumor-regression response to DMXAA treatment.
DMXAA completed Phase I clinical trials in New Zealand and the United Kingdom,
showing good tolerance (Rustin et al, 2003). A Phase II clinical trial that tested
DMXAA in combination with carboplatin and paclitaxel against non-small-cell lung
cancer demonstrated a greater tumor response rate, a reduction in median time to
tumor progression, and improved median survival (McKeage et al, 2009). However, a
Phase III trial in 2010 showed no survival benefit of DMXAA combination therapy in
patients with late-stage lung cancer. Given the effectiveness of DMXAA as a VDA,
efforts to elucidate novel mechanisms of action of this agent and explore new
administration approaches remain of paramount importance.
Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine
kinases involved in regulating a variety of cellular processes, including inflammation,
cell growth and survival, and cellular differentiation (Karin & Chang, 2001). Three
kinds of MAPKs have been identified: extracellular signal-regulated kinase (ERK),
c-Jun N-terminal kinase (JNK), and p38 MAPK. Of these, p38 MAPK appears to be
involved in the production of cytokines by stimulated monocytes (Saklatvala, 2004).
Inhibitors of p38 MAPK have been reported to block lipopolysaccharide-induced
TNF-α production at both transcriptional (Manthey CL, 1998) and post-transcriptional
(Young P, 1993) levels. p38 MAPK has also been shown to regulate actin
microfilament dynamics via activation of MAPKAP (mitogen-activated protein
kinase-activated protein) kinase 2/3 and phosphorylation of HSP27 (heat-shock
protein 27) (Freshney et al, 1994; Rouse et al, 1994) in endothelial cells, resulting in
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stress fiber formation. Previous research has also provided evidence that p38 MAPK
is necessary for combretastatin A4-induced endothelial cell blebbing (Kanthou &
Tozer, 2002) and involved in the action of DMXAA (Zhao et al, 2007).Very recently,
it has been reported that MAPKs, especially p38 MAPK, play an important role in
regulation of both TNF-a and IL-6 protein production induced by DMXAA in murine
macrophages at the post-transcriptional level (Sun et al, 2011). However, the direct
roles of p38 MAPK in the DMXAA’s in vivo anti-tumor activity, especially in the
DMXAA-induced tumor vascular shut-down, is still not known.
In this study, we examined the role of p38 MAPK in the antitumor activity of
DMXAA. In vitro, p38 MAPK was critically involved in DMXAA-induced actin
cytoskeleton reorganization in human umbilical vein endothelial cells (HUVECs) and
TNF-α production in macrophages. Consistent with this, in vivo inhibition of p38
MAPK significantly attenuated the tumor vascular shutdown by inhibiting the rapid
DMXAA-induced endothelial cell cytoskeleton reorganization and subsequent TNF-α
production. These results demonstrate that p38 MAPK is required for DMXAA-
induced disruption of the tumor vasculature.
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Materials and Methods
Reagents
DMXAA and PD0325901 were synthesized at Shanghai Institute of Materia
Medica, Chinese Academy of Sciences. SB202190, SP600125, Hoechst 33342, and
monoclonal antibodies specific for β-tubulin were obtained from Sigma-Aldrich (St.
Louis, MO, USA). Endothelial cell growth supplement (ECGS) was purchased from
Gibco (Grand Island, NY, USA). Monoclonal antibodies to phospho-ERK1/2,
ERK1/2, phospho-p38 MAPK, p38 MAPK, phospho-MAPKAPK2, and
phospho-MLC were from Cell Signaling (Beverly, MA, USA).Alexa Fluor
594-conjugated phalloidin were from Invitrogen (Carlsbad, CA, USA). Mouse TNF-α
neutralizing antibody and normal goat IgG were from R&D Systems (Abingdon, UK).
Cell lines
The murine macrophage cell line, Ana-1, was obtained from the Cell Bank of the
Shanghai Institute for Biological Sciences, Chinese Academy of Science (Shanghai,
China). Ana-1 was cultured in RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS). HUVECs were isolated from umbilical cord veins using
collagenase, as described previously (Jaffe et al, 1973). Briefly, cells were detached
by treatment with 0.25% type I collagenase, seeded on gelatin-coated culture plates,
and maintained in M199 medium containing 10% FBS, 20 μg/ml ECGS, and
50 μg/ml heparin. Cells were incubated at 37°C in a humidified atmosphere of 5%
CO2 in air. HUVECs were passaged 2–6 times before using in experiments.
Endothelial cell morphological examination
Changes in endothelial cell shape were examined using proliferating HUVECs.
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HUVECs were plated in 6-well plates preincubated with 1% gelatin. Cells were
grown for at least 24 hours before treatment. Photographs were taken using an
Olympus fluorescence microscope. For quantitation of cell area, images were
acquired from randomly selected areas of slides and then analyzed using Image Pro
Plus 6.0 software (Media Cybernetics, Silver Spring, Maryland, USA).
F-actin staining
HUVECs were plated on glass cover slips 24 hours before performing experiments.
Following DMXAA treatment with or without inhibitors, as indicated in the text, cells
were washed with phosphate-buffered saline (PBS) and then fixed for 20 minutes in
4% paraformaldehyde/PBS. Alexa Fluor 594-conjugated phalloidin (diluted 1:100 in
2% BSA/PBS) and 4', 6-diamidino-2-phenylindole (0.1 μg/ml) were used to stain
F-actin and nuclei, respectively. After extensive washes, cells were mounted and
visualized by fluorescence microscopy.
Western blotting
After drug treatment, cells were washed twice with ice-cold PBS and lysed in
sodium dodecyl sulfate (SDS) sample buffer. Cell lysates containing equal amounts of
protein were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to polyvinylidene difluoride membranes. After blocking in 5% non-fat
milk in Tris-buffered saline with 0.1% Tween-20, membranes were incubated at 4°C
overnight with the appropriate primary antibodies and then exposed to secondary
antibodies for 2 hours at room temperature. Immunoreactive proteins were visualized
using an enhanced chemiluminescence system (Pierce Chemical, Rockford, IL, USA).
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TNF-α determination
Removed tumor tissues were homogenized in culture medium using a tissue
homogenizer. The homogenates were centrifuged (2000 x g, 30 min, 4°C) and
supernatants were collected and kept at -70°C until use. TNF-α was assayed using a
TNF-α mouse enzyme-linked immunosorbent assay (ELISA) Kit according to the
manufacturer’s directions (Invitrogen, CA, USA).
RNA interference
Ana-1 cells were transfected with small interfering RNA (siRNA) duplexes (100
nM) against p38 using the TurboFect siRNA transfection reagent (Fermentas, St.
Leon-Roth, Germany), as described by the manufacturer. The cells were then allowed
to recover for 72 hours before stimulation. The siRNA sequence targeting mouse p38α
(NM_011951), synthesized by Genepharma (Shanghai, China), spans nucleotides
481–499 (target sequence, 5′-GAC TGT GAG CTC AAG ATT C-3′) and is specific
for mouse p38 MAPK based on a BLAST search of the NCBI database. The sense
p38-siRNA sequence was 5′-GAC UGU GAG CUC AAG AUU Ctt-3′ and the
antisense sequence was 5′-GAA UCU UGA GCU CAC AGU Ctt-3′. Negative control
siRNA sequences were 5′-UUC UCC GAA GGU GUC ACG Utt-3′ (sense) and 5′
-ACG UGA CAC GUU CGG AGA Att-3′ (antisense).
Mice and tumors
Six-to-eight-week-old female C57 mice weighing 16–18 g were purchased from
Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and raised under
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specific pathogen free conditions. Each animal was injected subcutaneously in the
right flank with Lewis lung tumor fragments. Experiments were carried out when
tumors had reached approximately 10 mm in diameter. Mice were administered a
single intraperitoneal injection of the indicated dose of DMXAA or sterile saline
alone (control). Animal experiments were conducted in accordance with the
Institutional Animal Care and Use Committee guidelines of the Animal Facility at the
Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
Blood perfusion determination
The perfused vasculature in Lewis lung tumors was identified by intravenous
injection of 100 μl (8 mg/ml) of Hoechst 33342 dye into the tail vein (Smith et al,
1988; Trotter et al, 1990). One minute after dye injection, mice were euthanized by
cervical dislocation. Tumors were quickly removed and rapidly frozen over liquid
nitrogen for subsequent sectioning (6-μm cryosections). Using this method, perfused
blood vessels could be visualized by the surrounding halo of fluorescent, Hoechst
33342-labeled cells. Images were captured using a fluorescence microscope and
analyzed using Image Pro Plus 6.0 software.
Assessment of hemorrhagic necrosis
Tumors were removed 24 hours after treatment with or without DMXAA and fixed in
10% formalin. Fixed tumors were then embedded in paraffin and sectioned. Sections
were stained with hematoxylin and eosin. Photographs were taken using an inverted
microscope. For quantitation of necrotic area, images were acquired from randomly
selected areas of slides and then analyzed using Image Pro Plus 6.0 software.
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Statistical analysis
The results are expressed as means ± SEMs. Paired Student’s t-tests were used to test
for significance, where indicated.
Results
p38 MAPK activation participates in DMXAA-induced morphological changes
and actin stress fiber formation in HUVECs.
Because endothelial cells lining tumor blood vessels are the main targets for VDAs,
such as combretastatin A4-P, we were interested in examining whether DMXAA had
an effect on endothelial cell morphology. HUVECs rapidly rounded up and retracted
from each other after treatment with 500 μM DMXAA for 5 minutes (Figure 1A),
morphological changes that lasted for up to 4 hours (data not shown). To investigate
the signaling pathways involved in these morphological changes, we pretreated cells
with inhibitors of ERK (PD0325901), p38 (SB202190), or JNK (SP600125) before
exposing them to DMXAA. Only pretreatment with SB202190 reversed
DMXAA-induced morphological changes, determined by HUVEC morphology and
the time course of changes in HUVEC cell area; PD0325901 and SP600125 were
without effect (Figure 1A, 1B). These results suggest that p38 MAPK, but not ERK or
JNK, is critically involved in mediating the effects of DMXAA on HUVEC
morphology.
Cell morphological changes are mainly associated with alterations in the cell
cytoskeleton, including actin and microtubules. Accordingly, we next examined
whether the HUVEC cytoskeleton was affected by DMXAA treatment. Compared
with control cells, there was a significant thickening of actin bundles and an increase
in the formation of stress fibers across HUVECs after treatment with 100 μM
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DMXAA for 30 minutes (Figure 1C), whereas microtubules were not affected (data
not shown). Furthermore, pretreatment with the p38 MAPK kinase inhibitor
SB202190, but not the ERK1/2 inhibitor PD0325901, significantly attenuated
DMXAA-induced actin cytoskeleton reorganization (Figure 1C), indicating that p38
MAPK is required for the DMXAA-induced actin cytoskeleton reorganization that
leads to changes in HUVEC morphology.
As expected, DMXAA significantly stimulated the activation of p38 MAPK, but
not ERK1/2, as early as 5 minutes in HUVECs (Figure 1D). Myosin light chain
(MLC), which regulates actin stress fiber assembly (Smith et al, 1988), was also
phosphorylated at Thr-18 and Ser-19 after treatment with DMXAA for 15 minutes
(Figure 1D). Furthermore, pretreatment with the p38 MAPK inhibitor SB202190
significantly inhibited DMXAA-induced phosphorylation of both p38 MAPK and
MLC (Figure 1E), suggesting that DMXAA-stimulated p38 MAPK activation is
upstream of MLC phosphorylation in HUVECs.
p38 MAPK activation is essential for TNF-α production in Ana-1 murine
macrophages following DMXAA exposure.
Several studies have shown that TNF-α, secreted from macrophages, is an
important regulator of the antitumor activity of DMXAA. Thus, we tested whether
p38 MPAK was also activated in murine Ana-1 macrophages during DMXAA
treatment. As shown in Figure 2A, p38 MAPK activation in Ana-1 cells was observed
as early as 30 minutes after DMXAA treatment (Figure 2A). MAPKAPK-2, a
downstream effector of p38 MAPK, was also phosphorylated 30 minutes after
exposure to DMXAA (Figure 2A). These results raise the possibility that DMXAA
stimulation of p38 MAPK activation is not cell-type specific. In addition, ERK1/2
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phosphorylation was also slightly increased after DMXAA treatment for 30 min in
Ana-1 macrophages (Figure 2A).
To explore whether DMXAA-induced p38 MAPK activation was related to TNF-α
production in Ana-1 cells, we pretreated cells with the p38 MAPK inhibitor
SB202190 before exposure to DMXAA. Figure 2B shows that DMXAA-stimulated
TNF-α production was significantly reduced (~60%) by SB202190 pretreatment for
30 minutes (Figure 2B), and was sligtly affected by pretreatment with the ERK1/2
inhibitor PD0325901 (~20%), but was unaffected by the JNK inhibitor SP600125
(data not shown), suggesting that MAPKs, especially p38 MAPK, participate in
DMXAA-induced TNF-α production in Ana-1 cells.
To confirm the role of p38 MAPK in TNF-α production, we used a more specific
approach, employing siRNA against p38 MAPK. As shown in Figure 2C, transfection
of Ana-1 cells with p38 siRNA significantly reduced p38 MAPK protein expression
as well as DMXAA-induced TNF-α production, confirming that p38 MAPK is a
critical mediator of DMXAA-induced TNF-α production in macrophages.
p38 MAPK activation is critically involved in DMXAA-induced tumor vascular
disruption in vivo.
The role of p38 MAPK in mediating DMXAA-induced antitumor activity was
examined in vivo using an established Lewis lung cancer model in C57 mice. Tumor
fragments were implanted in C57 mice and allowed to grow to 10 mm in diameter
before DMXAA administration. Consistent with previous reports (Joseph et al, 1999),
treatment of C57 mice with DMXAA at a dose of 20 mg/kg induced a time-dependent
production of intratumoral TNF-α, which increased from undetectable levels at 30
minutes and 2 hours to reach significant levels at 4 hours (Figure 3A). In contrast,
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increased levels of p38 MAPK phosphorylation in tumor tissues were detected as
early as 30 minutes after DMXAA treatment (Figure 3B), indicating that
DMXAA-stimulated p38 MAPK activation precedes TNF-α production in tumor
tissues.
To investigate the vascular disrupting effect of DMXAA, we visualized
blood-perfused vessels in tumor cryosections as revealed by surrounding Hoechst
33342-labeled cells. Consistent with previous reports, tumor blood flow was markedly
reduced as early as 30 minutes following DMXAA administration compared with
normal saline treatment (Figure 4A). Considering that the TNF-α level was not
affected within 30 minutes after DMXAA administration (Figure 3A, 4B), these
results clearly exclude the possibility that intratumoral TNF-α production is
responsible for the rapid early-stage of DMXAA-induced tumor blood flow reduction.
The aforementioned in vitro results demonstrate that DMXAA induces rapid actin
cytoskeleton reorganization in endothelial cells, an effect that is mediated by p38
MAPK activation. Accordingly, we next used the p38 MAPK inhibitor SB202190 to
examine whether p38 MAPK participated in the rapid reduction in tumor blood flow
induced in vivo by DMXAA. As shown in Figure 4 A and B, SB202190 pretreatment
significantly attenuated the DMXAA-induced reduction in tumor blood flow without
affecting intratumoral TNF-α production. Given that endothelial cells lining tumor
blood vessels are the main targets for VDAs, these results suggest that
DMXAA-induced, p38 MAPK-mediated rapid reorganization of the cytoskeleton in
endothelial cells may be responsible for the rapid reduction of tumor blood flow.
We next addressed the role of p38 MAPK in the later, sustained component of
DMXAA-induced blood flow reduction. SB202190 pretreatment dramatically
reversed the reduction in tumor blood flow induced by 4-hour DMXAA
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administration and inhibited DMXAA-induced p38 MAPK phosphorylation (Figure
4A, 4C). Because a large amount of intratumoral TNF-α has been produced at this
time point (Figure 3A, 4B), we determined whether SB202190 inhibited TNF-α
production in tumor tissues at it did in Ana-1 macrophage cells. As expected,
SB202190 significantly inhibited the induction of intratumoral TNF-α production by
4-hour DMXAA treatment (Figure 4B), indicating that TNF-α production is also
mediated by p38 MAPK. To further test the role of TNF-α production in the late-stage
(4 hours) vascular-disrupting effect of DMXAA, we used an anti-TNF-α neutralizing
antibody. The anti-TNF-α antibody, which effectively neutralized intratumoral TNF-α
(Figure 5B), partially reversed the reduction in tumor blood flow induced by
DMXAA treatment for 24 hours (Figure 5A), whereas a non-specific IgG did not,
raising the possibility that intratumoral TNF-α production plays an essential role in
maintaining the DMXAA-induced reduction in tumor blood flow. Collectively, the
results of in vivo experiments suggest that DMXAA-induced tumor vascular
disruption may be a two-step process: (1) a rapid reduction in tumor blood flow
induced by endothelial cell cytoskeleton reorganization, and (2) a sustained decrease
in blood flow maintained by intratumoral TNF-α production. In both cases, p38
MAPK is the key regulator and thus mediates DMXAA-induced disruption of the
tumor vasculature.
p38 MAPK activation is required for DMXAA-induced tumor necrosis in vivo.
Finally, we determined the fate of tumor cells after DMXAA treatment. Mice
bearing Lewis lung cancer tumors were treated with 20 mg/kg DMXAA for 24 hours,
after which tumor tissue was fixed, sectioned, and stained with hematoxylin and eosin.
The necrotic area of tumors was expressed as percentage of total area. As shown in
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Figure 6A and B, extensive necrotic areas within tumors were observed in mice
treated with DMXAA for 24 hours; in contrast, untreated mice showed large areas of
viable cells without significant necrosis. SB202190 treatment alone had no effect on
cell viability in tumor tissues; however, it remarkably decreased DMXAA-induced
tumor necrosis, demonstrating the critical role of p38 MAPK in DMXAA-induced
necrosis in vivo.
Discussion
DMXAA is a unique vascular disrupting agent with a mechanism distinct from that
of other known VDAs, such as the microtubule-disrupting agent combretastatin A4-P
and its derivatives. In this study, we explored the mechanism by which DMXAA
disrupts the tumor vasculature. We found that DMXAA induced actin cytoskeleton
reorganization in endothelial cells and TNF-α production in macrophage cells, with
both effects being mediated by p38 MAPK. More importantly, these in vitro activities
were translated into the in vivo antitumor effects of DMXAA. In this study, we
suggest for the first time that DMXAA-induced tumor vascular disruption consists of
at least two stages: an early stage (<30 min) in which tumor blood flow is rapidly
reduced due to endothelial cell cytoskeleton reorganization, and a late stage marked
by a sustained decrease in blood flow mediated by intratumoral TNF-α production.
Notably, both effects contribute to DMXAA-induced vascular disruption. Most
importantly, p38 MAPK plays an essential role in both DMXAA-induced endothelial
cell cytoskeleton reorganization and TNF-α production, and is thus a critical mediator
of the antitumor activity of DMXAA.
Endothelial cells lining tumor blood vessels are the main targets for VDAs, such as
combretastatin A4-P and its derivatives. Combretastatin A4-P induces rapid
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microtubule depolymerization, which stimulates MLC phosphorylation and thus
induces actin cytoskeleton reorganization and endothelial cell contraction, leading to a
rapid increase in vascular resistance and reduced tumor blood flow in vivo (Galbraith
SM, 2001 Jan-Feb). In the current study, we also suggest that the rapid shutdown of
tumor blood flow induced by DMXAA results from its action on the endothelial cell
cytoskeleton. This conclusion is based on several lines of evidence. First, DMXAA
induced rapid actin stress fiber formation and cytoskeleton reorganization in HUVECs
through p38 MAPK activation. Second, previous investigations of the
pharmacokinetics of DMXAA in Colon 38 tumor-bearing mice (Ching et al, 2002b)
showed that, after administration of DMXAA at a dose of 25 mg/kg, the maximum
plasma concentration of DMXAA was 530 μM, which is sufficient for DMXAA to
induce rapid actin cytoskeleton reorganization in endothelial cells. Third, the rapid
reduction in tumor flow induced by DMXAA occurred much earlier than the
intratumoral production of TNF-α, the rapid blood flow reduction was evident as early
as 30 minutes after DMXAA administration, a time at which TNF-α production could
not be detected. Thus, although we do not have direct evidence that DMXAA induces
rapid endothelial cell cytoskeleton reorganization in vivo, these observations
collectively indicate that the rapid shutdown of tumor blood flow induced by
DMXAA results from the effect of DMXAA on endothelial cell cytoskeleton, as is the
case for combretastatin A4. This may also be why DMXAA has been explored as a
VDA.
A number of early studies reported that, in vivo, DMXAA induces a prompt and
irreversible shutdown of blood flow that is not recovered by 24 hours, whereas the
disruption in blood flow induced by combretastatin A4 disruption recovers within
24 hours (Wilson et al, 2003). This suggests that, although DMXAA and other
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anti-tubulin VDAs, including combretastatin A4, share stimulation of actin
cytoskeleton reorganization and morphological change as common mechanisms for
inducing the rapid shutdown of tumor blood flow, the mechanisms underlying the
sustained reduction in blood flow are different. Our study indicated that the
DMXAA-induced sustained reduction of tumor blood flow is associated with
significant intratumoral TNF-α production. More importantly, the DMXAA-induced
sustained shutdown of tumor blood flow was blocked by inhibiting TNF-α production
using a neutralizing TNF-α antibody or by pretreating with a p38 MAPK inhibitor.
Given that TNF-α itself has been reported to destroy the tumor vasculature, blocking
blood flow and inducing hemorrhage in tumors, and thus acting as a VDA (Zhang et
al, 2009), it is not surprising that DMXAA exhibits a stronger and longer-lasting
tumor vessel-disrupting effect than combretastatin A4-P in a mouse model (Wallace et
al, 2007).
In this report, we demonstrated that p38 MAPK activation was involved in both
cytoskeleton reorganization in endothelial cells and TNF-α production in
macrophages; however, the mechanism by which p38 MAPK regulates actin
cytoskeleton reorganization and TNF-α production remains unclear. Our results
suggest that, in endothelial cells, DMXAA-induced p38 MAPK activation leads to
phosphorylation of MLC, triggering the formation of actin stress fibers. Alternatively,
p38 MAPK activation via MAPKAPK-2/3 may induce phosphorylation of the actin
polymerization modulator HSP27 to regulate actin microfilament dynamics (Guay et
al, 1997). In addition, the fact that p38 MAPK regulated both DMXAA-induced actin
cytoskeleton reorganization (data not shown) and TNF-α production in macrophages,
raises the possibility that there may be a connection between actin cytoskeleton
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reorganization and TNF-α production in macrophage cells. Indeed, it was found that
cytochalasin D pretreatment attenuated p38 MAPK activation induced by DMXAA
(data not shown).
Four p38 MAP kinase isoforms (α, β, γ, δ) have been identified, sharing about 60%
homology. p38α and p38β are ubiquitously expressed, p38γ is predominantly
expressed in skeletal muscle, whereas p38δ is found in the lung, kidney, testis,
pancreas, and small intestine (Kaminska, 2005). It has been reported that murine
macrophages express both p38α and p38δ, but only p38α mediates
lipopolysaccharide-induced inflammatory cytokine (such as TNF-α, IL-6, sTNF-RII,
and CXCL16) expression (Tang et al, 2006). Other reports also demonstrate that p38α,
but not p38β, was the major p38 isoform involved in the immune response in vivo
(Beardmore et al, 2005; O'Keefe et al, 2007). The siRNA used in our study is
designed to specifically target p38α (NM_011951). The p38α siRNA significantly
inhibited DMXAA-induced TNF-α production in murine macrophage Ana-1 cells, an
extent similar to the p38 MAPK inhibitor SB202190, which is known to inhibit both
p38α and p38β, suggesting p38α is the major p38 MAPK isoforms in mediating the
DMXAA’s pharmacological effect.
Consistently with the results reported recently (Sun et al, 2011), ERK1/2 was also
activated in Ana-1 cells after DMXAA treatment and it participated in TNF-α
production as p38 MAPK did (Fig. 2). However, the reduction of TNF-α production
induced by ERK1/2 inhibitor PD0325901 was much less than that of p38 MAPK
inhibitor SB202190 (20% vs 60%), and more importantly, ERK1/2 was not involved
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in DMXAA-induced actin cytoskeleton reorganization in HUVECs (Fig. 1).
Furthermore, in endothelial cells, the morphology change induced by DMXAA was
not reversed by pretreatment with 20 μM JNK inhibitor SP600125 (Fig 1A), and in
macrophage Ana-1 cells, DMXAA-induced TNF-α production was not affected by
SP600125 pretreatment. Thus, we believe that p38 MAPK, but not ERK1/2 or JNK, is
the key factor mediating actin cytoskeleton reorganization in tumor vascular
endothelial cells and TNF-α production in macrophages, both of which contribute to
DMXAA antitumor activity.
At present, p38 MAPK inhibitors are mainly used clinically as anti-inflammatory
agents. Because p38 MAPK plays such an essential role in regulating the antitumor
activity of DMXAA, and because p38 MAPK inhibition significantly suppresses
DMXAA-induced tumor vascular disruption and antitumor activity, we urge caution
in the clinical use of p38 MAPK-inhibiting drugs in patients being treated with
DMXAA or its derivatives, so as to avoid significantly reducing the antitumor activity
of DMXAA.
In summary, both endothelial cell actin cytoskeleton reorganization and intratumor
TNF-α production are critically involved in the DMXAA-induced shutdown of tumor
blood flow. More importantly, both effects are regulated by p38 MAPK. These
conclusions expand our knowledge about the mechanisms of DMXAA and may be of
great importance for the clinical application and further development of this agent.
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Authorship Contributions:
Participated in research design: Wu, Quan, and Lou.
Conducted experiments: Wu, Xu, and Li.
Contributed new reagents or analytic tools: Hu.
Performed data analysis: Wu, Quan, and Lou.
Wrote or contributed to the writing of the manuscript: Wu, Quan, and Lou.
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Zhao L, Marshall ES, Kelland LR, Baguley BC (2007) Evidence for the involvement
of p38 MAP kinase in the action of the vascular disrupting agent
5,6-dimethylxanthenone-4-acetic acid (DMXAA). Investigational New Drugs 25:
271-276.
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Footnotes:
This work was supported by the National Natural Science Foundation of China [Grant
90813009].
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Figure legends
Figure 1. DMXAA induces morphological changes and stimulates actin stress fiber
formation via p38 MAPK activation in endothelial cells. A and B. HUVECs were
exposed to 500 μM DMXAA for 30 minutes with or without pretreatment with 20 μM
SB202190, 20 μM PD0325901 or 20 μM SP600125 for 30 minutes. Cells were
photographed under an inverted microscope. The average cell-covered area was
quantified from four randomly sampled microscope fields using Image Pro Plus 6.0. C.
HUVECs were pretreated with vehicle, 20 μM SB202190, or 20 μM PD0325901 for
30 minutes, and then exposed to 100 μM DMXAA for 30 minutes. F-actin was
stained with FITC-phalloidin. Cells were photographed under a fluorescence
microscope. D. HUVECs were exposed to 100 μM DMXAA for the indicated periods
of time. E. HUVECs were preincubated with or without 20 μM SB202190 for 30
minutes before DMXAA exposure. Total cell lysates were analyzed by SDS-PAGE
and immunoblotted with the indicated antibodies. Data shown are representative of
three independent experiments.
Figure 2. DMXAA-induced TNF-α production in macrophages requires p38 MAPK
activation. A. Ana-1 cells were treated with 100 μM DMXAA for the indicated
periods of time. Total cell lysates were analyzed by SDS-PAGE and immunoblotted
with the indicated antibodies. B. Ana-1 cells were incubated with 100 μM DMXAA
for 4 hours with or without pretreatment with 20 μM SB202190 or 20 μM PD0325901
for 30 minutes. C. Ana-1 cells were transfected with p38-specific siRNAs or negative
control siRNAs. After 72 hours, cells were stimulated with 100 μM DMXAA for 4
hours. TNF-α in the supernatant was quantified by ELISA. Data are presented as
means ± SEMs (**p < 0.01,*p < 0.05, n=3).
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Figure 3. DMXAA induces intratumoral TNF-α production in vivo. A. Lewis lung
tumor-bearing mice were treated with DMXAA (20 mg/kg, injected intraperitoneally)
for the indicated periods of time. Tissues were homogenized, and the supernatant
obtained after centrifugation was assayed for TNF-α by ELISA. TNF-α activity in
tumor tissues is expressed as picograms per gram of tissue. Data are presented as
means ± SEMs (**p < 0.01, n=5). B. Western blot of tumor tissue extracts using
antibodies specific for phospho-p38 MAPK , total p38 MAPK and β-tubulin.
Figure 4. Inhibition of p38 MAPK attenuates both early and late-stage blood flow
reduction after DMXAA administration in vivo. A. C57 mice bearing Lewis lung
carcinomas were treated with DMXAA (20 mg/kg) for 30 minutes or 4 hours with or
without co-administration of SB202190 for 30 minutes, then were perfused in vivo
with Hoechst 33342 (800 μg/mouse). One minute later, tumor tissues were harvested
and cryosectioned. Images of tumor tissue cryosections were obtained by fluorescence
microscopy. Fluorescence intensity was quantified in eight randomly selected
microscope fields using Image Pro Plus 6.0. B. Tumor tissues were homogenized, and
TNF-α in the supernatant after centrifugation was assayed by ELISA and expressed as
picograms per gram of tissue. Data are presented as means ± SEMs (**p < 0.01, n=5).
C. C57 mice bearing Lewis lung carcinomas were treated with DMXAA (20 mg/kg)
for 4 hours with or without co-administration of SB202190. Western blot of tumor
tissue extract using antibodies specific for phospho-p38 MAPK and total p38 MAPK.
Figure 5. TNF-α maintains the decreased blood flow during late-stage
DMXAA-induced vascular disruption. A. Mice were treated with DMXAA for 24
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hours with co-administration of an anti-TNF antibody (120 μg/mouse) or IgG (120
μg/mouse), and then were perfused with Hoechst 33342. Photographs of tumor tissue
cryosections were obtained by fluorescence microscopy. Fluorescence intensity was
quantified in eight randomly selected microscope fields using Image Pro Plus 6.0. B.
Tumor tissues were homogenized, and TNF-α in the supernatant after centrifugation
was assayed by ELISA and expressed as picograms per gram of tissue. Data are
presented as means ± SEMs (*p < 0.05, n=5).
Figure 6. Inhibition of p38 MAPK attenuates DMXAA-induced tumor tissue necrosis.
Lewis lung cancer tumors were implanted in C57 mice and allowed to grow to 10 mm
in diameter. The mice were then treated with DMXAA at a dose of 20 mg/kg. Tumors
were removed after 24 hours, and tumor tissue was fixed, sectioned, and stained with
hematoxylin and eosin. A. Images were photographed under an inverted microscope.
B. The average necrotic area, expressed as percentage of total area, was quantified
from 7 randomly sampled microscope fields using Image Pro Plus 6.0 (**p < 0.01).
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Figure 1
A
B
+DMSO +SB202190 +PD0325901 +SP600125
Vehicle
DMXAA
0
10
20
30
40
50ControlDMXAA
SB202190+DMXAA
PD0325901+DMXAA
50 15 30 60
Time (min)
Cel
l are
a (%
)
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Figure 1
C
D
E
+DMSO +SB202190 +PD0325901
Vehicle
DMXAA
DMXAA 0 5 15 30 60 min
p-p38
p-MLC
p-ERK
p38
β-tubulin
SB202190 - + - +DMXAA - - + +
p-p38
p-MLC
β-tubulin
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Figure 2
A
B
DMXAA 0 30 60 120 240 min
p-p38
p38
p-MAPKAPK2
p-ERK
ERK
β-tubulin
0
50
100
150
200
250
300
DMXAA - + + +SB202190 PD0325901
**
*
TN
F-α
(pg/
ml)
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Figure 2
C Con NC p38 siRNA
p38
β-tubulin
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Figure 3
A
B
p-p38
p38
DMXAA (h) 0 0.5 4 12
0
500
1000
1500
2000
2500
3000
3500
Time (h)0 0.5 2 4 12
*
*
*
*T
NF
-α
(pg/
g)
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Figure 4
0
20
40
60
80
100
120
DMXAA - + + -SB202190 - - + +
**
Tum
or b
lood
per
fusi
on(%
)
0
20
40
60
80
100
120
DMXAA - + + -SB202190 - - + +
**
Tum
or b
lood
per
fusi
on(%
)
A30 min 4 h
+Vehicle
Vehicle
+SB202190
DMXAA
+Vehicle
Vehicle
+SB202190
DMXAA
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Figure 4
B
C
30 min 4 h
0
500
1000
1500
2000
2500
3000
3500
DMXAA - - + - SB202190 - + - +
TN
F-α
(pg/
g)
0
500
1000
1500
2000
2500
3000
3500
DMXAA - - + + SB202190 - + - +
**
TN
F-
α
(pg/
g)
DMXAA - - + +SB202190 - + - +
p-p38
p38
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Figure 5
A
B
Vehicle DMXAA
DMXAA+IgG DMXAA+anti-TNF
0
500
1000
1500
2000
2500
3000
3500
DMXAA - + + +anti-TNF IgG
*
TN
F-α
(pg/
g)
0
20
40
60
80
100
120
DMXAA - + + +anti-TNF IgG
*
Tum
or b
lood
per
fusi
on (%
)
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A
B
20 μm
+Vehicle
Vehicle
+SB202190
DMXAA
0
5
10
15
20
DMXAA - - + + SB202190 - + - +
**
Nec
roti
c ar
ea (
%)
Figure 6
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