p38 mapk is required for the antitumor activity of...

JPET # 191635

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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.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 13, 2012 as DOI: 10.1124/jpet.112.191635

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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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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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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

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DMXAA - + + +anti-TNF IgG

*

Tum

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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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