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Journal ArticleDOI

Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis

01 Apr 2009-The FASEB Journal (Federation of American Societies for Experimental Biology)-Vol. 23, Iss: 4, pp 1127-1137

TL;DR: It is concluded that PKCα might be an important target for the antiangiogenic effects of tubulin antagonism, and the data from the kinase array suggest that different tubulin antagonists might have individual intracellular actions.
Abstract: The aims of the study were to meet the demand of new tubulin antagonists with fewer side effects by characterizing the antiangiogenic properties of the experimental compound spongistatin 1, and to elucidate nonmitotic mechanisms by which tubulin antagonists inhibit angiogenesis. Although tubulin-inhibiting drugs and their antiangiogenic properties have been investigated for a long time, surprisingly little is known about their underlying mechanisms of action. Antiangiogenic effects of spongistatin 1 were investigated in endothelial cells in vitro, including functional cell-based assays, live-cell imaging, and a kinome array, and in the mouse cornea pocket assay in vivo. Spongistatin 1 inhibited angiogenesis at nanomolar concentrations (IC50: cytotoxicity>50 nM, proliferation 100 pM, migration 1.0 nM, tube formation 1.0 nM, chemotaxis 1.0 nM, aortic ring sprouting 500 pM, neovascularization in vivo 10 μg/kg). Further, a kinome array and validating data showed that spongistatin 1 inhibits the phosphorylatio...
Topics: Tube formation (52%), Angiogenesis (50%)

Summary (4 min read)

Introduction

  • Angiogenesis microtubules chemotaxis protein kinase C Because tumors can grow only to a size of 1–2 mm3 without being supplied with oxygen and nutrients by blood vessels, the inhibition of angiogenesis has gained clinical relevance in cancer therapy (1), also known as Key Words.
  • Thus, the application of cell-cycle-inhibiting compounds such as tubulin antagonists is promising in antiangiogenic therapy.
  • Thus, the determination of the functions of interphase microtubules in endothelial cells, especially those involved in angiogenic processes, is essential to conceive the mechanisms of microtubule inhibitors in angiogenesis.
  • In the anticancer test panel of the U.S. National Cancer Institute (NCI), spongistatin 1 has shown very strong effects on 20 cancer cell 1 Correspondence: Department of Pharmacy, Center for Drug Research, University of Munich, Butenandtstr.
  • E-mail: stefan.zahler@cup.unimuenchen.de doi: 10.1096/fj.08-117127 11270892-6638/09/0023-1127 © FASEB lines (15).

Compounds

  • Spongistatin 1 and CA4P were provided by George R. Pettit (Cancer Research Institute, Tempe, AZ, USA).
  • Paclitaxel, propidium iodide, and phorbol 12-myristate 13-acetate were purchased from Sigma (Taufkirchen, Germany), vinblastine from Hexal (Holzkirchen, Germany), nocodazole from AppliChem GmbH (Darmstadt, Germany), and staurosporine from Cayman Chemical (Ann Arbor, MI, USA).

Cell culture and media

  • Primary human umbilical vein endothelial cells were isolated by collagenase treatment of umbilical cords (17) and used at third passage.
  • Cells were cultivated on 0.001% collagen G in endothelial growth medium (Provitro, Berlin, Germany), containing 10% inactivated fetal calf serum (FCS) and growth factors (basic fibroblast growth factor 1.0 ng/ml, Heparin 0.004 ml/ml, and epidermal growth factor 0.1 ng/ml).
  • M199 medium (PAN Biotech, Aidenbach, Germany) was used as starvation medium.

Live-cell imaging

  • HUVECs were transfected with 7 g pEGFP-C1-tubulin (kindly supplied by Stefan Linder, Institute for Cardiovascular Diseases, University of Munich, Munich, Germany), using the Nucleofector II and the HUVEC Nucleofector Kit (Amaxa, Cologne, Germany).
  • At days 2 and 3 after transfection, cells were used for live-cell imaging (customized cell observation chamber; Emblem, Heidelberg, Germany; and confocal microscope (LSM 510 Meta; Zeiss, Jena, Germany).

Tubulin fractionation

  • The supernatants were collected (fraction of soluble tubulin).
  • After centrifugation (10,000 g for 10 min at 4°C), the supernatant contained the fraction of PIPES-insoluble tubulin.
  • Both fractions were prepared in 5 sodium dodecyl sulfate (SDS) sample buffer [312.5 mM Tris/HCl, pH 6.8; 50% glycerol; 5% SDS; 2% dithiothreitol (DTT); 0.025% Pyronin Y 5] for immunoblotting.
  • The anti-tubulin antibody (D-10) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the anti- -actin antibody (MAB1501R) from Chemicon International (Hofheim, Germany).

Immunoblotting

  • Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Eschborn, Germany) via tank blotting.
  • Protein levels were detected either by the ECLTM detection system (Amersham Pharmacia Biotech, Little Chalfont, UK), or by the Odyssey Infrared Imaging System version 2.1 (LI-COR Biosciences, Lincoln, NE, USA).

Cell cycle analysis

  • After stimulation, cell culture supernatants containing apoptotic cells were collected.
  • After another wash with PBS, cells were resuspended in hypotonic fluorochrome solution (HFS) (0.1% Na3-Citrat, 0.1% Triton X-100, and 50 g/ml propidium iodide).
  • Cell cycle phases were analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, Heidelberg, Germany).
  • Cell viability in HUVECs was measured by determination of the metabolical reduction of resazurin to resorufin, using the CellTiter-BlueTM assay (Promega, Madison, WI, USA).
  • Here 5000 and 15,000 HUVECs/well of a 96-well plate were seeded and treated with increasing concentrations of spongistatin 1 for 24 h.

Proliferation assay

  • The proliferation assay was performed according to the NCI protocols for angiogenesis.
  • Briefly, 1500 HUVECs/well were seeded into 96-well plates.
  • Then HUVECs were treated with increasing concentrations of spongistatin 1, vinblastine, CA4P, or paclitaxel for 72 h.
  • After stimulation, cells were stained with crystal violet solution (0.5% crystal violet in 20% methanol) for 10 min.
  • Next, crystal violet, which mainly binds to DNA, was eluted from cells with 0.1 M sodium citrate in 50% ethanol.

Migration scratch assay

  • HUVECs were seeded into 24-well plates and grown to confluency.
  • The wells were washed with PBS to remove detached cells and incubated for 16 h at 37°C in either starvation medium (no migration), culture medium (100% migration), or culture medium containing increasing concentrations of spongistatin 1, vinblastine, CA4P, or paclitaxel.
  • One image per well (center position) was taken on an inverted light microscope (Axiovert 200; Zeiss, Jena, Germany) using the Imago-QE camera system and the appending software (Till Photonics, Graefelfing, Germany).
  • For quantification, these images were analyzed with the S.CORE imaging analysis tool (S.CO LifeScience, Munich, Germany).
  • The cell-free area correlates to the ability of the HUVECs to migrate into the scratch.

Immunocytochemistry

  • HUVECs were seeded on 0.1% collagen A-coated glass coverslips, washed, fixed in 4% formaldehyde for 15 min, and permeabilized with 0.2% Triton X-100 in PBS.
  • F-actin was stained by incubating cells for 45 min with rhodamineconjugated phalloidin (Invitrogen, Eugene, OR, USA) diluted 1:400 in PBS containing 1% BSA.
  • Visualization and imaging were done on a confocal microscope (LSM 510 Meta; Zeiss).

Chemotaxis assay

  • Chemotaxis of spongistatin 1-treated HUVECs was investigated by -slide chemotaxis (IBIDI, Martinsried, Germany).
  • The chemotaxis assay was performed as described in the manual, “ -Slide Chemotaxis” (http://www.ibidi.de/applications/ap_chemotaxis.html).
  • Two opposing medium reservoirs were connected to the seeding chamber by a thin slit.
  • One of these reservoirs was filled with starvation medium without FCS; the other was filled with HUVEC culture medium containing 10% FCS, generating by diffusion a linear and stable ( 48 h) FCS gradient from 0 to 10% FCS in the seeding chamber of the -slide.
  • Cell movement was observed every 10 min over 24 h by live cell imaging in a cell observation chamber mounted to a confocal microscope (LSM 510 Meta; Zeiss).

Tube formation assay

  • MatrigelTM (BD Discovery Labware, Bedford, MA, USA) was placed into the lower chambers of -slide angiogenesis wells and hardened for 30 min at 37°C.
  • The images were analyzed with the tube formation module of S.CORE (S.CO LifeScience).
  • Aortic rings were incubated for 2 days in HUVEC culture medium before stimulation with spongistatin 1.
  • The controls received vehicle treatment only (n 5).
  • The length of new vessels in the cornea was measured from the inside margin of vessels around the limbus to the tip of the longest neovascular sprout.

Kinome chip analysis (PepChip)

  • PepChip performance and analysis of the results were done as described previously (20, 21) by Pepscan Presto BV (Lelystad, The Netherlands; for detailed protocol see http://www. pepscanpresto.com/files/PepChip%20Kinase%20Lysate% 20Protocol_v5.pdf).
  • On PepChip, 1152 different peptides with specific phosphorylation motifs for upstream kinases were spotted in triplicates.
  • Confluent HUVECs in 100 mm dishes were treated for 30 min with spongistatin 1 (5.0 nM), vinblastine (10 nM), or CA4P (20 nM).
  • Afterward, chips 1129THE TUBULIN ANTAGONIST SPONGISTATIN INHIBITS PKC were rinsed 3 times with distilled water and then air-dried.
  • The phosphorylation status of the chips with untreated or treated cells (spongistatin 1, vinblastine, or CA4P) was compared spot by spot.

PKC activity: (ser)-substrate phosphorylation

  • The supernatants were prepared in 5 SDS sample buffer for immunoblotting.
  • Phospho-(Ser) PKC substrate antibody was purchased from Cell Signaling Technology (CST, Frankfurt, Germany).
  • PKC in vitro activity assay Potential direct inhibition of PKC isozymes in vitro by spongistatin 1 was assessed using a micellar-based assay.
  • The samples were incubated for 10 min at 30°C and subsequently loaded onto phosphocellulose filter disks (Whatman, Dassel, Germany).
  • Then 2 ml of scintillation fluid (Ultima Gold; PerkinElmer) was added to each filter disk, and radioactivity was counted with a liquid scintillation counter.

PKC translocation

  • Briefly, HUVECs were preincubated with either 5.0 nM spongistatin 1 or 10.0 M nocodazole before stimulation with 10 nM phorbol 12-myristate 13-acetate (PMA) or stimulated with 10 nM PMA alone, washed twice with ice-cold PBS, and homogenized in lysing buffer (50 mM Tris/HCl, pH 7.5; 0.5 mM EDTA; 0.5 mm EGTA; 2.0 mM DTT; 7.0 mM glutathione; 10% glycerol; 1.0 mm PMSF; and complete medium).
  • The sediment was washed in lysing buffer containing 1.0 M NaCl and centrifuged at 100,000 g for 30 min.
  • The supernatant was discarded, and the pellet was solubilized with lysing buffer containing 20 mM 3-[(3-cholamidopropyl) diethylammonio]-1 propane sulfonate at 4°C for 30 min.
  • Both fractions were used for Western blotting.
  • Antibodies against PKC , PKC , and PKCε were purchased from Santa-Cruz, the anti-VE-Cadherin antibody from Cell Signaling Technology.

Statistical analysis

  • Data were expressed as means se, and analyzed using 1-way ANOVA, Student’s t test, or rank-sum test.
  • Values of P 0.05 were considered statistically significant.

RESULTS

  • To test whether spongistatin 1 causes microtubule disassembly in endothelial cells, 2 different approaches were pursued.
  • In migrating HUVECs (culture medium), F-actin is arranged in stress fibers within the cell body and lamellopodia at the leading edge.
  • Spongistatin 1 suppresses neovascularization of the mouse cornea Antiangiogenic properties of spongistatin 1 in vivo were determined in the mouse corneal micropocket assay.
  • To validate the PepChip data of reduced PKC activity, Western blot analysis of phosphorylated serine residues of PKC substrates was performed.
  • Spongistatin 1 inhibits the translocation of PKC to the membrane Because most of the PKC isoforms have to be translocated from the cytoplasm to membranes to become activated and translocation processes can depend on microtubules, the tubulin antagonist spongistatin 1 may influence the activity of specific PKC isoforms via inhibition of their transport to membranes.

DISCUSSION

  • In addition to classical cytostatic chemotherapy, antiangiogenic therapy has become a very important approach in cancer treatment.
  • Continuing investigations have shown that spongistatin 1 does not affect PKC activity by direct inhibition.
  • This is the first time that microtubule-dependent PKC translocation is shown in endothelial cells.
  • Further, it is known that VEGF-induced proliferation requires PKC (35), and it has been shown that PMA-induced angiogenesis depends on PKC activity (36).
  • The kinase inhibition profile of the different tubulin antagonists exhibits common features as well as differences between the single compounds, indicating that effects of microtubule-inhibiting drugs could be compound specific.

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The FASEB Journal Research Communication
Investigation of the marine compound spongistatin 1
links the inhibition of PKC translocation to
nonmitotic effects of tubulin antagonism
in angiogenesis
Andrea S. Rothmeier,* Ivan Ischenko,
Jos Joore,
Dorota Garczarczyk,
§
Robert Fu¨rst,*
Christiane J. Bruns,
Angelika M. Vollmar,* and Stefan Zahler*
,1
*Department of Pharmacy, Center for Drug Research, and
Department of Surgery, Klinikum
Großhadern, University of Munich, Munich, Germany;
Pepscan Systems BV, Lelystad, The
Netherlands; and
§
Division of Medical Biochemistry, Innsbruck Medical University,
Innsbruck, Austria
ABSTRACT The aims of the study were to meet the
demand of new tubulin antagonists with fewer side
effects by characterizing the antiangiogenic properties
of the experimental compound spongistatin 1, and to
elucidate nonmitotic mechanisms by which tubulin an-
tagonists inhibit angiogenesis. Although tubulin-inhibit-
ing drugs and their antiangiogenic properties have been
investigated for a long time, surprisingly little is known
about their underlying mechanisms of action. Antian-
giogenic effects of spongistatin 1 were investigated in
endothelial cells in vitro, including functional cell-based
assays, live-cell imaging, and a kinome array, and in the
mouse cornea pocket assay in vivo. Spongistatin 1
inhibited angiogenesis at nanomolar concentrations
(IC
50
: cytotoxicity>50 nM, proliferation 100 pM, mi-
gration 1.0 nM, tube formation 1.0 nM, chemotaxis 1.0
nM, aortic ring sprouting 500 pM, neovascularization
in vivo 10 g/kg). Further, a kinome array and validat-
ing data showed that spongistatin 1 inhibits the phos-
phorylation activity of protein kinase C (PKC), an
essential kinase in angiogenesis, and its translocation to
the membrane. Thus, we conclude that PKC might be
an important target for the antiangiogenic effects of
tubulin antagonism. In addition, the data from the kinase
array suggest that different tubulin antagonists might have
individual intracellular actions.—Rothmeier, A. S., Isch-
enko, I., Joore, J., Garczarczyk, D., Fu¨rst, R., Bruns, C. J.,
Vollmar, A. M., Zahler, S. Investigation of the marine
compound spongistatin 1 links the inhibition of PKC
translocation to nonmitotic effects of tubulin antagonism
in angiogenesis. FASEB J. 23, 1127–1137 (2009)
Key Words: angiogenesis microtubules chemotaxis protein
kinase C
Because tumors can grow only to a size of 1–2 mm
3
without being supplied with oxygen and nutrients by
blood vessels, the inhibition of angiogenesis has gained
clinical relevance in cancer therapy (1). The formation
of new vessels requires multiple endothelial cell division
cycles. Thus, the application of cell-cycle-inhibiting com-
pounds such as tubulin antagonists is promising in anti-
angiogenic therapy. Microtubule-inhibiting drugs turned
out to belong to the most potent antiangiogenic com-
pounds (2). Established tubulin antagonists such as pac-
litaxel or vinblastine have become part of clinical ap-
proaches for antiangiogenic standard chemotherapy.
Furthermore, promising new substances such as combret-
astatin A4 phosphate (CA4P) enter the scene, indicating
the persisting requirement for new and better com-
pounds with stronger effects and less toxicity (3–5).
Interestingly, it was found that interphase microtubules
of endothelial cells are particularly sensitive to tubulin
antagonists (6, 7). Therefore, nonmitotic effects of these
drugs seem to be very important in their antiangiogenic
action, in contrast to mitotic effects in classical cancer
therapy. Thus, the determination of the functions of
interphase microtubules in endothelial cells, especially
those involved in angiogenic processes, is essential to
conceive the mechanisms of microtubule inhibitors in
angiogenesis. Astonishingly, the characterization of the
role of interphase microtubules in angiogenesis and the
elucidation of antiangiogenic mechanisms of tubulin an-
tagonists has only just begun (8 –11).
In the present study, we investigated the antiangio-
genic potential of spongistatin 1 in vitro and in vivo.
Spongistatin 1 is a macrocyclic lacton polyether,
isolated from marine sponges (12, 13). It facilitates
the disassembly of microtubules by binding to a
specific site on -tubulin, which is just overlapping
the Vinca domain (14). In the anticancer test panel of
the U.S. National Cancer Institute (NCI), spongista-
tin 1 has shown very strong effects on 20 cancer cell
1
Correspondence: Department of Pharmacy, Center for
Drug Research, University of Munich, Butenandtstr. 5-13,
81377 Munich, Germany. E-mail: stefan.zahler@cup.uni-
muenchen.de
doi: 10.1096/fj.08-117127
11270892-6638/09/0023-1127 © FASEB

lines (15). Our own recent studies demonstrated
spongistatin 1 to be highly effective against a chemo-
therapeutic-resistant leukemia cell line without af-
fecting peripheral mononuclear blood cells, which
indicates the therapeutic aptitudes of spongistatin 1
(16). The uniqueness of its binding site on -tubulin
also gives reason to speculate that spongistatin 1 may
differ in its mode of action from other established
tubulin antagonitsts. Therefore, the investigation of
antiangiogenic properties of spongistatin 1 in com-
parison to already known drugs seemed to be most
promising.
In the present study we demonstrate strong antian-
giogenic effects of sponistatin 1 in vitro and in vivo.
These seem to be due to nonmitotic effects of
spongistatin 1 and hint toward an as yet unknown
mechanism of tubulin antagonism during angiogene-
sis: the inhibition of protein kinase C (PKC) trans-
location.
MATERIALS AND METHODS
Compounds
Spongistatin 1 and CA4P were provided by George R. Pettit
(Cancer Research Institute, Tempe, AZ, USA). Paclitaxel,
propidium iodide, and phorbol 12-myristate 13-acetate were
purchased from Sigma (Taufkirchen, Germany), vinblastine
from Hexal (Holzkirchen, Germany), nocodazole from Ap-
pliChem GmbH (Darmstadt, Germany), and staurosporine
from Cayman Chemical (Ann Arbor, MI, USA).
Cell culture and media
Primary human umbilical vein endothelial cells (HUVECs)
were isolated by collagenase treatment of umbilical cords
(17) and used at third passage. Cells were cultivated on
0.001% collagen G in endothelial growth medium (Provitro,
Berlin, Germany), containing 10% inactivated fetal calf se-
rum (FCS) and growth factors (basic fibroblast growth factor
1.0 ng/ml, Heparin 0.004 ml/ml, and epidermal growth
factor 0.1 ng/ml). M199 medium (PAN Biotech, Aidenbach,
Germany) was used as starvation medium.
Live-cell imaging
HUVECs were transfected with 7 g pEGFP-C1-tubulin
(kindly supplied by Stefan Linder, Institute for Cardiovascu-
lar Diseases, University of Munich, Munich, Germany), using
the Nucleofector II and the HUVEC Nucleofector Kit
(Amaxa, Cologne, Germany). At days 2 and 3 after transfec-
tion, cells were used for live-cell imaging (customized cell
observation chamber; Emblem, Heidelberg, Germany; and
confocal microscope (LSM 510 Meta; Zeiss, Jena, Germany).
Tubulin fractionation
HUVECs were lysed 20 min in 1,4-piperazinebis(ethane sul-
fonic acid) (PIPES) buffer [0.1 M PIPES, pH 6.9; 2.0 M
glycerol; 0.5% Triton X-100; 2 mM MgCl
2
; 2 mM K-EGTA salt;
5 M paclitaxel; 1 mM GTP; 1 mM PMSF; and complete
®
medium (Roche, Penzberg, Germany)]. Lysates were centri
-
fuged 45 min at 100,000 g, 4°C. The supernatants were
collected (fraction of soluble tubulin). The sediments were
incubated1hat4°Cwith Ca
2
Cl buffer (0.1 M Tris/HCl, pH
6.8; 1 mM MgCl
2
; 10 mM CaCl
2
; 1 mM PMSF; and complete
medium). After centrifugation (10,000 g for 10 min at 4°C),
the supernatant contained the fraction of PIPES-insoluble
tubulin. Both fractions were prepared in 5 sodium dodecyl
sulfate (SDS) sample buffer [312.5 mM Tris/HCl, pH 6.8;
50% glycerol; 5% SDS; 2% dithiothreitol (DTT); 0.025%
Pyronin Y 5] for immunoblotting. The anti-tubulin antibody
(D-10) was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA), the anti--actin antibody (MAB1501R) from
Chemicon International (Hofheim, Germany).
Immunoblotting
Proteins were separated by SDS-PAGE and transferred to
polyvinylidene difluoride membranes (Immobilon-P; Milli-
pore, Eschborn, Germany) via tank blotting. Protein levels
were detected either by the ECL
TM
detection system (Amer
-
sham Pharmacia Biotech, Little Chalfont, UK), or by the
Odyssey Infrared Imaging System version 2.1 (LI-COR Bio-
sciences, Lincoln, NE, USA).
Cell cycle analysis
Proliferating HUVECs (75,000 cells/well in a 12-well plate)
were treated for 24 h with increasing concentrations of
spongistatin 1, vinblastine, CA4P, or paclitaxel. After stimula-
tion, cell culture supernatants containing apoptotic cells were
collected. Cells were washed with PBS and harvested by
trypsine/EDTA treatment, resuspended in the cell culture
supernatant, and centrifuged at 600 g for 10 min at 4°C. After
another wash with PBS, cells were resuspended in hypotonic
fluorochrome solution (HFS) (0.1% Na
3
-Citrat, 0.1% Triton
X-100, and 50 g/ml propidium iodide). Cell cycle phases
were analyzed by flow cytometry on a FACSCalibur (Becton
Dickinson, Heidelberg, Germany). The intercalation of pro-
pidium iodide into DNA generates different fluorescence-
intensity peaks, depending on the DNA content of the cell.
Thus, cell cycle phases as well as apoptotic cells can be
determined (18).
Cell viability assay (relative metabolic activity)
Cell viability in HUVECs was measured by determination of
the metabolical reduction of resazurin to resorufin, using the
CellTiter-Blue
TM
assay (Promega, Madison, WI, USA). Here
5000 (proliferating) and 15,000 (confluent) HUVECs/well of
a 96-well plate were seeded and treated with increasing
concentrations of spongistatin 1 for 24 h. Also, 20 lof
Cell-Titer-Blue reagent was added to each well, and cells were
incubated for4hat37°C. The reduction of resazurin was
determined by recording fluorescence at 560 and 590 nm in
the SpectraFluor Plus plate reader (Tecan Trading AG,
Maennedorf, Switzerland) and by calculating the ratio of
fluorescence at 560/590 nm.
Proliferation assay
The proliferation assay was performed according to the NCI
protocols for angiogenesis. Briefly, 1500 HUVECs/well were
seeded into 96-well plates. The next day, control cells were fixed
and stained with crystal violet (see below) to determine the
initial cell number. Then HUVECs were treated with increasing
concentrations of spongistatin 1, vinblastine, CA4P, or paclitaxel
for 72 h. After stimulation, cells were stained with crystal violet
solution (0.5% crystal violet in 20% methanol) for 10 min.
1128 Vol. 23 April 2009 ROTHMEIER ET AL.The FASEB Journal

Unbound crystal violet was removed by rinsing with distilled
water, and cells were subsequently air dried. Next, crystal violet,
which mainly binds to DNA, was eluted from cells with 0.1 M
sodium citrate in 50% ethanol. The absorbance of crystal violet
is proportional to the cell number and was determined with the
Sunrise ELISA reader (Tecan Trading AG) at 540 nm.
Migration scratch assay
HUVECs were seeded into 24-well plates and grown to
confluency. A wound of 1 mm was inflicted to HUVEC
monolayers by scratching them with a pipette tip. The wells
were washed with PBS to remove detached cells and incu-
bated for 16 h at 37°C in either starvation medium (no
migration), culture medium (100% migration), or culture
medium containing increasing concentrations of spongistatin
1, vinblastine, CA4P, or paclitaxel. After incubation, cells
were washed with PBS and fixed in 4% formaldehyde. One
image per well (center position) was taken on an inverted
light microscope (Axiovert 200; Zeiss, Jena, Germany) using
the Imago-QE camera system and the appending software
(Till Photonics, Graefelfing, Germany). For quantification,
these images were analyzed with the S.CORE imaging analysis
tool (S.CO LifeScience, Munich, Germany). This software
tool is able to identify the cell-covered area by using an
algorithm based on brightness and contrast values as well as
morphological information. The cell-free area correlates to
the ability of the HUVECs to migrate into the scratch.
Immunocytochemistry
HUVECs were seeded on 0.1% collagen A-coated glass cover-
slips, washed, fixed in 4% formaldehyde for 15 min, and
permeabilized with 0.2% Triton X-100 in PBS. F-actin was
stained by incubating cells for 45 min with rhodamine-
conjugated phalloidin (Invitrogen, Eugene, OR, USA) di-
luted 1:400 in PBS containing 1% BSA. Visualization and
imaging were done on a confocal microscope (LSM 510 Meta;
Zeiss).
Chemotaxis assay
Chemotaxis of spongistatin 1-treated HUVECs was investi-
gated by -slide chemotaxis (IBIDI, Martinsried, Germany).
The chemotaxis assay was performed as described in the
manual, -Slide Chemotaxis” (http://www.ibidi.de/applica-
tions/ap_chemotaxis.html). Briefly, a HUVEC suspension
(7 l) of 5 10
6
cells/ml was seeded into the center chamber
of a -slide. Two opposing medium reservoirs were connected
to the seeding chamber by a thin slit. One of these reservoirs
was filled with starvation medium without FCS; the other was
filled with HUVEC culture medium containing 10% FCS,
generating by diffusion a linear and stable (48 h) FCS
gradient from 0 to 10% FCS in the seeding chamber of the
-slide. Cell movement was observed every 10 min over 24 h
by live cell imaging in a cell observation chamber (Emblem)
mounted to a confocal microscope (LSM 510 Meta; Zeiss).
Cell tracking and analysis were done using the manual
tracking plug-in (Fabrice Cordelieres, Orsay, France) and the
chemotaxis and migration tool (IBIDI) for ImageJ (U.S.
National Institutes of Health, Bethesda, MD, USA), as de-
scribed in the -slide chemotaxis protocol. The position of
cells was defined as a point at low magnification (10 lens).
Tube formation assay
Matrigel
TM
(BD Discovery Labware, Bedford, MA, USA) was
placed into the lower chambers of -slide angiogenesis wells
(IBIDI) and hardened for 30 min at 37°C. Then 12,000
HUVECs/well were seeded on the Matrigel and treated
overnight with 1.0 nM spongistatin 1, 5.0 nM vinblastine, 10.0
nM CA4P, or 50 nM pacliatxel. Images were taken on the
Axiovert 200 microscope (Zeiss) with the Imago-QE camera
system and the appending software (Till Photonics). The
images were analyzed with the tube formation module of
S.CORE (S.CO LifeScience). This software module identifies
cellular tubes on a multiparametric basis (depending on
brightness or contrast differences, length, and width of the
cell structure) and nodes of tubes (depending on a math-
ematic algorithm that interprets tubes and nontube cell
complexes).
Mouse aortic ring assay
Abdominal arteries of NMRI mice (Charles River Laborato-
ries, Sulzfeld, Germany) were prepared, cut in rings, and set
on Matrigel. Aortic rings were incubated for 2 days in HUVEC
culture medium before stimulation with spongistatin 1. En-
dothelial cell sprouting was documented 2 days after all
untreated rings had sprouted.
Mouse corneal micropocket assay
Male C57BL6 mice were purchased from Charles River Lab-
oratories, and micropockets were generated as described
(19). Spongistatin 1 was administered intraperitoneally at a
dose of 10 g/kg (dissolved in 2% dimethyl sulfoxide and
isotonic saline) daily for 5 days, beginning from the first
postoperative day (n5). The controls received vehicle treat-
ment only (n5). The maximal vessel length and clock hours
of circumferential neovascularization were measured on the
sixth day after corneal implantation. The length of new
vessels in the cornea was measured from the inside margin of
vessels around the limbus to the tip of the longest neovascular
sprout. The contiguous circumferential zone of neovascular-
ization was measured as clock hours (length of the limbal
vessel showing sprouts) with a 360° reticule (where 30° of arc
equals 1 clock hour, and 1 clock hour further corresponds to
1 mm limbal vessel length).
Kinome chip analysis (PepChip)
PepChip performance and analysis of the results were done as
described previously (20, 21) by Pepscan Presto BV (Lelystad,
The Netherlands; for detailed protocol see http://www.
pepscanpresto.com/files/PepChip%20Kinase%20Lysate%
20Protocol_v5.pdf). On PepChip, 1152 different peptides
with specific phosphorylation motifs for upstream kinases
were spotted in triplicates. Confluent HUVECs in 100 mm
dishes were treated for 30 min with spongistatin 1 (5.0 nM),
vinblastine (10 nM), or CA4P (20 nM). Native protein lysates
of these cells were generated by lysing cells with M-PER
Mammalian Protein Extraction Reagent (Pierce, Rockford,
IL, USA), containing 2.5 mM Na
4
-pyrophosphate, 2 mM
Na
2
--glycerophosphate, 1 mM Na
3
VO
4
, and 1 mM NaF.
Lysates were centrifuged 10 min at 13,000 rpm, 4°C, and
supernatants were frozen immediately in liquid nitrogen.
Here 70 l lysates/array was mixed with 10 l activation
solution (20 Ci -
33
ATP, 50% glycerol, 5 mM DTT, 50 mM
MgCl
2
, 50 mM MnCl
2
, 250 g/ml PEG 8000, and 250 g/ml
BSA) and centrifuged 5 min at 14,000 g. Next, 70 lofthe
supernatant was loaded onto the array and incubated for 2 h
at 37°C in saturated humidity. Chips were washed in 2 cycles,
first 5 min in 2 M NaCl containing 1% Triton X-100, followed
by 5 min in PBS containing 1% Triton X-100. Afterward, chips
1129THE TUBULIN ANTAGONIST SPONGISTATIN INHIBITS PKC

were rinsed 3 times with distilled water and then air-dried.
Phosphor-storage screens were exposed to the chip for 24 h to
determine and to quantify the phosphorylation status of
peptides (i.e., kinase substrates), which gave information
about the activity of the associated upstream kinase. The
phosphorylation status of the chips with untreated or treated
cells (spongistatin 1, vinblastine, or CA4P) was compared spot
by spot. The results were ranked by extent of inhibition of
phosphorylation. The kinases inhibited most according to
this score are shown in Table 1 for each tubulin antagonist
tested.
PKC activity: (ser)-substrate phosphorylation
HUVECs were treated with 5.0 nM spongistatin 1 for 30 min,
lysed in radioimmunoprecipitation assay buffer (150 mM
NaCl, 50 mM Tris/HCl, 1% Nonidet-P40, 0.25% Deoxycholat,
0.1% SDS, 1 mM PMSF, 1 mM NaF, 1 mM Na
3
VO
4
, and
complete medium), and centrifuged at 10,000 g, 10 min, 4°C.
The supernatants were prepared in 5 SDS sample buffer for
immunoblotting. Phospho-(Ser) PKC substrate antibody was
purchased from Cell Signaling Technology (CST, Frankfurt,
Germany).
PKC in vitro activity assay
Potential direct inhibition of PKC isozymes in vitro by
spongistatin 1 was assessed using a micellar-based assay. Here,
150 ng of recombinant glutathione S-transferase-tagged PKC
isozyme , I, II, , ε,or (kindly provided by Dr. M.
Kubbutat, ProQinase Ltd., Freiburg, Germany) was incubated
in a buffer containing 20 mM Tris-HCl, pH 7.5, and 20 mM
MgCl
2
, with 50 M PKC--19–31/Ser-25 substrate peptide
(NeoMPS, Strasbourg, France), 1 mM CaCl
2
,10M phospha
-
tidylserine (Sigma), 1 M 12-O-tetradecanoylphorbol-13-ace-
tate (Sigma), 40 M ATP, and 1 Ci -
33
ATP (PerkinElmer,
Fremont, CA, USA; 3000 Ci/mM) per 100 l as described
previously (22). In the assays for PKC and ε CaCl
2
, and for
PKC CaCl
2
, phosphatidylserine and 12-O-tetradecanoylphor
-
bol-13-acetate were omitted. Samples containing 100 nM of
spongistatin 1 were compared to controls. The samples were
incubated for 10 min at 30°C and subsequently loaded onto
phosphocellulose filter disks (Whatman, Dassel, Germany).
The membranes were washed 3 times with 1.5% phosphoric
acid and twice with distilled water. Then 2 ml of scintillation
fluid (Ultima Gold; PerkinElmer) was added to each filter
disk, and radioactivity was counted with a liquid scintillation
counter.
PKC translocation
HUVEC lysates were separated into a cytosolic and a mem-
branous fraction, as described previously by Li et al. (23).
Briefly, HUVECs were preincubated with either 5.0 nM
spongistatin 1 or 10.0 M nocodazole before stimulation with
10 nM phorbol 12-myristate 13-acetate (PMA) or stimulated
with 10 nM PMA alone, washed twice with ice-cold PBS, and
homogenized in lysing buffer (50 mM Tris/HCl, pH 7.5; 0.5
mM EDTA; 0.5 mm EGTA; 2.0 mM DTT; 7.0 mM glutathione;
10% glycerol; 1.0 mm PMSF; and complete medium). Lysates
were centrifuged at 100,000 g for 1 h. The supernatant
(cytosolic fraction) was collected. The sediment was washed
in lysing buffer containing 1.0 M NaCl and centrifuged at
100,000 g for 30 min. The supernatant was discarded, and the
pellet was solubilized with lysing buffer containing 20 mM
3-[(3-cholamidopropyl) diethylammonio]-1 propane sulfo-
nate at 4°C for 30 min. After centrifugation at 100,000 g for
1 h, the supernatant was kept as membranous fraction. Both
fractions were used for Western blotting. Antibodies against
PKC, PKC, and PKCε were purchased from Santa-Cruz, the
anti-VE-Cadherin antibody from Cell Signaling Technology.
Statistical analysis
Data were expressed as means se, and analyzed using 1-way
ANOVA, Student’s t test, or rank-sum test. Values of P 0.05
were considered statistically significant.
RESULTS
Spongistatin 1 causes disassembly of microtubules
in HUVECs
To test whether spongistatin 1 causes microtubule
disassembly in endothelial cells, 2 different ap-
proaches were pursued. First, live cell imaging of
green fluorescent protein (GFP)-tubulin-transfected
HUVECs, treated with 2.0 nM spongistatin 1 for 16 h,
was performed. At the beginning of the record, a
clearly structured microtubule skeleton could be
TABLE 1. Kinome array data showing kinases that were reduced most in their activity in tubulin-antagonist-treated HUVECs compared
to control cells
Spongistatin 1 Vinblastine CA4P
Peptide Kinase R.A. Peptide Kinase R.A. Peptide Kinase R.A.
RSKKNSLALSL PAK1 0.6 RKKKVSSTKRH PKC 0.4 DNSSDSDYDLH LCK, FYN 0.7
LLEDDSDEEED PKC 0.6 SWKENSPLNVS PKC 0.4 LCQAFSDVILA PLK1 0.6
KRRGASDLSSE CK2 0.5 RDKEVSDDEAE CK2 0.4 LERGNSGLGFS CaMKII 0.6
RKMKDTDSEEE c-SRC 0.4 NIIHGSDSVES NDK A 0.3 NYIPETPPPGY ERK 0.6
GLRRSSKFCLK EGFR 0.3 YFRYLSEVASG PKC 0.3 KERRRTESINS CK2 0.5
PTQPTSASPSL CK2 0.1 GGARASPATQP ERK 0.3 RKKKVSSTKRH RSK, PKA 0.5
LCQAFSDVILA AKT1 0 DEYNVTPSPPG GSK3 0.3 GFFSSSESGAP PKC 0.5
ERGQEYLILEK TEC kinase 0.3 LEPLCTPVVTC PKC 0.5
TDNEDYEHDDE SYK 0.3 PEPGPYAQPSV EGFR 0.4
QGISFSQPTCP ATR 0 KKKKGSLDSDN PKA 0.3
Some kinases are represented by multiple substrate peptides. R.A. residual activity.
1130 Vol. 23 April 2009 ROTHMEIER ET AL.The FASEB Journal

detected in the cells. With prolonged incubation with
spongistatin 1, tubulin fibers disassembled and left a
diffuse GFP signal distributed over the whole cell
(Fig. 1A; Supplemental Movie 1). Second, HUVECs
treated with 1.0 M paclitaxel or 0.1–2.0 nM spongista-
tin 1 for 4 h were fractionated into a PIPES buffer-
soluble fraction, containing tubulin heterodimers, and
a PIPES buffer-insoluble fraction, containing polymer-
ized microtubules. As was to be expected, the microtu-
bule-stabilizing compound paclitaxel caused an in-
crease of tubulin in the fraction of polymerized
microtubules. In contrast, the incubation with
spongistatin 1 results in dose-dependent decrease of
tubulin in this fraction as a result of microtubule
disassembly (Fig. 1B).
Spongistatin 1 is antiproliferative at
a nontoxic concentration
Antiproliferative effects of spongistatin 1 on endothe-
lial cells were investigated in a cell viability assay,
DNA-fragmentation analysis, proliferation assay, and
cell cycle analysis. Acute cytotoxicity of spongistatin 1
on proliferating cells was observed at a concentration as
low as 5.0 nM, as judged by the significant decrease in
the metabolic activity of spongistatin 1-treated cells. In
contrast, cytotoxic effects of spongistatin 1 on conflu-
ent HUVECs occurred at a concentration as high as
50.0 nM (Fig. 2A). At concentrations of 500 pM and 1.0
nM, spongistatin 1 induced a slight increase, up to 5%
apoptotic cells. At a concentration of 2.0 nM spongista-
tin 1, up to 20% of the cells were apoptotic. In
comparison to the established tubulin antagonists vin-
blastine, CA4P, and paclcitaxel, spongistatin 1 induced
apoptosis most efficiently (Fig. 2B). Astonishingly, pro-
liferation of HUVECs was already inhibited at a con-
centration as low as 100 pM spongistatin 1, whereas
more than 25–500 higher concentrations of vinblas-
tine (2.5 nM), C4AP (7.5 nM), or paclitaxel (50.0 nM),
respectively, were needed to inhibit endothelial prolif-
eration to a similar degree (Fig. 2C). Tubulin antago-
nists disrupt the mitotic spindle of dividing cells, cause
cell cycle arrest in the transition from G2 to M phase,
and consequently inhibit proliferation. As expected,
spongistatin 1 also caused G2/M arrest, again at lower
concentrations than vinblastine, CA4P, and paclitaxel.
However, significant inhibition of cell cycle progression
occurred starting at a concentration of 1.0 nM
spongistatin 1, a 10 higher concentration than that
needed to inhibit proliferation (Fig. 2D).
Spongistatin 1 inhibits migration of endothelial cells
and influences F-actin organization
Migration is a crucial step in angiogenesis. Therefore,
the influence of spongistatin 1 on endothelial cell
migration was investigated in a scratch assay. Spongista-
tin 1 effectively inhibited wound closure in a dose-
dependent manner, with an IC
50
of 1.0 nM, and was
therewith again more potent than the established tu-
bulin antagonists vinblastine (7.5 nM), C4AP (10.0
nM), or paclitaxel (25.0 nM) (Fig. 3A, B).
Actin and its motor proteins are supposed to gener-
ate the main force driving cells forward. Hence, alter-
ations in F-actin organization could be expected in cells
that are inhibited in migration. Growth-factor-deprived
cells (starvation medium) do not migrate. In these
cells, actin is organized in cortical rings, and lamellopo-
dia only barely exist (Fig. 3C, left panel). In migrating
HUVECs (culture medium), F-actin is arranged in
stress fibers within the cell body and lamellopodia at
the leading edge. Both were directed toward the wound
(Fig. 3C, middle panel). Spongistatin 1-treated cells
exhibited the combined characteristics of F-actin ar-
rangement in both migrating and nonmigrating cells.
Both actin fibers in the cell body and cortical rings were
formed. Furthermore, the appearance of lamellopodia
was reduced on spongistatin 1 treatment (Fig. 3C, right
panel).
Spongistatin 1 reduces chemotaxis,
but not chemokinesis
The influence of spongistatin 1 on directed migration
of endothelial cells was investigated in a chemotaxis
Figure 1. Live-cell imaging and tubulin fractionation proved the disassembly of microtubules by spongistatin 1.
A) GFP-tubulin-transfected HUVECs were stimulated with 2.0 nM spongistatin 1 for 16 h. Bottom panels show magnification of
microtubules of the HUVECs. B) Cell lysates of paclitaxel (pacl)- and spongistatin 1 (SP1)-stimulated HUVECs were separated
in PIPES buffer-soluble and -insoluble fractions. Tubulin heterodimers were detected in the soluble fraction, polymerized
microtubules in the insoluble fraction.
1131THE TUBULIN ANTAGONIST SPONGISTATIN INHIBITS PKC

Figures (9)
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  • ...Second, spongistatin 1 acts as a tubulin depolymerizing agent and is able to free the proapoptotic Bcl-2 family member Bim from its sequestration both by the microtubular complex and by the anti-apoptotic protein Mcl-1 [45,46] (Fig....

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