Protein kinase C mediates induced secretion of vascular endothelial growth factor by human glioma cells

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Protein kinase C mediates induced secretion of vascular

endothelial growth factor by human glioma cells

Jui-Chang Tsai,

a,b,*

Lee-Jene Teng,

c

Chin-Tin Chen,

b

Tse-Ming Hong,

d

Corey K. Goldman,

e

and G. Yancey Gillespie

e

a

Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei 100, Taiwan

b_{Center for Optoelectronic Biomedicine, National Taiwan University College of Medicine, Taipei 100, Taiwan} c_{School of Medical Technology, National Taiwan University College of Medicine, Taipei 100, Taiwan}

d_{School of Pharmacy, National Taiwan University College of Medicine, Taipei 100, Taiwan}

e_{Brain Tumor Research Laboratories, Division of Neurosurgery, Department of Surgery, The University of Alabama at Birmingham,}

Birmingham, AL 35294-0006, USA Received 3 August 2003

Abstract

To understand how vascular endothelial growth factor (VEGF) production is activated in malignant glioma cells, we employed

protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to evaluate the extent to which these protein kinases were

involved in signal transduction leading to VEGF production. PTK inhibitors blocked glioma proliferation and epidermal growth

factor (EGF)-induced VEGF secretion, while H-7, a PKC inhibitor, inhibited both EGF-induced and baseline VEGF secretion.

Phorbol 12-myristate 13-acetate (PMA), a non-speciﬁc activator of PKC, induced VEGF secretion by glioma cells, which was

enhanced by calcium ionophore A23187, but completely blocked after prolonged treatment of cells with 1 lM PMA, by presumably

depleting PKC. All inhibitors (genistein, AG18, AG213, H-7, prolonged PMA treatment) which inhibited EGF-induced VEGF

secretion in glioma cells also inhibited cell proliferation at similar concentrations. However, PKC inhibition only blocked 50% of the

VEGF secretion induced by growth factors (EGF, platelet-derived growth factor-BB, or basic ﬁbroblast growth factor). This reserve

capacity could be ascribed to a PKC-independent eﬀect, or to PKC isoenzymes not down-regulated by PMA. These ﬁndings extend

our previous assertion that VEGF secretion is tightly coupled with proliferation by suggesting that activation of convergent growth

factor signaling pathways will lead to increased glioma VEGF secretion. Understanding of signal transduction of growth

factor-induced VEGF secretion should provide a rational basis for the development of novel strategies for therapy.

Ó 2003 Elsevier Inc. All rights reserved.

Keywords: Vascular endothelial growth factor; Vascular permeability factor; Malignant glioma; Protein kinase C; Protein tyrosine kinase; Epidermal growth factor; Fibroblast growth factor; Platelet-derived growth factor; Isozymes

Vascular endothelial growth factor (VEGF) is a

po-tent and speciﬁc endothelial cell mitogen in vitro,

in-duces angiogenesis in vivo [1], and proin-duces a profound

increase in vascular permeability [2]. It is identical to

vascular permeability factor [3,4]. The biological eﬀects

of VEGF are mediated mainly by two tyrosine kinase

receptors, VEGFR-1 (Flt-1) and VEGFR-2

(KDR/ﬂk-1), which are expressed almost exclusively in endothelial

cells [5]. Two biologic characteristics of VEGF are

unique among angiogenesis factors. VEGF is a direct

and speciﬁc endothelial cell mitogen. Other angiogenesis

factors either support angiogenesis indirectly (epidermal

growth factor (EGF), tumor necrosis factor a,

trans-forming growth factor b1, or angiogenin) or are active

on non-endothelial cell types (acid ﬁbroblast growth

factor, aFGF; or basic ﬁbroblast growth factor, bFGF)

[6]. VEGF is also a secreted protein in contrast to

aFGF, bFGF, and platelet-derived endothelial cell

growth factor, which lack signal peptides required for

extracellular transport. Supposedly, these latter factors

are released by leakage from damaged cells or bound to

Biochemical and Biophysical Research Communications 309 (2003) 952–960

www.elsevier.com/locate/ybbrc

BBRC

*

Corresponding author. Fax: +886-2-2341-5487. E-mail address:[email protected](J.-C. Tsai).

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secreted extracellular matrix molecules. We have shown

that VEGF is detected in glioma cell cultures by ELISA

[7] and hypothesize that VEGF is the central mediator

of glioma angiogenesis because of its characteristics [8]

and its ability to initiate the sequence leading to

neo-vascularization [2].

Three of the major histopathologic features of

ma-lignant glioma (hypervascularity, tumor necrosis, and

peritumoral brain edema) may be related to VEGF.

Glioblastoma multiforme thus seems to be the prototype

tumor suitable for anti-angiogenic therapy [9]. Deﬁnitive

studies of regulatory pathways controlling

glioma-de-rived VEGF production may lead to development of

new therapeutic strategies for this common malignant

brain tumor. We have demonstrated that three tyrosine

kinase receptor growth factors, EGF, bFGF, and

PDGF-BB, are each capable of inducing VEGF

pro-duction from glioma cells [7]. The combined eﬀects of

these growth factors were additive at low concentrations

but reached a maximum stimulatory eﬀect at high

con-centrations implying a convergent pathway leading to

enhanced VEGF secretion.

Protein kinase C (PKC) encompasses a complex

family of closely related serine/threonine protein kinase

isozymes that mediate a wide range of signal

transduc-tion processes in cells [10]. Most isoforms of PKC can be

physiologically activated by cellular production of

dia-cyl glycerol (DAG) [11]. Free fatty acids also play a role

in activating PKC. There is evidence that growth

factor-mediated activation of receptor tyrosine kinases (e.g.,

PDGF or EGF receptors) initiates the cascade of events

(PLCc activation with cleavage of inositol triphosphate

producing phosphoinositol phosphate and DAG) which

results in activation of PKC [12]. Thus, inhibitors that

are directed selectively against PKC may have

wide-ranging therapeutic potential [13–15].

To understand how VEGF production is activated in

malignant glioma cells, we employed protein tyrosin

kinase (PTK) and PKC inhibitors to evaluate whether

PTK and PKC are involved in signal transduction

leading to VEGF production. The capacity of PMA to

down-regulate the DAG-dependent PKC isozymes

provided a means to evaluate the relative importance of

diﬀerent PKC isozymes in this process.

Materials and methods

Biochemical and biological reagents. Dulbecco’s modiﬁed Eagle’s medium mixed 50:50 with Ham’s nutrient mixture F-12 (DMEM/F12; Mediatech) supplemented to 2 mMLL-glutamine and 8% fetal bovine

serum (FBS) was used for culture of U-105MG glioma cells. FBS (Intergen, Purchase, NY) was heat inactivated (56°C, 45 min). EGF (human, receptor grade, Collaborative Research, Bedford, MA), re-combinant human PDGF-BB (R&D systems, Minneapolis, MN), and bFGF (Promega, Madison, WI) were all diluted to speciﬁed concen-trations in Dulbecco’s PBS (pH 7.2). Culture media and sera were

determined to have <100 pg/ml of endotoxin by the Limulus amoe-bocyte lysate assay. [3_{H]Thymidine was purchased from Amersham}

(Arlington Heights, IL).

Genistein (GIBCO, Grand Island, NY) was dissolved in dimethyl sulfoxide/ethanol (1:1) at a concentration of 25 mM. Tyrphostin AG18 and AG213 (from Dr. Alexander Levitzki, Hebrew University, Jeru-salem, Israel) [16] were dissolved in absolute ethanol at a concentration of 50 mM. The phorbol esters 4a-P and PMA (Sigma) were dissolved in absolute ethanol at a concentration of 1.6 mM. In addition, 7, H-8, and HA1004 (Seikagaku America, Rockville, MD) were dissolved in distilled water at a concentration of 10 mM. Chelerythrine (LC Lab-oratories, Woburn, MA) was dissolved in distilled water at a concen-tration of 10 mM. All stock solutions were diluted in culture medium or PBS to the desired concentrations at the time of addition.

Cell line. Human glioma cell line U-105MG has been described in detail [17]. The cell line has been passed >200 times and maintained in 150 cm2_{plastic tissue culture ﬂasks (Falcon Plastics, Lincoln Park, NJ)}

in complete culture media (DMEM/F12 + 2 mM LL-glutamine + 8%

FBS) at 37°C and 7.5% CO2. Antibiotics were not used routinely and

U-105MG cells were negative for mycoplasma by two-stage nested primer polymerase chain reaction test (ATCC, Rockville MD).

Antibodies and antisera. A4.6.1 anti-VEGF monoclonal antibody was from Genentech [18]. Polyclonal anti-VEGF antiserum was made in rabbit as has been reported [7,8].

Analysis of dose–response to PTK or PKC inhibitors. U-105MG cells were plated at 1 105_{cells/well in 24-well plates in 0.5 ml of}

DMEM/F12 + 2 mM LL-glutamine + 0.8% FBS and incubated (72 h,

37°C, 7.5% CO2) undisturbed. Supernates were aspirated and replaced

with 0.5 ml of DMEM/F12 + 2 mMLL-glutamine + 0.8% FBS contain-ing various concentrations of inhibitors of PTK or PKC with or without EGF in quadruplicate wells. EGF was added 30 min after adding inhibitors. After 72 h incubation (37°C, 7.5% CO2),

alamar-Blue (50 ll; Alamar Biosciences, Sacramento, CA) was added to each well, the plates incubated for 30 min, and cell viability based on met-abolic conversion of the deep blue dye to a pink color was assessed spectrophotometrically in situ. Absorbances of supernates were mea-sured at a wavelength of 562 nm and background absorbances at 590 nm were subtracted using a semiautomatic plate reader (model EL310, Bio-Tek Instruments, Winooski, VT). Conditioned media were subsequently assessed for VEGF by ELISA. Multiple experiments conﬁrmed that alamarBlue did not interfere with detection of VEGF by ELISA [7]. All experiments were repeated a minimum of three times.

Eﬀect of PMA pre-treatment on growth factor- and PMA-induced VEGF secretion. U-105MG cells were plated in 24-well plates as de-scribed above and incubated (37°C, 72 h) either with PMA (1.6 lM) or 4a-P (1.6 lM) to down-regulate conventional and novel isoforms of PKC. Supernates were aspirated, cells gently washed twice with warm PBS buﬀer, and incubated (37°C, 72 h) with fresh medium containing various concentrations of PMA, EGF, PDGF-BB, or bFGF. The degree of inhibition of induced VEGF secretion was calculated by the following formula:

100% ðCig CiÞ=ðCg C0Þ 100%;

where Cig is the VEGF concentration with PMA (or inhibitor)

pre-treatment and growth factor (or inducer), Ciis the VEGF

concentra-tion with PMA pre-treatment but without growth factor, Cg is the

VEGF concentration without PMA pre-treatment but with growth factor, and C0is the baseline VEGF concentration without PMA

pre-treatment or growth factor.

ELISA. A semiquantitative capture ELISA was used to detect amounts of VEGF in supernates as previously reported [7]. Brieﬂy, plates were coated with monoclonal anti-VEGF antibody A4.6.1, blocked, incubated (with intervening wash cycles) sequentially with: known or unknown samples, polyclonal anti-VEGF antiserum, bioti-nylated goat anti-rabbit antiserum, and avidin-biotibioti-nylated alkaline phosphatase complex (Vector Labs, Burlingame, CA). Polyclonal

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antibody binding to captured VEGF was detected by addition of p-nitrophenyl phosphate substrate (Sigma Chemical, St. Louis, MO) with absorbance (405 nm) determined using a semiautomated plate reader. VEGF amounts in samples were extrapolated from standard curves generated in each assay [7].

[3_{H]Thymidine incorporation. Proliferation of U-105MG cells was}

determined by [3_{H]thymidine incorporation. Cells were seeded in}

96-well plates at a density of 1 104_{cells/well in 90 ll of DMEM/}

F12 + 2 mMLL-glutamine + 0.8% FBS and incubated (37°C, 72 h, 7.5%

CO2) undisturbed. Various concentrations of inhibitors of PTK or

PKC were then added and incubated for 6 h. Cells were incubated with [3_{H]thymidine (speciﬁc activity 41 Ci/mmol; 0.5 lCi/well) for an}

addi-tional 15 h. Cells were detached by 30 min incubation at 37°C in cell release solution (trypsin 0.1%, EDTA 2.15 mM, sucrose 0.4 M, pH 7.5, 40 ll/well). Cells were then harvested with a semi-automatic cell har-vester (Skatron, Sterling, VA) to scintillant-impregnated Ready Filter (Beckman, Fuller, CA). Filter discs were transferred to scintillation vials and radioactivity measured in a liquid scintillation spectrometer (LKB Rackbeta 1211, Finland). Experiments were performed with at least four replicates and results were expressed as mean counts per minute (cpm).

Results

PTK inhibitors genistein and tyrphostin inhibited

EGF-induced VEGF secretion

Genistein, a broad action protein tyrosine kinase

in-hibitor, inhibited EGF-induced VEGF secretion (Fig. 1)

in a dose-dependent manner with IC

50

(concentration

causing a 50% inhibition) estimated to be about 25 lM.

Tyrphostin AG18 and AG213, which are eﬀective

blockers of EGF receptor kinase but extremely poor

inhibitors of serine/threonine kinases [19,20], also

in-hibited EGF-induced VEGF secretion with IC

50

for

each estimated to be about 15 lM (Fig. 2A). Baseline

secretion of VEGF was not inhibited by either

tyrpho-stin. AG18 and AG213 also inhibited cell proliferation

as assessed by [

3

_{H]thymidine incorporation (Fig. 2B);}

cell viability was not aﬀected.

PKC inhibitor H-7 inhibited EGF-induced VEGF

secre-tion as well as baseline level of VEGF secresecre-tion

PKC inhibitor H-7 began to inhibit EGF-induced

VEGF secretion at a concentration of 10 lM and

in-hibited baseline secretion of VEGF (Fig. 3A). HA1004

did not inhibit either EGF-induced or baseline secretion

of VEGF at the same or higher concentrations. HA1004

strongly inhibits PKA and PKG, but weakly inhibits

PKC with a Ki 7-fold higher than H-7 [21]. H-7 also

inhibited cell proliferation as assessed by [

3

_H]thymidine

incorporation, while HA1004 did not (Fig. 3C).

ala-marBlue dye conversion revealed decreased cell

metab-olism at H-7 concentrations above 25 lM (Fig. 3A). To

determine whether this inhibitory eﬀect was due to a

cytocidal action of the inhibitor, U-105MG cell viability

was assessed by trypan blue exclusion and found to be

96% and 90% at 10 and 25 lM, respectively. However,

cell viability was observed to decrease to 61% at 50 lM.

H-8, a PKG/PKA inhibitor, did not inhibit VEGF

secretion at concentrations below 20 lM, but inhibited

EGF-induced and baseline secretion of VEGF at

con-centrations above 30 lM. H-8 also inhibited cell

prolif-eration at higher concentration (50 lM) but did not

Fig. 1. Genistein inhibited EGF-induced VEGF secretion (closed cir-cle) in U-105MG cell line with an IC50 around 25 lM. alamarBlue

assay (triangle symbols) revealed decreased cell viability or cell me-tabolism at concentrations above 25 lM.

Fig. 2. PTK inhibitors inhibited VEGF secretion and cell proliferation in U-105MG glioma cells. (A) PTK inhibitor tyrphostins AG18 and AG213 inhibited EGF-induced VEGF secretion (closed symbols) in dose-dependent fashion with IC50values around 15 lM. Baseline

se-cretion of VEGF (open symbols) was not inhibited. Representative of three experiments. (B) AG18 and AG213 inhibited cell proliferation as assessed by [3_{H]thymidine incorporation. Each data point represents}

mean SD of quadruplicate cultures and these experiments were re-peated three times with similar results.

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interfere with cell metabolism as assessed by alamarBlue

dye reduction (Figs. 3B and C). H-8 has been reported

to be >1 log more eﬀective at inhibiting PKG (0.48 lM)

and PKA (1.2 lM) than PKC (15 lM) [22].

Chelerythrine inhibited baseline and EGF-induced

VEGF secretion only at concentrations of 30 lM or

higher but also inhibited cell metabolism and cell

pro-liferation in the same range (30 and 20 lM, respectively;

Figs. 4A and B). Trypan blue exclusion revealed that

most of these cells were not alive at 30 lM, and

there-fore, this was judged to be a toxic, rather than a speciﬁc

inhibitory, eﬀect.

Response of cultured U-105MG cells to PMA

The phorbol ester PMA, activates conventional and

novel forms of PKC, caused morphologic changes in

U-105MG glioma cells. Within several hours of

treat-ment, cells changed from a fusiform bipolar appearance

to a more ﬂattened, polygonal cell shape with multiple

short processes. Later, cells became larger and

cyto-plasmic vacuoles became evident. These morphologic

changes were induced by as little as 3.2 nM PMA,

whereas no noticeable changes in morphology were

in-duced by 4a-P (a phorbol ester that does not activate

PKC) under identical conditions.

PMA-induced VEGF secretion in a dose-dependent

manner with a signiﬁcant increase of VEGF secretion at

3.2 nM (P < 0:001, by ANOVA and Bonferroni

meth-od). VEGF secretion reached peak levels at about 50 nM

PMA and declined slightly as the dose of PMA was

increased (Fig. 5A). The control compound, 4a-P, did

not modulate VEGF secretion within the same range

(Fig. 5A). Pretreatment of cells with 1.6 lM PMA for 3

days, which down-regulates PKC in other cell types

[23,24], completely blocked a subsequent increase in

VEGF secretion after stimulatory challenge with PMA

(Fig. 5A). After pretreatment with PMA, however,

baseline secretion of VEGF was signiﬁcantly elevated

above that without PMA pretreatment or that with 4a-P

pretreatment (p < 0:01, unpaired Student’s t test).

Fig. 3. PKC inhibitors inhibited both baseline and EGF-induced VEGF secretion in U-105MG cells as well as DNA synthesis. (A) PKC inhibitor H-7 at 10 lM inhibited both EGF-induced and baseline secretion of VEGF (circles). AlamarBlue dye conversion revealed decreased cell metabolism at concentrations above 25 lM (triangles). HA1004, a negative control compound for H-7, had no eﬀect on secretion of VEGF at the same or higher concentrations (squares). Representative of three experiments. (B) H-8 inhibited both EGF-induced and baseline secretion of VEGF at concen-trations above 30 lM (circles) but did not interfere with cell metabolism as assessed by alamar blue (triangles). (C) H-7 and H-8 inhibited cell proliferation as assessed by [3_{H]thymidine incorporation, while HA1004 did not. Each data point represents mean}_{SD of quadruplicate cultures.}

These experiments were repeated three times with similar results.

Fig. 4. Inhibition of EGF-induced VEGF secretion by chelerythrine is due to a non-speciﬁc cytotoxic eﬀect. (A) Chelerythrine inhibited baseline and EGF-induced VEGF secretion at concentration of 30 lM. Representative of three experiments. (B) Chelerythrine also inhibited cell proliferation and cell metabolism at about the same concentration (20 and 30 lM, respectively) Each data point represents mean SD of quadruplicate cultures. These experiments were repeated three times with similar results.

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Incorporation of [

3

_{H]thymidine, used as an index of}

cell proliferation, demonstrated that as little as 3.2 nM

PMA signiﬁcantly inhibited DNA synthesis acutely in

U-105MG cells to 26% of baseline (Fig. 5B; p < 0:001,

by ANOVA and Bonferroni method). Higher

con-centrations of PMA exhibited no additional eﬀect.

However, after 72 h exposure to 1.6 lM PMA, cellular

proliferation appeared to have recovered as indicated by

the modest rate of [

3

_{H]thymidine uptake. Despite these}

eﬀects, U-105MG cells remained viable at high PMA

concentration as demonstrated by alamarBlue assay

(Fig. 5A) and trypan blue exclusion. By comparison,

4a-P exhibited no signiﬁcant eﬀect on U-105MG cell DNA

synthesis, even at 1.6 lM (Figs. 5A and B).

Calcium ionophore A23187, which activates some

isoforms of PKC by increasing intracellular calcium

concentration, enhanced VEGF secretion as eﬀectively as

PMA (80 nM) at a concentration of 1.25 lM (Table 1).

A23187 also potentiated PMA-induced VEGF secretion.

Down-regulation of PKC with PMA partially blocked

growth factor-induced VEGF secretion

We have shown previously that EGF, bFGF, or

PDGF-BB each are able to induce VEGF secretion in

U-105MG [7]. Here, we demonstrate that

down-regula-tion of PKC by pretreating the cells with PMA (1.6 lM

for 3 days) blocked VEGF secretion induced by EGF

(50 ng/ml), bFGF (50 ng/ml), or PDGF-BB (50 ng/ml) by

48%, 53%, or 47%, respectively (Fig. 6). In comparison,

PMA pretreatment eliminated the PMA induction of

VEGF secretion almost completely (97

7%). While

PMA pretreatment did not eliminate growth

factor-in-duced VEGF secretion completely, the diﬀerential

inhi-bition of PMA-induced and growth factor-induced

VEGF secretion was statistically signiﬁcant (p < 0:01, by

ANOVA and Bonferroni method). An analogous

rela-tion could also be demonstrated in that the PKC

inhib-itor, H-7 (25 lM), inhibited PMA-induced VEGF

secretion (83

9%) to a much greater extent than it did

EGF-induced VEGF secretion (45

4%) (Fig. 7,

p <

0:01).

Discussion

We have previously demonstrated that EGF, bFGF,

or PDGF-BB each are capable of inducing VEGF

se-cretion in U-105MG cells [7]. Receptors for EGF,

bFGF, or PDGF-BB are themselves protein tyrosine

kinases [25,26] and stimulation of these receptors with

Table 1

Eﬀects of calcium ionophore A23187 and PMA on VEGF secretion by U-105MG

Treatment condition VEGF (ng/ml) Alamar blue assay (absorbance)

Mean SD (% of Control) Mean SD (% of Control)

Control 9.0 0.6 (100) 0.33 0.02 (100)

A23187 (1.25 lM) 22 1.9 (240) 0.29 0.02 (88)

PMA (80 nM) 18 2.0 (200) 0.33 0.04 (100)

A23187 + PMA 28 1.0 (310) 0.36 0.04 (110) Fig. 5. Down-regulation of PKC by PMA pretreatment blocked

PMA-induced VEGF secretion in U-105MG cells. (A) PMA PMA-induced VEGF secretion in a dose-dependent manner achieving a plateau response at 64 nM (closed squares). The control compound, 4a-phorbol (4a-P), had no effect within the same range (closed circles). Pretreatment of the cells with PMA 1.6 lM for three days completely down-regulated the stimulatory effect of PMA on VEGF secretion (open squares). (B) As little as 3.2 nM PMA significantly inhibited DNA synthesis in U-105MG cells (closed squares). After three days of pretreatment (1.6 lM PMA), U-105MG DNA synthesis recovered modestly and was re-fractory to PMA inhibition (open squares). 4a-P had no significant effect on U-105MG DNA synthesis, even at a concentration of 1.6 lM (closed circles). Each data point represents mean SD of quadrupli-cate cultures; representative of three experiments.

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their cognate growth factors rapidly leads to receptor

autophosphorylation on tyrosine residues.

Ligand-stimulated autophosphorylation of tyrosine enhances

subsequent tyrosine kinase activity of the receptors and

creates binding sites for recruitment of speciﬁc enzymes,

which include phospatidylinositol (PtdIns) 3-kinase,

ras-GTPase activating protein, PtdIns-speciﬁc

phos-pholipase Cc, and pp74

c-raf

_{[27]. Ligand-activated}

PDGF receptors associate with all four enzymes.

Li-gand-activated EGF receptors appear to associate with

at least two of these four targets (PtdIns 3-kinase and

PtdIns-speciﬁc PLCc) and may associate with

ras-GTPase activating protein. Activated bFGF receptors

also associate with PLCc [25]. Activation of

PtdIns-speciﬁc PLCc would stimulate canonical PtdIns

turn-over pathways, leading to elevation of cytosolic calcium

and activation of PKC [28]. Thus, tyrosine kinase-type

growth factor receptors transduce signals through

PKC-dependent and PKC-inPKC-dependent pathways. While most

signaling pathways have yet to be completely deﬁned, it

may be possible to block a major signaling pathway if

the speciﬁc enzymes involved are known. PTK and PKC

mediate a wide range of signal transduction processes

and are considered more selective targets for

chemo-therapy than known targets of inhibitors of DNA or

RNA synthesis, or agents causing disruption of the

cytoskeleton [29,30]. In this study, we used inhibitors for

both PTK and PKC as probes to explore control points

in signal transduction of growth factor-induced VEGF

secretion. Given that induction of glioma cell

prolifer-ation and growth factor-induced VEGF secretion are

apparently inextricably linked, these control points may

lead to rational development of novel chemotherapeutic

strategies for malignant glioma.

Selective inhibition vs. cytotoxicity

Most compounds used to investigate regulatory roles

of PKC lack selectivity for PKC over several other

ki-nases [31]. For example, H-7 inhibits PKC, PKA, or

PKG at 6.0, 3.0, or 5.8 lM, respectively [32], and

therefore, is not a PKC-speciﬁc inhibitor. In order to

compensate for this lack of speciﬁcity, HA1004 was used

as a “negative” control compound for H-7. HA1004

inhibits PKA/PKG with comparable potency (2.3 and

1.3 lM, respectively) to that of H-7 but is about 1 log

less eﬀective in PKC inhibition (40 lM) [21]. In this

study, we showed that H-7 inhibited EGF-induced

VEGF secretion, while HA1004, used at similar

con-centrations, did not (Fig. 3A). We propose that the

in-hibition was most likely due to an eﬀect on PKC.

Although viability of U-105MG cells was still above

90% at 25 lM, it decreased to 61% at an H-7

concen-tration of 50 lM. Further, H-7 inhibited cell metabolism

Fig. 6. Down-regulation of PKC by PMA pretreatment blocked growth factor-induced VEGF secretion in U-105MG cells. U-105MG cells, incu-bated (72 h) either with (closed symbols) or without (open symbols) PMA (1.6 lM), were washed and incuincu-bated (72 h) with fresh media containing various concentrations of EGF (A), bFGF (B), or PDGF-BB (C). Supernates, assessed for VEGF by ELISA, revealed that down-regulation of PKC by PMA pretreatment blocked EGF-, bFGF-, or PDGF-BB-induced secretion of VEGF signiﬁcantly, but did not eliminate growth factor-induced VEGF secretion completely. Cell viabilities were unaﬀected by PMA pretreatment (triangles). Representative of three experiments.

Fig. 7. PMA pretreatment (PKC depletion) blocked EGF-, bFGF-, or PDGF-BB-induced VEGF secretion to 48%, 53%, and 47% of control, respectively. H-7, a speciﬁc PKC inhibitor, inhibited EGF-induced VEGF secretion to 45% of control, while it blocked PMA-induced VEGF secretion to 83%. PMA pretreatment almost completely blocked (97%) PMA-induced VEGF secretion.

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signiﬁcantly at concentrations above 25 lM as shown by

alamarBlue assay (Fig. 3A). Thus, the inhibitory eﬀect

of H-7 occurred at a concentration just below that which

caused a cytotoxic eﬀect.

As a further control for the inhibitory eﬀects of H-7

on PKA and PKG, we tested H-8, which inhibits PKG

(0.48 lM) and PKA (1.2 lM) much more eﬀectively than

it inhibits PKC (15 lM) [21]. As observed with HA1004,

H-8 did not inhibit EGF-induced VEGF secretion at

concentrations (10 lM) at which it should have inhibited

PKA and PKG. However, by increasing the

concen-tration above 20 lM, an inhibitory eﬀect was observed

which could be attributable to PKC inhibition. Like

HA1004, H-8 did not interfere with cell metabolism at

concentrations up to 50 lM.

While H-7 and H-8 are thought to inhibit PKC via

competition for the ATP binding site on PKC [33]

(Fig. 8), chelerythrine is a non-competitive inhibitor

with respect to ATP [34]. It inhibits the catalytic domain

of PKC, but not the regulatory domain [34] (which

binds DAG and PMA; Fig. 8). Chelerythrine is a potent

and speciﬁc inhibitor of PKC in vitro and showed

po-tent cytotoxic eﬀects against L-1210 tumor cells [34].

Our data demonstrated that chelerythrine inhibited both

EGF-induced and baseline VEGF secretion, as well as

metabolism at the same concentration (20 lM), which

we presume to represent a non-selective cytotoxic eﬀect,

rather than speciﬁc inhibition of PKC.

PMA binds to the DAG-speciﬁc regulatory domain

of PKC and activates PKC. An alternative technique to

assess the role of PKC in cell function relies on the

observation that prolonged treatment of cells with PMA

activates and eventually depletes PKC. Though PMA

signiﬁcantly inhibited the growth of U-105MG cells

acutely, DNA synthesis was able to proceed at a modest

rate (Fig. 5B). These cells remained viable and

meta-bolically active after treatment with a high PMA

con-centration (1.6 lM) for 3 days (Figs. 5A and B). PMA

acutely increased secretion of VEGF and this eﬀect

could be blocked completely either by PMA

pretreat-ment to deplete PKC or by inhibition of PKC with H-7,

thus supporting the notion that induced secretion of

VEGF is PKC dependent.

Growth factor-induced VEGF secretion depends at least in

part upon the activation of PKC

While down-regulation of PKC by prolonged

treat-ment of cells with PMA completely blocked

PMA-in-duced VEGF secretion, it did not completely block

EGF-, bFGF-, or PDGF-BB-induced VEGF secretion.

This implies that more than one signaling pathway is

involved in growth factor-induced VEGF secretion.

Notably, PMA pretreatment blocked growth

factor-(EGF, bFGF, or PDGF-BB) induced VEGF secretion

to about the same degree, i.e., about 50% of the induced

eﬀect was PKC dependent. The remaining capacity

could be due to so called “PKC-independent”

mecha-nisms, or simply due to PKC isozymes that are not

down-regulated by PMA [35].

Co-regulation of VEGF secretion and cell proliferation by

growth factors

Axiomatically, PTK and PKC exert major roles in

cell proliferation and diﬀerentiation. In this study, all of

the inhibitors which attenuated EGF-induced VEGF

secretion also diminished cell proliferation at similar

concentrations. PMA also inhibited proliferation of cells

in U-105MG, but this eﬀect was probably due to

down-regulation of PKC instead of activation of PKC,

be-cause PMA-induced DNA synthesis inhibition could

not be blocked by PKC inhibitors H-7 and H-8 (data

not shown). These ﬁndings suggest that regulation of

VEGF secretion is tightly coupled with cell

prolifera-tion, and further support the concept that normal tissue

growth and neovascularization are co-regulated by

normal growth signals, while tumor growth and tumor

angiogenesis are co-regulated by the inappropriate

presence of growth signals.

In summary, we have determined that growth

factor-induced VEGF secretion by U-105MG human

glio-blastoma cells in vitro depended in part upon activation

of PKC, could be blocked by inhibitors of PKC or by

broad spectrum down-regulation of conventional or

novel isoforms of PKC. It appears, however, that

Fig. 8. Schematic representation of protein kinase-C family members. PKC is currently comprised of a family of at least 11 isoenzymes thought to play critical roles in control of many cellular processes. These serine/threonine kinase isozymes are broadly divided into three classes: the “conventional” Ca2þ_{-dependent forms (a; b, and cÞ, the}

“novel” forms that lack calcium binding C2 domain (d; ; g, and hÞ

and do not require Ca2þ_{for activation, and the “atypical” forms that}

lack the Ca2þ_{binding domain and are not activated by DAG/PMA}

(f and sÞ. V1–V5 are variable or nonconserved regions of the PKC

molecule. C1–C4are conserved regions of the PKC molecule. The C1

region contains two sets of cysteine-rich sequences and is required for phorbol ester binding. PKCf has only one set of cysteine-rich se-quences and does not translocate or down-regulate in response to phorbol esters or diacylglycerol derivatives. Diﬀerent PKC isoen-zymes probably have distinct functions in the processing and mod-ulation of a variety of physiological and pathological responses to external signals. The existence of PKC isoenzymes can, in part, ex-plain why activation of PKC produces varied, and at times disparate, cell-speciﬁc responses.

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polypeptide growth factors (EGF and PDGF) and PMA

may utilize common elements of a signal transduction

pathway leading to the induction of cell proliferation

and VEGF secretion. This suggests that both glioma cell

growth and angiogenesis mediated by VEGF secreted

from proliferating glioma cells might be susceptible to

pharmacologic agents that interfere with these signal

transduction pathways. Further elucidation of these

important pathways and identiﬁcation of control points

may allow the development of focused therapies that

will be toxic to tumor cells but relatively benign with

respect to normal cells.

Acknowledgments

We thank Dr. Alexander Levitzki for the gift of tyrphostins AG18 and AG213 (Hebrew University of Jerusalem, Jerusalem, Israel) and Dr. Jin Kim (Genentech) for the gift of monoclonal VEGF anti-body A4.6.1. This work was supported in part by Grant NSC86-2314-B-002-048 from the National Science Council (Taipei, Taiwan).

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