8-chioro-cyclic amp inhibits autocrine and angiogenic ......vol. 3, 439-448, march 1997 clinical...

Vol. 3, 439-448, March 1997 Clinical Cancer Research 439

8-Chioro-cyclic AMP Inhibits Autocrine and Angiogenic Growth

Factor Production in Human Colorectal and Breast Cancer’

Caterina Bianco, Giampaolo Tortora,

Gustavo Baldassarre, Rosa Caputo,

Gabnelia Fontanini, Silvana Chine,

A. Raffaele Bianco, and Fortunato Ciardiello2

Cattedra di Oncobogia Medica, Dipartimento di Endocnnobogia eOncologia Molecolare e Clinica, Facolt#{224}di Medicina e Chirurgia,Universit#{224}degli Studi di Napoli Federico II, Via S. Pansini 5, 80131Napoli [C. B., G. I., G. B., R. C., A. R. B., F. C.], and Cattedra diAnatomia e Istologia Patobogica, Facolt#{224}di Medicina e Chirurgia,Universit#{224}di Pisa, 56100 Pisa [G. F., S. C.], Italy

ABSTRACT

8-Chboro-cyclic AMP (8-Cl-cAMP) is a cAMP analoguethat specifically down-regulates type I protein kinase A, a

signaling protein directly involved in cell proliferation andneoplastic transformation, and that causes growth inhibition

in a variety of human cancer cell types. In this report, wehave investigated the effects of 8-Cl-cAMP on the expressionof several growth factors in human colon (GEO andLS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines asignificant dose- and time-dependent inhibition in the ex-pression of various endogenous autocrine growth factors,such as transforming growth factor a, amphiregulin, andCRIPTO, and of two angiogenic factors, such as vascularendothelial growth factor and basic fibroblast growth factor,at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all threecell lines to invade a basement membrane matrix in a che-moinvasion assay. Finally, 8-Cl-cAMP-induced inhibition ofGEO tumor growth in nude mice was accompanied by asignificant suppression of transforming growth factor a,

amphiregulin, CRIPTO, basic fibroblast growth factor, andvascular endothelial growth factor production by the tumorcells, and of neoangiogenesis, as detected by factor VIIIstaining of host blood cells. These results demonstrate that

8-Cl-cAMP is a novel anticancer drug that inhibits theproduction of various autocrine and paracrine tumorgrowth factors that are important in sustaining autonomouslocal growth and facilitate invasion and metastasis.

INTRODUCTION

Growth factors are proteins that regulate normal cellular

proliferation and differentiation and that are important in initi-

ating and maintaining neoplastic transformation (1 , 2). Cancer

cells generally exhibit a decreased requirement for exogenous

growth factors as compared to normal cells. The relaxation in

growth factor dependency is due in part to the ability of tumor

cells to synthesize growth factors that can regulate their prolif-

eration through autocrine and paracrine mechanisms. Among

these, the EGF3-related peptides TGF-a, AR, and CRIPTO play

an important role in the pathogenesis of human colon and breast

cancers (3). TGF-a, AR, and CRIPTO are expressed by the

majority of human primary and rnetastatic breast and colorectal

cancers (4, 5). Inhibition of synthesis of these growth factors by

a specific antisense RNA or DNA approach inhibits human

colon and breast cancer cell growth (6-9). Angiogenesis, the

process leading to the formation of new blood vessels, plays a

central role in tumor survival and metastatic spreading (10, 1 1).

Several growth factors, such as bFGF, VEOF, TGF-a, and

P1GF, have been identified as potential regulators of angiogen-

esis (12-14). These proteins are produced by the tumor cells and

can stimulate normal endothelial cell growth through paracrine

mechanisms (10).

cAMP acts in mammalian cells by binding to either of two

distinct isoforms of PKA, termed PKAI and PKAII (15). PKAI

and PKAII have identical catalytic subunits but differ in the

regulatory subunits (defined as RI in PKAI and Rh in PKAII,

respectively; Ref. 15). Differential expression of PKAI and

PKAII has been correlated with cell differentiation and neoplas-

tic transformation. In fact, preferential expression of PKAII is

found in normal nonproliferating tissues and in growth-arrested

cells, whereas enhanced levels of PKAI are detected in tumor

cells and in normal cells following exposure to mitogenic stim-

uli (16). Moreover, we have shown recently that PKAI plays a

role in the transduction of mitogenic signals (17, 18). PKAI is

generally overexpressed in human cancer cell lines and primary

tumors (16). 8-Cl-cAMP is a site-selective cAMP analogue that

specifically inhibits PKAI by facilitating the degradation of the

RIa regulatory subunit while up-regulating at the transcriptional

level Rh expression (19). Decreased expression of RIa induced

by 8-Cl-cAMP treatment is associated with growth inhibition in

various human cancer cell lines in vitro and in vivo (20-24). We

have demonstrated recently in a Phase I clinical trial that 8-Cl-

Received 9/30/96; revised 12/9/96; accepted 12/18/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.1 This study was supported by grants from the Associazione Italiana per

Ia Ricerca sul Cancro and from Progetto Finalizzato ApplicazioniCliniche della Ricerca Oncologica, Consiglio Nazionale delle Ricerche.2 To whom requests for reprints should be addressed. Phone: 39-8 1-7462061; Fax: 39-81-7462066.

3 The abbreviations used are: EGF, epidermal growth factor: TGF,transforming growth factor; AR, amphiregulin; bFGF, basic fibroblastgrowth factor; P1GF, placenta growth factor; VEGF. vascular endothe-hal growth factor; cAMP, cyclic AMP; 8-Cl-cAMP, 8-chboro-cAMP;PKA, cAMP-dependent protein kinase; FBS, fetal bovine serum;HUVE, human umbilical vein endothelial; ECGF, endothelial cellgrowth factor; CM, conditioned medium; mAb, monoclonal antibody;EGFR, epidermal growth factor receptor.

Research. on August 28, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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440 Inhibition of Growth Factor Production by 8-Cl-cAMP

cAMP can be given to cancer patients at doses that allow

patients to reach plasma concentrations within the potential

therapeutic range for growth inhibition (25).

In this study, we have investigated the effects of 8-Cl-

cAMP on the expression and production of several autocrine

and angiogenic growth factors in human colon (GEO and

LS l74T) and breast (MDA-MB468) cancer cell lines. Treatment

with 8-Cl-cAMP determines a dose- and time-dependent down-

regulation in the production of TGF-a, AR, CRIPTO, VEGF,

and bFGF in all cell lines. Moreover, 8-Cl-cAMP treatment

interferes with the ability of these cells to invade the basement

membrane matrix in a chemoinvasion assay. Finally, 8-Cl-

cAMP is effective in inhibiting the growth of well-established

GEO tumor xenografts in nude mice with an almost complete

suppression of cancer cell production of endogenous growth

factors and angiogenic peptides.

MATERIALS AND METHODS

Cell Cultures. GEO and LS 174T cells were maintained

in McCoy’s medium supplemented with 10% heat-inactivated

FBS, 20 mM HEPES (pH 7.4), 100 lU/mb penicillin, 100 �.tg/ml

streptornycin, and 4 msi glutamine (ICN, Irvine, United King-

dom) in a humidified atmosphere of 95% air and 5% CO2 at

37#{176}C.MDA-MB468 cells were grown in a 1:1 (v/v) mixture of

DMEM and Ham’s F12 medium supplemented with 10% heat-

inactivated FBS, 20 mM HEPES (pH 7.4), 100 lU/mb penicillin,

100 �i.g/ml streptomycin, and 4 m�i glutamine (ICN). Early-

passage HUVE cells were kindly provided by Dr. G. Viglietto

(Istituto Nazionale Turnori-Fondazione Pascale, Naples, Italy).

HUVE cells were cultured in DMEM supplemented with 20%

heat-inactivated FBS, 100 p.g/rnl ECGF (Sigma Chemical Co.,

Milan, Italy), 100 p.g/rnl heparmn (Sigma), 20 mM HEPES (pH

7.4), 100 IU/ml penicillin, 100 �i.g/ml streptomycin, and 4 msi

glutarnine.

Monolayer Growth. Cells (104/well) were plated in six

multiwell cluster dishes (Becton Dickinson, Milan, Italy) and

treated every 48 h with different concentrations of 8-Cl-cAMP.

After 4 and 6 days of treatment, the cells were trypsinized and

counted with a hemocytorneter.

Soft Agar Growth. Cells (5 X 103/well) were seeded in

0.5 ml of 0.3% Difco Noble agar (Difco, Detroit, MI) in com-

plete culture medium. This suspension was layered over 0.5 ml

of 0.8% agar-complete medium in 24 multiwell cluster dishes

(Becton Dickinson) and treated with different concentrations of

8-Cl-cAMP. After 12 days, the cells were stained with nitroblue

tetrazolium (Sigma), and colonies were counted as described

previously (7).

Analysis of DNA Fragmentation. To evaluate the po-

tential induction of programmed cell death by 8-Cl-cAMP, cells

were treated every 48 h with various concentrations of 8-Cl-

cAMP. After 1, 3, or 4 days, both adherent and detached cells

were harvested and lysed, and DNA was extracted and electro-

phoresed as described (26).

RNA Isolation and Northern Blotting. Total cellular

RNA was isolated as described previously (23). Twenty p.g of

total RNA were electrophoresed through a denaturating 1.2%

agarose/2.2 M formaldehyde gel. Ethidium bromide staining of

the gels showed that equivalent amounts of RNA were con-

tamed in each lane. The samples were transferred to Hybond-N

membranes (Amersharn Corp., Arlington Heights, IL) and hy-

bridized with the following 32P-labeled human eDNA probes:

TGF-ca (27), CRIPTO (7), VEGF (28), KDR (29), FLT-l (30),

PIGF (31), �3-actin (27), and glyceraldehyde 3’-phosphate de-

hydrogenase (32). The cDNA probes for human VEGF, KDR,

FLT- 1 , and P1GF were kindly provided by Dr. G. Viglietto

(Istituto Nazionale Tumori-Fondazione Pascale).

Western Blotting. Protein lysates or concentrated CM

(50 p�g of total protein/lane) were separated by SDS-PAGE on

10% precast gels (Bio-Rad Laboratories, Milan, Italy), trans-

ferred to nitrocellulose filters, and incubated with either a rabbit

polycbonal anti-human VEGF antiserum (Preprotech, London,

United Kingdom) at 1 :500 dilution or a rabbit polycbonal anti-

human CRIPTO antiserum (kindly provided by Dr. R. Harkins;

Berlex Biosciences, Richmond, CA) at 1 :500 dilution, as de-

scribed previously (7). Irnmunoreactive proteins were visualized

by a chemiluminescence ECL Western blotting kit (Amersharn

Corp.).

Cell Cycle Distribution of HUVE Cells. HUVE cells

(105/dish) were plated in 35-mm dishes (Becton Dickinson) in

complete medium. After 24 h, the cells were washed twice with

PBS and incubated in DMEM containing 10% FBS without

ECGF and heparin. Under these conditions, HUVE cells ceased

to proliferate and underwent accumulation in the G0-G � phases

of the cell cycle within 48 h. HUVE cells were then washed

twice with PBS and incubated for an additional 24 h, either with

complete medium containing ECGF and heparin or with GEO

cell serum-free CM. For preparation of GEO cell CM, GEO

cells were grown in CM with or without different concentrations

of 8-Cl-cAMP for 96 h, rinsed twice with PBS (Sigma Chemical

Co.) and incubated for an additional 24 h in serum-free medium

without 8-Cl-cAMP. CM were then collected and centrifuged at

3000 rpm for 10 mm at 4#{176}Cto eliminate cell debris. CM were

diluted in a 2: 1 (v/v) acetone:serum-free medium and stored

overnight at -20#{176}C.The precipitates were resuspended in PBS

and added to HUVE cells at a final concentration of 200 p.g/ml

total proteins. HUVE cells were trypsinized, washed twice with

PBS, and fixed in 70% ethanol. Cells (106) were incubated for

30 mm at room temperature in 1 ml of propidiurn iodide staining

solution (50 p.g/ml propidium iodide in PBS). DNA and cell

cycle analysis were performed in duplicate with a FACScan

flow cytometer (Becton Dickinson) as reported previously (26).

Chemoinvasion Assay. The chemoinvasion assay was

performed using Biocoat Matrigel 35-mm dish invasion charn-

bers (Becton Dickinson), as described previously (33). GEO,

LS174T, and MDA-MB468 cells (l0� cells/dish) were plated in

35-nim culture dishes (Becton Dickinson) and treated every 48 h

with different concentrations of 8-Cl-cAMP. After 96 h, the

cells were trypsinized, washed twice with PBS, resuspended in

serum-free medium containing 0.1% BSA (Sigma Chemical

Co.), and placed (5 X l0� cells/dish) in the upper compartment

of the invasion chamber. Concentrated serum-free CM obtained

from NIH-3T3 mouse fibroblasts was used as chernoattractant in

the chamber lower compartment (33). After incubation for 18 h

in a humidified atmosphere of 95% air and 5% CO2 at 37#{176}C,the

cells on the upper surface of the filters were mechanically

removed, and the filters were fixed and stained. Ten fields per



Clinical Cancer Research 441

each experimental point were counted, as described previously

(34).

GEO Xenografts in Nude Mice. Five- to 6-week-oldfemale BALB/c athymic (nu+/nu+) mice were purchased from

Charles River Laboratories, Milan, Italy. The research protocol

was approved, and mice were maintained in accordance to

institutional guidelines of the University of Naples Animal Care

Committee. Animals were injected s.c. with i07 GEO cells that

had been resuspended in 200 p.1 of Matrigel (Collaborative

Biomedical Products). After 7 days, when well-established xe-

nografts were detectable with an approximate 0.30-0.35 cm3

tumor size, mice were treated twice weekly with i.p. injections

of different doses of 8-Cl-cAMP for the indicated period of

time. Tumor size was measured using the formula ir/6 X larger

diameter X (smaller diameter)2 (35).

Immunocytochemistry and Evaluation of Immunoper-oxidase Staining. Slides containing GEO, LS174T, andMDA-MB468 cells treated in vitro with different concentrations

of 8-Cl-cAMP or GEO tumor sections from GEO tumor xe-

nografts grown s.c. in nude mice treated i.p. with various doses

of 8-Cl-cAMP were incubated for 30 mm at 20#{176}Cwith methanol

containing 0.3% hydrogen peroxide to block any endogenous

peroxidase activity. After several washes with PBS, the sections

were blocked for 45 mm with 10% goat serum, washed with

PBS, and incubated overnight with the appropriate primary

antibody at 4#{176}C.The following antibodies were used. An anti-

CRIPTO rabbit antiserum raised against a l7-mer peptide cor-

responding to amino acid residues 97-1 13 of the human

CRIPTO protein was used at 1 :400 dilution (5). An anti-AR

rabbit antiserum generated against a 17-mer peptide correspond-

ing to amino acid residues 159-175 of the rat AR protein was

used at 1:200 dilution (5). Both the anti-CRIPTO and the an-

ti-AR rabbit antisera were kindly provided by Dr. W. J. Gullick

(Imperial Cancer Research Fund, London, United Kingdom).

An anti-human TGF-a mouse mAb (Ab-2; Oncogene Science,

Manhasset, NY) was used at 1 : 100 dilution. Each antibody was

specific for TGF-a, AR, or CRIPTO and did not cross-react

with the other two EGF-related peptides (5). An anti-VEGF

rabbit polycbonal antibody (Santa Cruz Biotechnology, Santa

Cruz, CA) was used at I :50 dilution. An anti-bFGF rabbit

polyclonal antibody (Santa Cruz Biotechnology) was used at

1:200 dilution. For proliferation cell nuclear antigen staining,

PC 10 mAb (Novocastra Laboratory, Newcastle, United King-

dom) was used at 1:200 dilution. To detect factor VIII, a rabbit

polyclonal antibody (Dakopatts, Gbostrup, Denmark) was used

at 1: 1000 dilution. Sections were then washed three times with

PBS and treated with an appropriate secondary biotinylated goat

antibody (1:200 dilution; Vectastain ABC kit; Vector Labora-

tory, Burlingame, CA) for 30 mm. Following several washes

with PBS, the slides were reacted for 30 mm with avidin

dehydrogenase-biotinylated horseradish peroxidase H complex,

rinsed twice in PBS, and incubated for 2 mm in 0.05% diami-

nobenzidine and in 0.01% hydrogen peroxide. The slides were

then rinsed in distilled water, counterstained with hematoxylin,

and mounted. Nonspecific staining was evaluated for each spec-

imen using either a similar concentration of preimmune rabbit

serum or IgG or by adsorbing the primary antibody with the

appropriate immunogenic peptide. Both intensity of staining (-

to + + +) and percentage of immunopositive cells were scored.

Two slides for each sample were evaluated by an investigator

(G. F.) blinded to the treatment code. At least 1000 cancer cells

per slide were counted and scored. Specific staining was semi-

quantitated by assigning a score based on color intensity of the

brown diaminobenzidine precipitate (+ , light brown staining;

+ + , a moderately brown color; + + + , an intense brown color),

as described previously (8).

RESULTS

We have shown previously that 8-Cl-cAMP determines

growth inhibition and differentiation in a wide variety of human

cancer cell lines in vitro and in vivo (20-24). Treatment with

8-Cl-cAMP for 4 or 6 days induced a dose-dependent inhibition

of monolayer growth in human breast (MDA-MB468) and colon

(LSI74T and GEO) cancer cell lines, with an IC50 of 10, 5, and

20 p.M. respectively. A comparable dose-dependent inhibition of

soft agar cloning efficiency was caused in all three cell lines by

the treatment with 8-Cl-cAMP (data not shown). The growth-

inhibitory effect induced by 8-Cl-cAMP was cytostatic and not

cytotoxic, as assessed by trypan blue dye exclusion. Further-

more, no evidence of apoptotic cell death was detected in the

three cancer cell lines treated with 8-Cl-cAMP at concentrations

up to 25 p.M for 6 days, as evaluated by gel electrophoresis

analysis of chromatin fragmentation into nucleosorne ladders

(data not shown; Ref. 26).

We next investigated the effects of 8-Cl-cAMP treatment

on the expression of several growth factors involved in tumor

growth and progression. GEO, LS I 74T, and MDA-MB648 cells

were treated with different concentrations of 8-Cl-cAMP for 6,

24, and 96 h, and then total RNA was isolated and analyzed by

Northern blotting. Control nontreated GEO and MDA-MB468

cells expressed specific TGF-a rnRNA transcripts of 4.8 kb and

of 4.8 and 1.6 kb, respectively (Fig. 1). Treatment with 8-Cl-

cAMP caused a marked reduction in TGF-a mRNA levels that

was time- and dose-dependent. In fact, an approximately 40-

60% inhibition in TGF-a mRNA expression was already detect-

able within 6 h of treatment with 10 p.M 8-Cl-cAMP in both

cancer cell lines (Fig. 1). An almost complete down-regulation

in TGF-a mRNA expression was observed in GEO and MDA-

MB468 cells treated with 25 p.M 8-Cl-cAMP for 96 h. A similar

effect on TGF-a mRNA levels was observed in LSI74T cells

treated with 8-Cl-cAMP (data not shown). The expression of

CRIPTO mRNA was also significantly reduced in GEO and

LS174T cells treated with the cAMP analogue (Fig. 2). In fact,

a reduction of 60-90% in CRIPTO mRNA expression was

detected after treatment of GEO and LS174T cells with 25 p.M

8-Cl-cAMP for 4 days as compared to control nontreated cells.

A 3.9-kb VEGF-specific mRNA was detectable in GEO,

LS174T, and MDA-MB468 cells (Fig. 3). In all three cell lines,

8-Cl-cAMP treatment determined a rapid and marked time- and

dose-dependent inhibition in VEGF mRNA levels as compared

to nontreated cells. In fact, an almost complete suppression in

VEGF mRNA expression was observed in all three human

cancer cell lines within 24 h of treatment with 10 p.M 8-Cl-

cAMP (Fig. 3). VEGF interacts with two high affinity distinct

cell membrane receptors, FLT- 1 and KDR, which possess an

intracellular tyrosine kinase activity (29-30). These receptors

are specifically expressed on endothelial cells and on some



A

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B

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I 234567

Fig. I Northern blot analysis of TGF-a mRNA expression in GEO (A)

and MDA-MB468 (B) cells. A: Lane 1, control nontreated GEO cells;

Lanes 2-4, GEO cells treated with 10 p.M 8-Cl-cAMP for 6, 24, and96 h, respectively; Lanes 5-7, GEO cells treated with 25 p.M 8-Cl-cAMPfor 6, 24, and 96 h, respectively. B: Lane 1, control nontreated MDA-MB468 cells; Lanes 2-4, MDA-MB468 cells treated with 10 p.M

8-Cl-cAMP for 6, 24, and 96 h, respectively; Lanes 5-7, MDA-MB468cells treated with 25 p.M 8-Cl-cAMP for 6, 24, and 96 h, respectively.

cancer cell lines (36). Recent studies have suggested that FLT-l

and KDR have different signal transduction properties and that

KDR may be the receptor involved in the regulation of neoan-

giogenesis (37). Both KDR and FLT-l rnRNAs were not de-

tectable in the three cancer cell lines before or after treatment

with 8-Cl-cAMP (data not shown). We next analyzed the

mRNA expression of PIGF, another angiogenic factor that is

closely related to VEGF (14). It has been suggested recently that

the activity of VEGF can be potentiated by P1GF (38). However,

no P1GF-specific mRNA transcript could be detected in the

three cancer cell lines, regardless of 8-Cl-cAMP treatment (data

not shown). Finally, as control we performed Northern blotting

analysis of G3PD mRNA expression in GEO, LS174T, and

MDA-MB468 cells treated with 8-Cl-cAMP. No significant

changes in G3PD mRNA (Fig. 4) or �3-actin (data not shown)

were observed in all three cell lines following 8-Cl-cAMP

treatment.

To determine whether the significant reduction in rnRNA

expression of mitogenic and angiogenic factors induced by

8-Cl-cAMP treatment is also accompanied by a decrease in the

synthesis of the corresponding proteins, Western blotting and/or

immunocytochemistiy were performed on all three cancer cell

lines that were treated with different concentrations of the

cAMP analogue. We first evaluated by immunoblotting the

2.2 kb - �. � *, �

I 234567

Fig. 2 Northern blot analysis of CRIPTO mRNA expression in GEO

(A) and LS174T (B) cells. A: Lane 1, control nontreated GEO cells;Lanes 2-4, GEO cells treated with 10 p.M 8-Cl-cAMP for 6, 24, and96 h, respectively; Lanes 5-7, GEO cells treated with 25 p.M 8-Cl-cAMPfor 6, 24, and 96 h, respectively. B: Lane 1, control nontreated LSI74Tcells; Lanes 2-4, LS174T cells treated with 10 p.M 8-Cl-cAMP for 6, 24,and 96 h, respectively; Lanes 5-7, LS174T cells treated with 25 p.M

8-Cl-cAMP for 6, 24, and 96 h, respectively.

levels of VEGF in the CM obtained from GEO, LS 174T, and

MDA-MB468 cells. CM were collected from control nontreated

cells and from cells treated for 4 or 6 days with 10 or 25 p.M

8-Cl-cAMP. VEGF may exist as four different isoforms con-

sisting of 121, 165, 189, and 20] amino acids, respectively (28).

VEGF165 is the more frequent isoform secreted by a variety of

normal and transformed cells, which has been implicated in

inducing angiogenesis (28). A major VEGF immunoreactive

species with an apparent molecular weight of 45,000, which is

consistent with VEGF165, was detected in all three tumor cell

lines (Fig. 5). An approximate 60-70% reduction in VEGF

protein levels was observed in GEO, LS174T, and MDA-

MB468 cells treated with 10 p.M 8-Cl-cAMP for 4 or 6 days as

compared to nontreated control cells. A more marked down-

regulation in VEGF protein levels was found in cells treated

with 25 p.M 8-Cl-cAMP for 4 or 6 days (Fig. 5). 8-Cl-cAMP

treatment determined also a significant inhibition in the expres-

sion of CRIPTO protein. In fact, as evaluated by Western

blotting on cell lysates, CRIPTO protein levels were reduced by

50-80% after treatment with 25 p.M 8-Cl-cAMP for 2 or 4 days

in GEO cells (Fig. 6) in LS174T and in MDA-MB468 cells (data

not shown). We next performed an immunocytochemical anal-

ysis for TGF-a, AR, CRIPTO, VEGF, and bFGF protein ex-

pression in all three cancer cell lines treated with 10 or 25 p.M

8-Cl-cAMP for 4 days. A mostly cytoplasmic-specific immu-

nostaining for all growth factors was observed in the majority of



A

3.9kb- #{149}#{248}#{216}ii�� #{149}

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I 234567Fig. 3 Northern blot analysis of VEGF mRNA expression in GEO (A).

LSI74T (B), and MDA-MB468 (C) cells. A: Lane 1, control nontreated

GEO cells: Lanes 2-4. GEO cells treated with 5 p.M 8-Cl-cAMP for 6.

24, and 96 h, respectively: Lanes 5-7. GEO cells treated with 10 p.si

8-Cl-cAMP for 6, 24, and 96 h. respectively: Lanes 8-10. GEO cellstreated with 25 p.M 8-Cl-cAMP for 6. 24, and 96 h. respectively. B: Lane

1. control nontreated LSI74T cells: Lanes 2-4, LS174I cells treatedwith 10 p.M 8-Cl-cAMP for 6, 24, and 96 h. respectively: Lanes 5-7.

L51741 cells treated with 25 p.51 8-Cl-cAMP ftr 6. 24. and 96 h.respectively. C: Lane I. control nontreated MDA-MB468 cells: Lanes

2-4. MDA-MB468 cells treated with 10 p.M 8-Cl-cAMP for 6, 24, and96 h, respectively: Lanes 5-7, MDA-MB468 cells treated with 25 p.M

8-Cl-cAMP for 6, 24, and 96 h, respectively.

GEO, LS 174T, and MDA-MB468 cells (Table I and data not

shown). A substantial dose-dependent reduction in both the

percentage of immunoreactive cells and the intensity of specific

cell staining for all growth factors was found in the three cancer

cell lines with a weak expression of each protein in only 5-25%

tumor cells after treatment with 25 p.M 8-Cl-cAMP for 4 days

(Table I).

Because 8-Cl-cAMP treatment was able to significantly

inhibit the production of various growth factors with angiogenic

activity, such as VEGF, bFGF, and TGF-a. we evaluated if this

reduction is biologically relevant using an in vitro assay of

endothelial cell proliferation. HUVE cells require a mixture of

1.4 kb- �1234567

Fig. 4 Northern blot analysis ofglyceraldehyde 3-phosphate dehydro-genase mRNA expression in GEO (A), LS174T (B). and MDA-MB468(C) cells. A: Lane 1, control nontreated GEO cells: Lanes 2-4. GEOcells treated with 10 p.M 8-Cl-cAMP for 6, 24, and 96 h. respectively:Lanes 5-7. GEO cells treated with 25 p.M 8-Cl-cAMP for 6. 24. and96 h, respectively. B: Lane I. control nontreated LSI74T cells: Lanes

2-4, LSI74T cells treated with 10 p.M 8-Cl-cAMP ftr 6, 24, and 96 h.respectively: Lanes 5-7. LS 174T cells treated with 25 p.si 8-Cl-cAMPfor 6, 24. and 96 h. respectively. C: Lane 1. control nontreated MDA-MB468 cells: Lanes 2-4. MDA-MB468 cells treated with 10 p.M

8-Cl-cAMP for 6. 24, and 96 h. respectively: Lanes 5-7. MDA-MB468cells treated with 25 p.M 8-Cl-cAMP for 6, 24. and 96 h. respectively.

20% FBS, heparin, and ECGF for optimal proliferation. HUVE

cells cultured for 24 h in a medium containing 10% FBS without

heparin and ECGF became quiescent and accumulated in the

G�-G1 phases ofthe cell cycle (Table 2). Incubation of quiescent

HUVE cells for 24 h with concentrated CM obtained from

control nontreated GEO cells stimulated their progression

through the cell cycle because the percentage of HUVE cells in

S phase raised from 5 to 20%. In contrast, incubation with

concentrated CM obtained from GEO cells treated for 4 days

with 10 or 25 p.M 8-Cl-cAMP determined only a moderate

increase in the percentage of HUVE cells in S phase ( I I and 8%,

respectively). These results suggest that 8-Cl-cAMP treatment

of GEO cells causes a significant inhibition in the secretion of

biologically active angiogenic growth factors.

Tumor cell invasion of the basement membrane is a critical

step in the multistage process that leads to metastatic spreading

(39). The invasive behavior of GEO, LS174T, and MDA-

MB468 cells was evaluated in a chemoinvasion assay using

Boyden chambers coated with Matrigel, a mixture of basement

membrane components (33). CM from mouse NIH-3T3 fibro-

blasts, which contains defined (fibronectin and collagen) as well

as undefined chemotactic factors, was used as chemoattractant



B

1 2 3 4 5Fig. 6 Western blot analysis of CRIPTO protein expression in GEOcells. Lane 1, control nontreated GEO cells; Lanes 2 and 3, GEO cellstreated for 2 or 4 days with 10 p.M 8-Cl-cAMP; Lanes 4 and 5, GEOcells treated for 2 or 4 days with 25 p.M 8-Cl-cAMP.


A

45 kDa-.�

I’�1��W� *

34 kDa - �-R � ��---i:L �

45 kDa-

C P�?!T�_45 kDa - _,

# -.

I 2345Fig. 5 Western blot analysis of VEGF protein expression in the con-centrated CM from GEO (A), LSI74T (B), and MDA-MB468 (C) cells.A: Lane 1, control nontreated GEO cells; Lanes 2 and 3, GEO cellstreated for 4 or 6 days with 10 p.M 8-Cl-cAMP; Lanes 4 and 5, GEOcells treated for 4 or 6 days with 25 p.M 8-Cl-cAMP. B: Lane 1, controlnontreated LS174T cells; Lanes 2 and 3, LSI74T cells treated for 4 or6 days with 10 p.M 8-Cl-cAMP; Lanes 4 and 5, LS174T cells treated for4 or 6 days with 25 p.M 8-Cl-cAMP. C: Lane 1, control nontreatedMDA-MB468 cells; Lanes 2 and 3, MDA-MB468 cells treated for 4 or6 days with 10 p.M 8-Cl-cAMP; Lanes 4 and 5, MDA-MB468 cellstreated for 4 or 6 days with 25 p.M 8-Cl-cAMP.

(40). GEO, LS l74T, and MDA-MB468 cells exhibited the abil-

ity to invade the Matrigel-coated filter (approximately 180, 150,

and 200 invasive cells/field, respectively; Fig. 7). A dose-de-

pendent 40 to 60% inhibition of the invasive activity was

observed in all three cancer cell lines after treatment with 10 or

25 p.M 8-Cl-cAMP for 4 days.

We finally investigated the in vivo antitumor activity of

8-Cl-cAMP. GEO cells (l0�) were injected s.c. into the dorsal

flank of nude mice. After 1 week, when established GEO

xenografts were palpable with a tumor size of approximately

0.30-0.35 cm3, the animals were treated twice weekly i.p. for 4

weeks with different concentrations of 8-Cl-cAMP (Fig. 8A).

Both control untreated and 8-Cl-cAMP-treated GEO tumor

groups grew as moderately differentiated human colon adeno-

carcinomas (data not shown). 8-Cl-cAMP determined a dose-

dependent inhibition of GEO tumor growth with an almost

complete suppression of tumor cell proliferation, as assessed by

‘-fl ,,w proliferation cell nuclear antigen staining at 2 mg/dose 8-Cl-

cAMP (Fig. 8). The in vivo growth-inhibitory effect of 8-Cl-

cAMP on GEO tumors was cytostatic rather than cytotoxic. In

fact, GEO tumors resumed a growth rate comparable to controls

within 10 to 14 days following termination of 8-Cl-cAMP

treatment (data not shown). To ascertain whether treatment with

8-Cl-cAMP was able to interfere with the production of growth

factors also in vivo, immunohistochemical evaluation of the

expression of TGF-a, AR, CRIPTO, bFGF, and VEGF was

performed on GEO tumors at the end of the 4 weeks of treat-

rnent. A marked reduction in both the percentage of positive

GEO cells and the average intensity of staining for all five

growth factors were observed (Fig. 8B). The growth inhibition

induced by 8-Cl-cAMP treatment was paralleled by a substantial

inhibition of angiogenesis, as detected by factor VHI staining of

mouse blood vessels.

DISCUSSION

Growth factors can regulate cancer initiation and progres-

sion through several mechanisms. These include: autonomous

uncontrolled growth as a result of the autocrine production of

endogenous growth factors by tumor cells; loss of sensitivity to

growth inhibitors; and stimulation of tumor vascularization due

to paracrine stimulation of normal host endothelial and mesen-

chymal cells by angiogenic growth factors secreted by cancer

cells (1, 2).

The EGF family of growth factors, such as EGF, TGF-a,

AR, and CRIPTO, plays a significant role in the development of

human breast and coborectal cancer (3). We have demonstrated

previously that inhibition of TGF-a, AR, or CRIPTO by anti-

sense oligonucleotide treatment or by infection with antisense

retroviral expression vectors is able to significantly block the

growth of human breast and colon carcinoma cell lines (6-9).



V4)

84)>C,)

C

.8Ez

250

200 -

150 -

100 -

50 -

0

CEO LSI74T MDA-MB468

bFGF and VEGF are potent angiogenic peptides that are

secreted in several types of human malignancies (12-13, 41). It

has been suggested that tumor cells secreting these growth

factors may have a growth advantage in vivo. In this regard,

overexpression of VEGF confers the ability of Chinese hamster

ovary cells to form vascularized tumors in nude mice, whereas

it does not have growth-stimulatory activity in vitro (42). A

more direct evidence of the VEGF role in tumor-induced an-

giogenesis has been obtained with the use of specific anti-VEGF

neutralizing mAbs. In fact, treatment of cancer cells with anti-

VEGF neutralizing antibodies does not affect their proliferation

in vitro, whereas it exerts a potent inhibitory effect on their

growth as xenografts in nude mice (43). Moreover, treatment of

nude mice bearing human colon carcinoma xenografts with an

anti-VEGF mAb results in a marked decrease in the number and

size of liver metastases (44). Similarly, tumor cells overexpress-

ing bFGF become more aggressive and highly metastatic in vivo

(45). However, treatment with an anti-bFGF neutralizing anti-

body induces a significant inhibition of primary and metastatic

tumor growth (45). Therefore, interference with the production

of angiogenic factors or with the signaling pathways regulated

by angiogenic factors has been proposed as a form of cancer

therapy that can be combined with more conventional cytotoxic

anticancer drugs (46, 47).

The cytostatic antiproliferative effects of 8-Cl-cAMP, a

specific cAMP analogue that inhibits PKAI expression and


Table I Growth factor expre ssion in human cancer cell lines following 8-C 1-cAMP treatment”

TGF-a AR CRIPTO VEGF bFGF

GEO cellsControl 75%

(+++)

70%(+++)

65%(+++)

50%(+++)

65%(+++)

8-Cl-cAMP10 p.M 25%

(++)

30%

(+)

25%

(++)

40%

(+)

35%

(++)

25 p.M 15%

(+)

5%

(+)

5%

(+)

20%

(+)

10%

(+)

LS174T cells

Control 60%(+++)

65%(+++)

75%(+++)

75%

(+++)

80%(+++)

8-Cl-cAMPlOp.M 25%

(++)

15%(+)

40%(++)

50%(+)

55%(++)

25 p.M 15%

(+)

5%

(+)

25%

(+)

10%

(+)

20%

(+)

MDA-MB468 cellsControl 70%

(+++)

65%(+++)

80%(+++)

60%(+++)

65%(+++)

8-Cl-cAMP10 p.M 20%

(+)

35%

(+)

60%

(++)

40%

(+)

40%

(++)

25 p.M 10%(+)

15%(+)

20%(+)

20%(+)

20%(+)

a The percentage of positive tumor cells and the average intensity of specific immunostaining was determined as described in “Materials and

Methods.”

Table 2 Cell cycle distribution of HUVE cells following incubationwith concentrated CM obtained from 8-Cl-cAMP-treated GEO cells

Incubation of HUVE cells with concentrated CM obtained fromGEO colon carcinoma cells treated with the indicated concentrations of8-Cl-cAMP was performed as described in “Materials and Methods.”Cell cycle distribution was assessed using propidium iodide staining andanalyzed with a FACScan flow cytometer. Data represent the average oftwo separate experiments, each performed in duplicate. SD was less than5%.

Cell eye le distribut ion (%)

G0-G1 S G2-M

Untreated 88% 5% 7%+GEO CM 74% 20% 6%+GEOCM+ 85% 11% 4%

8-Cl-cAMP (10 p.M)+GEOCM+ 86% 8% 6%

8-Cl-cAMP (25 p.M)

Fig. 7 Chemoinvasion assay of GEO, LS 1741, and MDA-MB468cells. Shown are control nontreated cells (�) and cells treated with 10p.M (�) or with 25 p.M (�) 8-Cl-cAMP for 4 days. The cell migrationassay through a Matrigel-coated filter was performed as described in“Materials and Methods.” Values represent the average of two differentexperiments, each performed in triplicate. SD was less than 10% foreach point.



7

6

5

4

3

2

I

0

A

EC)

a)EI

B

0 5 10 15 20 25 30 35 40

Days


4 F. Ciardiello and G. Tortora, unpublished observations.

PCNA TOFu AR CRIPTO VEGF bFGF FactorVIII

Control 65% 8O#{176}.’�(+++)

70%(+4+)

65%(+++)

40’.’o(+++)

50%(+-t-+)

+++

8-Cl-cAMP:o�5 mg/dose 63% 45%

(++)35%(++)

60%(+++)

30%(4-f)

20%(+4)

++

I mg/dose 35% 25%(+)

20%(++)

40%(4)

20%(+)

10%(4)

+

2mg/dose 15% 15%(+)

15%(+)

10%(4)

10%(+)

5%(4)

+1.

Fig. 8 A, antitumor activity of 8-Cl-cAMP treatment on establishedGEO human colon carcinoma xenografts. The mice were injected s.c.

into the dorsal flank with l0� GEO cells as described in “Materials andMethods.” After 7 days (average tumor size, 0.30-0.35 cm3) the micewere treated i.p. twice weekly with 8-Cl-cAMP at the following doses:L�, 0.25 mg/dose; 0, 0.5 mg/dose; U, 1 mg/dose; and E, 2 mg/dose for

4 weeks. #{149},control. Each group consisted of 15 mice. Bars, SD. B,immunohistochemical evaluation of growth factor expression and ofangiogenesis in GEO xenografts. Two mice per group were killed, andtumors were excised on day 35 after GEO cell injection. The percentageof positive cancer cells and the average intensity of specific immuno-staining were determined as described in “Materials and Methods.”

activity, have been widely demonstrated in a variety of human

cancer cell types (20-24). Furthermore, 8-Cl-cAMP has re-

cently entered clinical evaluation in cancer patients. In fact, a

Phase I trial in cancer patients refractory to standard therapies

has shown that 8-Cl-cAMP can be administered in several

cycles of continuous iv. infusion at doses that allow the drug

to reach plasma concentrations within a range potentially effec-

tive for tumor growth inhibition (25).

The results of this study demonstrate for the first time that

the antiproliferative effect of 8-Cl-cAMP is accompanied by a

marked inhibition in the synthesis and secretion of several

growth factors such as TGF-a, AR, CRIPTO, VEGF, and bFGF,

which are important modulators through autocrine and paracrine

pathways of human cancer cell growth and of tumor-induced

neoangiogenesis. The 8-Cl-cAMP-induced inhibition of growth

factors and angiogenic peptides in human colon and breast

cancer cells is time and dose dependent and probably occurs at

a transcriptional level. In fact, a significant reduction in mRNA

levels for these growth factors is detected within 6 to 24 h of

8-Cl-cAMP treatment and precedes the reduction in protein

levels. These results are in agreement and further extend our

previous observation of a specific down-regulation of TGF-a

mRNA and protein in mouse mammary epithelial cells overex-

pressing the TGF-a gene following treatment with 8-Cl-cAMP

(23). The reduction in the production of various peptide growth

factors induced by 8-Cl-cAMP treatment does not appear as a

nonspecific phenomenon due to cell growth inhibition. In fact, a

2- to 4-fold increase that is time and dose dependent in the

expression of the EGFR on the cell surface of GEO, LS174T,

and MDA-MB468 cells could be observed following treatment

with 8-Cl-cAMP (26).�

The results of the present study suggest that inhibition of

PKAI expression and activity by 8-Cl-cAMP treatment blocks

the autocrine mitogenic signaling sustained by EGF-related

growth factors. This has potential clinical relevance because

inhibition of TGF-a, AR, and CRIPTO, which are frequently

overexpressed in human colorectal and breast cancer as corn-

pared to normal colorectal and breast epitheliurn, may represent

an important goal of novel biological cancer therapies. In this

respect, several studies have demonstrated that blocking the

activation of EGFR, that is the specific receptor for EGF,

TGF-a and AR, by the use of anti-EGFR mAbs inhibits the in

vitro and in vivo growth of several human cancer cell lines (48,

49). We have demonstrated recently that 8-Cl-cAMP and an

anti-EGFR blocking mAb can be used in combination and

determine a synergistic growth-inhibitory effect on human colon

and breast cancer cell lines (26).

The inhibition in the synthesis and secretion of angiogenic

growth factors such as VEGF and bFGF obtained in 8-Cl-cAMP

treated cancer cells is functionally and biologically relevant. In

fact, analysis of the cell cycle distribution of growth factor-

depleted quiescent HUVE cells exposed to GEO cell CM re-

veals that GEO cells secrete factors that are able to stimulate

normal endothelial cell proliferation. In contrast, treatment of

GEO cells with 8-Cl-cAMP significantly inhibits the secretion

of HUVE cell growth-stimulating factors.

The decreased production of autocrine and paracrine

growth factors induced by 8-Cl-cAMP treatment is paralleled by

the reduced ability of the tumor cells to invade the basement

membrane in a chemoinvasion assay. In fact, a dose-dependent

40-60% inhibition in the capacity of GEO, LS 174T, and MDA-

MB468 cells to penetrate the Matrigel-coated filters is observed

after 8-Cl-cAMP treatment. These results suggest that 8-Cl-

cAMP may also interfere with the invasive behavior of cancer

cells, which is a critical event in the multistep process that leads

to the formation of distant metastases. Further studies will be

necessary to determine whether 8-Cl-cAMP may inhibit the

tumor cell production of various proteolytic enzymes, such as

type IV collagenase, that degrade basement membrane compo-

nents and that are involved in conferring the invasive phenotype

(39).

Finally, 8-Cl-cAMP is highly effective in inhibiting the

growth of GEO colon carcinoma cells in vivo. 8-Cl-cAMP

treatment for 4 weeks results in a dose-dependent suppres-




sion of GEO xenografts growth in the absence of toxicity in the

nude mice. The in vivo suppression of tumor growth is accom-

panied by a significant reduction in the expression of TGF-a,

AR, CRIPTO, VEGF, and bFGF in tumor cells and by a strong

inhibition of host neoangiogenesis, as measured by factor VIII

staining of endothelial cells.

In summary, the results of the present study demonstrate

that 8-Cl-cAMP is a novel anticancer drug with effects on the

production of several growth and angiogenic factors that have

potential therapeutic relevance. In fact, inhibition of endogenous

autocrine and paracrine growth factors that sustain autonomous

local growth and facilitate rnetastatic spreading of tumor cells is

obtained by treatment with an agent that has different intracel-

lular targets and that does not antagonize the effect of cytotoxic

antineoplastic drugs. In this respect, long-term treatment with

8-Cl-cAMP can be proposed in adjunct to conventional chem-

otherapy in cancer patients.

ACKNOWLEDGMENTS

We thank G. Borriello for the excellent technical assistance, Dr. P.

Tagliaferri for helpful discussions, and Dr. G. Viglietto for kindly

providing early-passage HUVE cells and the eDNA probes for humanVEGF, KDR, FLI- 1 , and PIGF.

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1997;3:439-448. Clin Cancer Res C Bianco, G Tortora, G Baldassarre, et al. factor production in human colorectal and breast cancer.8-Chloro-cyclic AMP inhibits autocrine and angiogenic growth

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