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Benzodiazepine receptors and diazepam-binding inhibitor in human cerebral tumors.

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Benzodiazepine Receptors and
Diazepam-Binding Ihbitor in Human
Cerebral Tumors
C. Ferrarese, MD, PhD, I. Appollonio, MD, M. Frigo, MD, S. M. Gaini, MD, R. Piolti, MD,
and L. Frattola, MD
Benzodiazepines can regulate neoplastic growth and immune response through specific peripheral benzodiazepine
receptors. We investigated the presence of peripheral and classic central benzodiazepine receptors as well as diazepambinding inhibitor, an endogenous ligand of both types of receptors, in different human cerebral tumors. Peripheral
benzodiazepine receptors were present in all the tumor types studied, whereas central benzodiazepine receptors and
diazepam-binding inhibitor were detectable in astrocytomas and glioblastomas and undetectable in meningiomas,
neurinomas, and metastases. The role of diazepam-binding inhibitor and of the different benzodiazepine receptors in
neoplastic cells is still to be defined.
Ferrarese C, Appollonio I, Frigo M, Gaini SM, Piolti R, Frattola L. Benzodiazepine receptors and
diazepam-binding inhibitor in human cerebral tumors. Ann Neurol 1989;26:564-568
Although benzodiazepines are extensively used clinically for their well-known anxiolytic, hypnotic, and
anticonvulsant activities, recent experimental evidence
has shown that they may also have other effects, such
as regulation of neoplastic growth and of the immune
response. In fact, they affect cell proliferation [l] and
stimulate monocyte chemotaxis [2}. Whereas their
clinical use is based on a central nervous system action
mediated through the classic central benzodiazepine
receptor (CBR) sites, which allostericdy modulate
gamma-aminobutyric acid-activated transmission, the
described experimental activities are based on interaction with a second class of benzodiazepine receptors,
called peripheral benzodiazepine receptors (PBRs) because they are present in several non-neuronal tissues.
Recently, an endogenous polypeptide capable of displacing benzodiazepine binding to both CBRs and
PBRs has been purified from the brain and liver of
different species and designated as diazepam-binding
inhibitor (DBI) {3]. There is evidence that in neurons
DBI can act as the endogenous modulator of GABAergic transmission {4]. On the contrary, very little is
known about non-neuronal DBI, which is highly concentrated in liver, kidney, and different endocrine tissues, where PBRs are also present in high concentrations [5}.
DBI and PBRs have been detected in primary c d tures of rat cerebral astrocytes; DBI is not released
from these cells but appears to act intracellularly by
binding to PBRs located on mitochondrial and nuclear
membranes [GI. Moreover, PBRs have been found in
mouse glioblastoma [7}, C6 ghoma [S], and NB41A3
neuroblastoma [9] and in human glioma cell line [10);
stimulation of these receptors may exert an antiproliferative action Ell}. These recent findings suggested
the possibility that benzodiazepine receptors and DBI
might play a role in cerebral neoplasia and led us to
investigate the presence of the two types of benzodiazepine receptors and of DBI in different human
cerebral tumors.
From the Neurological Clinic, University of Milan, Monza, Italy.
Address correspondence to Dr Ferrarese, Clinica Neurologica, Ospedale s. Gerardo, via Donizeni, 106, 20052 ~ o m (MI),
a
Italy.
~
~ N~~ 15,
~ 1988,~and in irevised ~form ~~b
~ 13, 1989.
d
cepted for publication Mar 29, 1989.
Materials and Methods
Pathological tissues were removed at surgery from patients
with cerebral tumor (18 men and 15 women; age range, 1376 years). For histopathological studies the tissues were
taken from various portions of the neoplasm, fixed in 10%
formalin, and embedded in paraffin. Tumor samples were
examined using special staining techniques for paraffin sections including hematoxylin-eosin,phosphotungstic acid and
hematoxylin, Masson stain, van Gieson's method, Gomori's
reticulum technique, periodic acid-Schiff stain with and
without diastase, and the Cajal technique. Tumors were
classified according to Zulch [12). Using this classification,
we separated grade 1 and 2 astrocytomas from glioblastomas
as expressions of different degrees of malignancy. Six normal
brain tissues were taken from patients who were operated on
for aneurysm (3 men and 3 women; age range, 35-64 years);
none of these patients had received therapy within the previous 15 days. The tissues of this group of patients were exam-
5 6 4 Copyright 0 1989 by the American Neurological Association
ined with the same procedures used for tumoral tissues and
were histologically normal.
Biochemical Studies
For biochemical studies the tissues were frozen immediately
in liquid nitrogen and preserved at - 80°C until use.
RAbIOIMMUNOASSAY FOR DIAZEPAM-BINDING INHIBITOR.
DBI-like immunoreactivity (DBI-LI) was determined in
bioptic tissue using a previously characterized rabbit antiserum [13}. For peptide extraction tissues were homogenized in 10 volumes (wh) of 1 N acetic acid by Polytron
(Brinkman Instruments, Westbury, NY), heated at 95°C for
10 minutes, and centrifuged at 20,000 g for 10 minutes.
Aliquots of the supernatants were lyophilized and resuspended in 250 t.1 of sodium phosphate buffer, p H 7.4,
containing: '251-labeled DBI (30,000 cpm, specific activity
200 Cdmmol; Bolton Hunter, Amersham, Buckinghamshire, UK) as tracer; 0.2 M sodium chloride; 12.5 mg of
bovine serum albumin; and 0.3 mg of calf thymus histone
type 11. Samples were incubated with the antiserum diluted
1:20,000, and the radioimmunoassay (RIA) was carried out
as previously described [14).
The specificity of the immunoreactive material detected
was determined by incubation of different aliquots of tissue
extracts that paralleled the standard curve and by the use of
reverse-phase high-pressure liquid chromatography (HPLC).
To characterize DBI-LI in bioptic
tissues, acetic acid extracts (approximately 2 mg of protein)
were filtered through 0.45-pm Millipore filters and applied
to a reverse-phase t.Bondapack-Cl8 HPLC column (30 cm
X 5 mm; Waters Associates, Milford, MA). A 0 to 60%
acetonitrile gradient was run over a 60-minute period with a
flow rate of 1 mumin. One-milliliter fractions were collected,
and different aliquots were lyophilized for DBI RIA.
REVERSE-PHASE HPLC.
Radio Receptor-Binding Assays
PBRs were studied using 3H-PK 11175 (85.0 Ciimmol; New
England Nuclear, Boswn, MA), an isoquinoline carboxamide derivative that binds to these receptors selectively,
with saturability and with high affinity Cl5, 161. For binding
assays, bioptic tissues were homogenized in 20 volumes (wiv)
of 50 mM Tris HCI, p H 7.4, centrifuged at 20,000 g, and the
pellet resuspended in Tris buffer up to a protein concentration of 1 mglml. Fifty-microliter aliquots were incubated with
40 pl of 3H-PK 11195, 5 nM final concentration, 10 pl of
10% dimethylsulfoxide (DMSO) or 10 pl of 10 pM unlabeled PK 11195 in 10% DMSO to assess the extent of
nonspechc binding. The mixtures were incubated for 1 hour
in an ice-water bath. Incubation was terminated by rapid
addition of 2 ml of ice-cold Tris buffer, followed immediately by vacuum filtration through Whatman GF/C
glass-fiber filters (Maidstone, England) which were presoaked in ice-cold Tris buffer. The filters were washed with 6
ml of ice-cold Tris buffer extracted in Aqwassure (Dupont,
Boston, MA) and counted in a scintillation beta-counter
(Beckman LS 1701, Berkeley, CA) with 60% efficiency.
In order to study CBRs selectively, 3H-flumazenil (75 Cli
mmol; New England Nuclear), a specific antagonist of these
sites, was employed with the same concentrations (5 nM)
and the same binding conditions as the PBR assay. Cold
flumazenil, 10 FM (Roche, Geneva, Switzerland), was used
to determine nonspecific binding.
PROTEIN AND DNA DETERMINATION. Fifty-microliter aliquots of acetic acid extracts and tissue homogenates were
used for protein determination, performed according to
Lowry's method.
DNA was determined according to the method of
Maniaus and colleagues { 17), with minor modifications.
Briefly, tissue homogenates were incubated at 37°C overnight in 10 mM Tris, p H 7.5, 100 mM sodium chloride, 1
mM ethylenediaminetetraacetate (EDTA) with 1% sodium
dodecyl sulfate, and 200 p,g/ml of proteinase K. After incubation they were extracted sequentially with phenol, phenol/
chloroform (1:l), and chloroform. DNA was precipitated
with cold ethanol ( - 20°C) and resuspended in 10 mM Tris,
p H 7.5, and 1 mM EDTA and determined by spectrophotometry at 260 nm ultraviolet light.
Statistical analysis was performed using Student's twotailed t test.
Results
The binding of 3H-flumazenil, specific ligand of CBR,
to normal gray and white matter and to different brain
tumors is shown in Table 1. The binding density of
normal gray matter was 10 times higher than that of
white matter (0.5 k 0.07 pmoVmg of protein or 17 2
3.2 pmoYmg of DNA in gray matter; 0.03
0.005
pmoYmg of protein or 0.15 ? 0.02 pmoYmg of DNA
in white matter).
Among the different brain tumors, CBRs were undetectable in the neurinomas, meningiomas, craniophaqngiomas, and metastases. In the astrocytomas and
glioblastomas we observed wide variability among the
individual specimens; CBRs were detected in 5 of 13
astrocytomas and in 2 of 5 glioblastomas. In the
tumors showing CBRs, the binding densities were
higher than in gray matter when values were expressed
per mg of protein but lower when expressed per DNA
content. These same tumors showed higher binding
densities than white matter when values were expressed both per mg of protein and per DNA content. No difference of binding affinities was revealed
among gray and white matter and the glial tumors
investigated; the binding was saturable and dissociation
constant ( K d ) values were similar in all tissues (see
Table 1).
Table 2 shows the binding of 3H-PK 11195 to normal and tumoral tissues. The specificity of the Iigand
was determined in these tissues by the use of diazepam, flumazenil, and PK 11195. Only diazepam and
PK 11195, but not flumazenil, displaced 3H-PK 11195
binding. The binding was saturable in all tissues, and
similar binding densities were present in normal gray
and white matter (1.5 ? 0.3 pmoYmg of protein or 5 3
*
Ferrarese et al: Benzodiazepine Receptors and DBI in Brain Tumors
565
Table 1. Central ('H-Flumazenil) Benzodiazepine Receptors in Human Brain and in Human Cwebval Tumors
3H-Flumazenil Bindingb
Bmax
Tissuea
pmoVmg of Protein
Normal tissue
Gray matter (6)
White matter (6)
Astrocytoma, grade 1-2 (13)
8 samples
5 samples
Glioblastoma (5)
3 samples
2 samples
Neurinoma ( 4 )
Meningioma (6)
Cranropharyngioma (1)
Metastases (3)
Renal adenocarcinoma (1)
Lung cancer (2)
pmoVmg of DNA
Kd
0.5
0.07
0.03 0.005'
17 ? 3.2
0.02'
0.15
3.7
3.5
ND
2.6 & O . F d
ND
ND
0.4
ND
ND
ND
ND
2.3
ND
ND
ND
ND
ND
ND
ND
*
*
*
10
_C
3.2'.d
(nM)
4.2
3.6
"Numbers of samples are in parentheses.
bValues are mean +. standard deviauon.
' p c 0.05 when values are compared with those for gray matter.
0.05 when values are cornpared with those for white matter.
Bmax
=
maximum binding density, & = dissociation constant, ND
=
not detectable.
Table 2. Peripheral (3H-PK I 1 195) Benzodiazepine Receptors in Human Brain and in Human Cerebral Tumors
'H-PK 11195 Bindingb
Bmax
Tissuea
Normal tissue
Gray matter (6)
White matter (6)
Astrocytoma, grade 1-2 (13)
Glioblastoma ( 5 )
Neurinoma (4)
Meningioma (6)
Craniopharyngioma (1)
Metastases (3)
Renal adenocarcinoma (1)
Lung cancer (2)
pmol/mg of Protein
1.5
1.7
0.3
0.4
7.6 -+ 2.7
8.4 ? 0.4'
12 _C 3.2'
20
10'
34
?
?
*
21
14
pmoVmg of D N A
*
53
15
56 -t- 18
37 t 19
39 i- 11
11 t 2.ld
23 k s . l d
74
120
37
Kd (nM)
15
13
12
12
16
12
22
14
14
"Numbers of samples are in parentheses
bValues are mean r?: standard deviation.
' p 0.05 when values are compared with those for normal tissue.
' p 0.002 when values are compared with those for normal tissue.
Bmax = maKlmum binding density, Kd = dissociation constant.
*
15 pmoVmg of D N A in gray matter; 1.7 i- 0.4
pmoVmg of protein or 5 6 ? 18 pmoL'mg of D N A in
white matter). PBR were observed in all the different
brain tumors investigated. When the binding densities
were expressed per protein content, all tumors showed
higher values than normal brain tissue. On the other
hand, when the maximum binding densities were expressed per D N A content, no significant difference in
binding density was revealed in glial tumors cornpared
566 Annals of Neurology Vol 26 N o 4 October 1989
with normal gray and white matter. As shown in Table
2, the binding density was signtficantly reduced in both
meningiomas and neurinomas. In the single case of
craniopharyngioma and metastasis of renal adenocarcinoma, an increase in the number of binding sites was
observed. The binding affinities were similar in normal
tissue and in the different brain tumors. The Kd values
are shown in Table 2.
The levels of DBI-LI in the investigated tissues are
Table 3. Levels of Diazepam-Binding Inhibitor-like Immunoreactivity in Human Brain and in Human Cerebral Tumors
DBI-LI~
Tissue"
pmoVmg of Protein
Normal tissue
Gray matter (6)
White matter (6)
Astrocytoma, grade 1-2 (13)
Glioblastoma (5)
Neurinoma (4)
Meningoma (6)
Craniopharyngioma (1)
Metastases
Renal adenocarcinoma (1)
Lung cancer (2)
480
430
330
370
ND
ND
ND
f
pmoVmg of DNA
3.8 & 0.9
3.5 2 0.7
75
* 85
2 150
* 100
1.1 & 0.5'
1.6 2 0.4'
ND
ND
ND
260
ND
1.5
ND
"Numbers of samples are in parentheses.
bValues are mean
standard deviation.
'p 0.05 when values are compared with those for normal tissue.
*
DBI-LI = diazepam-binding inhibitor-like immunoreactivity, ND = not detectable.
reported in Table 3. DBI concentrations were similar
in normal gray and white matter (480 rfr 75 pmoVmg
of protein or 3.8 rt 0.9 pmoVmg of DNA in gray
matter; 430 f 85 pmol/mg of protein or 3.5 f 0.7
pmoVrng of DNA in white matter). Among the different tumors, DBI was detectable in all gliomas and
ghoblastomas and in the single case of metastasis of
renal adenocarcinoma, whereas it was not detected in
the meningiomas, neurinomas, and other metastases.
In glial tumors DBI levels, expressed per DNA content, were significantly lower than in normal brain tissue (1.1 f 0.9 pmoVmg of DNA in gliomas; 1.6 2
0.4 pmoVmg of D N A in glioblastomas), although wide
variations among different tumors were observed.
The Figure shows that after reverse-phase HPLC,
only 1 peak of DBI irnmunoreactivity was detected in
human gliomas and also in normal brain tissue. In both
types of tissue the immunoreactivity peak had the
same retention time as standard DBI purhed from
human brain.
Discussion
This study demonstrated the presence of DBI, a putative endogenous modulator of the GABAergic system, in human gliomas. In the pathological tissues we
observed a reduced DBI concentration compared with
levels in normal white matter; among the different histological types the reduction did not correlate with the
degree of malignancy, Reverse-phase HPLC characterization revealed identical molecular forms of DBILI in normal and tumor tissues. The peptide was unmeasurable in nonglial tumors.
DBI has previously been detected in glial cells, in
which its processing and functions appear different
from neuronal DBI [GI. Although there is still no clue
0
m
.
L
L
GLIOMA
Reverse-phase high-pressure liquid chromatography projik of
diazepam-binding inhibitor-like immunoreactivity (DBI-LI) in
normal human brain and in human glioma. Filtered acetic acid
extracts were injected into the reverse-phase column and a linear
gradient ( 0 4 0 % ) of acetonitrile was applied, with a j w rate
of 1 mllmin. DBI-LI eluted as a single peak in both tissues.
Stanhrd DBI punjied from human autopsied brain had the
same retention time (arrow).
as to the function of DBI in astrocytes, it is known that
the peptide inhibits benzodiazepine binding to PBRs
located on mitochondrial and nuclear membranes {GI.
PBRs have already been detected in glial cells and in
different tumor cell lines. In our study they were present in aSl the different brain tumors of glial and nonglial origin invesugated. These binding sites were
saturable, with similar affinities in all tissues, and they
Ferrarese et al: Benzodiazepine Receptors and DBI in Brain Tumors 567
were speclfically displaced by peripherally active benzodiazepines.
Since we observed an increased density of receptors
in all tumors when the density was expressed per mg
of protein but normal or decreased densities when it
was expressed per DNA content, PBR could be a
marker of increased cellularity. Consistent with this
hypothesis, an increased number of binding sites for
PBRs has been observed in rat brain areas after local
injection of neurotoxins such as kainic acid and has
been explained by the increased number of gllal cells
induced by neurotoxin {lSl. In brain tumors, PBR
densities did not correlate with malignancy.
A possible role of PBRs in the regulation of cell
proliferation and differentiation has been suggested
previously, indicating that the receptors detected in
the different tumors may have a functional significance. Peripherally active benzodiazepines have been
shown to inhibit proliferation of mouse thymoma
cells 111 and of human gliomas 1111, and to induce
differentiation of Friend erythroleukemia cells 1191.
These effects appear to be mediated by a nuclear action of PBRs 1201. A possible interaction of DBI and
PBRs in glial tumors and the significance of these receptors in neoplastic growth warrant further investigation.
CBRs were detected only in a few astrocytomas and
ghoblastomas, where the binding densities were higher
than in normal white matter when values were expressed per mg of protein or per mg of DNA. It is
difficult to explain this characteristic of a few dial
tumors, but it might reflect particular pathological
properties and point toward specific clinical implications.
GABA-A-receptor complexes have previously
been found in some glioma cell lines {21] and in
human gliomas 1221. However, the physiological
significance of these receptors in neoplastic tissues has
not yet been defined. Since benzodiazepine receptors
and DBI are involved in stress and mood disorders, a
better understanding of their role in different tumors
could help to identify possible links between stress and
regulation of neoplastic growth and immune response.
This work was supported by the Consiglio Nazionale delle Ricerche,
Italy (grant 88:88.00559.44, Progetto Finalizzato Oncologia).
Unlabeled PK 11195 was given by Dr G. LeFu (Pham-~ukaLaboratories, Genevillers, France). Purified human DBI and DBI antibodies
were kindly supplied by Dr A. Guidotti (Fidia Georgetown Institute
for the Neurosciences, Georgetown University, Washington, DC).
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