Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma

Life Sciences, Vol. 63, No. 14, pp. 1269-1280, 1998 Copyrighte 1998 Elsevier Science Inc. Printed in the USA. All tights reserved C024-3205/B $19.00 + DO PII

ELSEVIEF

UP-REGULATION

sOO24-3205(98)00388-9

OF PERIPHERAL BENZODIAZEPINE RECEPTOR HEPATOCELLULAR CARCINOMA

SYSTEM

IN

I. Venturini”, M.L. Zeneroli”, L. Corsi’, R. Avallone’ F. Farina”, H. Alhob, C. Baraldi”. C. Ferraresec, N. Pecora’, M. Frigo’, G. Ardizzoned, A. Arrigod, R. Pellicci’, and M. Baraldi’. ‘Cattedra di Semeiotica e Metodologia Medica, Universita di Modena, bLab. of Neurobiology, Univ. Tampere and Dep. of Mental Health and Alcohol Research, National Public Health Inst., Helsinki, Finland, ‘Clinica Neurologica. Universita di Milano, (Monza), dDip. Trapianti d’organo, Universita di Genova. eChirurgia Generale. Ospedale S. Corona, Pietra Ligure, ‘Dip. Scienze Farmaceutiche, Universiti di Modena. 41100 Modena. Italy.

(Received in linal form July 21, 19%) Summary Increased number of peripheral benzodiazepine receptors (PBRs) have been found in some tumors outside the liver. The present study was to verify whether the PBR system is altered in hepatocellular carcinoma (HCC). The levels of endogenous benzodiazepine-like compounds (BZDs). measured by radioreceptor binding technique after HPLC purification and the endogenous ligand for PBRs. termed diazepam binding inhibitor (DBI). measured by radioimmunoassay utilizing a specific antibody for human DBI, were studied in the blood of 15 normal subjects, 12 liver cirrhosis and 10 patients with HCC. The levels of BZDs in serum were increased hundred fold in liver cirrhosis patients and slightly elevated in HCC patients. DBI was found to be increased in HCC patients. The binding recognition sites for PBRs (B,,,) were increased 4 to 7 fold in HCC tissue in comparison with that found in non-tumoral liver tissue (NTLT). On the contrary the concentrations of DBI were found to be significantly decreased in HCC tissue in comparison vvith the respective NTLT. These results seem to suggest an implication of PBRs and of their putative endogenous ligands in the metabolism of these neoplastic cells and possibly in their proliferation. The up-regulation of PBRs found in HCC tissue seems to indicate an increased functional activity of these receptors and opens up the possibility of new pharmacological and diagnostic approaches while the changes in the circulating endogenous ligands for the above receptors might be envisaged as early markers of tumorigenesis in liver cirrhosis. diazepam binding inhibitor, endogenous benzodiazepine-like compounds, hepatocellular carcinoma, liver cirrhosis, peripheral-type benzodiazepine receptors

Key lords:

Benzodiazepines are pharmacological anxiolytic, hypnotic. muscle-relaxant

agents extensively used clinically for their well known and anticonvulsant activities. There are two major

Correspondence to: Prof. Maria Luisa Zeneroli. Cattedra di Semeiotica e Metodologia Medica, Dip. di Medicina Intema, Universita di Modena. Via de1 pozzo 71 - 41100 Modena Italy. Phone: 39 59 422150, Fax: 39 59 424363, Email: Zeneroli 8%C220.UNIM0.1T

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classes of benzodiazepine recognition sites. The first class of these receptors mediates the pharmacological function of benzodiazepine on the central nervous system (CNS) by activating the so called central-type benzodiazepine receptors (CBRs), which arc part of the y-aminobutyric acid supramolecular receptor complex (GABA,) located in the CNS (1,2). The second class of recognition sites for benzodiazepines is termed peripheralbenzodiazepine receptors (PBRs) because of their initial discovery outside the CNS (3,4). Indeed PBRs are expressed as intra-cellular proteins located both within and outside the CNS mainly on the outer mitochondrial membranes and are widely distributed in peripheral organs including kidney, lung, heart. liver and endocrine tissues such as adrenal cortex. testis and pituitary (5). PBRs differ from the CBRs in their lack of coupling directly to GABA, receptors and in their ligand specificity. Commercial benzodiazepines which bind with high affinity to CBRs and exert anxiolytic properties also interact with PBRs (6-S). Putative endogenous ligands for PBRs such as porphyrins. especially the dicarboxylic porphyrins like protoporphyrin IX, competitively inhibit PBR binding (9). An endogenous polypeptide. diazepam binding inhibitor (DBI). capable of displacing benzodiazepine binding to both CBRs and PBRs has been purified from the brain and from the liver of different species (1 O12). Although the exact function of PBRs and of their endogenous ligands is not yet completely clarified, there is accumulating evidence suggesting that they might be involved in a number of intracellular functions (for a review see 13,14). Particularly in the brain, but also in peripheral tissues such as in adrenal glands. PBR ligands enhance steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane (15,16), which represents the ratedetermining step of steroid biosynthesis (17). Also DBI and some of its derivative products seem to stimulate steroidogenesis by interacting with PBRs. Several lines of evidence indicate that ligands which bind to PBR receptors may be involved in the regulation of cell growth and differentiation (18). Ligands for PBRs, in fact, mainly inhibit cell proliferation (19-21) \$hile low concentrations of diazepam or PK I I 195 induce mitosis and cell proliferation (22). We have shown that circulating BZDs in liver cirrhotic patients correlate with the severity of the disease (23). Surprisingly we noted a decrease in concentration of circulating BZDs when the liver cirrhosis was complicated by hepatocellular carcinoma (HCC). This finding together with the suggested implication of DBI and of PBRs in brain tumors (24,25). prompted us to investigate this receptor system. The results could be of particular interest since. despite the high concentrations of PBRs found in some tumors (for reviebv see 13.14). nothing is known about the status of this receptor system in HCC. Hence, the aim of the present study was to investigate the binding characteristics of the peripheral benzodiazepinc receptor system and of DBI in tumoral and non-tumoral liver tissue of patients with HCC. together with the levels of endogenous ligands for PBRs in the blood.

Materials Putient

and Methods

C’hcrructeristics

Liver tissue samples were collected from 10 patients with HCC (7 males and 3 females. mean age (i SE) 56 i 3 ys) during orthotopic liver transplantation or during surgery for partial hepatic resection at the Department of Surgery of Genoa University. From the same patients tissue samples of liver with non-tumoral tissue (NT1.T) morphology. confirmed by histological

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examination. was also collected. Only patients free of previous chemotherapy or medications. including hormones, benzodiazepines. and other drugs. were selected for this study. Anesthesia for surgery was done excluding the use of commercial benzodiazepines. The histological examination of the HCC fragments show a trabecular pattern of HCC with varying degrees of differentiation in 9 cases and 1 case of pseudoglandular tumor. The NTLT fragments show a normal liver in cases 1, 6, 10 and liver cirrhosis in the others (Table I). None of the patients who entered in this study received any pre-operative hormonal treatment or chemotherapy. Fragments of both samples were fixed in 10 % formalin, processed for histological examination with traditional staining techniques and classified according to Hokuda et al. (26). The remaining tissue was frozen immediately in liquid nitrogen and preserved at - 80°C until the day of the assay. Samples with a high degree of necrosis were not included in the study. The PBR binding was studied in all cases in both fragments while the diazepam binding inhibitor-like immunoreactivity (DBI-LI) was assayed only in the first seven cases. Fasting plasma samples were obtained from the same patients immediately before surgery. from another 12 patients with liver cirrhosis (10 males and 2 females. age range 30 to 69). and from 15 normal subjects (6 males and 9 females. age range 40 to 67). The 12 liver cirrhotic patients were selected with the same severity of the liver disease as the patients with liver cirrhosis complicated by HCC (cases 1.3-5.7-9). The study was carried out with the approval of the local ethical committee.

TABLE Histological

Assay

pattern

Sex

I

of the hepatocellular carcinomas tumoral liver tissues

Hepatocellular

carcinoma

and of the non-

Non-tumora tissue

Case

Age

n.

Y

1

60

m

2

46

m

3

48

m

4

62

m

5

60

f

6

75

m

Trabecular moderately

differentiated

Normal

7

37

m

Trabecular moderately

differentiated

Cirrhosis

8

55

f

Trabecular poorly differentiated

Cirrhosis

9

52

f

Trabecular well differentiated

Cirrhosis

10

65

m

Trabecular poorly differentiated Trabecular

moderately

Trabecular Trabecular

differentiated

Cirrhosis Normal

well differentiated

Cirrhosis

moderately differentiated

Cirrhosis

Trabecular

well differentiated

Pseudoglandular

poorly differentiated

Cirrhosis

Normal

ofendogenous BZD-like compounds in strum

The plasma samples (10 ml) were stored at -80°C until they were used for the assay. A preliminary extraction and HPLC purification was performed in order to eliminate endogenous allosteric modulators of GABA,, receptors such as DBI. R-carboline. inosine. and hypoxanthine. As previously described (3 1). aliquots of plasma samples (1 ml) were acidified

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with acetic acid (1 M) and centrifuged at 3000 x g for 10 min. The supernatant was passed through previously washed Sep-Pak CI8 cartridges (Millipore. Medford. MA, U.S.A.). The material was eluted from Sep-Pak with CH3CN/O.l % trifluoracetic acid (TFA) and then lyophilized. The lyophilized sample was reconstituted with 1 ml H20 and 200 ul was chromatographed in duplicate at 0.8 mlimin on a LiChrospher 100 RP-18 column (250 x 4.0 mm; 5 urn) equilibrated with 80 % water/O.l% TFA and 20 % acetonitrile. Absorbance was monitored at 230 nm. Samples were chromatographed using a water/O.1 % TFA and acetonitrile gradient at O.S%/min from 20 to 58% acetonitrile. Seventy five fractions of 1 ml were collected from each sample, lyophilized and reconstituted with water before radioreceptor assay. Known concentrations of diazepam, desmethyldiazepam and oxazepam. were run in parallel with the plasma samples. Unless otherwise indicated, all reagents were obtained from Sigma Chemical Co. (St. Louis, MI, U.S.A.) and were all HPLC grade. For the radioreceptor binding assay. aliyuots (100 ul) of the fractions obtained after HPLC purification were tested for their ability to inhibit [‘HlFlunitrazepam binding (1nM; specific activity 87 Ciimmol, NEN. Boston. MA, IJ.S.A.) to rat cerebellar membranes, as previously described (27) with light modifications. Briefly. the aliquots were incubated in 50 mM sodium phosphate buffer (pH 7.4) which contained 5 uM of GABA. 1 nM of [‘HlFlunitrazepam and rat cerebellar membranes (100 ul) in a final assay volume of 0.250 ml for 1 hr at 4°C. The reaction was terminated by vacuum filtration through glass fiber filters Whatman GFiC (Whatman Ltd.. Maidstone. UK). The filters were transferred to vials with scintillation cocktail and the radioactivity was measured by conventional spectrometer in a Beckman LS 1701 (Beckman Instruments. Palo Alto. CA). Data was expressed as diazepam equivalents (DE) based on extrapolation from standard displacement curves generated using diazepam. The total amount of BZDs present in the serum was evaluated from the sum of the concentrations found in the single active peaks. DBI-LI detection in serum To extract DBI, 1 ml of plasma was diluted with 1 ml of saline and 2 ml of 2 N acetic acid, heated at 90°C for 10 min and proteins were precipitated by the addition of NaOH (2 N). After centrifugation at 20,000 x g for 20 min, aliquots of supernatants were lyophilized in triplicate and used for radioimmunoassay. Antiserum raised in rabbits against human recombinant DBI was used to perform the radioimmunoassay, according to the method previously described (28). DBI-LI detection in tissues Tissues were homogenized in 10 volumes (wtivol) of 1 N acetic acid by Polytron, heated at 95°C for 10 min, and centrifuged at 20.000 x g for 10 min. Aliquots of the supematant were lyophilized for DBI radioimmunoassay. The protein content was determined with the method of Lowry et al. (30). For the detection of the DBI immunoreactivity in tissue extracts, 1 ml of supernatant was filtered and analyzed by reverse-phase high-pressure liquid chromatography (HPLC) using a u-Bondapack C-18 column (30 cm per 5 mm; Waters Associates, Milford. MA. U.S.A.). Peptides were eluted with a linear gradient from 0 to 60% acetonitrile for 60 min, with a flow rate of 1 mlimin. One-ml fractions were collected and lyophilized for DBIradioimmunoassay. DBI-LI was assayed by radioimmunoassay using rabbit antiserum against human recombinant DBI. that was incubated, diluted 1: 20,000 with the lyophilized samples in 0.25 ml of 0.05 M sodium-phosphate buffer pH 7.4 containing 121 Bolton Hunter-labeled DBI (30,000 cpm; specific activity, 200 Ci/mmol) as tracer; 0.2 M NaCl, 12.5 mg bovine serum

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albumin and 0.3 mg calf thymus histone type II. Different concentrations of purified DBI were used for a standard curve. After overnight incubation at 4”C, the [“‘IIDBI bound to the antibody was separated from free [‘?‘I]DBI by addition of 300 pl of protein A (Sigma) (2.5 mgiml of 0.05 M Tris buffer pH 8.0. containing 2 mM magnesium chloride). After 1 hour at 4°C. the protein A-DBI-antibody complex was precipitated by centrifugation at 3,000 x g for 10 min; the [“‘IIDBI in the supernatant was aspirated and the pellet counted in a gamma counter (28). The specificity and characteristics of this human recombinant DBI antiserum were studied by reverse-phase HPLC analysis, as previously described in detai1(28,29). DBI immunoreactivity was eluted with the same retention time as human recombinant DBI both in controls and in patients samples. Radioligand binding technique on liver tissue The subcellular fractions of the liver tissue were obtained according to the method of Anholt et al. (5). About 1 g wet weight of liver tissue was thawed and homogenized by a Brinkmann Polytron (setting 6 for 30 set) in 2 ml of ice-cold buffer A (2 mM HEPES, 70 mM sucrose, 0.21 M D-mannitol, pH 7,4). Then an additional 6 ml of ice-cold buffer A were added and the homogenate was centrifuged for 15 min at 635 x g at 4” C in the SM24 rotor of a refrigerated Sorvall RC-5B centrifuge. The resulting pellet was suspended in buffer A and designated the “nuclear fraction”. The supematant was centrifuged for 15 min at 6,500 x g at 4” C and the fraction”. In pellet, resulting from this centrifugation, was designated the “mitochondrial preliminary experiments the supernatatant was centrifuged at 3 10,000 x g for 1 h at 4” C to separate the “microsomal fraction”. The mitochondrial pellet was gently homogenized into a paste using a test tube filled with ice, and the final volume was adjusted to 2 ml with buffer A. After centrifugation of the mitochondrial suspension for 15 min at 10,000 x g at 4” C, the pellet was homogenized as described before with a final volume of 1 ml. After repeated centrifugation for 15 min at 10,000 x g the washed mitochondrial fraction was suspended in 1 ml of buffer A. The subcellular fractions and a sample of the initial homogenate were stored at -20” c. Receptor Binding Assays The characteristics of the PBRs were studied using the labeled isoquinoline carboxamide derivative [3H]PK 11195 (specific activity 85.0 Ci/mmol, NEN, Boston, MA, U.S.A.) as specific ligand with high affinity for PBRs. The binding was performed as previously described (5). Briefly, the subcellular fractions were diluted with 50 mM Tris HCl buffer, pH 7.7, to a concentration of 80-100 pg of protein/ml which were assayed by the method of Lowry et al. (30). Aliquots of these suspensions (320 ul) were incubated with 40 ~1 of [‘H]PK 11195 at the desired concentration and 40 ul of distilled water with or without 10 uM unlabeled PK 11195 to assess the extent of nonspecific binding. The mixtures were incubated for 1 hour in an ice-water bath. After incubation for 60 min at 4°C samples were filtered under vacuum through Whatman GF/C glass-fiber filters (Whatman, Maidstone, U.K.) and washed 3 times with 2 ml of ice cold Tris HCl buffer. The filters were placed directly in vials preloaded with 8 ml of scintillation liquid and the radioactivity on the filters was counted after 12 h in a scintillation beta-counter (Beckman LS 1701, Berkeley, CA) with 60 % efficiency. All the assays were performed in triplicate. Nonspecific binding was defined as binding of [‘H]PK 11195 in the presence of 1 uM unlabeled PK 11195 (RBI, Natick. MA) and was usually less than 15 % of the total binding. [‘H]PK 11195 specific binding was calculated by subtracting

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Peripheral

in the presence

Benzodiazepine

Receptor

of 1 uM unlabeled

System in HCC

Vol. 63, No. 14, 1998

PK 11195 from total binding

determined

in its

Statistical analysis The data was reported as mean + SE. Significant Student’s t-Test and one-way analysis of variance.

differences

of data

were determined

using

Results BZD-like compounds in serum As shown in Fig. 1 (left panel). BZDs in normal subjects were under the detection limit (2 pmol DE/ml) in 6 of the 15 subjects. In the others the mean (* SE) value was 14 + 3 pmol DE/ml. The concentrations of BZDs in patients with liver cirrhosis were significantly higher (1368 + 463 pmol DE/ml. p < 0.001 vs controls). In the seven patients with liver cirrhosis complicated by HCC. the mean (% SE) value vvas 147 i 84 pmol DE/ml (n.s. vs controls, p < 0.001 vs liver cirrhosis). It must be underlined that in patients with liver cirrhosis complicated by HCC the concentrations of BZDs fell to values IO-fold lower than those found in patients with uncomplicated liver cirrhotic. As previously described by us and by others (23.27.3 1.32) analytical studies showed that the BZD-like materials are represented by halogenated BZDs such as diazepam and N-desmethyldiazepam but also by non-halogenated molecules termed “endozepines”.

DBI-LZ levels in xerum The levels of DBI-LI (Fig. 1. right panel) in normal subjects were 0.91 + 0.12 pmoliml (mean * SE) while in liver cirrhosis the levels were significantly lower 0.34 + 0.05 pmol/ml (p < 0.001). Interestingly patients with HCC showed. on the contrary. a rise of these levels with a 2fold increase in comparison with controls (1.73 f 0.16 pmoliml: p < 0.001 vs liver cirrhosis and p < 0.01 vs controls)

2 0

F

1.5

5 E a 2

1

B E 0.5

0

Normal subjects

h9 Liver cirrhosis cl HCC

* L

Fig. 1

Concentrations of diazepam binding inhibitor-like immunoreactivity (DBI-LI) (left panel) and of benzodiazepine-like compounds (BZDs) (right panel) in serum of normal subjects. of patients uith liver cirrhosis and of patients with hepatocellular carcinoma. The values are given as mean f SE. Student’s r-Test: * p < 0.001 vs controls. . p < 0.001 controls.

vs controls.

o p < 0.01 vs controls and r

p < 0.001

and p < 0.001 vs liver cirrhosis: vs cirrhosis.

not significant

vs

Peripheral

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DBI-LI in HCC and in NTLT The levels of DBI-LI were measured in tissues of 7 HCC patients whose livers were normal in two cases (1 and 6) and cirrhotic in the others. The concentrations (Table II) of DBI-LI in HCC tissues were significantly lower than in NTLT (mean f SE: 2652 k 168 and 3894 f 219 pmol/mg prot., respectively; Student’s t-Test: p < 0.001). The one-way ANOVA showed a significant difference (p < 0.01) in the DBI values found in NTLT and in HCC indicating that in the tumoral tissue there is a constantly decreased concentration of this polypeptide. Incidentally, no difference in DBI content was found in normal or cirrhotic NTLT.

TABLE

II

Concentrations of diazepam binding inhibitor-like immunoreactivity (DBI-LI) in non-tumoral and tumoral tissues of patients with HCC

r Cases

Mean + SE

DBI-LI (pmol/mg

prot.)

NTLT

HCC

4841

2050

3214

2709

4150

3090

3170

2141

3888

3140

4150

2490

3850

2950

3894*219

2652 * 168 *

Student’s t-Test : * p < 0.001; one-way ANOVA: p < 0.001. The non-tumoral tissues of cases N. 2 and 6 were normal livers whereas the others were liver cirrhosis.

PBRS in HCC and in NTLT As shown in Fig. 2, the saturation curves of [‘H]PK 11195 binding to mithocondrial preparations from HCC and from NTLT, performed in triplicate, demonstrate the presence of specific binding in both tissues. The specific binding was constantly higher in the HCC tissue when compared with the respective NTLT. The Scatchard plots derived from the saturation curves performed on the 10 HCC tissues and on the 10 NTLT, revealed the presence of one population of PBRs in both types of tissue. As reported in Table III, the B,,, mean value (5 SE) was 1462 * 246 fmol/mg prot. inHCC tissue, showing a significant increase in comparison with the Bmax value of NTLT (Mean f SE: 254 * 42 fmol/mg prot.). Despite the presence of a high interindividual variation, the value of the Bm3, was constantly 4 to 7 fold higher in the HCC than in the respective NTLT. The one-way ANOVA indicated a significant difference in the B,,, values (p < 0.0001) between the NTLT and the tissue from HCC. The K, level (Mean

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+ SE) was 0.27 + 0.04 nM in the NTLT and 0.28 + 0.05 nM in the HCC without any substantial difference. It is noteworthy that as far as regards both B,,,,, and K, values there were no significant differences (cases 1.3-5.7-9).

between

the two types of NTLT.

normal

(cases 2.6.10).

and cirrhotic

Experiments performed on nuclear and on microsomal fractions obtained from NTLT showed that the densities (B,,,,,) of PBRs in these two fractions are lower than that found in the mithocondrial fraction being respectively 87 + 21 and 52 i 11 fmolimg prot. In HCC tissues the densities were 244 + 43 and 115 * 38 fmolimg prot respectively (one-way ANOVA: p < 0.01); thus suggesting that PBRs undergo to a similar increase found in mithocondrial fraction even if less pronounced.

Discussion In this study we provide the first demonstration of an increased density of the PBRs in HCC tissue. Together with this observation there was the finding that in the same HCC tissues there

-

2,500

‘0 ‘a

n HCC tissue 0

Cirrhotic NTLT

0

Normal NTLT

F ‘E =.

1,500

E?

Fig. 2 Typical saturation curves of [‘H]PK 11195 binding to PBRs present on mithocondrial membranes of HCC tissue and of the surrounding nontumoral tissue from normal or cirrhotic liver. Each point represents the mean value of the data obtained from experiments done in triplicate.

was a decreased concentration of DBI. An increased concentration of PBRs has been reported previously in brain tumors (for a review see 14) and in other malignant tumors such as in ovarian carcinoma. colonic carcinoma. and colonic adenocarcinoma (33,34). In brain tumors. a significant correlation between PBR expression and the degree of malignancy and patient survival has been reported (35.36). Also, malignant ovarian tumors show an increased number of receptors whereas benign types of ovarian tumors do not (34). In the present study the increase in the number of PBRs found in HCC tissue ranged from 4 to more than 7 fold in comparison with the surrounding liver tissue of the same patient. The increase in well and moderately differentiated HCC ranged from 5.5 to 7.3 fold while the poorly differentiated

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HCC showed an increase from 4.3 to 4.8 fold. The number of PBRs is high in cells undergoing a rapid turnover and containing high densities of mitochondria with a highly active oxidative phosphorilation (13.14) and this might be the case in the HCC cells with an active and rapid replication. Alternatively the increase in PBR density in liver tumors observed here may be related to enhanced metabolic rates in the neoplastic cells or it might reflect alterations of the tine mitochondrial structures in cell tumors. the so called mitochondrial pyknosis. The up-regulation of PBRs is associated with a selective local reduction in the concentration of their endogenous ligand. DBI, in this tumoral tissue. This finding is at variance with the demonstration that DBI-LI and DBI mRNA levels are increased in malignant brain tumors (25,36). As yet. we do not have any explanation for this difference, we can simply surmise that the homeostasis of this peptide is differently regulated in the periphery and in the CNS. TABLE Binding

characteristics

from tumoral

III

of [3H]PK 11195 on mitochondrial

and non-tumoral

liver tissues of patients

fractions

with hepatocellular

carcinoma

Non-tumoral

HCC tissue

tissue K*

B ,“a\

fmolimg prot.

nM

fmolimg prot.

1

246

0.18

1110

0.57

2

516

0.13

3195

0.47

3

130

0.15

715

0.16

4

240

0.33

1290

0.15

5

230

0.17

1699

0.15

6

109

0.33

635

0.32

7

315

0.20

2090

0.25

8

430

0.50

1870

0.45

9

165

0.35

1180

0.18

10

175

0.36

840

0.15

Mean f SE

254 + 42

0.27 * 0.04

1462 + 246*

0.25 * 0.05

Cases

B ma\

KCI nM

Binding characteristics were calculated from Scatchard plots derived from [‘H]PK 11195 binding saturation curves performed in triplicate. Student’s ITest: * p < 0.001 vs non-tumoral tissue: one-way ANOVA of the B,,,: p < 0.0001. The non-tumoral tissues N. 2, 6 and 10 were normal livers whereas the others were liver cirrhosis

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The decreased presence of DBI-LI in HCC seems not to depend on a reduced synthesis since its level in blood is increased in patients with liver cirrhosis complicated by HCC. Hence this reduced concentration must be ascribed to an increased metabolic rate of the peptide in this tissue. The reasons for the opposite changes of DBI in plasma and HCC tissue might lie in an increased synthesis by other organs. such as adrenal glands. and respectively in an increased metabolism in HCC cells. Since DBI is invol\,ed in the regulation of multiple biological processes such as steroidogenesis. aq I-coA metabolism and glucosemediated insulin secretion (13.14). its decreased presence in HCC could affect the mitochondrial metabolism and induce the up-regulation phenomenon of PBRs. As regards the BZD-like materials in the blood. these compounds usually increase in patients with liver cirrhosis and their levels rise with the progression of the disease (23). However. this seems not to be the case in patients with liver cirrhosis complicated by HCC. Considering that the metabolic dysfunction of the liver persists or is worsened by the development of the HCC, the decreased levels of circulating BZD-like compounds might be due to an enhanced local utilization by the increased number of PBR receptors. The finding that there is an increased amount of DBI in the blood of HCC patients together with a fall of the BZDs supports the view that there is a reciprocal regulation between BZDs and DBI. In fact. in liver cirrhosis decreased. as previously described liver cirrhosis (37-39).

where BZDs in consumers

prevail the peptide content in the blood is of commercial BZDs and in patients with

From the clinical point of view a better understanding of the role of PBRs and their endogenous ligands in the development of liver tumors might be useful for new therapeutic approaches. The variations described in levels of DBI and BZDs could be used as an early marker of tumorigenesis in liver cirrhosis patients. Finally. the use of radiolabeled ligand for PBRs might allow an early recognition of liver tumors by imaging studies as already attempted for brain tumors with Positron Emission Tomography (40).

Acknowledgments This work was supported

by M.l.R.A.A.F.(Rome)

grant and by grant of Modena

University.

References 1. 2. 3. 4.

A. GUIDOTTI. M. BARALDI, E. COSTA. Pharmacology, 19 267-277 (1979). J.F. TALLMAN, D.W. GALLAGER. Annu. Rev. Neurosci.. 8 21-44 (1985). C. BRAESTRUP and R.F SQUIRES. Proc. Natl. Acad. Sci. USA. 74 3805-3809 (1977). L. ANTKIEWICZ-MICHALUK, A. GUIDOTTI, K.E. KRUEGER, Mol. Pharmacol.. 34 272-278 (1988). 5. R.R. ANHOLT. P.L. PEDERSEN, E.B. DE SOUZA. S.H. SNYDER. J. Biol. Chem., 261 576-583 (1986). 6. P.J. MARANGOS, J. PATEL, J.P. BOULENGER, R. CLARK-ROSENBERG, Mol. Pharmacol., 22 26-32 (1982). 7. H. SCHOEMAKER, R.G. BOLES, W.D. HORST. H.I. YAMAMURA, J. Pharmacol. Exp. Ther., 225 61-69 (1983). 8. J.K. WANG. T. TANIGUCHI: S. SPECTOR, Mol. Pharmacol.. 25 349-351 (1984) 9. A. VERMA. J.S. NYE. S.H. SNYDER. Proc. Natl. Acad. Sci. USA, 84 2256-2260 (1987). IO. A. GUIDOTTI. C.M. FORCHETTI. M.G. CORDA. D. KONKEL. C. D. BENNETT, E.

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