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Peripheral benzodiazepine receptor ligands induce apoptosis and cellcycle arrest in human hepatocellular carcinoma cells and enhance
chemosensitivity to paclitaxel, docetaxel, doxorubicinand the Bcl-2 inhibitor HA14-1
Andreas P. Sutter1, Kerstin Maaser1, Patricia Grabowski1, Gesine Bradacs1, Kirsten Vormbrock1,Michael Hopfner1, Antje Krahn1, Bernhard Heine2, Harald Stein2, Rajan Somasundaram1,
Detlef Schuppan3, Martin Zeitz1, Hans Scherubl1,*
1Medical Clinic I, Charite-Universitatsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany2Institute of Pathology, Charite-Universitatsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
3Department of Internal Medicine, Friedrich Alexander University, Erlangen, Germany
Background/Aims: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer deaths worldwide.
Thus, novel therapies are urgently needed. A promising approach is the use of peripheral benzodiazepine receptor
(PBR) ligands which inhibit the proliferation of various tumors.
Methods: PBR expression both in human HCC cell lines and in tumor specimens of HCC patients was analyzed by
RT-PCR and immunostaining. To evaluate PBR ligands for the treatment of HCC, we tested their effects on human
HCC cells.
Results: PBR was localized to the mitochondria both of HCC cell lines and tumor tissues of HCC patients. In contrast,
normal liver did not express PBR. PBR ligands inhibited the proliferation of HCC cell lines by inducing apoptosis andcell cycle arrest. Apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase-3
activation and nuclear degradation. Furthermore, pro-apoptotic Bax was overexpressed while anti-apoptotic Bcl-2 and
Bcl-XL were suppressed. Cell cycle was arrested both at the G1/S- and G2/M-checkpoints. Synergistic anti-neoplastic
effects were obtained by a combination of PBR ligands with cytostatic drugs (paclitaxel, docetaxel, doxorubicin), or
with an experimental Bcl-2 inhibitor.
Conclusions: This is the first report on the induction of apoptosis and cell cycle arrest by PBR ligands in HCC cells.
Moreover, PBR ligands sensitized HCC cells to taxans and doxorubicin.
q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Bax; Bcl-2; Chemoresistance; Chemotherapy; Peripheral benzodiazepine receptor; Apoptosis; Cell cycle
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Pub
doi:10.1016/j.jhep.2004.07.015
Received 11 February 2004; received in revised form 21 June 2004;
accepted 12 July 2004; available online 29 July 2004
* Corresponding author. Tel.: C49-30-8445-3534; fax: C49-30-8445-
4481.
E-mail address: [email protected] (H. Scherubl).
Abbreviations: FGIN-1-27, N,N-di-n-hexyl-2-(4-fluorophenyl)indole-3-
acetamide; HCC, hepatocellular carcinoma; PBR, peripheral benzo-
diazepine receptor; PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-
propyl)-3-isoquinolinecarboxamide; PTP, permeability transition pore.
1. Introduction
Hepatocellular carcinoma (HCC) is the fifth most
common malignancy in the world and is estimated to
cause half a million deaths annually. The incidence of HCC
is dramatically increasing in the USA, Europe and Asia,
most probably due to the increasing prevalence of hepatitis
C [1,2]. Unfortunately, treatment of HCC is unsatisfactory.
Curative ablation or resection of HCC, or liver transplan-
tation can be achieved only in a minority of patients. Local
tumor destruction, chemoembolisation or systemic
Journal of Hepatology 41 (2004) 799–807
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Table 1
Clinicopathological characteristics of the patients
Patients (n) 32
Gender
Male 20
Female 12
Age (years)
Average 59.8
Range 16–79
Grading (of the HCC)
G1 12
G2 12
G3 7
N/A 1
A.P. Sutter et al. / Journal of Hepatology 41 (2004) 799–807800
chemotherapy are the remaining treatment options in
advanced HCC. However, overall survival is poor [3].
Therefore, innovative treatment approaches are urgently
needed.
Venturini et al. were the first to show an overexpression
of the peripheral benzodiazepine receptor (PBR) in human
hepatocellular carcinoma cells; they suggested the use of
PBR ligands as an innovative treatment of HCC [4]. PBR
ligands have been shown to inhibit the proliferation of
esophageal, colorectal and breast cancer cells by induction
of apoptosis and cell cycle arrest [5–7]. Moreover, PBR
ligands have been shown to overcome Bcl-2-mediated
chemoresistance in lung cancer [8].
The PBR was initially discovered as a binding site for
benzodiazepines in peripheral tissues. It is structurally and
pharmacologically distinct from the central-type benzo-
diazepine binding site which is associated with the GABAA
receptor [9]. PBR is mainly localized in the outer
mitochondrial membrane [5,6] but has also been detected
in the plasma membrane [10] and nucleus [11]. PBR is
overexpressed in tumors of the liver, colon, and breast
[11–13], thus offering the possibility of a tumor-targeted
therapy [14].
PBR has not yet been studied functionally in hepatocel-
lular carcinoma. Here we report on the mitochondrial
expression of PBR in hepatocellular carcinoma. We
demonstrate the anti-proliferative and pro-apoptotic actions
of PBR ligands in HCC cells. Moreover, we provide new
insight into PBR-ligand-mediated apoptosis and evidence
for a synergistic anti-neoplastic action of PBR ligands and
cytostatics.
2. Materials and methods
2.1. Cell lines and drugs
The human HCC cell lines Huh-7 [15] and HepG2 [16] were cultured inRPMI1640 medium containing 10% fetal bovine serum and 100 U/mlpenicillin and streptomycin. Primary human keratinocytes [17] were grownin Keratinocyte SFM (Gibco, Paisley, United Kingdom). Cells were kept ina humidified atmosphere (5% CO2) at 37 8C.
Cells were incubated with culture medium containing FGIN-1-27, PK11195 (Tocris, Bristol, United Kingdom), or FGIN-1-52 [18]. Forcombination treatment, cells were incubated simultaneously with PBRligands and one of the following agents: Paclitaxel (Sigma), docetaxel(Fluka Chemie, Buchs, Switzerland), doxorubicin (Sigma), or HA14-1(Alexis, Grunberg, Germany).
2.2. Analysis of peripheral benzodiazepine
receptor expression
Semi-quantitative analysis of PBR mRNA expression was carried outby RT-PCR with the number of cycles at which the band intensity increasedlinearly with the amount of mRNA used. Total RNA was extracted withRNAClean (Hybaid, London, United Kingdom) and subsequently digestedwith DNAse I (Gibco, Karlsruhe, Germany). Oligo-dT-primers and theSuperScript Preamplification-Kit (Gibco) were used for cDNA synthesis.PCR reactions were performed as previously described [5,19]. The identityof the PCR product as being a fragment of PBR was confirmed by
sequencing (Invitek Sequencing Service GmbH, Berlin, Germany). PBRgene expression was standardized to the expression of the housekeepinggene b-actin.
For analysis of PBR protein expression, cells were immunostained asdescribed [5]. In brief, samples were fixed and permeabilized andsubsequently incubated with a polyclonal anti-PBR antibody (1:50, R&Dsystems, Wiesbaden, Germany), or isotypic control rabbit IgG1 (DAKO,Hamburg, Germany). Cells were then incubated with a secondary FITC-labeled goat-anti-rabbit IgG antibody (BD Pharmingen, Heidelberg,Germany). Fluorescence was detected by flow cytometry on a FACSCa-libur (Becton Dickinson, Heidelberg, Germany) and analyzed usingCellQuest software. PBR expression was further studied immunohisto-chemically both in normal liver and in HCCs of 32 patients whohad undergone surgery for HCC. For detailed clinicopathological datasee Table 1. Microsections were deparaffinized, rehydrated, and automati-cally stained, as described [5]. In brief, sections were stained with the anti-PBR antibody 8D7 [20]. After washing, samples were incubated with asecondary anti-mouse antibody and subsequently with the alkalinephosphatase-anti-alkaline phosphatase (APAAP) complex (DAKO). Stain-ing was detected using the ‘fast-red system’ (DAKO), and samples werecounterstained in Mayer’s haematoxylin. The subcellular localization ofPBR in HCC cells was analyzed by immunofluorescence microscopy, aspreviously described [5]. In brief, cells were stained with the mitochondrialdye CMTMRos, fixed, permeabilized, and subsequently incubated with aprimary anti-PBR polyclonal antibody (R&D systems). Thereafter, cellswere incubated with a secondary FITC-labeled goat-anti-rabbit antibody.Imaging was performed using the inverted confocal microscope LSM 510with a 63!/1.2 W Korr objective (Zeiss, Oberkochen, Germany).
2.3. Radioligand binding assays
Cells were homogenized in ice-cold PBS using a small potter-typemechanical homogenizer. Protein concentration was determined using theBradford assay (BioRad, Hercules, CA). Total [3H]PK 11195 (specificactivity 83.5 Ci/mmol; NEN Life Science Products) binding wasdetermined using serial dilutions of the labeled ligand (0.5–15 nM). Non-specific binding was determined by adding 13.3 mM unlabeled PK 11195.Each sample contained 50–100 mg protein in PBS in a final volume of300 ml. Samples were incubated at 4 8C overnight, and each was transferredby vacuum filtration onto 25 mm Whatman filters using a Brandel harvester(Brandel, Gaithersburg, MD). Bound [3H]PK 11195 was quantified byliquid scintillation spectrometry. Dissociation constants (KD) and thenumber of binding sites (Bmax) were determined by Curve-Fit (Prismversion 3.0; Graph Pad Software Inc., San Diego, CA).
2.4. Determination of cell number
Cell number was evaluated by crystal violet staining, as described [21].In brief, cells in 96-well plates were fixed with 1% glutaraldehyde. Thencells were stained with 0.1% crystal violet. The unbound dye was removedby washing with water. Bound crystal violet was solubilized with 0.2%
Fig. 1. Expression of PBR in hepatocellular carcinoma (HCC) cells. (A)
PBR mRNA expression in Huh-7 (lane 1) and HepG2 cells (lane 2)
detected by RT-PCR. The expression of the housekeeping gene b-actin
(amplicon: 822 bp) in Huh-7 (lane 3) and HepG2 cells (lane 4) was
analyzed for standardization. (B) PBR protein expression in HCC cells
was shown by flow cytometry. Huh-7 (left panel) and HepG2 cells (right
panel) were stained with a polyclonal anti-PBR antibody with (gray
area) or without (gray line) previous membrane permeabilization.
Black line: isotypic control.
A.P. Sutter et al. / Journal of Hepatology 41 (2004) 799–807 801
Triton-X-100. Light extinction which increases linerarly with the cellnumber was analyzed at 570 nm using an ELISA-Reader.
2.5. Detection of apoptosis
Changes in DJM were assessed as described [21]. In brief, cells werestained with 5,5 0,6,6 0-tetrachloro-1,10,3,3 0-tetraethyl-benzimidazolylcarbo-cyanine iodide (JC-1, 1 mg/ml, Molecular Probes, Eugene, OR) for 15 minat 37 8C in the dark, prior to analysis by flow cytometry.
Caspase-3 activity was assessed as described [22]. The activity ofcaspase-3 was calculated from the cleavage of the fluorogenic substrateDEVD-AMC (Calbiochem-Novabiochem, Bad Soden, Germany).
The proportion of apoptotic cells was determined by quantifying thepercentage of sub-G1 (hypodiploid) cells after flow cytometric analysis ofpropidium iodide-stained isolated nuclei.
2.6. Western blotting
Western blotting was performed as described [19]. Blots were blockedin 1% non-fat dry milk for 30 min, and then incubated at 4 8C overnightwith anti-human Bcl-2 (1:200, Novo Castra Laboratories, Newcastle uponTyne, United Kingdom), Bcl-XL (1:200, Santa Cruz Biotechnology, CA),Bax (1:1000, Santa Cruz) or b-actin (1:5000, Sigma). Band intensities wereanalyzed densitometrically using TINA software (raytest Isotopenmessge-rate, Straubenhardt, Germany).
2.7. Cell cycle analysis
Cell cycle analysis was performed by the method of Vindelov andChristensen [23]. Cells were trypsinized, washed, and the nuclei wereisolated using CycleTest PLUS DNA Reagent Kit (Becton Dickinson).DNA was stained with propidium iodide according to the manufacturers’instructions. The DNA content of the nuclei was detected by flow cytometryand analyzed using CellFit software (Becton Dickinson).
2.8. Statistical analysis
If not stated otherwise, means of four independent experimentsGSEMare shown. Individual drug treatment and patient groups were compared bythe unpaired, two-tailed Mann-Whitney U-test. Dichotomized variableswere compared using the chi square test. P values were considered to besignificant at !0.05.
Fig. 2. Localization of PBR in HCC tissues and cells. (A, B)
Immunohistochemical detection of PBR (red) in HCC tissue and in
adjacent non-neoplastic tissue (NO). (A) BarZ200 mm; (B) BarZ100 mM. (C–E) For immunocytochemistry, PBR was immunostained
with a polyclonal antibody (C), and mitochondria were marked with
CMTMRos (D). Superposition of both fluorescence images resulted in a
bright yellow color (E), indicating a co-localization of PBR and
mitochondria in Huh-7 cells. BarZ10 mM. (For interpretation of the
references to color in this figure legend, the reader is referred to the
web version of this article).
3. Results
3.1. Expression and subcellular localization of PBR
in hepatocellular carcinoma cells
Both Huh-7 and HepG2 cells expressed mRNA tran-
scripts of PBR (Fig. 1A). Expression of PBR protein was
detected in permeabilized Huh-7 and HepG2 cells, whereas
in non-permeabilized cells, no PBR-specific fluorescence
was observed, indicating an intracellular localization of PBR
(Fig. 1B). The expression levels both of PBR transcript and
protein did not notably differ between the two cell lines. In
11 of 32 analyzed HCC patients, PBR was expressed in HCC
tissues, whereas the respective non-neoplastic liver tissues of
the same patients were negative for PBR. In this small group
of patients PBR expression did neither correlate with age,
gender, histopathological grading, nor with underlying liver
disease (also see Table 1). In all 11 PBR-positive HCCs, the
specific PBR staining was observed to be unevenly
distributed within the cytoplasm. No specific PBR staining
was observed in the plasma membrane nor in the cell nuclei,
suggesting that the PBR is located in the mitochondria of
HCC cells (Fig. 2A and B). The mitochondrial localization
Fig. 3. Anti-proliferative effects of PBR ligands. The PBR ligands
FGIN-1-27 and PK 11195 induced a time- and dose-dependent growth
inhibition in Huh-7 (A) and HepG2 (B) cells. Growth inhibition was
significant for 10–100 mM FGIN-1-27, and for 25–100 mM PK 11195
(P!0.05). Means of four independent experiments are shown. (C)
PBR- and tumor cell specificity of the growth inhibitory effects. In
contrast to PBR ligands, FGIN-1-52 did not inhibit the growth of Huh-7
(white columns) or HepG2 cells (hatched columns; Fig. 2C, left panel).
Non-malignant human primary keratinocytes remained nearly unaf-
fected by incubation with 50 mM FGIN-1-27 (white columns) or 75 mM
PK 11195 (black columns), in contrast to their inhibitory actions in
Huh-7 and HepG2 cells (Fig. 2C, right panel).
Table 2
Growth inhibition induced by the PBR ligands FGIN-1-27 and PK 11195, th
inhibitor HA14-1 in Huh-7 and HepG2 cells
Drug Huh-7
IC10 IC50 IC90
FGIN-1-27 4!103G1!103 2!104G3!103 O1!105
PK 11195 1!104G2!103 4.3!104G4!103 8.9!104G5
Paclitaxel 1.7G0.9 7.1G1 O25
Docetaxel 2.1G1.1 7.9G2 O25
Doxorubicin 140G60 652G57 O1!103
HA14-1 8!103G3!103 2.5!104G3!103 4.3!104G2
The IC10, IC50, and IC90 values (nM) were calculated from interpolations of the d
experiments.
A.P. Sutter et al. / Journal of Hepatology 41 (2004) 799–807802
of PBR was further confirmed for Huh-7 cells
by simultaneously staining mitochondria and PBR
(Fig. 2C–E). Green fluorescence of PBR was detected within
the cytoplasm, but neither in the cell membrane nor in the
nucleus (C). The staining pattern was comparable to the red
one obtained by staining of mitochondria (D). Superposition
of both images resulted in a yellow color, indicating a
co-localization of PBR and mitochondria (E).
3.2. [3H]PK 11195 binding properties of PBR in
hepatocellular carcinoma cells
PBR expressed in liver cancer cell lines binds with high
affinity [3H]PK 11195 (Huh-7: KDZ0.42G0.02 nM,
HepG2: KDZ1.04G0.36 nM), in good agreement with a
previous study in liver cancer tissues [24]. Saturation
isotherm analysis revealed that [3H]PK 11195 binding
capacity (Bmax) amounted to 234.05G18.69 fmol/mg in
Huh-7 and to 97.47G23.90 fmol/mg in HepG2 cells.
3.3. Inhibition of cell proliferation by PBR ligands
The PBR ligands FGIN-1-27 and PK 11195 (10–
100 mM) dose-dependently inhibited the proliferation of
Huh-7 (Fig. 3A) and HepG2 cells (Fig. 3B). After 96 h of
incubation with FGIN-1-27 or PK 11195, a maximal
decrease in cell growth by 80–100% was observed. The
IC10, IC50 and IC90 values are given in Table 2. While PK
11195 (100 mM) decreased original cell numbers after 96 h,
FGIN-1-27 was predominantly cytostatic.
Despite the indoleacetamide structure of FGIN-1-52
being similar to FGIN-1-27, it displays almost no affinity
to PBR [18,25]. Accordingly, FGIN-1-52 did not inhibit
the proliferation of HCC cells, even at concentrations as
high as 100 mM (Fig. 3C, left panel), suggesting PBR
specificity of growth inhibition. Next, we analyzed the
tumor specificity of growth inhibition using primary
human keratinocytes as a model of non-malignant
epithelial tissue. FGIN-1-27 or PK 11195 (10–75 mM)
did not inhibit the growth of PBR-expressing [5]
keratinocytes (Fig. 3C, right panel).
e cytostatic drugs paclitaxel, docetaxel and doxorubicin, and the Bcl-2
HepG2
IC10 IC50 IC90
5!103G1!103 2!104G2!103 O1!105
!103 2.9!104G3!103 5.5!104G3!103 9!104G6!103
1G0.1 4.5G0.9 O25
1G0.3 6.5G1 O25
38G10 457G100 O 1!103
!103 2.2!104G2!103 2.8!104G1!103 4!104G3!103
ose–response relationships. Data are derived from at least four independent
Fig. 4. Mitochondrial alterations and apoptosis induction by PBR
ligands. Huh-7 (black symbols) and HepG2 cells (open symbols) cells
were incubated with 10–100 mM FGIN-1-27 (squares) or PK 11195
(circles). FGIN-1-27 and PK 11195 dose-dependently decreased the
DJM after 24 h of incubation (A), induced caspase-3 activation after
48 h of incubation (B), and increased the proportion of apoptotic cells
measured as subdiploidy after 96 h of incubation (C). Data are shown
as meansGSEM of 4 independent experiments. *Statistical significance
(P!0.05) compared to untreated controls.
A.P. Sutter et al. / Journal of Hepatology 41 (2004) 799–807 803
3.4. PBR ligands induced DJM disruption and apoptosis
After 24 h, FGIN-1-27 and PK 11195 dose-dependently
induced a significant decrease of the mitochondrial
membrane potential (DJM) in HCC cells (Fig. 4A).
Furthermore, PBR ligands induced an increase in caspase-
3 activity after 48 h (Fig. 4B). They also increased the
incidence of nuclear apoptosis after 96 h of treatment (Fig.
4C). While a modest but significant apoptotic peak was
observed after treatment with FGIN-1-27, PK 11195
induced a stronger increase of apoptotic cells. This could
explain the cytotoxic effects of PK 11195 but not of FGIN-
1-27 after 96 h (Fig. 3).
3.5. Modulation of Bcl-2, Bcl-XL and Bax expression
by PBR ligands
As mitochondrial alterations occurred during PBR
ligand-induced apoptosis, the expression levels of mito-
chondrial pro-(Bax) and anti-apoptotic (Bcl-2 and Bcl-XL)
proteins were analyzed after 96 h of treatment. In HepG2
cells, Bcl-2 and Bcl-XL expression were downregulated,
whereas Bax expression was increased (Fig. 5). Due to these
alterations, the Bax/Bcl-XL ratio increased upon PBR ligand
treatment. Similarly, this ratio was dose-dependently
elevated by either FGIN-1-27 (up to 1.5-fold) or PK
11195 (up to 8.5-fold) in Huh-7 cells.
3.6. PBR ligands induce cell cycle arrest in the G0/G1
and G2/M phase
Treatment with PBR ligands (24 h) dose-dependently
arrested Huh-7 cells in the G0/G1-phase of the cell cycle
(Fig. 6A). In HepG2 cells, both a dose-dependent G0/G1
arrest and, at high concentrations (100 mM), a G2/M arrest
was observed (Fig. 6B).
Fig. 5. PBR ligands modulate the expression of pro- and anti-apoptotic
proteins. Modulation of protein expression in HepG2 cells was assessed
by Western blot analysis after 96 h of treatment with PBR ligands. The
anti-apoptotic proteins Bcl-XL and Bcl-2 were dose-dependently
downregulated by PBR ligands, whereas the pro-apoptotic protein
Bax was upregulated. A representative result out of three independent
experiments is shown.
Fig. 6. Induction of cell cycle arrest in the G0/G1 and G2/M phases by
PBR ligands. After 24 h of incubation of Huh-7 (A) and HepG2 cells (B)
with PBR ligands, cells accumulated in the G0/G1 phase (white
columns) of the cell cycle, while the proportion of cells in the S phase
(hatched columns) decreased. Means of four independent experiments
are shown. When compared to control, significant differences of the
proportion of cells in the G0/G1 phase of the cell cycle were observed in
response to FGIN-1-27 and PK 11195 from 50–100 mM (P!0.05).
Additionally, a significant (P!0.05) arrest in the G2/M phase (black
columns) was observed in HepG2 cells after treatment with FGIN-1-27
or PK 11195 (100 mM).
Fig. 7. Synergistic growth inhibition of HCC cells by cytostatic drugs or
the Bcl-2 inhibitor HA14-1 when combined with the PBR ligand FGIN-
1-27. Combination treatment with sub-IC50 concentrations of cytostatic
agents (paclitaxel, docetaxel, doxorubicin) or the Bcl-2 inhibitor
HA14-1 and the PBR-ligand FGIN-1-27 for 96 h led to synergistic
growth inhibitory effects in Huh-7 (A) and HepG2 cells (B). Black bars
indicate the values of the calculated additive growth inhibition. Data
are given as percentage of untreated controls (meansGSEM of at least
3 independent experiments).
A.P. Sutter et al. / Journal of Hepatology 41 (2004) 799–807804
3.7. PBR ligands chemosensitize HCC cells to treatment
with paclitaxel, docetaxel, doxorubicin, or a Bcl-2 inhibitor
Next, we investigated whether the combination of PBR
ligands with either cytostatic drugs or the Bcl-2 inhibitor
HA14-1 was superior to the anti-proliferative treatment with
single agents. First, the IC10, IC50 and IC90 values of
the taxans paclitaxel and docetaxel, the intercalating agent
doxorubicin, and HA14-1 were determined after 96 h of
treatment (Table 2). The IC50 values of taxans were the
lowest (low nanomolar range), followed by doxorubicin
(high nanomolar range), and HA14-1 (micromolar range).
For combination treatments, individual drugs were used
at two different concentrations between their respective IC10
and IC50 values. The combination of either cytostatic drug
or HA14-1 with FGIN-1-27 acted in a synergistic or additive
way (Fig. 7). Similar results were obtained with PK 11195.
Paclitaxel or docetaxel or doxorubicin at concentrations
inducing 10–50% growth inhibition on its own caused
growth inhibition by 50–90% when each of them was
combined with one of the PBR ligands. In Huh-7 cells, the
most effective synergism (20% observed versus 53%
calculated growth) was seen for paclitaxel plus PK 11195
(10 mM), whereas in HepG2 cells PK 11195 (10 mM) most
dramatically enhanced doxorubicin-induced growth inhi-
bition (28% observed versus 69% calculated growth). In
contrast to those synergisms, the combination of either PBR
ligand with HA14-1 proved to be less than additive in Huh-7
cells.
Based on the synergism between PBR ligands and
paclitaxel, we next investigated whether this combination
led to synergistic induction of apoptosis and/or cell cycle
arrest in Huh-7 cells. PBR ligands (at 50 mM) or paclitaxel
(at 2.5 nM) by themselves induced only a modest apoptotic
response after 96 h. However, the combination of PBR
ligands and paclitaxel led to a dramatic increase of apoptotic
cells compared to treatment with either agent alone (Fig. 8).
Fig. 8. Synergistic apoptotic effects of a combination of PBR ligands
and paclitaxel in Huh-7 cells. The combination of either the PBR ligand
FGIN-1-27 or PK 11195 with paclitaxel led to a synergistic increase of
hypodiploid Huh-7 cells after 96 h of treatment. The percentage of sub-
G1 apoptotic cells is noted on each histogram. Representative data
from three independent experiments are shown.
A.P. Sutter et al. / Journal of Hepatology 41 (2004) 799–807 805
4. Discussion
In this study we show that the growth of human
hepatocellular carcinoma (HCC) cells is inhibited by PBR
ligands. In addition, PBR ligands sensitized HCC cells to
cytostatic agents.
As the overexpression of mitochondrial PBR in HCC
[12] has recently been discussed controversially [26], we
evaluated PBR expression in HCCs of 32 patients. No PBR
expression was observed in normal liver. Yet, PBR was
found to be expressed in 11 out of 32 HCCs. Thus, one third
of HCC patients may respond to a PBR ligand-based
therapy. In contrast to our findings, PBR expression had
been described in earlier reports [12,26] to occur even in
normal liver. The reasons for this discrepancy are unclear.
In esophageal, colorectal and breast cancer, PBR protein is
expressed both in tumoral and normal tissues. It is
overexpressed in 33% (esophageal) or 88% (colorectal) of
the tumor tissues [5,6]. In breast cancer, PBR expression
correlates with the tumor stage [26].
The PBR ligands FGIN-1-27 and PK 11195 induced both
a dose- and time-dependent growth inhibition and apoptosis
in the PBR-expressing HCC cell lines HepG2 and Huh-7,
suggesting that PBR ligands may antagonize the mitogenic
and anti-apoptotic intrinsic functions ascribed to PBR
[27,28]. The fact that the anti-proliferative effects of PBR
ligands were observed in different cancer types [5,6,29]
suggests that PBR ligands interfere with a common
signaling pathway. As the anti-proliferative effects of PBR
ligands were found in tumor cells but not in non-malignant
keratinocytes (Fig. 3) or hepatocytes [30,31], PBR ligands
appear to be tumor-selective agents [5,32]. The reason why
PBR ligands induced growth inhibition of tumor cells but
not of keratinocytes is yet unknown.
Furthermore, we show that PBR ligands sensitize HCC
cells to cytostatic drugs. The addition of PBR ligands led to
a synergistic anti-neoplastic action with either paclitaxel, or
docetaxel, or doxorubicin. Thus, the dose effectiveness of
doxorubicin was doubled, the one of paclitaxel or docetaxel
was increased five-fold. The synergistic enhancement of
paclitaxel-induced growth inhibition of Huh-7 cells was
shown to be due to a dramatic enhancement of apoptosis.
These findings argue for combination therapies of doxor-
ubicin or taxans with PBR ligands. By inhibiting Bcl-2
expression, PBR ligands may also allow to overcome Bcl-2-
mediated chemoresistance in HCC [8,33].
We further showed that a cell cycle arrest contributed to
the anti-proliferative effects of PBR ligands. The proportion
of HCC cells in the G0/G1 phase significantly increased
upon treatment with PBR ligands. In addition, a G2/M arrest
was observed in HepG2 cells. Thus, our results suggest that
PBR ligands can act both at the G1/S and G2/M checkpoints
in HCC cells, which has previously been found for breast
cancer cells [7]. In colorectal and esophageal cancer cells,
however, only a G1/S arrest was found [5,6]. The exact
mechanisms by which PBR ligands interfere with cell cycle
regulation are still under investigation. Recently, we
identified the p38MAPK pathway as a key regulator of
PBR-ligand-mediated G1/S arrest and apoptosis in esopha-
geal cancer cells [19].
Moreover, we observed a significant rise of apoptotic cells
after treatment with PBR ligands. Mitochondrial alterations
are well-known initiating events in the process of apoptosis
[34]. PBR was found to be localized in the mitochondria of
all PBR-expressing HCCs studied. Within the mitochondrial
membrane, PBR has been suggested to contribute to the
formation of the permeability transition pore [35]. The
opening of this pore, leading to DJM breakdown, is
considered to play a critical role in the initiation of apoptosis
[36]. Similar to our findings in colorectal and esophageal
cancer cells [5,6], our present results suggest that a drop in
DJM and the activation of caspase-3 are involved in PBR
ligand-induced apoptosis of HCC cells. The permeability
transition pore is known to interact with proteins of the Bcl-2
family [37,38]. PBR expression correlates positively with
Bcl-XL and negatively with Bax expression during apopto-
sis, suggesting an interaction between Bcl-2 proteins and
PBR [30,39]. We herein demonstrate the induction of Bax
and the suppression of Bcl-2 and Bcl-XL by PBR ligands.
The PBR ligand-induced suppression of Bcl-2 may
sensitize Bcl-2-overexpressing cells to chemotherapy [8,
40,41]. Another strategy against Bcl-2-mediated
A.P. Sutter et al. / Journal of Hepatology 41 (2004) 799–807806
(chemo)resistance is the administration of the Bcl-2
inhibitor HA14-1, a useful parental compound for the
future development of clinically applicable agents [42]. The
down-regulation of Bcl-2 by PBR ligands combined with
its inhibition by HA14-1 proved to inhibit the growth of
HepG2 cells synergistically. Thus, we conclude that Bcl-2
is an attractive target for innovative treatment strategies of
hepatocellular carcinoma which can be effectively com-
bined with PBR ligands.
Although PK 11195 binding affinity in HCC cells was
determined to be in the low nanomolar range, the fact that
FGIN-1-52 [18] did not have any anti-proliferative effects
on HCC cells suggests that the effect of FGIN-1-27 was
PBR-specific. PBR-specificity of pro-apoptotic and anti-
mitotic signaling was previously addressed in esophageal
and colorectal carcinoma cells [5,6]. The quantitative
discrepancy between the micromolar ligand concentrations
necessary to induce apoptosis and cell cycle arrest and the
nanomolar binding affinities of PBR ligands has been
extensively discussed before [5,6].
Liver toxicity of alpidem, another PBR ligand, in
patients has been suggested to be due to its interaction
with mitochondrial permeability transition [43]. More-
over, the inhibitory actions of PBR ligands on tetra-
pyrrole transport may lead to long-term liver cytotoxicity
[44]. However, PBR ligands have been safely adminis-
tered in in vivo studies showing no short-term toxicity
and being well-tolerated [8,45]. In line with these results,
no acute toxicity on rat hepatocytes was found [30].
Nevertheless, long-term effects of the PBR ligands
applied in this study should be addressed in future
studies, potentially requiring the development of ligands
lacking this property.
In conclusion, in this report we demonstrate PBR-ligand-
mediated growth inhibition of HCC cells by induction of
apoptosis and cell cycle arrest. PBR ligands qualify for the
development of novel tumor-specific therapies. Further-
more, we suggest the chemosensitization of HCC cells by
PBR ligands as a new approach for enhancing the
susceptibility of HCC cells to chemotherapy.
Acknowledgements
We are indebted to Pierre Carayon who generously
provided us with the anti-PBR antibody 8D7. We thank
Bastian Gerst for expert technical assistance and Yelda
Oezdem for preparing the immunohistochemical slides.
We are indebted to Carola Muller and Holger Seltmann for
kindly providing us with primary human keratinocytes. This
study was supported by grants of the Deutsche Krebshilfe
and Berliner Krebsgesellschaft. Gesine Bradacs was
supported by a scholarship from the DFG, Graduiertenkol-
leg 276/3, ‘signal transduction and recognition’.
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