Chlorpromazine and apigenin reduce adenovirus replication and decrease replication associated toxicity.

Abstract

Adenoviruses can cause severe toxicity in immunocompromised individuals. Although clinical trials have confirmed the potency and safety of selectively oncolytic adenoviruses for treatment of advanced cancers, increasingly effective agents could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary.
We analyzed the effect of chlorpromazine, an inhibitor of clathrin-dependent endocytosis and apigenin, a cell cycle regulator, on adenovirus replication and toxicity. First, we evaluated the in vitro replication of a tumor targeted Rb-p16 pathway selective oncolytic adenovirus (Ad5/3-Delta24) and a wild-type adenovirus in normal cells, fresh liver samples and in ovarian cancer cell lines. Further, we analyzed the in vitro cell killing efficacy of adenoviruses in the presence and absence of the substances. Moreover, the effect on in vivo efficacy, replication and liver toxicity of the adenoviruses was evaluated.
We demonstrate in vitro and in vivo reduction of adenovirus replication and associated toxicity with chlorpromazine and apigenin. Effective doses were well within what would be predicted safe in humans.
Chlorpromazine and apigenin might reduce the replication of adenovirus, which could provide a safety switch in case replication-associated side effects are encountered in patients. In addition, these substances could be useful for the treatment of systemic adenoviral infections in immunosuppressed patients.

Full text

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THE JOURNAL OF GENE MEDICINE
J Gene Med 2007; 9: 3–9.
Published online 6 December 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.984
RESEARCH ARTICLE
Chlorpromazine and apigenin reduce adenovirus
replication and decrease replication associated
toxicity
Anna Kanerva,1,2,3Mari Raki,1,2
Tuuli Ranki,1,2Merja S¨ arkioja,1,2
Jarmo Koponen,1,2Renee A.
Desmond,4Aija Helin,5
Ulf-H˚ akan Stenman,5Helena
Isoniemi,6Krister H¨ ockerstedt,6
Ari Ristim¨ aki,7Akseli
Hemminki1,2*
1Cancer Gene Therapy Group, Rational
Drug Design Program and Haartman
Institute, University of Helsinki, Finland
2Department of Oncology, Helsinki
University Central Hospital (HUCH),
Helsinki, Finland3Department of
Obstetrics and Gynecology, HUCH,
Helsinki, Finland4Department of
Medicine, University of Alabama at
Birmingham, Birmingham, Alabama, USA
5Department of Clinical Chemistry,
HUCH, Helsinki, Finland6Transplantation
and Liver Surgery Unit, Department of
Surgery, HUCH, Helsinki, Finland
7Department of Pathology, HUCH, and
Molecular and Cancer Biology Research
Program, University of Helsinki, Finland
*Correspondence to: Akseli Hemminki,
Cancer Gene Therapy Group, Haartman
Institute and Rational Drug Design
Program, Biomedicum Helsinki,
Department of Oncology, Helsinki
University Central Hospital, PO Box 63,
00014 University of Helsinki, Finland.
E-mail: akseli.hemminki@helsinki.fi
Received: 6 April 2006
Revised: 27 July 2006
Accepted: 6 October 2006
Abstract
Background
mised individuals. Although clinical trials have confirmed the potency and
safety of selectively oncolytic adenoviruses for treatment of advanced can-
cers, increasingly effective agents could result in more toxicity and therefore
it would be useful if replication could be abrogated if necessary.
Adenoviruses can cause severe toxicity in immunocompro-
Methods
dependent endocytosis and apigenin, a cell cycle regulator, on adenovirus
replication and toxicity. First, we evaluated the in vitro replication of a tumor
targeted Rb-p16 pathway selective oncolytic adenovirus (Ad5/3-?24) and a
wild-type adenovirus in normal cells, fresh liver samples and in ovarian cancer
cell lines. Further, we analyzed the in vitro cell killing efficacy of adenoviruses
in the presence and absence of the substances. Moreover, the effect on in vivo
efficacy, replication and liver toxicity of the adenoviruses was evaluated.
We analyzed the effect of chlorpromazine, an inhibitor of clathrin-
Results
replication and associated toxicity with chlorpromazine and apigenin.
Effective doses were well within what would be predicted safe in humans.
We demonstrate in vitro and in vivo reduction of adenovirus
Conclusions
of adenovirus, which could provide a safety switch in case replication-
associated side effects are encountered in patients. In addition, these
substances could be useful for the treatment of systemic adenoviral infections
in immunosuppressed patients. Copyright  2006 John Wiley & Sons, Ltd.
Chlorpromazine and apigenin might reduce the replication
Keywords
adenovirus; gene therapy; virus replication; reduced toxicity
Introduction
The efficacy and safety of adenoviral gene therapy as an antitumor
approach has been validated in recent randomized [1–3] and non-
randomized [4] trials. However, when faced with bulky advanced disease,
effective tumor transduction continues to be the limiting step for
achieving clinical benefits. Replication-competent viruses such as oncolytic
adenoviruses might prove useful in this regard [5]. The replicative
life cycle of the virus results in cancer cell destruction (‘oncolysis’),
while modifications in the viral genome reduce replication in normal
tissues.
Copyright  2006 John Wiley & Sons, Ltd.

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4
A. Kanerva et al.
An increasing understanding of adenovirus biology and
interactions with cellular proteins has allowed creation of
more effective viruses for cancer gene therapy. However,
with more potent agents an increase in side effects is
also possible [6]. The potential for adenovirus-associated
toxicity is corroborated by reports of severe toxicity
in immunocompromised individuals [7] and a fatality
reported in an adenoviral gene therapy trial [8]. We
hypothesized that we may be able to reduce adenovirus
replication with pharmacological intervention.
Initial binding of adenovirus occurs via interaction of
the fiber knob domain with the primary receptor. Binding
is followed by internalization, which triggers endocytosis
via clathrin-coated pits, viral disassembly, endosomal
lysis and transport into the nucleus. Endosomal uptake
into clathrin-coated pits seems a universal feature
regardless of the virus receptor [9]. The antipsychotic
agent chlorpromazine inhibits the assembly of clathrin
adapter protein AP2 on clathrin-coated pits, which
causes the pits to disappear from the cell surface
and reappear on endosomal membranes [10]. Another
general requirement for effective DNA virus replication is
induction of S-phase, which can be inhibited with G2/M
arrest mediated by the natural bioflavonoid apigenin
[11].
Materials and methods
Cell lines and agents
293 cells were purchased from Microbix (Toronto,
Canada), while lung adenocarcinoma cell line A549 was
obtained from the American Tissue Culture Collection
(ATCC, Manassas, VA, USA). Ovarian adenocarcinoma
cell lines SKOV3.ip1, Hey and OV-4 were obtained from
Dr. Price, Dr. Wolf (both M. D. Anderson Cancer Center,
Houston, TX, USA) and Dr. Eberlein (Harvard Medical
School, Boston, MA, USA), respectively. All cell lines
were cultured in recommended conditions. Chlorpro-
mazine and apigenin were purchased from Sigma-Aldrich
Finland (Helsinki, Finland). Gemcitabine (Gemzar)
was obtained from Eli Lilly and Co. (Indianapolis, IN,
USA).
Recombinant adenoviruses
Ad5/3-?24 was previously described [12]. Ad300wt, a
wild-type human Ad5, was obtained from ATCC. Both
viruses were propagated on A549 cells, and purified on
cesium chloride gradients. The viral particle (vp) con-
centration was determined at 260 nm, and standard
plaque assay on 293 cells was performed to determine
infectious particles. The ratio of vp/infectious particles
was 10.3 and 10 for Ad5/3-?24 and Ad300wt, respec-
tively.
Replication in fresh human liver tissue
and cell monolayers
Fresh liver samples were obtained with the signed
informed consent and ethical committee permission from
patients undergoing surgery at the Helsinki University
Central Hospital. Precision-cut (250 µm) slices were
cut with a Vibratome 1000 Plus sectioning system
(Vibratome, St. Louis, MO, USA), and preincubated
with chlorpromazine (5 µg/ml), apigenin (2.5 µg/ml) or
growth medium (mock) for 1 h before adding 107vp of
wild-type or Ad5/3-?24. Liver sections do not adhere and
thuswashingwasnotperformed. Atindicated time points,
liver slices and supernatant were frozen separately. After
gradualthawonice,theliverslicesweremincedintosmall
pieces, combined with supernatant and subsequently
freeze-thawed three times. After centrifugation, the
supernatant was titered on 293 cells by TCID50 assay.
Further, released liver aspartate transaminase (AST) level
was measured from supernatant after 24 h infection
with a photometric method (Roche Hitachi MODULAR,
Hitachi Ltd., Tokyo, Japan). Cell line monolayers were
preincubated with chlorpromazine (5 µg/ml), apigenin
(2.5 µg/ml) or growth medium (mock) for 1 h. The
viruses (10 vp/cell) were added on supernatant, which
was replaced by fresh growth medium ± reagents 1.5 h
later. Replication was analyzed after three freeze/thaw
cycles as above. Time points for analysis were selected
based on assumptions on maximal replication on one
hand and limitations on liver tissue availability on the
other.
Cell killing assay
Cell monolayers were preincubated with chlorpromazine
(5 µg/ml) or apigenin (2.5 µg/ml) for 1 h. Thereafter,
cells in quadruplicate were infected with Ad300wt,
Ad5/3-?24 or no virus for 1.5 h at 37◦C, and fresh
growth medium ± reagents was added. Cell viability was
measured using the CellTiter 96 AQueousOne Solution cell
proliferation assay (MTS assay, Promega, Madison, WI,
USA), when any combination at 100 vp/cell displayed
complete cell killing.
In vivo models of efficacy, replication
and toxicity
All animal protocols were reviewed and approved by
the Experimental Animal Committee of the University
of Helsinki and the Provincial Government of Southern
Finland. Mice were obtained from Taconic (Ejby,
Denmark) at 4 weeks of age and quarantined at least
for 1 week prior to the study. Subcutaneous (s.c.) human
ovarian cancer (Hey cell) tumors were established in
female NMRI CD-1 nude (n = 10 tumors/group), and
treated with intratumoral (i.t.) injections of 3 × 108vp of
Ad5/3-?24 or no virus on days 0, 2 and 4. Mice received
Copyright  2006 John Wiley & Sons, Ltd.
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Reduction of Adenovirus Replication
5
intraperitoneal (i.p.) injections of phosphate-buffered
saline (PBS), chlorpromazine (200 µg) or apigenin
(250 µg) every day. Tumor growth and body weight
were followed. Tumor volume is calculated from the
formula 0.5 × length × width2, and the data is expressed
as % of the tumor volume at the initiation of therapy
(32–88 mm3) to allow correction for variation in initial
tumor volume.
In replication assay, the s.c. Hey cell tumors were
established as above, and treated with a single i.t.
injection of 3 × 108vp of Ad5/3-?24 on day 0. Mice
received i.p. injections of PBS, chlorpromazine (200 µg)
or apigenin (250 µg) every day (n = 12 tumors/group).
Four tumors/group were harvested at indicated time
points, homogenized, and the virions were released
by three freeze/thaw cycles. The amount of infectious
particles was analyzed by TCID50.
We utilized a previously described murine toxicity
model analyzing the liver damage after combination
treatment of Ad5/3-?24 and gemcitabine [6]. Briefly,
107SKOV3.ip1 cells were injected i.p. into CB17 SCID
mice on day 0. On day 10 mice were injected i.p.
with 3 × 107vp of Ad5/3-?24 and (1) PBS (n = 7),
(2) chlorpromazine (200 µg, n = 8), or (3) apigenin
(250 µg, n = 8). PBS, chlorpromazine and apigenin were
thereafter administered daily. Further, all mice received
80 mg/kg gemcitabine i.p. on days 11, 14, 21 and 24.
The mice were followed daily and killed when there
was any evidence of pain or distress. Livers and residual
tumors were harvested and fixed in buffered formalin.
Serial paraffin-embedded sections were taken and stained
with H&E. Histopathology was scored blinded by an
independent pathologist.
Statistics
The effects of CPZ and apigenin on replication or cell
killing efficacy were analyzed using bootstrap multiple
comparisons of means in analysis of variance (ANOVA)
(PROC MULTTEST SAS v9.1, SAS Institute, Cary, NC,
USA). The results with virus + reagent combinations were
compared to the other groups. For statistical analyses
the levels of viral replication were log transformed for
normality. An adjusted bootstrap P value of <0.05 was
deemed statistically significant.
Theanalysisofthetumorsizedatawasperformed using
a repeated measures growth model with PROC MIXED
(SAS v.9.1), which treated the within-mouse effect of
time as a continuous variable and the treatment group as
a fixed effect. The tumor size data was log transformed for
normality. The effects of treatment group, time and the
interaction of treatment group and time were evaluated
by F tests. Baseline tumor size was included as a covariate
in all models and flank as a covariate. The a priori
plannedcomparisonsofdifferencesinpredictedtreatment
means were computed by t-statistics at study’s end and
averaged over all time points. Tukey-Kramer adjustment
was utilized to allow for multiple comparisons.
Liver toxicity data was analyzed using the χ2test. Only
preplanned comparison was made.
Results and discussion
We analyzed the in vitro replication of a tumor targeted
Rb-p16 pathway selective oncolytic adenovirus (Ad5/3-
?24) [12] and a wild-type adenovirus in fresh human
liver explants. Wild-type virus displayed replication over
48 h. Interestingly, chlorpromazine reduced the titer 8-
fold (Figure 1A, P = 0.0271 at 48 h). Further, lower
liver aspartate transaminase (AST) levels were measured
from chlorpromazine-treated livers suggesting reduced
hepatocyte damage (Figure 1E). Importantly, as Ad5/3-
?24 is in development for human trials, it did not
replicate productively in the liver explants (Figure 1B).
Nevertheless, even marginal replication was further
attenuated by chlorpromazine as evidenced by decreasing
titer at later time points. In non-malignant 293 cells both
viruses replicated avidly as expected, and chlorpromazine
reduced replication up to 1960-fold (Figures 1C and 1D,
wild-type: P = 0.0278, 0.0056 and 0.0433; Ad5/3-?24:
P = 0.0248, 0.0040 and 0.0259 at 24, 36 and 48 h,
respectively).
In ovarian adenocarcinoma Hey cells apigenin reduced
replication 100-fold (Figures 2A and 2B, wild-type: P =
0.0156; Ad5/3-?24: P = 0.0478 at 72 h), but the effect
was less pronounced in another ovarian cancer cell
line OV-4 (Figures 2C and 2D, P = 0.0428 at 24 h).
Presumably, genetic heterogeneity between OV-4 andHey
resulted in variation in replication attenuation with the
drugs. The close association of replication to cell killing
was corroborated in a longitudinal assay where apigenin
reduced the activity of Ad5/3-?24 (Figure 2E, P =
0.0680). Carette et al. reported 3 to 4 orders of magnitude
reduction in oncolytic adenovirus DNA copy number with
apigenin treatment in a quantitative polymerase chain
reaction (PCR) assay, and also the expression of transgene
was reduced up to 4 logs in various cancer cell lines [13].
Of note, in those experiments the apigenin concentration
was 8 times higher than the concentration we utilized.
In our experiments higher concentrations displayed cell
toxicity (data not shown).
As human adenoviruses do not replicate productively
in murine normal tissues [14], human xenografts in
mice were utilized for in vivo studies. Both apigenin
and chlorpromazine were found to reduce the antitumor
efficacy of Ad5/3-?24 (both P < 0.0001, Figure 3A), due
to reduced replication (Figure 3B). Production of new
viruses was reduced 36-fold with chlorpromazine (P =
0.0286 and 0.0312 on days 3 and 7, respectively). An 11-
fold decrease was seen also with apigenin (P = 0.1380).
Neither apigenin nor chlorpromazine had antitumor
activity per se. We do not fully understand the reason for
the discrepancy in the effect of chlorpromazine on Hey
cells with Ad5/3-?24 in vitro versus in vivo. However,
becausethisvirusreplicatesveryrapidlyin vitro,thespeed
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A. Kanerva et al.
Figure 1. Reduction of replication of wild-type adenovirus (A, C) and tumor-selective oncolytic Ad5/3-?24 (B, D) in vitro in normal
tissues. (A, B) Replication was analyzed in precision-cut 250 µm human liver slices, which were infected with 107viral particles (vp)
± chlorpromazine (5 µg/ml) or apigenin (2.5 µg/ml). Later, slices and supernatant were homogenized and freeze/thawed three
times. Infectious particles were titered on 293 cells by TCID50assay. (C, D) Non-malignant but adenovirus transcomplementing
293 cells. (E) Following infection of human liver slices, AST levels were measured from the supernatant. Data is presented as mean
± SE, n = 2–4,∗P < 0.05,∗∗P < 0.01 (ANOVA, PROC MULTTEST)
of replication may actually become counterproductive
to packaging of functional virions. Theoretically, cells
might be lysed before producing the maximum number
of virions. Thus, slowing of the speed of replication may
not necessarily be sensitively seen as reduction of in vitro
virion production. Nevertheless, tumor penetration and
intratumoral dissemination might be improved by rapid
replication and release, and these aspects cannot be
assessed in vitro. Therefore, we feel that the in vivo data
gives a more complete and relevant picture of the actions
of the drugs.
To assess the general toxicity of the treatment regimens
and the overall health of the mice, their body weight was
followed (Figure 4A). There was no weight loss in any
treatment group, and the lowest body weights were in
groups not treated with virus.
The liver is the most relevant organ for adenovirus-
associated toxicity [8]. Previously, we developed a murine
model of adenovirus replication associated liver toxicity
[6]. Peritoneally disseminated human ovarian cancer
is first inoculated and then cured by injecting Ad5/3-
?24 and gemcitabine. However, the increase in cancer-
free survival uncovered treatment-related toxicity. Many
mice died due to fulminant liver necrosis caused by
persistent virus replication and subsequent sustained liver
damage that was exacerbated by gemcitabine. Here,
this model was repeated and mice succumbed with
liver necrosis, foamy degeneration and steatosis. Further,
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Reduction of Adenovirus Replication
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Figure 2. Reduction of replication of wild-type adenovirus (A, C) and tumor-selective oncolytic Ad5/3-?24 (B, D) in vitro in cancer
tissues. Infectious particles were titered on 293 cells by TCID50assay. (A, B) Ovarian adenocarcinoma cell line Hey. (C, D) Ovarian
adenocarcinoma cell line OV-4. (E) To corroborate infectious virus production with a longitudinal cell killing assay (MTS), Hey cell
monolayers were preincubated with the reagents and infected with Ad5/3-?24
the few surviving hepatocytes displayed large nuclei
(Figure 4B). Of note, apigenin- or chlorpromazine-treated
mice displayed less liver damage (Figure 4C and 4D).
When all evaluable livers were analyzed in a blinded
manner, liver toxicity was present more often in the
PBS group compared to mice that received apigenin or
chlorpromazine (P = 0.0213).
Heretofore, oncolytic adenoviruses have been well
tolerated in cancer trials at doses up to 2 × 1013vp
[5]. However, the most widely studied oncolytic agent
is dl1520 (a.k.a. ONYX-015), which is rather attenuated
even in tumor cells, when compared to wild-type
adenovirus [5]. Newer generation agents replicate
similarly or even more rapidly than wild-type adenovirus
[5,12]. More potent oncolytic adenoviruses could mean
more side effects.
The data presented here suggest that apigenin and
chlorpromazine can reduce the replication of aden-
oviruses in vitro and in vivo which could thereby provide
a safety switch in case of side effects. Compounding the
need for an antidote, virus replication may persist for
weeks [4], as opposed to most conventional medicines
that have short half-lives. Importantly, chlorpromazine
has been used for decades in millions of humans and the
safety and side effect profile are well understood [15].
Apigenin is present in many vegetables, such as pars-
ley and onion, which suggests that patients participating
in oncolytic adenovirus trials might benefit from dietary
advice to avoid attenuation of antitumor activity. Also
apigenin has been studied in clinical trials with good
safety data [16]. In this study, both substances were used
at concentrations previously reported to translate into
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A. Kanerva et al.
Figure 3. Reduction of replication in vivo. (A) Human ovarian cancer (Hey) tumors (n = 10) were established in female nude
mice, and treated with intratumoral (i.t.) injections of 3 × 108viral particles (vp) of Ad5/3-?24 or no virus on days 0, 2 and 4.
Mice received intraperitoneal (i.p.) injections of PBS, chlorpromazine (200 µg) or apigenin (250 µg) every day. Tumor volume is
expressed as % of day 0 volume. (B) To evaluate virus replication, Hey tumors (n = 12) were established and treated with a single
i.t. injection of 3 × 108vp of Ad5/3-?24 on day 0. Mice received daily i.p. injections of PBS, chlorpromazine or apigenin. Four
tumors/group were harvested at indicated time points, homogenized, and freeze-thawed three times. Infectious particles were
determined by TCID50(n = 8). Data present mean ± SE,∗P < 0.05 (ANOVA),∗∗∗P < 0.0001 (PROC MIXED, Tukey-Kramer adjusted)
Figure 4. Reduction of replication-associated toxicity in vivo. (A) Lack of weight loss suggested good tolerability. (B–D) In a murine
model of adenovirus replication associated toxicity [6], reduced toxicity was seen with chlorpromazine or apigenin (P = 0.0213,
χ2test). 107SKOV3.ip1 ovarian cancer cells were injected i.p. into CB17 SCID mice. Ten days later, mice (n = 7–8/group) were
injected i.p. with 3 × 107vp of Ad5/3-?24. PBS, chlorpromazine and apigenin were administered daily. Typical histopathological
appearance of the livers of (B) PBS-, (C) chlorpromazine- and (D) apigenin-treated mice (H&E, magnification ×20)
well-tolerated and active human serum concentrations
[15,16].
In addition, these substances could be useful for the
treatment of systemic adenoviral infections in immuno-
suppressed patients, particularly pediatric bone marrow
transplant recipients. Intriguingly, the mechanisms tar-
geted by these drugs are central for replication of many
other types of viruses, including herpes- and parvoviruses.
Therefore, this approach may have applicability beyond
adenoviruses.
Acknowledgements
This work was supported by HUCH Research Funds (EVO),
Academy of Finland, Sigrid Juselius Foundation, Emil Aaltonen
Foundation, Finnish Cancer Society, University of Helsinki,
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Sohlberg Foundation, Research and Science Foundation of
Farmos, Biocentrum Helsinki, Instrumentarium Research Fund,
and Finnish Oncology Association.
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