RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Synergism Between Propolis and Hyperthermal Intraperitoneal

Chemotherapy with Cisplatin on Ehrlich Ascites Tumor in Mice
NADA OR

SOLI

C,
1
NIKOLA CAR,
2
DUJE LISI

CI

C,
1
VESNA BENKOVI

C,
1
ANICA HORVAT KNE

ZEVI

C,
1
DOMAGOJ

DIKI

C,
1
J

OZSEF PETRIK
3
1
Department of Animal Physiology, Faculty of Science, University of Zagreb, Zagreb 10000, Croatia
2
Pliva Croatia Ltd., Zagreb 10000, Croatia
3
Department of Medical Biochemistry and Haematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb 10000,
Croatia
Received 23 January 2013; revised 17 September 2013; accepted 25 September 2013
Published online 17 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23755
ABSTRACT: We investigated antitumor, genotoxic, chemopreventive, and immunostimulative effects of local chemoimmunotherapy and
hyperthermal intraperitoneal chemotherapy (HIPEC) in a mouse-bearing Ehrlich ascites tumor (EAT). Mice were treated with water-soluble
derivative of propolis (WSDP) at a dose of 50 mg kg
1
, 7 and 3 days before implantation of EAT cells, whereas cisplatin (5 or 10 mg kg
1
)
was injected 3 days after implantation of EAT cells at 37

C and 43

C. The following variables were analyzed: the total number of cells,

differential count of the cells present in the peritoneal cavity, functional activity of macrophages, comet assay, and micronucleus assay.
The combination of WSDP + CIS 5 mg kg
-1
at 37

C resulted in tumor growth inhibition and increased the survival of mice by additional
115.25%. WSDP with HIPEC increased the survival of mice by additional 160.3% as compared with HIPEC. WSDP reduced cisplatin
toxic and genotoxic effect to normal cells without affecting cisplatin cytotoxicity on EAT cells. In addition, WSDP with HIPEC increased
the cytotoxic actions of macrophages to tumor cells. Water-soluble derivative of propolis increases macrophage activity and sensitivity
of tumor cells to HIPEC and reduces cisplatin toxicity to normal cells.
C
2013 Wiley Periodicals, Inc. and the American Pharmacists
Association J Pharm Sci 102:43954405, 2013
Keywords: hyperthermia; chemotherapy; immunomodulation; propolis; cisplatin; Ehrlich ascites tumorp; cancer chemopreveention;
immunotherapy; natural products; toxicity; food interaction; hyperthemia; cisplatin; ehrlich ascites tumor
INTRODUCTION
Peritoneal carcinomatosis (PC) is the dissemination and im-
plantation of tumor cells into the abdominal cavity, often with-
out systemic metastases, with the origin of the tumor can be
local or distant organs. PC is a very bad prognostic sign and
a frequent cause of death in patients with cancer of the colon,
stomach, as well as other gastrointestinal and gynaecological
cancers.
1,2
Intraperitoneal (i.p.) drug administration results in high lo-
coregional drug concentrations, increased exposure of drug to
the microscopic or small macroscopic peritoneal disease, and
high peritoneal tumor concentrations, whereas systemic toxi-
city is limited. Intraoperative i.p. chemotherapy allows better
exposure of the entire seroperitoneal surface to chemothera-
peutic agents, minimizes the number of viable exfoliate tu-
mor cells after resection and avoids complications related
to intra-abdominal catheters. Moreover, intraoperatively i.p.
chemotherapy may be combined with hyperthermia.
24
Hyper-
thermia enhances the cytotoxicity of some chemotherapeutic
agents including cisplatin.
14
Cisplatin [cis-diamminechloroplatinum (II)] is one of the
most effective chemotherapeutic agents for cancer treatment.
It is generally accepted that the cytotoxic activity of cisplatin
results from its interactions with DNA to form DNA adducts,
Correspondence to: Nada Or soli c (Telephone: +385-1-4877-735; Fax: +385-1-
482-6260; E-mail: norsolic@yahoo.com)
Journal of Pharmaceutical Sciences, Vol. 102, 43954405 (2013)
C
2013 Wiley Periodicals, Inc. and the American Pharmacists Association
primarily intrastrand cross-link adducts, which activate sev-
eral signal transduction pathways and culminate in the acti-
vation of apoptosis.
5,6
The major limitations associated with
the treatment of human patients with cisplatin are side effects
(nephrotoxicity, hapatotoxicity, myelotoxicity, and hematotoxi-
city) and resistance of tumor cells to this drug.
713
Oxidative
stress is one of the most important mechanisms involved in
cisplatin-induced toxicity.
9
Because of its dose-limiting toxic
side effects and drug resistance, there is a clinically important
need to develop procedures that enhance the therapeutic ef-
cacy of cisplatin.
In response to this need, we examined the usefulness of pre-
ventive treatment of Ehrlich ascites tumor (EAT) by local im-
munotherapy with water-soluble derivative of propolis (WSDP)
combined with cisplatin chemotherapy in normal (37

C) and
hyperthermic (43

C) intrapersonal conditions. Hyperthermia

is known to enhance the therapeutic efcacy of cisplatin when
given simultaneously.
2,4,14
In addition, i.p. chemotherapy has
the advantage of a high local concentration of the cytostatic
drug in tumor tissue with less systemic side effects. Further-
more, antioxidants can enhance the efcacy of chemother-
apy and/or minimize the side effects when combined with
chemotherapeutic treatment.
2,1317
Propolis (bee glue) is the generic name for the resinous sub-
stance collected by honeybees from various plant sources. It
is masticated, partially digested with honeybee salivary en-
zymes, mixed with beeswax, and used by bees to seal holes
in their hives, smooth out the internal walls, and protect the
entrance against intruders.
13,16
For propolis production, bees
use materials resulting from a variety of botanical processes in
Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013 4395
4396 RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology
different parts of plants. These substances are actively secreted
by plants as well as substances exuded from wounds in plants.
Chemical composition of propolis depends on the specity of the
local ora at the site of collection and thus on the geographic
and climatic characteristic of the site. As a consequence, more
than 300 constituents of propolis have been identied so far.
18
In spite of the great differences in the chemical composition,
propolis from all regions exhibit similar biological properties.
It has been suggested that the therapeutic activities of propo-
lis depend mainly on the presence of avonoids.
13,16,19
It was
demonstrated that propolis has pronounced immunomodula-
tory effect,
2026
antioxidant effect,
17,2729
and antitumor effect
both in vitro and in vivo.
2024,29,30
Chemopreventive and direct
antitumor effect of propolis is based on preventing carcinogen
metabolic activation, preventing tumor cell proliferation by in-
activation or downregulation of prooxidant enzymes or signal
transduction enzymes, and induction of apoptosis.
13,16,24,30
In-
direct antitumor effect of propolis is based on its antioxida-
tive properties via scavenging of free radicals and increasing
the activities of antioxidant enzymes, and immunomodulating
properties via macrophage activation.
16,20,21,2527
Much information can be gained by investigating the conse-
quences of hyperthermia on individual cell populations in vitro;
however, the precise effects of such a therapeutic modality in
vivo depend on the tumor microenvironment and the cellular
composition therein. Thus, the selection of mouse model with
the EAT is suitable for studying the effectiveness of HIPEC in
the presence of ascites in peritoneal carcinogenesis. Although
the direct cytotoxic effects of hyperthermia on tumor tissue can
lead to an immediate reduction in tumor volume, long-term
benets to local and distal tumor recurrence will very much de-
pend on the induction of immunity and the capacity of effector
cells to trafc to tumors and elicit their cytotoxic functions. The
aim of this study was to examine antitumor, genotoxic, chemo-
preventive, and immunostimulative effects of WSDP combined
with anticancer drug cisplatin in mice bearing EAT in physi-
ological (37

C) and hyperthermic (43

C) intraperioneal condi-
tions. So, we hypothesized that immunostimulation comprising
macrophages plays a major part in the eradication of tumors
by hyperthermal intraperitoneal chemotherapy (HIPEC).
MATERIALS AND METHODS
Animals
Male Swiss albino inbred mice, weighing 2025 g, approxi-
mately 2-month old, obtained from the Department of Animal
Physiology, Faculty of Science, University of Zagreb, were used
in this study. In all the experiments, mice were of the same sex.
The animals were kept not more than ve per cage and were
maintained on a pellet diet (standard diet 4RF 21; GLP cer-
ticate, Mucedola, Italy) and water ad libitum. Experimental
groups comprised 715 mice each. Animal studies were per-
formed in compliance with the guidelines in force in the Repub-
lic of Croatia (Law on the Welfare of Animals, N.N. #135, 2006;
Regulations for the Environmental Conditions of Experimental
Animals, Special Conditions for the Facilities and Experiment
Categories, N.N. #176, 2004) and according to the Guide for
the Care and Use of Laboratory Animals [DHHS Publ. (NIH)
86123, 1985].
Tumor Cells
Ehrlich ascites tumor is transplantable, poorly differentiated,
and fast-growing malignant tumor that appeared originally as
a spontaneous breast carcinoma in a mouse. EAT cells were
maintained in male Swiss albino mice in ascitic form by serial
i.p. inoculation at 7 or 9 days intervals. After harvesting and
preparation of cells, their total number and viability were de-
termined by counting in B urkerT urk chamber using Trypan
Blue dye. The desired concentration of tumor cells (2 10
6
cells
per 0.5 mL) was obtained by dilution with saline (0.9% sodium
chloride solution).
Hyperthermia
Intraperitoneal hyperthermia was induced by i.p. inoculation
of 2 + 2 mL saline (0.9% sodium chloride solution) preheated
in water bath to 43

C. Hyperthermia treatment was performed

immediately before cisplatin application. Systemic body tem-
perature was determined using an electronic thermometer
(BAT-10; Physitemp Instruments, NewJersey, USA) before and
during hyperthermia procedure. Intraperitoneal temperature
was measured by the needle probe introduced to a depth of
1 cminto peritoneumcavity of mice treated with hyperthermia.
Cisplatin was applied immediately after the last treatment of
hyperthermia. The temperature was recorded in each experi-
mental group every 2.5 min during the heating phase and every
2.5 min during the cooling phase. Hyperthermia with applied
procedure was well tolerated by the animals.
2,4
Cisplatin
The anticancer drug cisplatin [cis-diamindikloroplatinum (II)]
was supplied by Pliva (Zagreb, Croatia). It was injected into
mice intraperitoneally at doses of 5 and 10 mg kg
1
.
Water-Soluble Derivative of Propolis
Water-soluble derivative of propolis was prepared by the
method described in our previous paper.
20
Briey, Croatian
propolis from beehives kept at the outskirts of Zagreb was ex-
tracted with 96%ethanol, and then the preparation was ltered
and evaporated to dryness in vacuumevaporator. The resultant
resinous product was added to a stirred solution of 8% L-lysine
(Sigma, Deisenhofen, Germany) and freeze-dried to yield the
WSDP, a yellow-brown powder.
The chemical prole of propolis from the northern hemi-
sphere, often named as poplar-type propolis can be char-
acterized by the three analytical parameters: total avonol
and avone content, total avanone and dihydroavonol con-
tent, and total polyphenolics content.
19
According to the
spectrophotometric assay,
16,19
WSDP contains: avones and
avonols (2.13%), avanones and dihydroavonols (9.06%), to-
tal avonoids (11.19%), and total polyphenols (70.48%). Accord-
ing to the HPLC analysis, WSDP contains 1.28% caffeic acid,
4.5% chlorogenic acid, 1% p-coumarinic acid, 4.7% isoferulic
acid, 30.29% ferulic acid, and 38.11% lysine.
The WSDP was stored under sterile conditions at 20

C
to minimize bacterial contamination. Before use, WSDP was
dissolved in distilled water and injected into mice intraperi-
toneally at dose 50 mg kg
1
.
Experimental Procedure
Mice were divided into 12 experimental groups. Tumor was pro-
duced by i.p. implantation of EAT cells (2 10
6
) to all groups,
Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013 DOI 10.1002/jps.23755
RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology 4397
including control groups. Mice were preventively treated with
WSDP (i.p. 50 mg kg
1
) 7 and 3 days before i.p. inoculation of
2 10
6
EAT cells, whereas cisplatin (i.p. 5 or 10 mg kg
1
) was
applied 3 days after inoculation of EAT cells at 37

C and 43

C.
Intraperitoneal hyperthermia was induced by i.p. inoculation
of 2 + 2 mL saline (0.9% sodium chloride solution) preheated
in water bath to 43

C immediately before cisplatin application.

This procedure was performed to all experimental groups, re-
gardless of cisplatin application. The same procedure was per-
formed for experimental groups treated at 37

C, whereas saline
was preheated in water bath to 37

C.
Intraperiotoneal liquid samples from preventively treated
mice were collected fromeachgroup 3 days after tumor cell inoc-
ulation and approximately 1 h after i.p. application of cisplatin.
The following variables were analyzed: tumor cells viability and
mortality in the intraperitoneal liquid, differential count of the
cells present in the intraperitoneal liquid, and determination
of functional activity of macrophages in the intraperitoneal liq-
uid. Simultaneously, peripheral blood, liver, and kidney sam-
ples were collected for comet assay. Peripheral blood samples
for micronucleus test were collected 48 h after i.p. application
of cisplatin. Before sample collection, mice were sacriced with
ether anesthesia and by cervical dislocation. Blood samples for
comet assay were collected after mice exsanguinations from
axillary blood vessels. After disinfection of external abdominal
region, each animal was inoculated with 5 mL of saline solu-
tion, and after gentle agitation of abdominal well, the solution
containing peritoneal cells was removed for cellular evalua-
tion. The liver and kidney tissues were collected from mice and
pressed through the cloth (Miracloth, Falcon) in the precooled
(4

C) homogenization buffer solution at pH 7.5 (0.075 M NaCl

and 0.024 M Na
2
EDTA) in 1 g tissue:1 mL buffer ratio. Periph-
eral blood samples for micronucleus test were collected from a
tail vein.
Survival Analysis
Animal life span was evaluated by surveillance of spontaneous
death or by selective killing of animals showing signs of pain or
suffering according to established criteria. Long surviving mice
(living more than 90 days) were euthanized on the 90
th
day by
an overdose of anesthetic and cervical dislocation. Results were
expressed as a percent of mean survival time of the treated an-
imals over mean survival time of the control group (treated vs.
control, T/C%). The percentage of increased lifespan (ILS%) was
calculated by the following formula: ILS% = (T C)/C 100,
where T represents mean survival time of the treated animals
and C represents the mean survival time of the control group.
Long-term survivors (LTS%) were mice surviving more than
90 days after i.p. implantation of EAT cells. According to the
criteria of the National Cancer Institute, T/C above 125% and
ILS above 25% mean that treatment had signicant antitumor
effect.
Tumor Cells Viability and Mortality in the Intraperitoneal Liquid
The viability and mortality of tumor cells was determined by
counting in a B urkerT urk chamber by observing the ability of
intact cells to exclude Trypan Blue dye and by phase contrast
microscopy.
Differential Count of the Cells Present in the Intraperitoneal
Liquid
The cells in the peritoneal cavity of mice were harvested,
stained with May Gr unwald and Giemsa water solution
(1 part Giemsa:2 part water), and later differentiated into
macrophages, lymphocytes, and neutrophiles and tumor
cells.
16,24
Differential cell counts were determined by counting
at least 800 cells in each sample.
Functional Activity of Macrophages in the Intraperitoneal Liquid
Functional activity of macrophages in the peritoneal cavity was
determined by the spreading technique described in Refs.16,24
Thus, 10
3
cells in 0.1 mL of the cellular suspension obtained
in the peritoneal cavity were placed over glass cover slips at
room temperature for 15 min. The nonadherent cells were re-
moved by washing with phosphate-buffered saline, and the ad-
herent cells were incubated in culture medium 199 containing
10 nM Hepes at 37

C for 1 h. Following incubation, the cul-

ture medium was removed and the cells were xed with 2.5%
glutaraldehyde for 10 min. Then, the cells were stained with
5% Giemsa solution and examined under microscope where the
percentage of spread cells were determined under 400 mag-
nications. Spread cells were those that presented cytoplasmic
elongation, while the nonspread cells were rounded.
16,24
Comet Assay
The in vivo comet assay (single cell gel electrophoresis) under
alkaline conditions (pH > 13), as a simple visual method for
the measuring of DNA damage, was used to investigate the
genotoxicity of testing compounds. The basic principle of the
comet assay is the migration of DNA in an agarose matrix
under electrophoretic conditions. Two slides per animal of pe-
ripheral blood, liver, kidney, and tumor samples were prepared.
The samples were analyzed using a modication of the method
of Singh et al.,
31
as described in the following procedure. Fully
frosted slides were coated with 1% and 300 :L of 0.6% normal
melting point agarose (Sigma). A freshly prepared cell suspen-
sion, obtained as a mixture of 5 :L cells with 100 :L of 0.5%
lowmelting point (LMP) agarose (Sigma), were placed onto pre-
cleaned microscope slides. After cooling on ice for 10 min, slides
were covered with 100 :L 0.5% LMP agarose (Sigma). After so-
lidication of the agarose gel, slides were immersed for 2 h in
ice-cold lysis solution, consisting of 100 mM Na
2
EDTA, 2.5 M
NaCl, 10 mM TrisHCl, and 1% sodium sarcosinate, adjusted
to pH 10 with 1% Triton X-100 and 10% dimethyl sulfoxide
apprior to use. Prior to electrophoresis, slides were removed
from the lysing solution and placed for 20 min in a horizon-
tal electrophoresis unit (near the anode) lled with an alkaline
buffer, to allow the unwinding of DNA and to express alkali
labile damage. The electrophoresis alkaline solution (pH 13)
consisted of 1 mM Na
2
EDTA and 300 mM NaOH. After un-
winding of DNA, electrophoresis was carried out in the same
solution for 20 min at 25 V(300 mA). Alkali unwinding and elec-
trophoresis were performed at 4

C under dimmed light. After

electrophoresis, the slides were washed with a neutralization
buffer (0.4 M TrisHCl, pH 7.5) three times at 5 min intervals.
After neutralization, slides were stained with the uorescent
dye, ethidium bromide (20 :g mL
1
), covered and stored in
sealed boxes at 4

C for analyzing. A total of 400 cells of the

same kind per each experimental group (100 from each sample
in four replicates) were analyzed using a 400 magnication
DOI 10.1002/jps.23755 Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013
4398 RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology
uorescence microscope (Olympus, Tokyo, Japan) equipped
with an excitation lter of 515560 nm and a barrier lter
of 590 nm. Electrophoresis at high pH results in structures re-
sembling comets, as observed by uorescence microscopy. When
viewed under a microscope, a cell has the appearance of a comet,
with a head (the nuclear region) and a tail containing DNA
fragments or strands migrating in the direction of the anode.
The intensity of the comet tail relative to the head reects the
number of DNA breaks.
32,33
Despite modern evaluation soft-
ware for precise estimates of DNA damage, the human eye
also provides the successful determination of damage degree
according to comet appearance. DNA migration can be deter-
mined visually by the categorization of comets into different
classes of migration, or by using an eyepiece micrometer to
estimate image or tail length.
32,34
For quantifying DNA dam-
age, comets were classied into ve categories (04 arbitrary
units) based on the tail length.
6,33
The average extent of DNA
damage among cells was expressed as total comet score (TCS)
and calculated according to following formula: TCS = [0 N
0
(number of comets from class 0) + 1 N
1
(number of comets
fromclass 1) +2 N
2
(number of comets fromclass 2) +3 N
3
(number of comets from class 3) + 4 N
4
(number of comets
from class 4)].
Micronucleus Assay In Vivo
Peripheral blood samples were collected from the tail vein af-
ter a brief warming period under a heat lamp. Acridine or-
ange supravital staining of blood smears was made according
to the method described in Ref.16. To determine a number of
micronuclei a total of 8000 reticulocytes per each experimental
group (2000 from each sample in four replicates) were analyzed
using a 400 magnication uorescence microscope (Olympus)
equipped with an excitation lter of 502525 nm.
Statistics
Data were analyzed using statistical software STATA 7.0
(Stata Press, College station, Texas). Results were expressed
as means SD. Statistical signicance was evaluated using
ANOVA. A p value <0.05 was considered to be signicant.
Treatment and dose-specic survival curves were calculated
by KaplanMeier method,
35
and comparison between survival
curves was made by log-rank test.
36
RESULTS
WSDP Enhances Cisplatin Hyperthermal Antitumor Effect and
Life Span of Mice
These experimental studies showed statistically signicant
(p< 0.05; log-rank test) differences in the survival times of
mice preventively treated with test compounds at 37

C and
43

C as compared with control (Table 1, Fig. 1). It is important

to emphasize that signicantly increased survival time was ob-
served after preventive treatment performed only with WSDP
at 37

C and especially at 43

C in relation to control group

(Table 1, Fig. 1, II); ILS% at 37

C = 17.21; ILS% at
43

C =273.09; four mice treated with hyperthermia were long-

term survivors (p = 0.0054, KaplanMeier analysis). Further-
more, signicant survival time was observed after preventive
treatment with combination of WSDP and cisplatin at dose
5 mg kg
1
at 43

C in relation to group treated only with

cisplatin at dose 5 mg kg
1
; WSDP + cisplatin 5 mg kg
1
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Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013 DOI 10.1002/jps.23755
RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology 4399
Figure 1. KaplanMeier survival curves of Swiss albino mice-bearing EAT after preventive treatment with WSDP and cisplatin in physiological
(a) and hyperthermal condition (b). Mice were treated with WSDP (50 mg kg
1
, i.p.) 7 and 3 days before i.p. inoculation of EAT (2 10
6
) cells.
Cisplatin was injected (5 or 10 mg kg
1
, i.p.) 3 days after inoculation of EAT cells under physiological and hyperthermal condition. Signicant
difference (p < 0.05; log-rank test) from control group under physiological condition (WSDP, p = 0.0037; cisplatin 5 mg kg
1
, p = 0.0052; cisplatin
10 mg kg
1
, p = 0.0004; cisplatin 5 mg kg
1
+ WSDP, p = 0.0029; cisplatin 10 mg kg
1
+ WSDP, p = 0.0020 under hyperthermal condition
(WSDP, p = 0.0054; cisplatin 5 mg kg
1
, p = 0.0040; cisplatin 10 mg kg
1
, p = 0.0004; cisplatin 5 mg kg
1
+ WSDP, p = 0.0004; cisplatin 10 mg
kg
1
+ WSDP, p = 0.0004).
ILS%= 346.87; cisplatin 5 mg kg
1
ILS%= 186.54; seven mice
treated with WSDP + cisplatin 5 mg kg
1
versus two mice
treated only with cisplatin 5 mg kg
1
were long-term survivors
(p =0.0074, KaplanMeier analysis). The most pronounced an-
titumor effect was achieved after preventive treatment by cis-
platin at dose 10 mg kg
1
at 37

C and 43

C and after treatment

with combination of WSDP and cisplatin at doses 5 and 10 mg
kg
1
at 43

C (Table 1, Fig. 1). In these groups, all mice were

long-term survivors (p = 0.0004, KaplanMeier analysis). It is
important to mention that during the heating phase intraperi-
toneal temperature was stable, ranging from 42.5

C to 43

C,
whereas rectal temperature was not signicantly affected. Dur-
ing cooling phase, the intraperitoneal temperature of treated
mice decreased to normal values approximately 15 min after
hyperthermal procedure.
The analysis of the number of alive tumor cells present in
the peritoneal cavity revealed that all experimental groups in-
oculated with tumor cells in the presence of test compounds,
except groups treated only with WSDP, exhibited a signicant
(p < 0.05; ANOVA) reduction of alive tumor cells in peritoneal
cavity as compared with control group (Table 2). The combina-
tion treatment in physiological condition resulted in substan-
tial inhibition of growth of EAT cells in relation to treatment
with WSDP or cisplatin alone (4.61 10
6
1.89 10
6
vs.
19.23 10
6
2.36 10
6
or 7.28 10
6
1.14 10
6
). Ad-
ditional inhibition of EAT cells was observed in hyperthermal
condition (0.95 10
6
0.59 10
6
vs. 9.57 10
6
1.53 10
6
or 1.17 10
6
0.97 10
6
).
Activated Macrophages Are Responsible for the Reduction of
Tumor Growth
The reduction of tumor cells and increased macrophage con-
tent in total number of cells present in peritoneal liquid are
observable in preventively treated mice with test components
at 37

C and 43

C in relation to control groups. Furthermore,

the reduction of tumor cells and increased macrophage con-
tent are present in all groups treated with the combination of
WSDP and cisplatin at doses 5 and 10 mg kg
1
at 37

C and
43

C as compared with groups treated only with cisplatin at

doses 5 and 10 mg kg
1
(Table 3). Apart fromthe increase in the
DOI 10.1002/jps.23755 Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013
4400 RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Table 2. Tumor Cells Viability and Mortality in the Intraperitoneal Cavity of Swiss Albino Mice-Bearing EAT After Preventive Treatment
with WSDP and Cisplatin in Physiological and Hyperthermal Condition
Normal Intraperitoneal Temperature (37

C) Intraperitoneal Hyperthermia (43

C)
Experimental Groups
a
Alive Cell Number
(N 10
6
) X SD
Dead Cell Number
(N 10
6
) X SD
Alive Cell Number
(N 10
6
) X SD
Dead Cell Number
(N 10
6
) X SD
Control (EAT) 21.10 3.79 0.92 1.14 13.72 1.42 0.20 0.15
WSDP 19.23 2.36 2.93 4.05 9.57 1.53 0.57 0.39
Cisplatin 5 mg kg
1
7.28 1.14
b
2.34 1.57 1.17 0.97 0.04 0.08
Cisplatin 5 mg kg
1
+ WSDP 4.61 1.89
b
1.28 4.91 0.95 0.59 0.43 0.17
c
Cisplatin 10 mg kg
1
8.67 1.22
b
2.34 0.79 1.50 1.36 0.18 0.21
Cisplatin 10 mg kg
1
+ WSDP 7.44 1.68
b
0.88 1.51 0.50 0.28 0.16 0.27
a
Mice were treated with WSDP (50mg kg
1
, i.p.) 7 and 3 days before i.p. inoculation of EAT (2 10
6
) cells. Cisplatin was injected (5 or 10 mg kg
1
, i.p.) 3 days
after inoculation of EAT cells. The number of alive and dead tumor cells in the intraperitoneal liquid was determined 3 days after inoculation of EAT cells. The
results are expressed as mean value (X SD) of each experimental group (n = 7).
b
Signicant difference (p < 0.05; ANOVA) from control group.
c
Signicant difference (p < 0.05; ANOVA) from group treated with cisplatin 5 mg kg
1
.
percentage of macrophages, macrophage spreading test showed
increased functional activity of macrophages, particularly in
the hyperthermal conditions (Table 4). A signicant increase
in physiological condition was observed in group treated with
WSDP + CIS
10
in relation to control, whereas in hyperthermal
condition, a signicant increase was observed in group treated
with WSDP +CIS
5
and WSDP +CIS
10
; macrophage spreading
activity was higher in WSDP +CIS
10
group (84.71 6.92) than
in WSDP + CIS
5
(65.50 8.85).
WSDP Can Reduce Cisplatin-Induced Genotoxic Effect to Normal
Cells Without Loos of the Antitumor Capacity Cisplatin
As shown in Table 5, cisplatin administration induces a dose-
dependent marked damage DNAof peripheral blood leukocytes,
liver, and kidney and tumor cells compared with control groups
in physiological and hyperthermal condition. HIPECtreatment
increased genotoxic effect cisplatin to normal cells. WSDP in
combination with CIS decreased cisplatin-induced genotoxic
effect in normal cells in both physiological and hyperthermal
conditions. Following TCS, WSDP has a greater protective ef-
fect on peripheral blood leukocytes compared with the cells of
the kidney and liver (Table 5). There is no evidence on the effect
of WSDP reduction in TCS tumor cells of mice treated with the
combination of WSDP and cisplatin compared with cisplatin
alone in physiological and hyperthermal conditions (Table 5).
The results of micronucleus assay are represented in
Table 6. Signicant (p < 0.05; ANOVA) increase in the number
of micronuclei was observed in groups treated only with cis-
platin at doses 5 and 10 mg kg
1
at 37

C compared with control

group (cisplatin 5 mg kg
1
, p = 0.0238; cisplatin 10 mg kg
1
,
p = 0.0283). Simultaneously, signicant (p < 0.05; ANOVA)
decrease in the number of micronuclei was observed in groups
preventively treated with WSDP in combination with cisplatin
at doses 5 and 10 mg kg
1
at 37

Ccompared with groups treated

only with cisplatin at doses 5 and 10 mg kg
1
(cisplatin 5 mg
kg
1
+ WSDP, p = 0.0169; cisplatin 10 mg kg
1
+ WSDP,
p = 0.0156). In groups treated with cisplatin at doses 5 and
10 mg kg
1
and with combination of WSDP and cisplatin
at doses 5 and 10 mg kg
1
at 43

C, there was a signicant

(p < 0.05; ANOVA) increase in the number of micronuclei in
relation to control group (cisplatin 10 mg kg
1
, p = 0.000008;
cisplatin 5 mg kg
1
, p = 0.0002; cisplatin 10 mg kg
1
+ WSDP,
p = 0.0091; cisplatin 5 mg kg
1
+ WSDP, p = 0.0279).
DISCUSSION
Chemotherapy improves the disease-free and overall survival
in cancer patients, but its clinical applications are limited by
profound side effects. Thus, strategies for cancer treatment
using combined therapies or combined agents with distinct
molecular mechanisms are considered to be more promising
for higher efcacy and/or lower toxicity, thereby resulting in
superior survival rates. There has been a growing amount of
interest in investigating the effects of chemopreventive agents
on the inhibition of cancer cell growth and toxicity in combina-
tion with chemotherapeutic agents.
Cisplatin is one of the most active cytotoxic agents used
in the treatment of cancer, and induces mitochondrial dys-
functions, particularly the inhibition of the electron transfer
system, thereby resulting in enhanced reactive oxigen species
(ROS) production
3739
and subsequent tissue damages. There-
fore, the administration of antioxidants prior to cisplatin treat-
ment has been used to protect against nephrotoxicity and
hepatotoxicity.
13,16,3739
In an effort to protect normal cells and to improve local tumor
control, i.p chemotherapy has the advantage of a high local con-
centration of the cytostatic drugs with less systemic exposure
compared with conventional intravenous drug administration
and limited systemic side effects. The efcacy of i.p. chemother-
apy is synergistic with hyperthermia.
14
This research was performed to evaluate antitumor, geno-
toxic, chemopreventive, and immunostimulative effects of
WSDP combined with anticancer drug cisplatin after preven-
tive treatment of Swiss albino mice bearing EAT in normal
(37

C) and hyperthermic (43

C) i.p. conditions.
We have proposed the use of EAT cells in vivo as a very re-
liable system for the investigations of possible cytotoxic effects
of combinations of chemoimmunotherapy and HIPEC, consid-
ering indirect inuence of different reparatory and immune
system mechanisms. Animal models of PC are important in the
evaluation of new treatment modalities.
In the present study, the results of the survival analysis,
tumor cells viability, and mortality in the intraperitoneal liq-
uid and differential count of the cells present in the intraperi-
toneal liquid indicate the synergistic effect among cisplatin,
WSDP, and hyperthermia, where WSDP enhances cytotoxic ef-
fect of cisplatin while hyperthermia increases the sensitivity of
tumor cells to cisplatin (Tables 13, Fig. 1). The combination
Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013 DOI 10.1002/jps.23755
RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology 4401
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treatment resulted in substantial inhibition of growth of EAT
cells in relation to treatment with WSDP or cisplatin alone and
increased the survival of mice by additional 115.25 with low
concentration of cisplatin. The combined treatment of WSDP
with HIPEC with CIS (5 or 10 mg kg
1
) increased the survival
of mice by additional 160.3% (ILS, CIS
5, 10
+ WSDP = 346.87)
as compared with single treatments of mice with HIPEC
(186.54%).
The mechanism(s) by which hyperthermia kills cells re-
mains controversial,
2,40
as does the means by which elevated
temperatures increase the cytotoxicity of anticancer drug.
Four general possibilities that have been proposed are: (a)
increased levels of drugs within cells,
41
(b) increased ef-
ciency of lesion formation,
42
(c) occurrence of different lesions
at hyperthermic temperatures, and (d) heat-induced inhibi-
tion of DNA repair.
43,44
Moreover, the same ndings indicate
that heat shock proteins induced in tumor cells under hyper-
thermic stress are able to elicit specic T- and NK-cell im-
mune response.
4547
Some of our previous studies
2,13,16,24,25,48
have examined the ability of chemoimmunotherapy to inhibit
tumor growth and metastases formation. A general nding
of these studies
2,13,16,24,25,48
is that propolis preparation and
avonoids present in propolis and different plants exhibit
an important immunomodulatory effect and could decrease
chemotherapeutic-induced toxic and genotoxic effect to nor-
mal cells without effecting cytostatic cytotoxicity in EAT or
mammary carcinoma cells. Thus, this study also suggested that
HIPECwith WSDP could be used to maximize enhanced immu-
nity of mice with EAT while potentially reducing the adverse
effects of drug-heat agents (chemotherapeutic) on normal cells
with equal or increased efcacy to tumor cells.
Increased efcacy and selective tumor cell killing effect in
vivo are achieved at temperatures between 40

C and 44

C,
which is related to a characteristic difference between normal
and tumor physiology. The architecture of the vasculature in tu-
mors is chaotic, resulting in regions with hypoxia and low pH,
which is not found in normal tissues. These environmental fac-
tors make tumor cells more sensitive to hyperthermia.
14
Hyper-
thermic potentiating of cisplatin cytotoxic effect is associated
with increased cellular accumulation of cisplatin,
40
enhanced
adduct formation with critical structures,
49
occurrence of dif-
ferent lesions at hyperthermic temperatures, and heat-induced
inhibition of DNA repair.
50
Hyperthermia enhances cisplatin
solubility and interactions with DNA to form DNA adducts,
primarily intrastrand cross-link adducts, which culminate in
the activation of apoptosis, reduced cell number, and increased
life span (Table 2, Fig. 1). Furthermore, hyperthermia causes
structural and functional changes in cells. Enhanced passive
or active transport across the damaged cell membranes con-
tributes to increasing intracellular amounts of cisplatin. Hy-
perthermia inhibits protein and enzyme synthesis and destroys
lysosomal membranes. The disruption of mitotic spindle and
enzymes, particularly DNA and RNA polymerase, inhibits the
synthesis and repair of DNA, which further enhances the ac-
tivity of cisplatin. In addition, it has been demonstrated that
WSDP is capable of inducing positive changes in the immune
response and producing antitumor activity.
16,2125
In our previ-
ous study, WSDP exerted a signicantly longer survival of mice
with mammary carcinoma and reduced articial lung metas-
tases; antitumor activity was a result of increased activity of
T lymphocytes and NK cells.
24,25
This study also conrmed
that preventive applications of WSDP to activate the immune
DOI 10.1002/jps.23755 Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013
4402 RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Table 4. Functional Activity of Macrophages in the Intraperitoneal Cavity of Swiss Albino Mice-Bearing EAT After Preventive Treatment
with WSDP and Cisplatin in Physiological and Hyperthermal Condition
Normal Intraperitoneal Temperature (37

C) Intraperitoneal Hyperthermia (43

C)
Experimental Groups
a
Activated
Macrophages (%)
Nonactivated
Macrophages (%)
Activated
Macrophages (%)
Nonactivated
Macrophages (%)
Control (EAT) 51.33 12.40 48.67 12.40 45.88 9.61 54.13 9.61
WSDP 66.50 21.00 33.50 21.00 67.00 13.3 33.00 13.3
Cisplatin 5 mg kg
1
54.50 17.63 45.50 17.63 49.00 7.00 51.00 7.00
Cisplatin 5 mg kg
1
+ WSDP 52.40 13.45 47.60 13.45 65.50 8.85
b,c
34.50 8.85
b,d
Cisplatin 10 mg kg
1
57.80 6.38 42.20 6.38 40.00 12.28 60.00 12.28
Cisplatin 10 mg kg
1
+ WSDP 64.44 10.16
b
35.56 10.16
b
84.71 6.92
b,d
15.29 6.92
b,d
a
Mice were treated with WSDP (50 mg kg
1
, i.p.) 7 and 3 days before i.p. inoculation of EAT (2 10
6
) cells. Cisplatin was injected (5 or 10 mg kg
1
, i.p.) 3 days
after inoculation of EAT cells under physiological and hyperthermal condition. Functional activity of macrophages in the intraperitoneal cavity was determined
3 days after inoculation of EAT cells. The results are expressed as mean value (X SD) of each experimental group (n = 4).
b
Signicant difference (p < 0.05; ANOVA) from control group.
c
Signicant difference (p < 0.05; ANOVA) from group treated with cisplatin 5 mg kg
1
.
d
Signicant difference (p < 0.05; ANOVA) from group treated with cisplatin 10 mg kg
1
.
anticancer response in combination with certain cytostatics ex-
erted a remarkable anticancer effect.
13,16
WSDP is known to di-
rectly activate macrophages and dendritic cells, which in turn
stimulate NK cells through interleukin-12 secretion.
2127
It is
known that natural killer cells and macrophages cooperate in
tumor rejection,
16,2026
whereas hyperthermia may have either
a direct suppressive effect on tumor cells or it may act indirectly
via enhanced or host immune function.
51,52
It has been theorized that following tumor heating, absorbed
fragments of necrotic cancer cells provide the antigenic stimu-
lus needed to enhance the host immune system that then lead
to the destruction of tumor cells.
53,54
In the present study, the
most pronounced effect on animal survival was achieved with
the combination of WSDP and cisplatin when combined with
hyperthermia; the enhancement of cytotoxicity in combination
with hyperthermia and synergistic activity of WSDP and cis-
platin should be considered to be the case as was suggested by
Orsoli c et al.
2
According to other ndings by Lindegaard et al.,
55
the synergistic effect between hyperthermia and cisplatin is the
highest during simultaneous application as was also shown in
this study. From our results, it is obvious that hyperthermia
increased the sensitivity of tumor cells to cisplatin (chemo sen-
sitization effect).
In the present study, reduction of tumor cells (Table 2) and
increased macrophage content in the intraperitoneal cavity
(Table 3) indicate that stimulation of the immune system, par-
ticularly macrophages, is a signicant mechanism that inhibits
tumor growth and leads to tumor destruction. The reduction
of tumor cells is a result of direct cytotoxic effect of propolis
and cisplatin, and immunostimulative effect of propolis. The
variable macrophage spreading revealed that treatment with
WSDP affects the functional state of macrophages, the major
cells involved in tumor rejection (Tables 2 and 3). Macrophage
activation might induce the production and release of several
cytokines, especially in hyperthermal condition, such as IL-1,
IL-6, IL-12, TNF-", and NO.
2127,54
Some of these cytokines have
direct cytotoxic effect on tumor cells, whereas others activate
other cells of immune system: cytotoxic T cells, B cells, and NK
cells. In addition, these cytokines might stimulate production
of C-reactive protein and complement factor C3 that would act
as opsonins on tumor cells.
23,24
The combination of these effects
might impede tumor growth and lead to elimination of tumor
cells.
Furthermore, this study showed that WSDP may reduce the
toxicity of cisplatin on normal cells (liver, kidney, and blood).
Cisplatin therapy induces oxidative stress, principally involv-
ing ROS, in renal tubular and liver cells. The interaction of ROS
withcellular components may result indamage to biomolecules,
including DNA, proteins, and lipids (Table 5). Our results
showed that the repeated i.p. administration of WSDP in com-
bination with cisplatin treatment prevented cisplatin-mediated
increases in the DNAdamage in normal cells without loos of the
antitumor capacity cisplatin (Table 5). Genotoxic effect of cis-
platin is enhanced after treatment at dose 10 mg kg
1
compared
with 5 mg kg
1
. Furthermore, the number of DNA damage on
normal and tumor cells is increased at 43

C compared with
treatment at 37

C (Table 5). Increased cytotoxic effect on nor-

mal and tumor cells after simultaneous application of high-dose
cisplatin and hyperthermia conrms the chemosensitive effect
where sensitivity of tumor cells to cisplatinis increased. Accord-
ing to that, it seems that the haematological, liver, and kidney
toxicity because of the common drugs such as cisplatin may be
avoided by the use of the antioxidants such as WSDP
17,24,28,3739
or by reducing doses of cytostatics. These data are consistent
with the data,
17,37,39
showing that caffeic acid phenethyl ester
(CAPE), a phenolic component of propolis, has a protective role
against cisplatin-induced damage to the kidney, liver, or other
normal tissues.
These data were also conrmed by the results of the micronu-
cleus test. Micronucleus assay (Table 6) indicates that propolis
preparation used in the physiological condition as addition to
chemotherapy could decrease genetic damages, number of mu-
tations, and teratogenic effect of toxic metabolites and reactive
radicals generated during metabolism of cytostatics and nally
contribute to better functional status of organism. However, in
hyperthermal condition, it seems that hyperthermia enhanced
the clastogenicity of alkylating agents (Table 6) as suggested
by Asanami and Shimono.
56
The mechanism(s) whereby WSDP provides protection
against cisplatin-induced renal and liver injury remains a
matter of speculation. The principal pharmacological effect
of propolis, rich with avonoids, and certainly the best doc-
umented, is a reduction of capillary (or venular) permeabil-
ity, resulting in reduction of various forms of experimental
edema.
5759
This information is, unfortunately, insufcient to
adequately explain the protective mechanism against cisplatin
Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013 DOI 10.1002/jps.23755
RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology 4403
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DOI 10.1002/jps.23755 Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013
4404 RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology
renal injury, the mechanism of which is only poorly under-
stood. Nevertheless, several possible explanations of propolis-
mediated protection can be suggested. It is well established,
for example, that chemopreventive effect of WSDP could result
from increased activity of antioxidant enzymes and scaveng-
ing of free radicals, the production of which is enhanced during
chemotherapy with cisplatin. The scavenging of chemotherapy-
induced free radicals could also be one of the important mech-
anisms of chemoprevention by WSDP, which was conrmed by
Chen et al.
30
and Refs.16,29,60,61, showing that propolis and
related polyphenolic compounds exercise their activity through
the scavenging of hydroxyl, superoxide free radicals, and lipid
peroxides. Thus, it has been suggested that an active compo-
nent of propolis, including CAPE, is a potent exogenous cytopro-
tective and antigenotoxic agents against cell oxidative damage
that could be used as a template for designing novel drugs to
combat diseases induced by oxidative stress components, such
as various types of cancer.
17,37,39
By increasing the activities
of antioxidant enzymes, polyphenols from propolis reduce the
number of free radicals and ROS and increase the production
of molecules capable of protecting against oxidative stress. It is
possible that propolis may inuence the survival of the mice-
bearing EAT via the free radical scavenging ability, tissue re-
generation properties, and immunostimulatory effects.
CONCLUSIONS
The results of these studies suggest that propolis preparations
used as addition to chemotherapy could enhance cytotoxic ef-
fect of cytostatic drugs on tumor cells and/or decrease their
toxic effect on normal cells. In addition, propolis stimulates
macrophages, the major cells involved in tumor rejection. The
combination of hyperthermia and cytostatic drugs offers the
possibility of chemotherapy dose reduction to minimize the side
effects and decrease cancer treatment expenses. In addition,
chemopreventive effect of WSDP on normal cells is conrmed
without negative interference with cytotoxic effect of cisplatin
on tumor cells. These results suggest that hyperthermia and
propolis may be a promising adjunct to cisplatin chemotherapy.
ACKNOWLEDGMENTS
This work was supported by the Ministry of Sciences, Education
and Sports of the Republic of Croatia project No. 119-0000000-
1255.
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DOI 10.1002/jps.23755 Or soli c et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:43954405, 2013