Original research article [602902]

Original research article
Effect of propranolol on IL-10, visfatin, Hsp70, iNOS, TLR2,
and survivin in amelioration of tumor progression and
survival in Solid Ehrlich Carcinoma-bearing mice
Amany A. Abdina,*, Nema A. Solimanb, Eman M. Saiedc
aDepartment of Pharmacology, Faculty of Medicine, Tanta University, Tanta, Egypt
bDepartment of Medical Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egypt
cDepartment of Pathology, Faculty of Medicine, Kafr El-Sheikh University, Tanta, Egypt
Introduction
Breast cancer is the most frequently diagnosed cancer and the
leading cause of cancer death in females worldwide. About half the
breast cancer cases and 60% of the deaths are estimated to occur in
economically developing countries [1,2] . Epidemiological studies
have associated psychosocial factors such as chronic stress with
cancer progression and, to a lesser extent, cancer onset [3].Sympathetic nervous system (SNS) regulation of cancer cell
biology and tumor microenvironment has clarified the molecular
basis for long-suspected relationships between stress and cancer
progression and now suggest a highly leveraged target for
therapeutic intervention [4]. This has led to hypothesis that b-
blockers may favorably impact cancer progression [5]. The
expression of the three subtypes of b-adrenergic receptor (b1,
b2, and b3) was found to reach maximal concentration even before
the actual increase in tumor mass with higher b2concentrations
(74%) than b1(36%) in tumor tissues [6–8] . b-Adrenergic signaling
regulates multiple cellular processes that contribute to cancer
initiation and progression, including inflammation, angiogenesis,
apoptosis/anoikis, cell motility and trafficking, DNA damage repair,Pharmacological Reports 66 (2014) 1114–1121
A R T I C L E I N F O
Article history:
Received 18 April 2014
Received in revised form 21 July 2014
Accepted 23 July 2014
Available online 8 August 2014
Keywords:
Propranolol
Solid Ehrlich Carcinoma
Apoptosis
Angiogenesis
ImmunomodulationA B S T R A C T
Background: b-Adrenergic signaling could contribute to initiation and progression of breast cancer. This
research investigated some potential mechanisms of propranolol in amelioration of progression and
survival in breast cancer.
Methods and results: Solid Ehrlich Carcinoma (SEC) xenograft model was induced in 30 mice divided into
3 groups; where group I served as untreated SEC group. In groups II and III, propranolol treatment i.p. in
low (5 mg/kg) and high dose (10 mg/kg) caused significant increase in interleukin-10 (IL-10) and
decrease in heat shock protein 70 (Hsp70) and inducible nitric oxide synthase (iNOS) activity with non
significant change in visfatin in tumor tissues compared to untreated SEC. In untreated SEC, tumor
volume (V) exhibited significant negative correlation with IL-10 levels and toll like receptor 2 (TLR2)
expression with significant positive correlation with Hsp70 levels and iNOS activity. While propranolol
in either doses caused reduction of tumor volume (V), and improved percentage tumor growth inhibition
(% TGI) only its high dose exhibited significant impact on survival rate. Propranolol dose-dependent
effect was evident for IL-10 and Hsp70, and even only the high dose significantly increased and decreased
TLR2 and survivin, respectively. This comes in favor of recommending high dose of propranolol in cancer
therapy. Nonetheless, use of low dose cannot be ignored when benefit to risk balance have to be
considered.
Conclusions: Propranolol could provide palliative effects in progression and survival of breast cancer that
are mainly mediated via direct immunomodulatory and apoptotic mechanisms and probably associated
with indirect anti-angiogenic activity.
/C223 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.
z o.o. All rights reserved.
*Corresponding author.
E-mail addresses: amanyabdin@med.tanta.edu.eg , amanynhr@hotmail.com
(A.A. Abdin).Contents lists available at ScienceDirect
Pharmacological Reports
jou r nal h o mep ag e: w ww .elsevier .co m /loc ate/p h arep
http://dx.doi.org/10.1016/j.pharep.2014.07.010
1734-1140/ /C223 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

cellular immune response [4,9] . On molecular basis, cAMP is
thought to mediate their effect on growth and differentiation of
malignant cells [10]. Thus, b-blockade that has been routinely used
for treatment of many cardiovascular disorders [11], could now
provide an appropriate starting point in oncology [4]. In breast
cancer, it has been observed that the optimal b-antagonist
regimens were achieved by the non-selective b-blockers rather
than b1-selective agents [12,13] . In this context, propranolol as a
prototype non-selective b-blocker that has no intrinsic sympa-
thetic activity (ISA) would be expected to provide the broadest
biological leverage and minimize the risk of missing an active
b-receptor target [4]. Many emerging pre-clinical and clinical
studies on breast cancer have focused only on impact of b-blockers
in improving survival, progression and metastasis outcome
[12,14–17] , while the underlying mechanisms are still in need
to be fully elucidated. Among these mechanisms, the cross-talk of
immuno-apoptotic-angiogenic pathways is considered the corner
stone in pathogenesis of breast cancer. Therefore, this research
aimed to disclose some potential mechanisms of propranolol in
low and high doses using biochemical markers as interleukin-10
(IL-10), visfatin, heat shock protein 70 (Hsp70), and inducible-
nitric oxide synthase (iNOS) as well as immunohistochemical
markers such as toll like receptor 2 (TLR2) and survivin that related
to these pathogenic pathways and could contribute to ameliora-
tion of tumor progression and survival outcome in tumor-bearing
mice using Solid Ehrlich Carcinoma (SEC) model.
Materials and methods
Drugs and chemicals
Propranolol (Mayestrotense, 1 mg/ml ampoule) was purchased
as a product of Alex. Co. for Egypharma, Egypt. Other chemicals are
of analytical gradient were obtained as products of Sigma–Aldrich
Chemical Co., unless indicated otherwise.
Cell Line and induction of Solid Ehrlich Carcinoma (SEC) xenograft
model
Ehrlich Ascites Carcinoma (EAC) cell line was obtained from the
Pharmacology and Experimental Oncology Unit of the National
Cancer Institute (NCI), Cairo University, Egypt. EAC cells are of
mammary origin. The viability of the cells was 99% as judged by
trypan blue exclusion assay. The xenograft model of Solid Ehrlich
Carcinoma (SEC) was induced in male Swiss albino mice by viable
EAC cells 2.5 /C2 106in 0.2 ml isotonic saline implanted subcutane-
ously (s.c.) into the right thigh of the hind limb of each mouse. The
tumor was developed in 100% of the mice with a palpable solid
tumor mass (/C21100 mm3) was achieved within 12 days post-
implantation [18,19] .
Animals groups and treatment protocol
Thirty adult male Swiss albino mice weighing 18–20 g, were
allowed ad libitum to fed water and standard pellet chow (EL-Nasr
Chemical Company, Cairo, Egypt) through the whole period of
the experiment. Animals were housed and allowed to become
acclimatized to laboratory conditions for 1 week prior to the
experiment. The experiment was conducted in accordance to
the National Institutes of Health guide for the care and use of
Laboratory animals (NIH Publications No. 8023, revised 1978). All
mice were rendered tumor-bearing and divided randomly into 3
equal groups; where group I served as untreated control group and
received a vehicle of isotonic saline. The tumor-bearing mice in
group II and group III were treated 5 days/week by intraperitoneal
(i.p.) injection of propranolol in a dose of 5 mg and 10 mg/kg,respectively [20]. The treatment by either vehicle or propranolol
was started from 12th day to 42nd day post-implantation. Based
on its low oral bioavailability due to first pass hepatic metabolism,
propranolol was administered parenteral to ensure high non
fluctuating concentrations. The used doses are representative of
/C243–6-folds the allowed intravenous dosage for human that is
0.15 mg/kg [21]. Nevertheless, these doses are still within tolerable
limit away from intraperitoneal LD50 for mice (80 mg/kg).
Recording of survival rate
The day of implantation was considered zero point of the
experiment for recording and analysis of the survival rate weekly
for 6 weeks (by recording number of the survived mice in each
group at the end of each week).
Tumor volume (V) and percentage tumor growth inhibition (% TGI)
Tumor volumes were recorded from the start point at 12th day
post-implantation and thereafter every 4 days till the last record at
42nd day post-implantation just prior to scarification of the
survived mice. Using a Vernier caliper, tumor volume (V) was
calculated as V(mm3) = (a2/C2 b)/2, where a (small diameter), and b
(large diameter) are perpendicular, expressed in millimeters (mm).
Drug efficacy was expressed as the percentage tumor growth
inhibition calculated as % TGI = 100 /C0 (T/C /C2 100), where T is the
mean relative tumor volume (RTV) of the treated tumor and C is the
mean RTV in the control group. RTV is defined as Vx/V1, where Vx is
the tumor volume at the end point of the experiment before
scarification of the animals and V1 is the tumor volume at the start
point of the treatment [22].
Processing of tumor tissue samples
At the end point of the experiment (the 42nd day post-
implantation), all the survived mice were sacrificed. The tumor
was excised, washed immediately with ice-cold saline, blotted dry
on a filter paper, and divided into two parts. One part was
preserved in 4% buffered paraformaldehyde and further processed
for histopathological and immunohistochemical examination. The
other part was homogenized in 10 volumes of ice-cold 50 mM
phosphate-buffered saline (PBS), pH 7.4 using a Potter–Elvenhjem
tissue homogenizer. The resultant supernatant was frozen at -80/C14C
for further assay of tissue levels of interleukin-10 (IL-10), visfatin,
heat shock protein (Hsp70), and iNOS activity. Protein content in
tumor tissue (mg/g wet tissue) was measured by method of Lowry
et al. (1951) [23].
Enzyme-linked immunosorbent assay (ELISA) of visfatin, IL-10, and
Hsp70 levels in tumor tissues
The supernatants were quantitatively assayed for visfatin, IL-10
and Hsp70 levels using ELISA kits supplied by (RayBiotech, Inc.
Cat#: EIA-VIS-1), (BOSTER BIOLOGICAL TECHNOLOGY Co., Inc.
Catalog No. EK0417), and (Uscn Life Science Inc. Wuhan Cat. No.
E90873Bo), respectively, following the manufacture’s protocol. The
results were expressed as ng/mg protein, pg/mg protein and ng/mg
protein for visfatin, IL-10 and Hsp70, respectively.
Measurement of inducible-nitric oxide synthase (iNOS) activity in
tumor tissues
iNOS activity was measured according to the method of
Ryoyama et al. [24], whereby L-arginine and molecular oxygen
were catalyzed by NOS to generate nitric oxide (NO). The rate of NO
production by NOS in 1 min was determined with the GriessA.A. Abdin et al. / Pharmacological Reports 66 (2014) 1114–1121 1115

reaction. The iNOS activity was determined by spectrophotometric
assay at 540 nm. iNOS activity is expressed as U/mg protein.
Histopathological examination
For histopathological characteristics of Solid Ehrlich Carcinoma,
tumor specimens were fixed in 10% formalin, embedded in
paraffin, and 5 mm sections were stained with hematoxylin and
eosin (H&E). Sections prepared from different organs (liver, lung,
brain, lymph nodes) and bone of the studied groups for detection of
metastatic lesions.
Immunohistochemical expression and scoring of toll like receptor-2
(TLR-2) and survivin
For immunohistochemistry, 3 mm sections from tumor tissues
were deparaffinized in xylene for 30 min and rehydrated with
graded alcohol series. Sections were then processed for further
immunohistochemical staining using the UltraVision Detection Kit
(TP-015-HD, Lab Vision, USA) according to the manufacturer’s
protocol. Rabbit polyclonal anti-Toll-like Receptor 2 (TLR2/CD282)
antibody (Cat. No. PA5-20020, Lab Vision, USA), in a dilution of
2 mg/ml, and rabbit polyclonal anti-survivin antibody (Cat. No. RB-
9245-R7, Ready to use, Lab Vision, USA) were used for detection of
TLR2 and survivin, respectively. As positive controls, sections from
a case of prostatic carcinoma known to show over-expression of
survivin were used, while sections from normal human spleen
were used for TLR2. Negative controls were prepared by omission
of the primary antibodies.
TLR2 positivity was indicated by cytoplasmic staining [25],
while positivity for survivin was considered when distinct nuclear
and/or diffuse cytoplasmic immunohistochemical reaction was
found [26]. Quantification of TLR2 [27] and survivin [28]
expression was performed on a 3-point scale, where 0 = no
expression, (+) = weak expression, (++) = moderate expression,
and (+++) = strong expression taking into account the percentage
of positive epithelial cells and the intensity of expression.
Statistical analysis
Values of the measured parameters were expressed as mean
/C6 SEM. One Way-ANOVA test (F value) was used to detect significance
of the difference among more than two arithmetic means, followed by
post hoc Scheffe test to detect the difference between each two means.
Fisher’s exact test was used to detect the difference between
categorical data. The paired-samples t-test was applied to detect
difference in the tumor volume monitored at the start point and at the
end point of the experiment. The cumulative survival curve was
plotted and the overall and pairwise comparisons of survival rate
were analyzed by applying Breslow test (generalized Wilcoxon) using
the Kaplan–Meier method. The difference was considered significant
at values of p < 0.05. The statistical analysis was processed using the
Statistical Program of Social Sciences (SPSS) for windows, version
14.0.
Results
Results of survival rate
In regard to survival rate (Table 1), the overall survival
comparison among groups was significant (p < 0.05). The pairwise
comparisons showed significant difference between the untreated
group I and group III that treated by propranolol 10 mg/kg
(p < 0.05). When group II treated by propranolol 5 mg/kg was
compared to either the untreated group I or group III treated by
propranolol 10 mg/kg, the pairwise comparisons showed nonsignificant difference (p > 0.05). The cumulative survival function
of the different studied groups was displayed in Fig. 1.
Results of tumor volume (V) and percentage tumor growth inhibition
(% TGI)
Compared to the untreated group I, there was significant decrease
in tumor volume of group III (treated by 10 mg/kg propranolol) at all
recording points, while tumor volume of group II (treated by 5 mg/kg
propranolol) exhibited significant decrease only at the recording
points at the 16th and 20th days (Fig. 2). The % TGI was determined to
be 14.9% in group II treated by propranolol 5 mg/kg, and 43.6% in
group III treated by propranolol 10 mg/kg (Fig. 3).
Results of biochemical markers
Propranolol treatment of SEC either in a dose of 5 mg/kg or
10 mg/kg caused significant increase in IL-10 levels with signifi-
cant decrease in Hsp70 and iNOS activity in tumor tissues
compared to the untreated SEC. Treatment by propranolol
10 mg/kg resulted in significant higher levels of IL-10, significant
decrease in Hsp70 levels, and non significant difference in iNOS
activity when compared to treatment by propranolol 5 mg/kg.
There was non significant difference in visfatin levels between all
the studied groups (Table 2).
In the untreated SEC (group I), the tumor volume (V) exhibited
significant negative correlation with IL-10 levels and significant
positive correlation with Hsp70 levels and iNOS activity in tumor
tissues, while visfatin levels showed non significant correlation
(Table 3).Table 1
Comparative statistics for survival rate in the studied groups at the end point of the
experiment (the 42nd day post-implantation).
Groups Survival rate (%)#Survival duration
(weeks; % confidence interval (CI),
lower bound to upper bound)
Group I 50% (5.3 /C6 0.3 weeks; 95% CI, 4.74–5.93)
Group II 70%, P1: NS (5.9 /C6 0.1 weeks; 95% CI, 5.67–6.13)
Group III 90%, P1< 0.05, P2: NS (6.0 /C6 0.0 weeks; 95% CI, 6.00–6.00)
Values expressed as mean /C6 SEM; NS, non significant. Breslow test: #, significant
overall survival comparison (p < 0.05). P1: pairwise comparison of group I (untreated
Solid Ehrlich Carcinoma, SEC) vs group II (SEC treated by propranolol 5 mg/kg) and
group III (SEC treated by propranolol 10 mg/kg). P2: pairwise comparison of group III
(SEC treated by propranolol 10 mg/kg) vs group II (SEC treated by propranolol 5 mg/kg).
Fig. 1. The cumulative survival curve plotted by Kaplan–Meier method showing the
censored cases (+) of each group recorded by the 6th week. 1: group I (untreated
Solid Ehrlich Carcinoma, SEC). 2: group II (SEC treated by propranolol 5 mg/kg). 3:
group III (SEC treated by propranolol 10 mg/kg).A.A. Abdin et al. / Pharmacological Reports 66 (2014) 1114–1121 1116

Results of histopathological examination and immunohistochemical
expression of toll like receptor 2 (TLR2) and survivin
Histopathological examination of the studied groups revealed
the typical picture of Solid Ehrlich carcinoma (Fig. 4A and B).
Examination of sections prepared from the organs (liver, lung,
brain, lymph nodes) and bone of the studied groups revealed no
metastatic spread of the primary tumors.
Positive TLR 2 expression was detected as diffuse cytoplasmic
staining (Fig. 5A–C). Positive survivin expression was detected as
diffuse cytoplasmic staining. None of the studied cases showed
nuclear survivin expression (Fig. 6A–C). The score of TLR 2 and
survivin expression was significantly higher and lower, respec-
tively in group III compared to group I, while there was non
significant difference for both in group II when compared to either
group I or group III (Table 4).
In the untreated SEC (group I), the tumor volume (V) exhibited
significant negative correlation with TLR2 expression and non
significant correlation with survivin expression in tumor tissues
(Table 3).
Discussion
b-Adrenergic signaling regulates multiple cellular processes
that contribute to initiation and progression of breast cancer
mainly immune responses [9], apoptosis [29,30] , and angiogenesis
[31,32] . However, there is limited information about the mecha-
nisms by which b-blockers could impact the course and survival incases of breast cancer. In this research work, SEC-bearing model in
mice was established as a valid model that was frequently used to
investigate chemotherapeutic strategies for breast cancer [33].
This model is typically of high virulence, quick development and
infiltrative nature, reflecting its high-grade malignancy [34]. The
tumor caused high lethality where survival rate was only 50% in
the group with untreated SEC by the 6th week post-implantation.
Treatment by propranolol resulted in a dose-dependent improve-
ment in survival rate, reduction in tumor volume, and increase in
the % TGI. These results confirmed the previously established
impact of b-blockers in the clinical outcome of breast cancer
[12,14–17] . The biomarkers that have been assessed in this study
revealed that propranolol caused a dose-dependent increase in IL-
10 with decrease in Hsp70 and iNOS, while there was non
significant change in visfatin in tumor tissues compared to the
untreated SEC. Such effects could be attributed to the b-blockade
activity of propranolol based on the fact that b-adrenoceptors
signaling plays an important role in regulation of tumor-directed
immune responses. Among these responses is the immune-
stimulated biosynthesis of NO, distinctive modulation of secretion
of pro-inflammatory and anti-inflammatory cytokines such as IL-
10 [35]. The data are complex regarding influence of IL-10 on
cancer where it favors or inhibits tumor progression [36]. Inspit of
this complexity, it is observable how well the present study
provided negative correlation between tumor progression and IL-
10 levels in tumor tissues. This finding indicates that increasing IL-
10 levels by propranolol is one of its mechanisms in amelioration of
breast cancer progression. The favor effects of IL-10 in cancer
include suppression of angiogenesis either directly on tumor cells
or indirectly enhances antitumor immunity by influencing
infiltrating immune cells [37–39] , modulation of apoptosis [40].
Accordingly, propranolol suppresses tumor growth by immuno-
regulatory role mediated by the optimal level of IL-10 as supported
by previous studies of Loppnow et al. [41] and Gage et al. [42]. In
contrast, IL-10 may exert pro-tumorigenic effect via suppression of
adaptive immune responses that lead to tumor escape from
immune surveillance [43]. These opposing effects of IL-10 might
depend on interactions with either cytokines or factors found in
the tumor microenvironment. In this context, IL-10 has been
reported to inhibit NO production and regulate the inducible
NO synthase (iNOS) activation [44,45] . The iNOS-derived NO has
been recognized as one of the most versatile players in immune
system and pathogenesis of various diseases including cancer
[46,47] . In consistent, the current results revealed a significant
positive correlation of iNOS activity with ESC progression that
reflects importance of inhibiting iNOS enzyme as a new target by
beta blockers in alleviate stress associated tumors as reported by
Powe et al. [15]. Visfatin is an adipocytokine that beside its
biological role in many metabolic, energetic and stress responses, it
Fig. 2. Tumor volume of the studied groups at the recording points every 4 days
from the 12th day (start point of propranolol treatment) to the last record at the
42nd day post-implantation. Paired-samples t-test: significant difference of tumor
volume between start point and end point in each group,+p < 0.05 and++p < 0.01.
Scheffe test: significant difference of group II (SEC treated by propranolol 5 mg/kg)
or group III (SEC treated by propranolol 10 mg/kg) vs group I (untreated SEC),
*p < 0.05, **p < 0.01 and ***p < 0.001. SEC; Solid Ehrlich Carcinoma.
Fig. 3. Percentage tumor growth inhibition (% TGI) in group II (SEC treated by propranolol 5 mg/kg) and group III (SEC treated by propranolol 10 mg/kg) relative to group I
(untreated SEC). SEC; Solid Ehrlich Carcinoma.A.A. Abdin et al. / Pharmacological Reports 66 (2014) 1114–1121 1117

exhibits proliferative, anti-apoptotic, pro-inflammatory, pro-an-
giogenic, and immunomodulating properties [48]. In contrast to
previous studies [49,50] , the present result showed non significant
effect of propranolol on visfatin levels in tumor tissues denoting
that its anti-angiogenic effect could be contributed to another
indirect pathways such as immunomodulatory mechanism and
arise a possibility that the effects of b-adrenoceptor signaling for
angiogenesis in breast cancer may be independent of b-
adrenoceptors expression by breast tumor cells. In context, other
previous studies reported that propranolol mediates anti-angio-
genic effect via inhibition of vascular endothelial growth factor
(VEGF) [51]. Recently, b-adrenoceptors expression has been found
to not correlate with the improved outcomes in breast cancer [52].
Thus, the effect of their agonists and antagonists may beparadoxical on cell proliferation and tumor growth [53]. Contrary
to the present results, stimulation of b2-adrenoceptors induced
significant tumor growth suppression and tumor regression in
mice bearing MDA-MB-231 human breast tumors [54]. However,
this could be attributed to the nature of these cells that expressed
high b-adrenoceptors levels, and when stimulated by an agonist,
they evoked immediate and robust reductions in DNA synthesis
[55]. In regard to the tumorigenic role of Hsp70 and its an anti-
apoptotic capacity [56,57] , the present findings showed that
propranolol treatment decreased Hsp70 levels when compared to
their allied untreated mice, reflecting role of propranolol in protein
homeostasis under stress. Hsp70 expression is influenced by b-
adrenergic receptor intermediates including cyclic AMP (cAMP)
[58]. When over-expressed in cancers, Hsp70 is implicated in
tumor cell proliferation, differentiation, metastasis, and recogni-
tion by the immune system [59]. In consistence, targeting Hsp70
may open new possibilities for treatment of resistance cancers
[60,61] . Immune cells constitute a major cell population in tumor
microenvironment and essential to provide signals for growth,
anti-apoptosis, angiogenesis, and metastasis [62,63] . Among
immune components, TLRs are critical in bridging innate and
adaptive immune responses with significant role in many cancers
immunosurveillance including breast cancer [64,65] . TLRs role in
apoptosis is controversial, some studies suggested pro-apoptotic
properties (especially TLR2 and TLR4) [65,66] , while others
suggested anti-apoptotic functions [67,68] . However, the current
result favors the anti-tumorigenic role of TLR2 as its expression in
tumor tissues exhibited significant negative correlation with
tumor volume in the untreated SEC group. In addition, treatment
by propranolol in high dose (10 mg/kg) caused significant increaseTable 2
Comparative statistics for levels of interleukin-10 (IL-10), visfatin, heat shock protein 70 (Hsp70), and inducible-nitric oxide synthase (iNOS) activity in tumor tissues from the
survived mice of the studied groups at the end point of the experiment (the 42nd day post-implantation).
Parameter Group I (n = 5) Group II (n = 7) Group III (n = 9) F value (p value)
IL-10 (pg/mg tissue protein) 36.3 /C6 0.76 39.1 /C6 0.41
P1< 0.0541.7 /C6 0.71
P1< 0.001
P2< 0.0515.478 (p < 0.001)
Visfatin (ng/mg tissue protein) 13.8 /C6 0.63 12.5 /C6 0.23
P1: NS14.0 /C6 0.47
P1: NS
P2: NS3.219 (p: NS)
Hsp70 (ng/mg tissue protein) 68.3 /C6 1.2 57.4 /C6 0.99
P1< 0.0149.4 /C6 1.8
P1< 0.001
P2< 0.0133.320 (p < 0.001)
iNOS activity (U/mg tissue protein) 67.9 /C6 1.31 40.4 /C6 1.7
P1< 0.00144.6 /C6 0.99
P1< 0.001
P2: NS98.889 (p < 0.001)
Values expressed as mean /C6 SEM; n, number; NS, non significant. Scheffe test: P1: group I (untreated Solid Ehrlich Carcinoma, SEC) vs group II (SEC treated by propranolol 5 mg/kg)
and group III (SEC treated by propranolol 10 mg/kg). P2: group III (SEC treated by propranolol 10 mg/kg) vs group II (SEC treated by propranolol 5 mg/kg).
Table 3
Correlation of tumor volume (V) with levels of interleukin-10 (IL-10), visfatin,
heat shock protein 70 (Hsp70), inducible-nitric oxide synthase (iNOS) activity,
and expression score of toll like receptor 2 (TLR2) and survivin in tumor
tissues from the survived mice of untreated SEC group at the end point of the
experiment (the 42nd day post-implantation).
Parameter r value
IL-10 (pg/mg tissue protein) /C00.935*
Visfatin (ng/mg tissue protein) 0.522NS
Hsp70 (ng/mg tissue protein) 0.968**
iNOS activity (U/mg tissue protein) 0.886*
TLR2 expression /C00.894*
Survivin expression 0.577NS
NS, non significant; SEC, Solid Ehrlich Carcinoma.
*Significance at p < 0.05.
**Significance at p < 0.01.
Fig. 4. Histopathological findings of untreated Solid Ehrlich Carcinoma (group I). [A] Tumor tissue composed of large necrotic centers surrounded by undifferentiated
carcinomatous cells (H&E 100/C2). [B] Higher magnification of Solid Ehrlich Carcinoma showing the cellular details of the tumor, the cells are spherical in shape, containing
relatively large, highly chromatophilic nuclei with one or more prominent nucleoli, giant forms are also seen (H&E 400/C2).A.A. Abdin et al. / Pharmacological Reports 66 (2014) 1114–1121 1118

in immunohistochemical expression of TLR2 pointing to its role in
amelioration of tumor progression. TLR2 or TLR4 agonists have
been mentioned to stimulate the MyD88 signaling pathway in
antigen presenting cells (APCs) with subsequent downstreamactivation of cytokines, chemokines, and their receptors, including
IL-10 [66]. This comes in consistent with the anti-tumoral effect of
b-blockers via improvement of the immune competence [69].
Survivin belongs to the inhibitor of apoptosis proteins (IAP) family
Fig. 5. Immunohistochemical expression of TLR2 in tumor tissues of the studied
groups. [A] TLR2 staining of a section from group I (untreated Solid Ehrlich
Carcinoma, SEC) showing weak (+) cytoplasmic expression (immunoperoxidase
400/C2). [B] TLR2 staining of a section from group II (SEC treated by propranolol 5 mg/
kg) showing moderate (++) cytoplasmic expression (immunoperoxidase 400/C2). [C]
TLR2 staining of a section from group III (SEC treated by propranolol 10 mg/kg)
showing strong (+++) cytoplasmic expression (immunoperoxidase 400/C2).
Fig. 6. Immunohistochemical expression of survivin in tumor tissues of the studied
groups. [A] Survivin staining of a section from group I (untreated Solid Ehrlich
Carcinoma, SEC) showing strong (+++) cytoplasmic expression (immunoperoxidase
400/C2). [B] Survivin staining of a section from group II (SEC treated by propranolol
5 mg/kg) showing moderate (++) cytoplasmic expression (immunoperoxidase
400/C2). [C] Survivin staining of a section from group III (SEC treated by propranolol
10 mg/kg) showing weak (+) cytoplasmic expression (immunoperoxidase 400/C2).A.A. Abdin et al. / Pharmacological Reports 66 (2014) 1114–1121 1119

and has been found to over-express in breast cancer and associated
with more aggressive behavior and decreased survival [70] by
enhancing cell proliferation, inhibition of apoptosis, and promo-
tion of angiogenesis [26,28] . Thus, reduction in survivin expression
in tumor tissues by propranolol in a high dose (10 mg/kg) confirms
the beneficial effects of propranolol. Herein, the cytoplasmic
survivin expression comes in consistent with the undifferentiated
high grade nature of SEC, and is in line with the suggestion that
cytoplasmic expression of survivin is associated with unfavorable
prognosis in breast cancer, while nuclear expression is an indicator
of good prognosis [26]. Considering efficacy of propranolol, low
(5 mg/kg) and high (10 mg/kg) doses were adopted in this study.
The dose-dependent effect of propranolol was evident for IL-10
and Hsp70, while the other biomarkers iNOS showed non
significant difference between the low and the high dose, and
even more only the high dose caused significant effect on TLR2 and
survivin. In addition, treatment by propranolol in high dose
exhibited higher impact in improvement of survival rate, tumor
volume (V), and percentage tumor growth inhibition (% TGI) than
its low dose. This comes in favor of recommending high dose of
propranolol in cancer therapy. Nonetheless, use of the low dose
cannot be ignored when the benefit to risk balance have to be
considered.
Concluding these results, they indicate that propranolol could
provide palliative effects in progression and survival of breast
cancer that are mainly mediated via direct immunomodulatory
and apoptotic mechanisms that probably associated with indirect
anti-angiogenic activity.
Authors contribution
All the named authors participated sufficiently in this research
work according to the specialty and expert of each one as follows:
Dr. Amany A. Abdin designed the research protocol, conducted the
handling of animals and treatment protocol of the research,
recorded the clinical outcome such as survival rate, tumor volume
and percentage tumor growth inhibition (% TGI), collected
samples and participated in the assay of the biochemical
parameters, conducted the statistical analysis, presented the
results as tables or figures and participated in writing and revised
the manuscript. Dr. Neama A. Soliman participated in the assay of
the biochemical parameters, wrote and revised the manuscript.
Dr. Eman M. Saied conducted the histopathological and immu-
nohistochemical examination, participated in writing and revised
the manuscript.
Funding
This is a declaration that this work has no funding.Conflict of interest
This is a declaration that this work has no conflict of interest.
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Weak (+) Moderate (++) Strong (+++)
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