Poloxamer P85 increases anticancer activity of Schiff base against prostate cancer in vitro and in vivo

Article abstract of DOI:10.1097/CAD.0000000000000528

Prostate cancer is the second most common cancer among men and the leading cause of death after lung cancer. Development of hormone-refractory disease is a crucial step for prostate cancer progression for which an effective treatment option is currently unavailable. Therefore, there is a need for new agents that can efficiently target cancer cells, decrease tumor growth, and thereby extend the survival of patients in late-stage castration-resistant prostate cancer. In the current study, a novel heterodinuclear copper(II)Mn(II) Schiff base complex combined with P85 was used to evaluate anticancer activity against prostate cancer in vitro and in vivo. Cell proliferation and cytotoxicity were evaluated by cell viability, gene, and protein expression assays in vitro. Results showed that the heterodinuclear copper(II)Mn(II) complex-P85 combination decreased cell proliferation by upregulating the apoptotic gene expressions and blocking the cell proliferation-related pathways. Tramp-C1-injected C57/B16 mice were used to mimic a prostate cancer model. Treatment combination of Schiff base complex and P85 significantly enhanced the cellular uptake of chemicals (by blocking the drug transporters and increased life time), suppressed tumor growth, and decreased tumor volume steadily over the course of the experiments. Overall, heterodinuclear copper(II)Mn(II) complex-P85 showed remarkable anticancer activity against prostate cancer in in vitro and in vivo.

Full text of DOI:10.1097/CAD.0000000000000528

Preclinical report
1
Poloxamer P85 increases anticancer activity of Schiff base
against prostate cancer in vitro and in vivo
Selami Demircia, Ayşegül Doğana, Neşe Başak Türkmend, Dilek Telcia,
Ahmet B. Çağlayanc, Mustafa Ç. Bekerc, Ertuğrul Kılıçc, Ferda Özkanb,
Bülent Dedee and Fikrettin Şahina
Prostate cancer is the second most common cancer among
men and the leading cause of death after lung cancer.
Development of hormone-refractory disease is a crucial
step for prostate cancer progression for which an effective
treatment option is currently unavailable. Therefore, there is
a need for new agents that can efficiently target cancer
cells, decrease tumor growth, and thereby extend the
survival of patients in late-stage castration-resistant
prostate cancer. In the current study, a novel
heterodinuclear copper(II)Mn(II) Schiff base complex
combined with P85 was used to evaluate anticancer activity
against prostate cancer in vitro and in vivo. Cell proliferation
and cytotoxicity were evaluated by cell viability, gene, and
protein expression assays in vitro. Results showed that the
heterodinuclear copper(II)Mn(II) complex–P85 combination
decreased cell proliferation by upregulating the apoptotic
gene expressions and blocking the cell proliferation-related
pathways. Tramp-C1-injected C57/B16 mice were used to
mimic a prostate cancer model. Treatment combination of
Schiff base complex and P85 significantly enhanced the
cellular uptake of chemicals (by blocking the drug
course of the experiments. Overall, heterodinuclear
copper(II)Mn(II) complex–P85 showed remarkable
anticancer activity against prostate cancer in in vitro and
in vivo. Anti-Cancer Drugs 00:000–000 Copyright © 2017
Wolters Kluwer Health, Inc. All rights reserved.
Anti-Cancer Drugs 2017, 00:000–000
Keywords: castration-resistant prostate cancer, P85, poloxamer,
prostate cancer, Schiff base
aDepartment of Genetics and Bioengineering, Faculty of Engineering,
bDepartment of Pathology, Faculty of Medicine, Yeditepe University,
cDepartment of Physiology, Faculty of Medicine, Istanbul Medipol University,
Istanbul, dDepartment of Pharmaceutical Toxicology, University of Inonu, Malatya
and eDepartment of Chemistry, Faculty of Sciences and Arts, Süleyman Demirel
University, Isparta, Turkey
Correspondence to Ayşegül Doğan, PhD, Department of Genetics and Bioengineering,
Faculty of Engineering, Yeditepe University Kayisdagi, 34755 Istanbul, Turkey
Tel: + 90 216 578 0619; fax: + 90 216 578 0829;
e-mails: aguldgn@gmail.com, aysegul.dogan@nih.gov
Present address: Selami Demirci: National Heart, Lung, and Blood Institute
(NHLBI), NIH, Bethesda, Maryland, USA.
Present address: Ayşegül Doğan: National Cancer Institute, CDBL, NIH,
Frederick, Maryland, USA.
Received 9 January 2017 Revised form accepted 21 May 2017
transporters and increased life time), suppressed tumor
growth, and decreased tumor volume steadily over the
Introduction
tumor response variability to chemotherapy underscoring
the challenge [6]. Although a combination of chemother-
apeutic agents is considered to be an effective treatment
strategy for prostate cancer, there is a scarcity of data on
the enhanced survival rates or improved quality of life of
patients. Besides, patient survival rates are lower
compared with the higher toxicity associated with
combinatorial treatment [7]. There are several available
Food and Drug Administration approved chemother-
apeutics such as estramustine, mitoxantrone, docetaxel,
and cabazitaxel for the prostate cancer treatment [4].
Docetaxel and cabazitaxel are taxane group che-
motherapeutics, and act on the microtubule structure,
P-glycoprotein, and multidrug-resistance protein-1 (MRP-
1) [3]. The activity of these chemotherapeutics is impaired
because of the amplification of these drug transporters [8].
Although docetaxel is the most promising available
chemotherapeutic in the market, acquired resistance is a
major problem and response in CRPC is not satisfactory.
Therefore, development of new chemotherapeutics,
Prostate cancer is the second most frequently diagnosed
cancer among men and also the leading cause of death
(9% mortality ratio) after lung cancer [1]. Regulation of
prostate cancer is a multistep process controlled by
androgens at the cellular and molecular levels through
androgen and tyrosine kinase receptor mutations,
amplifications, or enhancement the sensitivity of recep-
tors to growth factors [2]. Development of androgen
independency is one of the biggest problems during the
treatment of prostate cancer. Androgen-deprivation
therapy as a standard treatment can enhance the sensi-
tivity of cells to chemotherapy and radiotherapy.
Development of castration-resistant prostate cancer
(CRPC) is a major challenge for prostate cancer therapy
when hormone-deprivation therapy cannot prevent cancer
growth [3]. Surgical removal, antiandrogens, chemother-
apy, and radiotherapy are the treatment options generally
applied for advanced-stage prostate cancer [4,5]. The
heterogenic nature of prostate cancer is exemplified by the
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DOI: 10.1097/CAD.0000000000000528
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2
Anti-Cancer Drugs 2017, Vol 00 No 00
particularly for late-stage metastatic prostate cancer, is an
increasingly attractive field.
Cu(II)-Mn(II) complex was performed in six steps.
4-(Chloroacetyl)biphenyl was prepared from chloroacetyl
chloride and biphenyl in the presence of aluminum chloride
according to Friedel–Crafts acylation. Chloroketooxime was
obtained by reacting 4-(chloroacetyl)biphenyl with alkyl
nitride in the presence of dry HCl gas. Ligand was prepared
by the condensation reaction of chloroketooxime with
p-toluidine and 1,3-propanediamine, respectively. The oily
mononuclear Cu(II) complex was obtained by the addition of
metal salts of Cu(II) to the solution of the ligand dissolved in
acetone. Finally, the heterodinuclear Cu(II)–Mn(II) complex
of the iminooxime ligand was precipitated by the reaction of
the mononuclear Cu(II) complex with Mn(II) salt and
1,10-phenanthroline. The structure of the Schiff base used in
this study is shown in Fig. 1a. The complex was stored at
room temperature in a light-protected tube in a dry place.
Pluronic P85 (BASF Corporation, Badische Anilin und
Soda-Fabrik, Florham Park, New Jersey, USA) was prepared
in PBS (Pan-Biotech, Aidenbach, Germany) at a stock
concentration of 10% (w/v) by incubation at 4°C overnight
for complete dissolution. The main stock solution (10%) was
diluted to 1% in Dulbecco’s modified Eagle’s medium
(DMEM; Invitrogen, Gibco, UK) containing 10% fetal
bovine serum (FBS; Invitrogen) and 1% penicillin/strepto-
mycin/amphotericin (PSA; Invitrogen) for cell culture
experiments. P85 (0.05% w/v) was combined with the
selected concentrations of the Schiff base in DMEM
supplemented with 10% FBS and 1% PSA for in-vitro
analysis. The main stock of P85 (10%) was diluted to
0.05% (w/v) in PBS for in-vivo experiments.
Schiff bases are derived as a result of the condensation
reaction of primary amine and carbonyl compounds [9].
Different Schiff base derivatives or their metal com-
plexes were identified for their anti-inflammatory [10],
antifungal [11], antimicrobial [12], and antihypertensive
[13] activities. In addition to their broad range of biolo-
gical activities, the anticancer properties of various Schiff
bases have been reported previously [14–16]. Their
DNA-binding and cleavage activities favor the usage of
these compounds as potential drugs in cancer treatment.
The antitumor activity of Schiff bases has been attributed
to DNA breakage through hydrogen bond formation with
nitrogen atoms of DNA or copper binding to the DNA
and/or to the proteins [17]. In the current study, we
evaluated the anticancer activity of a novel Schiff base
derivative, referred to as the heterodinuclear copper(II)
Mn(II) complex, in combination with the poloxamer P85.
In our previous studies, we have documented the anti-
tumor activity of this Schiff base derivative against liver,
colorectal cancer, and prostate cancer [18–20].
Polymer-dependent drug delivery to tumor tissues, on
the basis of the encapsulation of drug molecules into
stable micelles, is a new modality in chemotherapy
research. Micellar encapsulation of drug molecules
decreases the systemic degradation of drug molecules in
the blood stream and increases the drug uptake by tumor
cells [21]. Poloxamers, also known as Pluronics, arranged
in an A–B–A triblock structure that consist of hydrophilic
poly(ethylene oxide-PEO) and hydrophobic poly(propy-
lene oxide-PPO) units have been used for the delivery of
chemotherapeutics to cancer cells [22]. P85 among these
copolymers have been combined with different mole-
cules to inhibit ATP-dependent efflux pumps, increase
membrane permeabilization [23], and overcome drug
resistance in various cancer cells [24]. Their ability to
transport drugs through blood barriers makes them
potential candidates for chemotherapeutic delivery. P85
has increased anticancer activity of several chemother-
apeutic drugs in the previous studies [25,26]. We have
already evaluated the anticancer activity of the hetero-
dinuclear copper(II)Mn(II) in complex with P85 for an
early-stage prostate cancer model in vivo [18]. In the
current study, we have conducted in-vivo experiments
using a prostate cancer model in C57/Bl6 mice mimicking
the late-stage disease, and in-vitro experiments, to
mechanistically analyze the cytotoxic effect of the Schiff
base–P85 complex by defining the molecular pathways
involved.
Cell lines
Tramp-C1 (CRL 2730, mouse prostate cancer cells), DU
145 (HTB 81, human prostate cancer cells), and LNCaP
(CRL 1740, human prostate cancer cells) were purchased
from ATCC (Rockville, Maryland, USA). All cell lines
were incubated in DMEM (Invitrogen) supplemented
with 10% FBS (Invitrogen) and 1% PSA (Invitrogen) in a
humidified chamber at 37°C and 5% CO2.
Cell viability analysis
The cell viability of prostate cancer cell lines (Tramp-C1,
DU 145, and LNCaP) was measured using the
3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phe-
nyl)-2-(4-sulfo-phenyl)-2H-tetrazolium
(MTT)-assay
(#G3582; CellTiter96 Aqueous One Solution; Promega,
Southampton, UK) as described previously [28]. The
Schiff base was dissolved in dimethyl sulfoxide (DMSO;
Sigma-Aldrich, St. Louis, Missouri, USA) at a stock
concentration of 1 mg/ml and diluted to 10 µg/ml in
DMEM supplemented with 10% FBS and 1% PSA.
Materials and methods
Cells were exposed 2 µg/ml of the Schiff base in
complete growth medium and its P85 combination.
Working solution for P85 was selected as 0.05% (w/v).
Cell viability was analyzed at three different time inter-
vals (24, 48, and 72 h). At the end of each time point,
Preparation of drug combination
The heterodinuclear copper(II)Mn(II) complex (Schiff base)
was synthesized and characterized by our group as
described previously [27]. Preparation of the heterodinuclear
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Schiff base–P85 shows anticancer activity Demirci et al.
3
Fig. 1
Structure of Schiff base and toxicity analysis. (a) Chemical structure of the heterodinuclear copper(II)Mn(II) complex. Cytotoxicity of the Schiff
base–P85 combination on (b) DU 145, (c) LNCaP, and (d) Tramp-C1 at day 1, 2, and 3. Cell viability was evaluated by comparison with NC. NC,
negative control (growth medium); P, Pluronic P85 (0.05% P85); PC, 20% dimethyl sulfoxide; S, Schiff base (2 µg/ml); SP, Schiff base–P85 complex
(2 µg/ml S–0.05% P85). *P < 0.05.
absorbance at 490 nm was detected using an ELISA plate
reader (Biotek, Winooski, Vermont, USA).
experiments were conducted using the CFX96 RT-PCR
system (Bio-Rad, Hercules, California, USA).
Western blot analysis
Real-time quantitative reverse transcription PCR
analysis
All chemicals used in immunoblotting assays were pur-
chased from Bio-Rad Laboratories (Richmond, California,
USA). Primary antibodies against Akt (#9272), nuclear
factor-κB (NF-κB) (#8242) and GAPDH (#8884), all
purchased from Cell Signaling Technology (Beverly,
Massachusetts, USA), were used to detect marker proteins
for cancer cells treated with drug compositions.
Nitrocellulose membranes were incubated with primary
antibodies (dilution 1 : 5000) at 4°C for 16 h. After washing
with TBS-T three times, the membranes were incubated
with the anti-rabbit secondary antibody (sc-2004, dilution
1 : 5000; Santa Cruz Biotech Inc., Dallas, Texas, USA)
prepared in blocking buffer for 1 h at room temperature.
GAPDH was used as an internal control and images were
obtained using the luminometer system (Bio-Rad). Band
intensities were calculated using NIH Image J software
(NIH Image, Bethesda, Maryland, USA) and normalized to
the respective GAPDH band intensities. Results were
represented as fold change of control.
RT-PCR experiments were conducted according to the
previously described protocol [29]. Primers for Bcl-
2-associated X protein (BAX), B-cell lymphoma 2 (Bcl-2),
ATP-binding cassette subfamily G member 2 (BCRP),
caspase 3, epidermal growth factor receptor (EGFR),
MRP, and p53 were designed using Primer-BLAST
software from the National Center for Biotechnology
(Bethesda, Maryland, USA) and synthesized by
Macrogen (Seoul, Korea). β-Actin was used as the
housekeeping gene. Total RNA from drug-treated cancer
and healthy cells were isolated using the RNeasy plus
mini kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. cDNA was synthesized
using the High Fidelity cDNA synthesis kit (Roche,
Indianapolis, Indiana, USA). RT-PCR with the SYBR
Green (Foster City, California, USA), method was used
to detect mRNA levels of the target genes. All RT-PCR
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4
Anti-Cancer Drugs 2017, Vol 00 No 00
Animals
Statistical analysis
Healthy male C57/Bl6 mice (total 32; n = 8/group)
weighing 20 2 g were obtained from Yeditepe
University (Istanbul, Turkey). The animals were housed
individually in disinfected cages and acclimated to a
constant temperature of 23 1°C and a relative humidity
of 60 10%. The mice were maintained on a 12-h light/
dark cycle and fed with food and water ad libitum. All
respective procedures were approved by the Yeditepe
University Ethics Committee of Experimental Animal
Use and the Research Scientific Committee.
The data were statistically analyzed using one-way ana-
lysis of variance and the Tukey post-hoc test, with a
P-value less than 0.05 considered statistically significant.
Results
Cytotoxicity of Schiff base–Pluronic P85 in prostate
cancer cell lines
The impact of various Schiff base concentrations and its
Pluronic combinations on prostate cancer cells was eval-
uated to identify the minimum effective dose for further
experiments [34]. A combination of 2 µg/ml Schiff base–
0.05% P85 (SP) was used for cytotoxic analysis for 24, 48,
and 72 h. Although stand-alone Schiff base application
(S) decreased the viable cell ratio approximately to 65%
compared with the baseline, Schiff base–0.05% P85
decreased cell viability to 30, 42, and 42% for DU 145
(Fig. 1b), LNCaP (Fig. 1c), and Tramp-C1 (Fig. 1d) cells,
respectively, at the end of the 72 h incubation period.
P85-only treatment remained ineffective on the viability
of cancer cells, unless combined with the Schiff base
chemical (Fig. 1).
Development of the tumor model and drug application
Prostate cancer was developed on the dorsal side of C57/
Bl6 mice by a Tramp-C1 cell injection according to the
previously described protocol [18,30]. Briefly, 2 × 107
Tramp-C1 cells were injected subcutaneously under
moderate anesthesia and the tumor development was
monitored daily. For the examination of the Schiff base’s
anticancer activity, specified concentrations of Schiff base
(0.5 mg/kg) and Pluronic P85 (0.05%) combination were
administered intraperitoneally every 4 days after tumor
volumes became ∼ 0.34 cm3. Control groups received the
same volume (200 µl) of sterile PBS. Mice were exam-
ined for mortality and morbidity throughout the study
and weight changes were recorded. Experiments were
terminated for each group individually when the animals
started to die. Mice were killed by cervical dislocation at
the end of the experiment and tumor tissues were
maintained in a 10% formaldehyde solution for histo-
pathological examinations. The experimental design is
shown in Table 1.
Gene expression analysis
Apoptotic gene expression levels were examined in the
drug-treated group to evaluate the mechanism of cyto-
toxicity. The results showed that Bax, caspase 3, and p53
expressions were significantly upregulated in the Schiff
base–P85 (SP) group, indicating the onset of apoptosis
in the Schiff base and P85 combination group. Although
there was no significant change in the expression
levels of antiapoptotic Bcl-2 gene in DU 145 cells for
the experimental groups, LNCaP and Tramp-C1 cells
showed a significant reduction of Bcl-2 expression
(Fig. 2). Although stand-alone Schiff base increased the
expression of some of the apoptotic genes, the mono-
treatment with the Schiff base or P85 did not generally
affect the apoptotic gene expression levels. EGFR
expression, a vital gene in the prostate cancer progression
during hormone-refractory disease emergence, reduced
significantly in the S and SP groups (Fig. 3). BCRP and
MRP expression levels were also found to be low in the
S and SP groups with respect to the vehicle-treated
control. The most significant reduction was observed in
the SP group (Fig. 3).
Tumor volume measurements
Throughout the experiments and after resection of
tumors, their sizes were measured by a caliper according
to the formula given below [31]:
Lengthꢀwidthꢀheight
Tumor volume ¼
:
2
Pathological analysis
Paraffin slides were obtained and stained with hema-
toxylin and eosin for Gleason score analysis according to
the previously described protocol [32,33]. Multiple
organs were analyzed for metastasis.
Table 1 In-vivo experimental design
Groups
Treatment
Period (tumor formation) (days)
Control
S (0.5 mg/kg)
SP (0.5 mg/kg S–0.5% P85)
P (0.5% P85)
20 × 106 Tramp-C1 injection + PBS solution injection
48
48
48
48
20 × 106 Tramp-C1 injection + S solution injection
20 × 106 Tramp-C1 injection + SP solution injection
20 × 106 Tramp-C1 injection + SP solution injection
S, Schiff base, administration of S at 0.5 mg/kg of body weight; SP, Schiff base–P85 complex, administration of SP complex at 0.5 mg/kg S–0.05% P85 of body weight;
P, Pluronic P85 (0.05% P85).
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Schiff base–P85 shows anticancer activity Demirci et al.
5
Fig. 2
Comparison of mRNA expression levels of Bax, Bcl-2, caspase 3, and p53 in the experimental groups. Gene expression was evaluated by comparison
with NC. NC, negative control (growth medium); P, Pluronic P85 (0.05% P85), S, Schiff base (2 µg/ml); SP, Schiff base–P85 complex (2 µg/ml S–
0.05% P85). *P < 0.05.
Protein expression analysis
and Tramp-C1 cells. Interestingly, Akt expression did
not change in Tramp-C1 cells for either S or SP groups
(Fig. 4).
Akt and NF-κB protein expression levels were detected
to observe the proliferative state of cancer lines treated
with the Schiff base and its P85 combination. SP-exposed
cells showed reduced levels of Akt and NF-κB proteins,
which might be the primary explanation for decreased
cell viability and enhanced cytotoxicity. S application also
decreased the protein expressions in DU 145 cells for
both proteins, and NF-κB protein expression in LNCaP
Animal weights, life time, and tumor volumes
Forty-eight days after a subcutaneous Tramp-C1 cell
injection was administered, tumors appeared on the
dorsal side and animals were divided randomly into four
groups as shown in Table 1. The maximum tolerated
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Anti-Cancer Drugs 2017, Vol 00 No 00
Fig. 3
Comparison of mRNA expression levels of BCRP, MRP, and EGFR in the experimental groups. Gene expression was evaluated by comparison with
NC. EGFR, epidermal growth factor receptor; MRP, multidrug-resistance protein; NC, negative control (growth medium); P, Pluronic P85 (0.05%
P85); S, Schiff base (2 µg/ml); SP, Schiff base–P85 complex (2 µg/ml S–0.05% P85). *P < 0.05.
dose of the Schiff base and Pluronic combination was
selected for animal experiments on the basis of our pre-
viously published toxicology analysis [18]. Schiff base
(0.5 mg/ml) and P85 (0.05% P85) combination or vehicle
(PBS) in 0.2 ml volume were applied intraperitoneally
every 4 days during the experimental period. Weight
changes were recorded periodically and observed daily to
evaluate morbidity and mortality. Animals in each group
were killed by cervical dislocation when they started to show
tumor formation. The initial average weight was ∼24 g for
each experimental group. However, weights started to
increase during the experimental period because of the
rapidly growing tumors, except for the SP and P groups.
The average weight was 32, 30, 26, and 24 g for the control,
S, SP, and P, respectively (Fig. 5a). The mean life time
during the experimental period was 58, 60, 85, and 70 days
for the control, S, SP, and P groups, respectively (Fig. 5b).
The animals treated with the SP combination lived 37 days
after tumor formation was observable (drug application-day
48), whereas the mean life time was only 10 days for
the control animals (Figs 5c and 6c). All animals in the
experimental group had visible solid tumors. Figure 5d
shows the exponential tumor growth for the control, S, and P
groups. In SP animals, tumors started to grow after 16 days of
drug application and increased slowly over the course of the
experiment (Fig. 5d). Tumor volumes were ∼5.4, 10, 3, and
5.7 cm3 for the control, S, SP, and P groups, respectively, at
the end of the experiment (Fig. 5e). Highly vascularized
solid tumors and animals carrying these tumors on the dorsal
site close to tail are shown in Fig. 6a.
Histopathological analysis
Gleason scores of tumors in the experimental groups
were determined by histopathological analyses (Fig. 6b).
Although high Gleason scores (5 + 5) and aggressive
phenotypes were diagnosed in all experimental groups
(Fig. 6b and Table 2), only the control and P groups
showed tumor metastasis to the distant organs. Metastatic
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Schiff base–P85 shows anticancer activity Demirci et al.
7
Fig. 4
Akt and NF-κB protein expression levels of DU 145, LNCaP, and Tramp-C1 cells. NC, negative control (growth medium); NF-κB, nuclear factor-κB;
P, Pluronic P85 (0.05% P85); S, Schiff base (2 µg/ml); SP, Schiff base–P85 complex (2 µg/ml S–0.05% P85). *P < 0.05.
foci were observed in the kidney for control animals and
the P group (Fig. 7a and b). In addition, metastasis was
recorded in the testis of the P group (Fig. 7c and
Table 2).
investigated the anticancer activity of a Schiff base deri-
vative and its P85 combination both in-vitro and in-vivo
using a late-stage Tramp-C1 prostate cancer model. More
recently, we have shown that the Schiff base and P85
combination inhibited tumor growth for an early-stage
prostate cancer model in C57/B16 mice without exerting
any tissue or organ toxicity, suggesting its potential use in
metastatic cancer [18].
Discussion
Prostate cancer is a common malignancy among men,
which can be responsive to chemotherapy before devel-
oping into androgen-independent CRPC and acquiring
an aggressive phenotype [1,35]. Hormone-ablation ther-
apy efficiency is similar to that of chemotherapy or
radiotherapy for prostate cancer in the early stages;
however, cancer cells can generally induce an androgen-
independent stage and metastasize to other body parts,
primarily to bone, which reduces the overall survival at
the late stages [36]. Because treatment options are lim-
ited for the late-stage prostate cancer because of acquired
resistance, new treatment modalities and pharmaceutical
compounds are highly warranted. Although several che-
motherapeutics have been approved for CRPC, high
survival rates have not been observed so far, and the side
effects of the chemicals prevent the usage in elderly
patient population [37]. In the present study, we have
Using three different prostate cancer cell lines in vitro
(DU 145, LNCaP, Tramp-C1), we showed that treatment
with Schiff base and P85 suppressed the cell viability
more significantly compared with the stand-alone Schiff
base application. Similarly, the Schiff base and its P85
combination could induce the expression of apoptotic
genes including BAX, caspase 3, and p53, while decreasing
the antiapoptotic Bcl-2 expression. To understand the
possible molecular events occurring after the adminis-
tration of Schiff base–P85, we carried out protein
expression analyses. It was reported that the activation of
Akt and its downstream pathways such as NF-κB are
major regulators for cancer cell survival, growth, pro-
liferation, and chemoresistance [38]. The combination of
Schiff base and P85 decreased the Akt and NF-κB gene
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8
Anti-Cancer Drugs 2017, Vol 00 No 00
Fig. 5
Animal weight, mean life time, and tumor measurements of the experimental groups. (a) Average animal weights during the experiment, (b) mean life
time from the beginning of the experiment, (c) mean life time after drug application, (d) tumor growth rate after drug application, (e) mean tumor
volume at the end of the experiment. Control, animals received 0.2 ml PBS; P, Pluronic P85 (0.05% P85); S, Schiff base, administration of S at
0.5 mg/kg of body weight; SP, Schiff base–P85 complex, administration of the SP complex at 0.5 mg/kg S–0.05% P85 of body weight. *P < 0.05.
and protein levels in the prostate cancer cells in vitro,
which provides a possible explanation for the cytotoxic
activity.
A prostate cancer model in subcutaneous Tramp-
C1-injected C57/B16 mice that mimics the advanced
stage of cancer was created by cell transplantation to the
flank region and following cancer progression until tumor
growth reached ∼ 0.34 cm3 volume. Although control
and Schiff base-administered animals gained weight
throughout the course of the experiment possibly
because of the development of large tumors, animal
weights in the Schiff base and P85 combination or stand-
alone P85 groups were normal. The Schiff base–P85
treatment improved the survival rate and increased the
overall life time of tumor-bearing mice compared with
single treatment or control groups. Tumor growth was
markedly diminished and tumor volume was significantly
reduced in the Schiff base–P85 group, suggesting a
potential efficiency of this formulation in the treatment
of late-stage prostate cancer tumors. Although the Schiff
base–P85-treated animals had 3 cm3 tumors at the end of
the experiment, the animals that received Schiff base or
P85 had 10 and 6 cm3 visible tumors, respectively.
Control group animals that were carrying ∼5.4 cm3
tumors died rapidly after 10 days of saline application. All
tumors derived from this prostate cancer model had high
Gleason scores, explaining the aggressive tumor pheno-
type. Metastatic tumors in the distant organs were
Drug efflux pumps (P-glycoprotein, BCRP, MRP-1, and
MRP-2), potent target sites for P85 on the membrane,
are expressed on both healthy and tumor tissues. The
expression levels of these multidrug resistance pumps
on the cancer cell membranes are significantly high
compared with that of normal cells [39]. The inhibitory
role of P85 on these drug transporters could be an
advantage for in-vitro and in-vivo drug-delivery
approaches compared with conventional therapies.
The gene expression analysis showed a marked
decrease in MRP and BCRP gene levels for the prostate
cancer cells treated with Schiff base–P85, indicating a
strong correlation between Schiff base and P85. AR-
dependent growth of prostate cancer cells is mediated
by activating Elk-1-binding sites and promoter activa-
tion through phosphorylation and EGFR activation,
which subsequently enables cell growth [40]. In the
current study, the expression levels of EGFR were
lower in the Schiff base–P85 group than the Schiff
base or P85-alone groups, showing synergistic activity
between the Schiff base and P85.
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Schiff base–P85 shows anticancer activity Demirci et al.
9
Fig. 6
Tumor gross appearance at the end of the experiments and histopathological analysis. (a) Tumors resected from animals and animals with large
tumors on the flank region. (b) Histopathological appearance of tumor samples. All tumors have a high Gleason score, with a differentiated phenotype.
(c) Timing plan for experimental groups of in-vivo analysis. Control, animals received 0.2 ml PBS; P, Pluronic P85 (0.05% P85); S, Schiff base,
administration of S at 0.5 mg/kg of body weight; SP, Schiff base–P85 complex, administration of the SP complex at 0.5 mg/kg S–0.05% P85 of body
weight; magnification × 100.
Table 2 Gleason score analysis and organ metastasis after histopathologic examination
Metastasis to organs
Groups
Gleason score
Liver
Heart
Kidney
Testis
Intestine
Lung
Control
S (0.5 mg/kg)
SP (0.5 mg/kg S–0.5% P85)
P (0.5% P85)
5 + 5
5 + 5
5 + 5
5 + 5
–
–
–
–
–
–
–
–
+
–
–
+
–
–
–
+
–
–
–
–
–
–
–
–
S, Schiff base, administration of S at 0.5 mg/kg of body weight; SP, Schiff base–P85 complex, administration of the SP complex at 0.5 mg/kg S–0.05% P85 of body
weight; P, Pluronic P85 (0.05% P85).
detected in kidney and testis tissues of control or
Pluronic administered animals. Although the activity of
stand-alone P85 has not been identified as yet, it is clear
that P85 also played a protective role in the survival of
animals by the stimulation of antigen-producing cell
production in the immune system [41]. Moreover, the
rapid degradation of Schiff base in the circulation and
excretion through the kidney might be the reason for the
unsatisfactory results observed in animals that received
stand-alone Schiff base.
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10 Anti-Cancer Drugs 2017, Vol 00 No 00
Fig. 7
Histopathological examinations of metastatic foci. (a) Metastasis in the kidney of the control group (b) and the P group. (c) Testis metastasis of the P
group. Control, animals received 0.2 ml PBS; P, Pluronic P85 (0.05% P85); magnification (a, b) × 100, (c) × 40.
These preclinical data showed that the Schiff base–P85
combination functionally decreased tumor growth and,
therefore, is a promising candidate for prostate cancer
chemotherapy. In agreement with the published studies
[23,24], the combination of P85 caused micellar encap-
sulation of the Schiff base, which enabled the easy
transport of Schiff base through the membrane by
blocking drug transporters. Transport of the Schiff base
in P85 micelles plausibly provided stability in blood
stream and prevented elimination [42]. P85, when used
under a critical micelle concentration, forms unimers and
interacts with the lipid membrane to enhance flexibility
and block the ATP-dependent drug transporters [43].
The concentration of P85 (0.05%) used in in-vitro ana-
lysis alters the fatty acid content of the membrane and
inhibits the drug transporters on cancer cells [44].
Schiff base, when combined with P85, could be protected
in the circulation and could diffuse from the barriers
(blood–brain barrier, testis–blood, and prostate–blood
barriers) easily to obstruct tumor cell growth.
Acknowledgements
The authors thank Laxminath Tumburu for his helpful
comments and valuable suggestions during language
editing.
This study was funded by Yeditepe University.
Conflicts of interest
There are no conflicts of interest.
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