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Anti-angiogenic and anti-inflammatory effects of long-circulating liposomes

co-encapsulating curcumin and doxorubicin on C26 murine colon cancer cells

Article  in  Pharmacological reports: PR · October 2017

DOI: 10.1016/j.pharep.2017.10.004

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 Pharmacological Reports 70 (2018) 331–339

Contents lists available at ScienceDirect

Pharmacological Reports
journal homepage: www.elsevier.com/locate/pharep

Original article

Anti-angiogenic and anti-inflammatory effects of long-circulating

liposomes co-encapsulating curcumin and doxorubicin on C26 murine
colon cancer cells
Alina Sesarmana,b,c , Lucia Tefasc , Bianca Sylvesterc, Emilia Licaretea,b , Valentin Raucaa,b ,
Lavinia Luputa,b , Laura Patrasa,b , Manuela Banciua,b,* , Alina Porfirec
a
Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babes-Bolyai University, Cluj-Napoca, Romania
b
Molecular Biology Centre, Institute for Interdisciplinary Research in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, Romania
c
Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-
Napoca, Romania

A R T I C L E I N F O A B S T R A C T

Article history: Background: Emerging treatment options for colon cancer are needed to overcome the limitations
Received 9 February 2017 regarding the side effects of current chemotherapeutics and drug resistance. The goal of this study was to
Received in revised form 19 September 2017 assess the antitumor actions of PEGylated long-circulating liposomes (LCL) co-delivering curcumin
Accepted 13 October 2017
(CURC) and doxorubicin (DOX) on murine colon carcinoma cells (C26).
Available online 16 October 2017
Methods: The cytotoxicity of CURC and DOX, administered alone or in combination, either in free or LCL
form, was evaluated with regard to antiproliferative effects on C26 cells and to protumor processes that
Keywords:
might be affected.
Doxorubicin
Curcumin
Results: Our results indicated that PEGylated LCL-CURC-DOX exerted strong antiproliferative effects on
Liposomes C26 cells, slightly exceeding those induced by free CURC-DOX, but higher than either agent administered
Cytotoxicity alone in their free form. These effects of LCL-CURC-DOX were due to the inhibition of the production of
Colon cancer angiogenic/inflammatory proteins in a NF-kB dependent manner, but were independent of ROS
production or AP-1 c-Jun activation. Notable, the anti-angiogenic actions of LCL-CURC-DOX appeared to
be much stronger than those induced by the co-administration of CURC and DOX in their free form, on
C26 colon cancer cells.
Conclusion: LCL-CURC-DOX demonstrated enhanced cytotoxicity on C26 murine colon cancer cells by
inhibiting the production of the majority of factors involved in tumor-associated angiogenesis and
inflammation and is now being evaluated in vivo regarding its efficacy towards tumor growth in colon
cancer.
© 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights
reserved.

Abbreviations: AP-1, activator protein 1; AP-1 c-Jun, c-Jun subunit of activator protein 1; bFGF, basic fibroblast growth factor; BrdU, bromodeoxyuridine; CURC, curcumin;
DOX, doxorubicin; DPPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; FasL, Fas ligand; FCS, fetal calf serum; G-CSF, granulocyte-colony stimulating factor; HPLC, high-
performance liquid chromatography; IFN-g, interferon g; IGF, insulin-like growth factor; IGF-II, insulin-like growth factor II; IL-1a, interleukin-1a; IL-1ß, interleukin-1ß; IL–
4, interleukin-4; IL–6, interleukin-6; IL–8, interleukin-8; IL–9, interleukin-9; IL–10, interleukin-10; IL–12, interleukin-12; IL-12p40, interleukin-12 subunit p40; IL-12p70,
interleukin-12 subunit p70; IL–13, interleukin-13; LCL, long-circulating liposomes; M-CSF, macrophage-colony stimulating factor; MCP–1, monocyte chemoattractant
protein-1; MDA, malondialdehyde; NC, nitrocellulose; NF-kB, nuclear factor kB; NF-kB p65, p65 subunit of nuclear factor kB; pAP-1 c-Jun, phosphorylated c-Jun subunit of
activator protein 1; PBS, phosphate buffered saline; PEG, polyethylene glycol; DSPE, 1,2-distearoyl-sn-glycero-3 phosphoethanolamine sodium salt; pNF-kB p65,
phosphorylated p65 subunit of NF-kB; PF–4, platelet factor 4; ROS, reactive oxygen species; SD, standard deviation; TAC, total antioxidant capacity; TBS-T, tris-buffered saline
containing 0.1% Tween-20; TIMP–1, tissue inhibitor of metalloproteinases 1; TIMP–2, tissue inhibitor of metalloproteinases 2; TNF-a, tumor necrosis factor a; TPO,
thrombopoietin; VEGF, vascular endothelial growth factor.
* Corresponding author.
E-mail address: manuela.banciu@ubbcluj.ro (M. Banciu).

https://doi.org/10.1016/j.pharep.2017.10.004
1734-1140/© 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
332 A. Sesarman et al. / Pharmacological Reports 70 (2018) 331–339
Introduction
Systemic administration of chemotherapeutic drugs (5-fluoro-
uracil, capecitabine, oxaliplatin, irinotecan or doxorubicin) for
colon cancer therapy is not that efficient as the drugs do not target
the tumor tissue in an effective concentration [1]. Additionally,
some patients also develop resistance to chemotherapy or adverse
effects such as pain, nausea, diarrhea, hair loss or skin reactions [2].
Co-administration of conventional anticancer agents with natu-
rally occurring drugs, may overcome chemotherapy limitations.
Therefore, the antitumor activity of doxorubicin, an anthracycline
antibiotic conventionally administered in liver, colorectal, breast,
lung, ovarian or thyroid cancers [3] might be improved by using
curcumin, a polyphenol with proven antineoplastic actions in
gastric, cervical, skin, genitourinary, breast, esophageal, lung,
neurological, hematological and intestinal cancers [4,5]. Moreover,
CURC was shown to potentiate the effects of several drugs such as
DOX in lung cancer [6], of 5-FU in colon cancer [7] and of
camptothecin in colon cancer cells, respectively [8]. Despite their
proven efficiency in preclinical studies, the clinical usefulness of
CURC or DOX is hampered. DOX induces severe myocardial toxicity,
hepatotoxicity and chemoresistance, while systemic administra-
tion of CURC is hindered by its low bioavailability. To overcome
these drawbacks and to further exploit the anticancer effects of
CURC and DOX, in vivo, various nanocarriers such as PEGylated
long-circulating liposomes (LCL), were developed [9,10]. PEGylated
LCL have many advantages over conventional liposomes since they
can evade the reticulo-endothelial system uptake, accumulating
into tumors, and offering an overall increase in the concentration
of the drug delivered to the tumor site [11].
Therefore, in this study we aimed to assess the cytotoxic effects
of PEGylated LCL-CURC-DOX on C26 murine colon cancer cells and
to investigate the protumor processes that might be affected as a
consequence of the inhibition of cell proliferation, such as tumor
associated-inflammation, angiogenesis and oxidative stress. Our
findings suggest that co-delivery of CURC and DOX in PEGylated
LCL might be of great potential for colon cancer therapy.
Materials and methods
Reagents
DPPC and PEG-2000-DSPE sodium salt were purchased from
Lipoid GmbH (Ludwigshafen, Germany). Cholesterol, curcumin,
doxorubicin hydrochloride, fetal calf serum, formic acid, cell
proliferation ELISA kit, proteases and phosphatases inhibitor
mixtures were acquired from Sigma-Aldrich Chemie GmbH
(Munich, Germany). C26 murine colon carcinoma cells were
purchased from Cell Lines Service GmbH (Eppelheim, Germany).
RPMI-1640 medium and supplements were purchased from Lonza
Group AG (Basel, Switzerland). HPLC-grade ethanol was purchased
from Chemical Company (Iasi, Romania). Methanol and acetoni-
trile were purchased from LGC Standards GmbH (Wesel, Germany).
Preparation of long-circulating liposomes
All LCL were prepared from 40 mM phospholipids, of which
38 mM DPPC and 2 mM PEG-2000-DSPE, and a 10:1 phospholipid:
cholesterol molar ratio. For LCL-DOX the lipids were dissolved in
ethanol, dried using a rotary evaporator at 60  C for 20 min, the
resulting lipid film being rehydrated at 60  C, for 20 min with a
1 mM DOX solution in PBS, pH = 4.5. For LCL-CURC and LCL-CURC-
DOX, the lipids and 5 mM CURC were dissolved in ethanol, the
organic solvent evaporated under reduced pressure and the lipid
film was then hydrated for 30 min, at 45  C with PBS or 0.5 mM
DOX in PBS, pH = 4.5, respectively. LCL size was reduced by
multiple extrusion steps through polycarbonate membranes
(Whatman International Ltd, Maidstone, UK). The LCL-DOX and
LCL-CURC-DOX were subjected to dialysis against PBS to remove
the non-entrapped drug. LCL-CURC were purified by centrifugation
at 2000g for 30 min prior to extrusion, and the remaining
sediment was re-dispersed in PBS, pH = 4.5. The size of the
liposomes (expressed as the intensity of scattered light), the size
distribution and the zeta potential of the liposomes were
determined by dynamic light scattering method using a Zetasizer
Nano ZS (Malvern Instruments, Malvern, UK). The entrapment
efficiencies of CURC and DOX in the LCL were determined by HPLC
analysis on a Zorbax C18 column (3.5 mm) using a gradient elution
with a mobile phase composed of 0.2% formic acid and acetonitrile,
with fluorescence detection. The excitation wavelengths were
490 nm for DOX and 425 nm for CURC, while the emission
wavelengths were 560 nm for DOX and 533 nm for CURC,
respectively. All data were expressed as mean  SD of triplicate
measurements.
Cell culturing conditions
C26 murine colon carcinoma cells were cultured in RPMI-1640
medium with 10% heat-inactivated FCS, at 37 C in a humidified
atmosphere containing 5% CO2 [12].
Cell proliferation assay
5  103 C26 cells/well were allowed to adhere overnight to a 96-
well cell culture plate. Then, the cells were incubated, for 48 h, with
CURC diluted from stock solutions in the range of 0–40 mM or with
DOX diluted in the range of 0–10 mM. To test the efficacy of the
combined administration of CURC-DOX on C26 cells proliferation,
the concentrations of 20 mM CURC and 0.15 mM DOX, were chosen.
LCL incorporating CURC, DOX or CURC-DOX, at the same
concentration as mentioned above, were also prepared. Untreated
C26 cells were used as control. The effects of ethanol on C26 cells
proliferation was assessed at similar concentrations as those used
for the preparation of working concentrations of free CURC. The in
vitro cytotoxicity of different drugs applied on C26 cells was
evaluated by using ELISA BrdU-colorimetric immunoassay as
previously described [12]. To determine the nature of the
interaction between CURC and DOX when co-administered (free
or encapsulated) C26 cells were co-treated with various concen-
trations of CURC ranging between 0 and 42 mM and with DOX
ranging between 0 and 0.25 mM. The combination index (CI) which
determines the nature of the interaction between two pharmaco-
logical agents such as the synergism, additivity or antagonism, was
calculated according to the Chou-Talalay method [13] and detailed
in the Statistical analysis subsection below.
Preparation of cell culture lysates
C26 cells exposed for 48 h to various treatments were lysed
with specific lysis buffers, containing phosphatases and proteases
inhibitor mixtures, in order to obtain proteins from whole cell
lysates and nuclear fractions, as previously described [12,14].
Protein concentration was measured by the Bradford assay (Sigma-
Aldrich Chemie GmbH, Munich, Germany).
Western blot analysis
Proteins (25 mg) from each total or nuclear lysates, prepared
from C26 cells treated with various agents, were fractionated by
SDS-PAGE, transferred on a NC membrane and probed with specific
primary (cat# SC-56735, SC-45, SC-7981-R, SC-33039) and HRP-
labeled antibodies (cat# SC-2004, SC-2005) (Santa Cruz
 A. Sesarman et al. / Pharmacological Reports 70 (2018) 331–339
Biotechnology, Dallas, Texas, USA). Equal protein loading was
confirmed by using a polyclonal rabbit anti-mouse b-actin
antibody (cat#A2103, Sigma-Aldrich Chemie GmbH, Munich,
Germany). Formed immunocomplexes were detected by chemilu-
minescence using a ClarityTM Western ECL kit (Bio-Rad Laborato-
ries, Hercules, USA) as described [12].
Protein microarray analysis
Changes in the expression level of 24 inflammatory/angiogenic
proteins in untreated C26 cells or treated with CURC-DOX in free or
LCL form, were investigated as previously published [15].
Assessment of oxidative stress parameters
MDA concentration was determined from cell lysates by HPLC
as previously described [12]. TAC was measured based on the
method described by Erel [16]. The data were expressed as mg
MDA/mg proteins or mM Trolox equivalents/mg of protein in
whole cell lysates and presented as mean  SD of duplicate
measurements.
Statistical analysis
For statistical analysis, Student's t-test for independent means
was used. The differences between the effect of drug treatments on
cell proliferation, NF-kB and AP-1 activation and on the level of
MDA and TAC, were analyzed by one-way ANOVA. Correlations
between different drug concentrations and their cytotoxic effects
on cell proliferation were evaluated using Spearman correlation
coefficient, r. The IC50 values for all experimental treatments were
determined by non-linear regression of the sigmoidal dose-
response curves using GraphPad Prism version 6 for Windows.
The nature of the interaction between CURC and DOX was
evaluated according to the Chou-Talalay method. A CI = 1 is
indicative of additivity, while a CI<1 or a CI>1, indicates synergism
or antagonism between the drugs, respectively [13,17]. The
differences between the effects induced by CURC-DOX in free or
LCL form on inflammatory/angiogenic factors were analyzed by
two-way ANOVA with Bonferroni correction for multiple compar-
isons. All statistical analyses were performed by using GraphPad
Prism version 6 for Windows, GraphPad Software (San Diego, CA,
USA). Significance was considered at values of p < 0.05. (ns,
p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****p < 0.0001).
Results
Characterization of liposomal formulations
The results regarding the LCL size, the polydispersity index, the
zeta potential, the entrapment efficiencies, and final concentration
of antitumor agents were summarized in Table 1. Moreover a
detailed characterization of all liposomal formulations used within
these studies was previously presented by Tefas et al. [18].
Table 1
Characterization of LCL formulations encapsulating CURC or/and DOX.
LCLs Size (nm) PI Zeta potential (mV)
LCL-CURC 180.10  3.06 0.08  0.01 38.10  1.76
LCL-DOX 161.60  2.98 0.11  0.03 39.9  0.50
LCL-CURC-DOX 182.00  1.22 0.07  0.01 42.30  0.20
333
The effects of CURC or/and DOX, in free or LCL form, on C26 cell
proliferation
As shown in Fig. 1, treatment with increasing concentrations of
free (A) CURC (ranging between 0 and 40 mM) or (B) DOX (ranging
between 0 and 10 mM) resulted in concentration-dependent
antiproliferative effects (Spearman correlation coefficient
r = 0.9762, p = 0.0004, for CURC, Spearman correlation coefficient
r = 1, p < 0.0001, for DOX) on C26 cells. For all the experiments
where CURC and DOX (in free or LCL form) were administered in
combination to C26 cells, we used the following concentrations:
20 mM CURC and 0.15 mM DOX. They were selected based on the
determination of IC50 values (see Table 2). Our results showed that
treatment with CURC-DOX (20 mM + 0.15 mM) (Fig. 1C) significant-
ly reduced cell proliferation compared to that induced by either
CURC (p < 0.0001) or DOX treatment alone (p < 0.0001). However,
when both LCL formulations (individual or co-encapsulated) of
CURC or DOX were compared in terms of antiproliferative effects
on C26 cells, we observed an enhanced cytotoxicity of LCL-CURC-
DOX only when compared to LCL-DOX (p < 0.01). There was a
modest, but statistically significant increase in the extent of
antiproliferative effects on C26 cells, when CURC and DOX were co-
administered in LCL form rather than in the free form (p = 0.029).
To evaluate the interaction type between CURC and DOX when
they are used in combination (in free or LCL form) we have
calculated the combination index (CI) according to the Chou-
Talalay method [13,17]. Our results showed that co-encapsulation
of CURC and DOX in LCL (LCL-CURC-DOX) exerted a moderate
synergistic inhibitory effect (CI = 0.732) on colon carcinoma cells
proliferation while co-administration of free CURC with free DOX
(CURC-DOX) induced an additive cytotoxic effect (CI = 1.007) on
cancer cells. The values of IC50 and CI were summarized in Table 2.
Empty liposomes did not show any relevant toxic effect on C26 cell
proliferation (data not shown). We have tested ethanol effects on
C26 colon carcinoma cells and found no cytotoxicity at the
concentrations used in this assay (data not shown), as previously
reported [19].
The effects of LCL-CURC-DOX on the activation level of the
transcription factors NF-kB and AP-1 in C26 cells
Our next effort was to investigate whether changes in the level
of expression and activation of two transcription factors, NF-kB
and AP-1, occurred upon treatment of C26 cells with CURC, DOX or
CURC-DOX in free as well as in LCL form. As shown in Fig. 2A, none
of our CURC/DOX treatments modified the level of expression of
total NF-kB p65 but interestingly, as seen in Fig. 2B we found a
strong reduction in the expression level of pNF-kB p65 when cells
were treated with 20 mM CURC (p < 0.0001) or with CURC-DOX
(20 mM +0.15 mM, p < 0.0001) compared to its level in untreated
cells. DOX alone in a concentration of 0.15 mM did not significantly
altered the level of expression of pNF-kB p65 (p > 0.05). When cells
were exposed to LCL-CURC-DOX, the expression level of pNF-kB
p65 was only slightly reduced (p < 0.05) compared to that
EE (%) Antitumor agent concentration (mM)
CURC DOX CURC DOX
66.78  0.58 – 4.75  0.04 –
– 83.16  2.02 – 0.52  0.16
87.48  1.86 38.41 0.13 4.50  0.06 0.02  0.00
334 A. Sesarman et al. / Pharmacological Reports 70 (2018) 331–339

Fig. 1. The effects of CURC or/and DOX treatment on the proliferation of C26 cells. The antiproliferative effects of CURC (A) or DOX (B) on C26 cells, were determined after
exposure of cells to indicated concentrations of anticancer agents. (C) C26 cells were treated with 20 mM CURC, 0.15 mM DOX or with a combination of CURC-DOX, in free as
well as LCL form. Cell proliferation after 48 h of incubation with these agents, was measured. Drug-induced antiproliferative effects were expressed as percentage of inhibition
of cell proliferation compared to untreated cells. Data are presented as the mean  SD of triplicate measurements.

measured in control lysates. In contrast to the effect exerted on the

Table 2 activation level of NF-kB p65 transcription factor, LCL-CURC-DOX
Summary of CURC and DOX IC50 (mM) and combination index (CI) values of
or the other CURC/DOX treatments, did not influence the
experimental treatments.
expression (Fig. 3A), neither the activation (Fig. 3B) level of the
IC50 (mM) CI c-Jun subunit of AP-1 (p > 0.05).
Free drugs CURC 23.220 –
DOX 0.203 – The effects of free or liposomal CURC-DOX on the production of
CURC-DOX 13.69/0.081 1.007
angiogenic/inflammatory factors in C26 cells
LCL encapsulated drugs CURC 13.890 –
DOX 0.092 –
CURC-DOX 9.939/0.059 0.732 To determine the effects of CURC-DOX in free or LCL form on the
expression level of inflammatory and angiogenic proteins in C26
cells, a screening of 24 proteins in cell lysates was performed and
compared to the expression level of the same proteins in control
 A. Sesarman et al. / Pharmacological Reports 70 (2018) 331–339 335

Fig. 2. The effects of LCL-CURC-DOX on the activation level of NF-kB in lysates from C26 cells. (A) Western blot analyses showing the effects induced by CURC (20 mM), DOX
(0.15 mM) or CURC-DOX (20 mM + 0.15 mM) in free as well as LCL form, on the expression and activation level of NF-kB p65. b-actin was used as loading control. (B)
Percentages of the active form of NF-kB p65 (pNF-kB p65) calculated from the amount of total NF-kB p65. The results represent the mean of percentage of pNF-kB p65 from
total amount of NF-kB of two independent measurements  SD.

lysates. Our results showed that when exposed to free CURC-DOX
(20 mM + 0.15 mM), the expression level of 23 out of 24 proteins
were reduced compared to their production in untreated cells
(Table 3). More specifically, the levels of IFN-g, IL-6, Leptin, VEGF,
IL-12p40 were strongly reduced by 75–100%, the levels of M-CSF,
TNF-a, TPO, IL-9, Eotaxin, MIG were reduced by 50–75%, the levels
of TIMP-2, IL-12p70, IL-1ß, IGF-II, G-CSF, PF-4, GM-CSF were
moderately reduced (25–50%) and the levels of IL-1a, bFGF, IL-13,
Fas-L and MCP1 were slightly decreased (0–25%). Similarly, LCL-
CURC-DOX treatment was able to significantly reduce the
expression level of 23 proteins out of 24 (Table 3) compared to
their production in untreated cells. More specifically, the levels of
TNF-a, TIMP-2, IL-6 were strongly reduced (75–100%)the levels of
IL-13, Leptin, MCP1, GM-CSF, IL-9, IL-12p40, G-CSF, IFN-g, IGF-II,
M-CSF, VEGF were moderately reduced (50–75%), and the levels of
IL-12p70, IL-1ß, Eotaxin, TPO, bFGF, IL-1a, PF-4, FasL were slightly
reduced (25–50%). The level of expression of MIG-1 was reduced
only by 18% compared to its level of expression in control lysates.
CURC-DOX in both free as well as LCL form of administration, was
able to moderately stimulate TIMP-1 production (by 41.03% and
39.75%, respectively) compared to its production in control lysates.
Interestingly, for 3 out of 17 pro-angiogenic/pro-inflammatory
proteins inhibited, namely IL-1a (p < 0.05), MCP-1 (p < 0.05) and
TIMP-2 (p < 0.01), a significantly stronger degree of inhibition was
achieved when cells were exposed to LCL-CURC-DOX rather than to
free CURC-DOX.
The effects of LCL-CURC-DOX on oxidative stress markers produced in
C26 cells
To determine whether the antiproliferative effects of LCL-
CURC-DOX on C26 cells was due to their modulatory action on
intracellular oxidative stress, the concentration of MDA, a marker
of lipid peroxidation and TAC, comprising the total concentration
of non-enzymatic anti-oxidants, were measured and showed in
Fig. 4. We found that LCL-CURC-DOX slightly reduced the MDA
concentration compared to its concentration in control lysates,
although treatment with CURC, but not with DOX, significantly
increased (p < 0.0001) the concentration of MDA (Fig. 4A). Notably,
the treatment with CURC-DOX (20 mM + 0.15 mM) slightly in-
creased (p < 0.05) the MDA concentration compared to that
measured in control lysates. TAC showed an overall enhancement
(more than 50% compared to TAC measured in untreated cells),
irrespective of the pharmacological treatment applied on C26 cells
(Fig. 4B).
Discussion
In this study we assessed the cytotoxic effects exerted by
PEGylated LCL co-encapsulating CURC and DOX on C26 murine
colon cancer cells. First we demonstrated that CURC or DOX
administered alone inhibited C26 cells proliferation in a concen-
tration-dependent manner (Fig. 1A and B) and that enhanced
336 A. Sesarman et al. / Pharmacological Reports 70 (2018) 331–339

Fig. 3. The effects of LCL-CURC-DOX on the activation level of AP-1 c-Jun in lysates from C26 cells. (A) Western blot analyses showing the effects induced by CURC (20 mM),
DOX (0.15 mM) or CURC-DOX (20 mM + 0.15 mM) in free as well as LCL form, on the expression and activation level of AP-1 c-Jun. b-actin was used as loading control. (B)
Percentages of the active form of AP-1 c-Jun (pAP-1 c-Jun) calculated from the amount of total AP-1 c-Jun. The results represent the mean of percentage of pAP-1 c-Jun from
total amount of AP-1 c-Jun of two independent measurements  SD.

antiproliferative effects were obtained when these agents were
administered in combination, compared to those induced by each
agent alone. Despite its high efficiency in vitro, combined
administration of free CURC and DOX would not be advantageous
in vivo, as the therapeutic potential of these drugs is limited by the
poor bioavailability of CURC or by DOX-induced toxicity towards
normal tissues. Therefore, to obtain an efficient and durable
therapeutic response in vivo, we encapsulated CURC and DOX,
alone or in combination, in PEGylated LCL, in a molar ratio in which
CURC concentration exceeded that of DOX to favor synergistic
inhibitory effects on cell proliferation [20]. The present drug
formulations were designed based on previous in vitro and in vivo
studies that demonstrated higher efficacies of the antitumor
agents after their encapsulation in PEGylated liposomes than after
their free administration [9,21–25]. In line with previous studies
where CURC or DOX were loaded in other PEGylated nano-
formulations such as polymeric micelles [26,27] our results proved
that encapsulation of CURC and DOX in PEG-coated liposomes
enhances their cytotoxic potential on cancer cells. An explanation
for these findings might be the fact that PEG-liposomal encapsu-
lation enabled internalization of a higher amount of CURC or DOX
in C26 cells possibly as a result of endocytosis of the lipid particles
by these cells. Moreover, while co-delivery of CURC with DOX in
PEGylated LCL, further increased DOX cytotoxic potential when
compared to its individual administration in LCL, it only slightly
exceeded CURC-DOX antiproliferative effects on C26 cells (Fig. 1C).
To elucidate the manner of the interaction of these anticancer
agents in combination, the CI was calculated according to the
Chou-Talalay method. Thus, while co-administration of CURC and
DOX (CURC-DOX) as free agents exerted an additive antiprolifer-
ative effect (CI = 1.007) on colon carcinoma cells, their co-
encapsulation in LCL (LCL-CURC-DOX) induced a moderate
synergistic action (CI = 0.732) on C26 cell proliferation.
The enhanced cytotoxicity, the physicochemical characteristics,
and the PEGylated surface support the fact that LCL-CURC-DOX
would be a promising candidate allowing passive delivery and
accumulation of CURC and DOX to colon tumors, in vivo. Thus, to
gain insights into the underlying mechanisms of LCL-CURC-DOX
toxicity on C26 cells, we investigated protumor processes that may
account for the inhibition of cell proliferation, such as tumor
associated-inflammation, angiogenesis, and oxidative stress. It has
been previously shown that the transcription factors AP-1, NF-kB
or STATs are constitutively active in many cancer cell lines,
including those of colon, fostering all protumor processes.
Specifically, there are some reports indicating that the transcrip-
tion factor AP-1 regulates the proliferation, invasion and meta-
static ability of colon cancer cells [28]. In C26 cells, we found that
AP-1 c-Jun was constitutively active, yet its level of expression and
activation was not modified by any of CURC/DOX-based treatments
tested here (Fig. 3A and B). NF-kB is a key transcription factor
which in its inactive form localizes in the cytoplasm, being
associated with the inhibitory IkBa protein. Upon activation, IkBa
is degraded and the NF-kB subunits p65-p50 translocate to the
nucleus where they modulate the transcription of specific genes
involved in tumor inflammation, angiogenesis, invasion, and
metastasis [28,29]. In line with previously published data [7,30–
32], ours demonstrated that treatment of C26 cells with free CURC
and free CURC-DOX strongly reduced the level of expression of
active pNF-kB p65 in nuclear lysates, while free DOX did not
significantly affected NF-kB activation (Fig. 2A and B) [33].
Surprinsingly, co-administration of CURC and DOX in LCL slightly
diminished NF-kB activation in C26 cells compared to control,
 A. Sesarman et al. / Pharmacological Reports 70 (2018) 331–339 337

Table 3
The effects of CURC-DOX administered in the free or LCL form on the expression level of angiogenic/inflammatory proteins in C26 cells.

Angiogenic/ Percentage of inhibition ( ) and stimulation (+) of Statistical Percentage of inhibition ( ) and stimulation (+) of Statistical
inflammatory angiogenic/inflammatory proteins in C26 cells + CURC-DOX significance angiogenic/inflammatory proteins in C26 cells + LCL-CURC- significance
proteins compared to untreated cells DOX compared to untreated cells
G-CSF 40.50  3.84 **** 66.25  3.62 ****
GM-CSF 49.63  8.43 **** 62.90  4.78 ****
M-CSF 51.60  4.50 **** 72.54  2.68 ****
IGF-II 37.81  7.71 *** 69.83  9.89 ****
IL-1a 10.18  0.77 ns 40.16  8.09 ****
IL-1ß 36.21  1.90 *** 32.48  12.04 **
IL-6 78.57  5.15 **** 81.53  1.04 ****
IL-9 54.89  0.15 **** 64.56  3.22 ****
IL-12p40 81.90  0.92 **** 65.32  0.59 ****
IL-13 19.60  16.97 ns 58.72  2.55 ****
TNF-a 52.86  9.41 **** 79.83  1.39 ****
MCP-1 20.17  9.55 ns 62.56  0.04 ****
Eotaxin 60.51  3.13 **** 35.33  13.32 ***
FasL 17.86  12.08 ns 44.71  7.33 ****
bFGF 16.88  4.25 ns 36.44  3.80 ***
VEGF 79.67  0.63 **** 74.31  0.00 ****
Leptin 79.13  1.85 **** 59.74  4.74 ****
TPO 54.43  14.63 **** 36.12  4.93 ***
TIMP-1 41.03  1.52 **** 39.75  7.58 ****
TIMP-2 28.53  1.27 ** 80.91  2.51 ****
PF-4 42.49  0.68 **** 42.75  16.83 ****
IL-12p70 32.39  1.88 ** 31.40  0.98 **
IFN-g 75.84  2.70 **** 67.49  1.73 ****
MIG 74.99  4.53 **** 18.02  9.87 ns

The angiogenic/inflammatory protein levels after CURC-DOX and LCL-CURC-DOX treatment are compared to control levels of the same proteins. The results are expressed as%
of the average inhibition ( ) or stimulation (+)  SD of two independent measurements. Statistical significance was evaluated by using two-way ANOVA analysis of variance
with Bonferroni correction for multiple comparisons.

Fig. 4. The effects of LCL-CURC-DOX on oxidative stress parameters, in lysates from C26 cells. (A) MDA concentration expressed as mg MDA/mg protein and (B) TAC expressed
as mM Trolox equivalents/mg protein were measured in lysates from C26 cells incubated for 48 h with CURC (20 mM), DOX (0.15 mM) or CURC-DOX (20 mM + 0.15 mM) in free
as well as LCL form. Data are presented as mean SD of duplicate measurements.

despite showing enhanced cytotoxicity on C26 cells over free
CURC-DOX These disparate effects of free and co-encapsulated
CURC-DOX on NF-kB activation might be the result of differences
between the uptake rates of free vs. liposomal CURC-DOX by cancer
cells [34]. Liposomes are usually taken up by endocytosis, a process
relatively slower compared to that of transmembrane diffusion of
free drugs. Moreover, the effect induced by CURC and DOX (slightly
increased by co-encapsulation of CURC and DOX in LCL compared
to their free combined administration, p = 0.026, Fig. 1C) on cell
proliferation is a direct cytotoxic effect determined predominantly
by DOX and only enhanced by the presence of CURC. The effect of
the combination treatment on NF-kB activation is an indirect
338 A. Sesarman et al. / Pharmacological Reports 70 (2018) 331–339
effect, possibly determined mainly by CURC [31,35]. We envisage
that longer incubation times (> 48 h) with LCL-CURC-DOX would
allow stronger effects in terms of inhibiting NF-kB activation in C26
cells.
Further, we sought to investigate if treatment with both free
and liposomal CURC-DOX affects the production of proteins
involved in tumor inflammation, angiogenesis and apoptosis in
C26 cells. Interestingly, both treatments significantly inhibited the
overall production of most of the studied proteins (Table 3),
particularly of primary modulators of inflammation and angio-
genesis (VEGF, TNF-a, IL12p-40, IL-6). Stronger inhibitory effects of
LCL-CURC-DOX over CURC-DOX were noticed for 3 out of 17 tested
pro-inflammatory/pro-angiogenic proteins (IL-1a, MCP-1 and
TIMP-2). Our data are in line with previously published studies,
showing an enhanced anti-angiogenic potential of CURC or DOX
encapsulated or co-encapsulated in liposomes or in other lipid
nanoparticles [6,20,26,27,36] and suggest that the cytotoxicity of
LCL-CURC-DOX might be related to its ability to block early events
of angiogenesis or recruitment of inflammatory cells. Interestingly,
the production of the anti-inflammatory/anti-angiogenic proteins
were also strongly to moderately reduced by LCL-CURC-DOX
treatment, except for TIMP-1, whose expression is most likely
differentially regulated in colon cancer cells. Since LCL-CURC-DOX
had a reduced impact on oxidative stress markers assayed in this
study, although administration of CURC alone or combined with
DOX suggested pro-oxidant effects [37,38] (Fig. 4A and B) we
assume that the extent of this mechanism accounts insufficiently
for LCL-CURC-DOX-induced cytotoxicity on C26 cells.
Conclusion
Overall, our results showed that PEGylated LCL-CURC-DOX
exerted strong antiproliferative effects on C26 cells slightly
exceeding those induced by free CURC-DOX, but significantly
higher than each agent administered alone, in their free form. The
LCL-CURC-DOX toxicity on C26 cells was attained through
inhibition of the production of angiogenic/inflammatory proteins
in a NF-kB-dependent manner. Currently, given the physicochem-
ical characteristics and enhanced anti-angiogenic/anti-inflamma-
tory potential, the LCL-CURC-DOX is being evaluated in vivo, for its
efficacy in inhibiting tumor growth. This newly developed LCL
formulation, co-encapsulating CURC and DOX, holds great poten-
tial as a future therapy for colon and other related types of cancer.
Conflict of interest
None.
Financial support
This work was supported by a grant of the Romanian National
Authority for Scientific Research and Innovation, CNCS-UEFISCDI,
project number PN-II-RU-TE-2014-4-0220.
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