Cytotoxic effects of gemcitabine-loaded liposomes in human anaplastic thyroid carcinoma cells.

Abstract

Identification of effective systemic antineoplastic drugs against anaplastic thyroid carcinomas has particularly important implications. In fact, the efficacy of the chemotherapeutic agents presently used in these tumours, is strongly limited by their low therapeutic index.
In this study gemcitabine was entrapped within a pegylated liposomal delivery system to improve the drug antitumoral activity, thus exploiting the possibility to reduce doses to be administered in cancer therapy. The cytotoxic effects of free or liposome-entrapped gemcitabine was evaluated against a human thyroid tumour cell line. ARO cells, derived from a thyroid anaplastic carcinoma, were exposed to different concentrations of the drug. Liposomes formulations were made up of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-MPEG (8:3:1 molar ratio). Cell viability was assessed by both trypan bleu dye exclusion assay and fluorimetric analysis of cell DNA content.
A cytotoxic effect of free gemcitabine was present only after 72 h incubation (ARO cell mortality increased of approximately 4 fold over control at 1 microM, 7 fold at 100 microM). When gemcitabine was encapsulated in liposomes, a significant effect was observed by using lower concentrations of the drug (increased cell mortality of 2.4 fold vs. control at 0.3 microM) and earlier exposure time (24 h).
These findings show that, in vitro against human thyroid cancer cells, the gemcitabine incorporation within liposomes enhances the drug cytotoxic effect with respect to free gemcitabine, thus suggesting a more effective drug uptake inside the cells. This may allow the use of new formulations with lower dosages (side effect free) for the treatment of anaplastic human thyroid tumours.

Full text

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BioMed Central
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BMC Cancer
Open Access
Research article
Cytotoxic effects of Gemcitabine-loaded liposomes in human
anaplastic thyroid carcinoma cells
Marilena Celano1, Maria Grazia Calvagno1, Stefania Bulotta1,
Donatella Paolino1,2, Franco Arturi1, Domenicoantonio Rotiroti1,
Sebastiano Filetti3, Massimo Fresta1 and Diego Russo*1
Address: 1Department of Pharmacobiological Sciences, University of Catanzaro "Magna Græcia", 88100 Catanzaro, Italy, 2Department of
Pharmaceutical Sciences, University of Catania, 95100 Catania, Italy and 3Department of Clinical Sciences, University of Roma "La Sapienza",
00100 Roma, Italy
Email: Marilena Celano - celano.donato@tiscali.it; Maria Grazia Calvagno - marycalv@yahoo.com; Stefania Bulotta - bulotta@unicz.it;
Donatella Paolino - paolino@unicz.it; Franco Arturi - arturi@unicz.it; Domenicoantonio Rotiroti - rotiroti@unicz.it;
Sebastiano Filetti - sebastiano.filetti@uniroma1.it; Massimo Fresta - fresta@unicz.it; Diego Russo* - d.russo@unicz.it
* Corresponding author
Abstract
Background: Identification of effective systemic antineoplastic drugs against anaplastic thyroid
carcinomas has particularly important implications. In fact, the efficacy of the chemotherapeutic
agents presently used in these tumours, is strongly limited by their low therapeutic index.
Methods: In this study gemcitabine was entrapped within a pegylated liposomal delivery system to
improve the drug antitumoral activity, thus exploiting the possibility to reduce doses to be
administered in cancer therapy. The cytotoxic effects of free or liposome-entrapped gemcitabine
was evaluated against a human thyroid tumour cell line. ARO cells, derived from a thyroid
anaplastic carcinoma, were exposed to different concentrations of the drug. Liposomes
formulations were made up of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-
distearoyl-sn-glycero-3-phosphoethanolamine-MPEG (8:3:1 molar ratio). Cell viability was assessed
by both trypan bleu dye exclusion assay and fluorimetric analysis of cell DNA content.
Results: A cytotoxic effect of free gemcitabine was present only after 72 h incubation (ARO cell
mortality increased of approximately 4 fold over control at 1 µM, 7 fold at 100 µM). When
gemcitabine was encapsulated in liposomes, a significant effect was observed by using lower
concentrations of the drug (increased cell mortality of 2.4 fold vs. control at 0.3 µM) and earlier
exposure time (24 h).
Conclusion: These findings show that, in vitro against human thyroid cancer cells, the gemcitabine
incorporation within liposomes enhances the drug cytotoxic effect with respect to free
gemcitabine, thus suggesting a more effective drug uptake inside the cells. This may allow the use
of new formulations with lower dosages (side effect free) for the treatment of anaplastic human
thyroid tumours.
Published: 13 September 2004
BMC Cancer 2004, 4:63doi:10.1186/1471-2407-4-63
Received: 02 March 2004
Accepted: 13 September 2004
This article is available from: http://www.biomedcentral.com/1471-2407/4/63
© 2004 Celano et al; licensee BioMed Central Ltd.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
At present, the prognosis of anaplastic thyroid carcinomas
is very poor [1,2]. Since they are usually unable to concen-
trate radioiodine, the therapeutical approach is based on
combination of aggressive surgery, external beam radia-
tions and chemotherapy. Only rarely, however, this treat-
ment results effective especially for treating metastatic
disease [1,2]. A major limit of the chemotherapeutic
agents presently used in these tumours, including doxoru-
bicin, paclitaxel and various drug combinations [1,2], is
represented by their low therapeutic index. Thus, the iden-
tification of systemic antineoplastic drugs effective against
these carcinomas has particularly relevant implications.
Gemcitabine is a new fluorinated nucleoside analogue
provided with a potent anti-tumour activity, tested in
vitro and in vivo against a variety of solid malignancies [3-
6]. In addition, gemcitabine is a potent radiosensitizing
agent [7]. In a preclinical study, gemcitabine showed a
marked cytotoxic activity against poorly differentiated
human thyroid carcinoma cell lines [8]. In contrast, no
appreciable response was observed in four patients with
aggressive thyroid anaplastic carcinoma treated with com-
bination of gemcitabine and vinorelbine [9]. Since hema-
tological and other toxicities have been reported when
effective anti-tumour doses of gemcitabine were tested
[10], the aim of this study was to verify the possibility of
maintaining the drug cytotoxicity at lower doses, by using
a liposomal drug delivery system. Our data demonstrate
that incorporation of gemcitabine in liposomes enhances
the cytotoxic effect of the drug against a well-established
human thyroid cancer cell line, as compared to the free
drug.
Methods
Chemicals
Gemcitabine (2,21-difluorodeoxycytidine) hydrochloride
(HPLC purity >99%) was a kind gift of Eli-Lilly Italia
S.p.A. (Sesto Fiorentino, Firenze, Italy) and was used with-
out further purification. Cholesterol was obtained from
Sigma Chemicals Co. (St. Louis, USA). 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine monohydrate and N-(carb-
onyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-
sn-glycero-3-phosphoethanolamine sodium salt (MPEG-
2000-DSPE) were purchased from Genzyme products
(Suffolk, United Kingdom). Double-distilled pyrogen-free
water from Sifra S.p.A. (Verona, Italy) was used. Sterile
saline was a product of Frekenius Kabi Potenza S.r.l.
(Verona, Italy). All other materials and solvents were of
analytical grade (Carlo Erba, Milan, Italy).
Liposome preparation
Liposome formulations were made up of DPPC/Chol/
MPEG-2000-DSPE (8:3:1 molar ratio). To maximize the
entrapment efficiency of a hydrophilic molecule such as
gemcitabine, various liposome preparation procedures
[11,12] were applied together. Namely, liposome colloi-
dal suspensions were prepared by dissolving the lipid
mixture (40 mg) in chloroform-methanol (3:1 v/v).
Organic solvent was removed by a rotavapor thus allow-
ing the formation of a thin lipid film on the inner surface
of a pyrex glass vial. To remove any trace of organic sol-
vent, lipid films were stored overnight in a Büchi T-50
under high vacuum at 30°C in the dark. Lipid films were
hydrated with a solution (2 ml) of 250 mM ammonium
sulphate by vortexing at 45°C for 15 min. The resulting
colloidal suspension of multilamellar vesicles was sub-
mitted to ten cycles of freeze in liquid nitrogen and thaw
in warm (35°C) water in order to allow the homogenous
distribution of the ionic species. The entrapped ammo-
nium sulphate solution was removed by centrifugation at
14000 rpm (IEC, International Equipment Company,
mod MP4R, equipped with a mod. 851 rotor) for 1 h at a
temperature of 4°C. The pellet was re-suspended in 400 µl
of a 1 mM gemcitabine aqueous solution and stored at
room temperature for 3 h. Double-distilled pyrogen-free
water (1.6 ml) was added to the liposome suspension and
then a freeze-drying procedure was carried out (Edwards
freeze-dryer Modulayo). Then, the non-incorporated gem-
citabine was removed and the freeze-dried liposomes
were re-suspended just before the experiments with 2 ml
of the culture medium. Scalar dilutions were prepared in
the same medium to obtain the final concentrations used
in the experiments.
Liposome loading capacity
The loading capacity of liposomes was evaluated after
removing the free gemcitabine (centrifugation at 14000 ×
g for 1 h at 4°C) from the vesicular colloidal suspension
coming from the re-suspension of freeze-dried liposomes.
The amount of gemcitabine in the supernatant was spec-
trophotometrically determined at 268.8 nm (Shimadzu
UV-1601). UV calibration straight-line (y = 6.958 × 10-3 +
0.3971x, where y is the UV adsorbance and x is the drug
concentration) presented a r2 value of 0.9993. The
amount of gemcitabine entrapped in liposomes was
determined as a difference between the amount of drug
added during liposome preparation and the amount of
the entrapped drug present in the supernatant. The encap-
sulation efficiency was expressed as percentage of the total
amount of gemcitabine that became entrapped.
Cell cultures and cell viability
The ARO cells, from a human anaplastic thyroid carci-
noma, were grown as previously described [13]. All exper-
iments were performed in 12-well culture dishes, when
cells reached approximately 50% confluence. Cell viabil-
ity was evaluated by trypan bleu dye exclusion assay.
Briefly, after incubation with gemcitabine or liposomes
containing the drug at different doses, cells were

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trypsinized and the pellet re-suspended in a 0.4% trypan
bleu buffer and counted in the hemocytometric chamber.
Cell mortality was calculated as the percentage of stained
cells over the total and expressed as the ratio between
treated and untreated (control) cells. DNA cell content
was assayed by using a fluorimetric DNA assay kit (Bio-
rad laboratories, Segrate, Milano, Italy).
Results
We first analysed the effects of different concentrations of
gemcitabine on the viability of ARO thyroid cancer cells.
As shown in Figure 1 the cytotoxic effect was observed
only after 48 or 72 h incubation of the drug. After 72 h,
the cell mortality increased 4.6 and 7.9 fold over control
using 1 and 100 µM of gemcitabine. In accordance with
previous reports [8], no significant effect was visible after
24 h exposure (Fig. 1).
In order to improve the drug entry into the cells, gemcit-
abine entrapment in a liposome capsule was next per-
formed, using a combination of various liposome
preparation procedures. In particular, a pH gradient
method was used [14]. Namely, the presence of ammo-
nium sulphate in the internal compartments of liposomes
provide an acidic environment that elicit the protonation
of gemcitabine in order to drastically reduce the drug
back-diffusion (leakage) from liposomes (Figure 2). The
combination of various liposome preparation procedures
together with the application of a pH gradient provided
an encapsulation efficiency of ~90%.
The gemcitabine-loaded liposomes were then tested for
their cytotoxic activity against ARO cell, and the results
compared with those of gemcitabine alone. As shown in
Figure 3, a cytotoxic effect appeared after 24 h incubation,
with an initial increased cell mortality at 0.3 µM (2.4 fold
over control), and a maximum at 10 µM (death of almost
all the cells) (Figure 3). Similar results were obtained by
analysing the DNA content of ARO cells treated with free
or liposome-encapsulated gemcitabine (data not shown).
Discussion
Despite an intense application of aggressive multimodal
therapeutic regimens, no standardized protocol has
shown success in the treatment of anaplastic thyroid car-
cinoma [2]. A critical need for new approaches is therefore
vital for the systemic therapy of metastatic anaplastic thy-
roid carcinoma. At present one promising option is pro-
vided by the gene therapy. Studies are currently in
progress attempting to: a. reintroducing iodine uptake by
viral-driven Iodide/sodium symporter gene expression
[15,16]; b, inducing tumour cell death by expressing p53
or by suicide gene prodrug systems [17,18]. An alternative
approach is represented by increasing the effectiveness of
chemotherapy, using novel agents, combination therapy
or enhancing drug delivery inside the tumour cell.
The latter option has been taken advantages in the
progress related to liposome research. Traditional dosage
forms of anticancer drugs have the limitation of many
potential obstacles (barriers) before they reach their target
site, one of these is the large volume of distribution after
i.v. administration. This situation elicits a low therapeutic
index and a high toxicity in healthy tissues [19]. After
encapsulation into liposomes similar to those used in our
investigation the volume of distribution of a drug is
reduced, and its concentration in the tumour tissue is
increased [20]. In fact, liposomes can protect drugs from
metabolic inactivation, and, due to size limitation, they
are not able to transport encapsulated molecules across
healthy endothelium, thus avoiding anticancer drug accu-
mulation in a large extent in healthy tissues and hence a
decrease of side-effects [21]. On the other hand, the
increased permeability of the tumour endothelium allows
Cytotoxic effect of gemcitabine
Figure 1
Cytotoxic effect of gemcitabine. ARO cells were grown to
50% confluence and treated with increasing concentrations
of gemcitabine for different incubation time. Cell mortality
was evaluated by trypan bleu dye exclusion assay (see Meth-
ods). In control cells, mortality was always lower than 5%.
The values, expressed as the ratio over control, represent
the mean ± SEM of at least three different experiments in
triplicates.

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the extravasation of liposomes, with a consequent
accumulation of anticancer drugs in the site of action [22].
Furthermore, it should be considered that liposomes are
non-toxic being mostly constituted by naturally occurring
lipids.
In the last years, gemcitabine has emerged as a very potent
anti-tumour drug and it is currently used, alone or in com-
bination, in the treatment of patients with different malig-
nancies, including ovarian, pancreatic, non-small cell
lung and other cancers [3-6]. Despite its promising effec-
tiveness derived from a preclinical study [8], no apprecia-
ble response was observed in a clinical study including
four patients with aggressive thyroid anaplastic carcinoma
treated with combination of gemcitabine and vinorelbine
[9]. Although low, compared to other anti-neoplastic
drugs, hematological and other toxicities appeared when
effective anti-tumour doses of gemcitabine were tested.
In this study, we prepared a liposome formulation with a
high entrapment efficiency of gemcitabine and tested its
efficacy against a well established human poorly differen-
tiated thyroid carcinoma cell line. Our data demonstrate
that, in ARO cells, incorporation of gemcitabine in lipo-
somes enhances the cytotoxic effect of the drug as com-
pared to free gemcitabine, suggesting a more effective
uptake of the drug inside the cells. This activity was dem-
onstrated at concentrations even lower than serum levels
obtained in clinical trials. In our knowledge, no data are
available in the literature concerning the relationship
between gemcitabine uptake and its cytotoxicity, since
neither liposomes or other carrier system have been tested
against tumour cells. Our preliminary experiments
(unpublished observations) show that a similar effect
(improved uptake and cytotoxicity at lower doses) may be
obtained also in colon cancer cells. It is note worthing that
the various component used for the preparation of the
liposomal delivery device were already approved by regu-
latory offices to be used in the preparation of liposome-
based formulations, i.e. Doxil®.
Conclusion
The clinical predictive value of the in vitro cell line pre-
clinical cancer model is well established [23] and the ARO
cells are one of the cell model of anaplastic thyroid cancer
more exploited both for investigating the mechanism of
tumour progression and for testing new molecules with
antiproliferative effect. However, these promising obser-
vations need to be confirmed in studies using in vivo
xenograft cancer models, prior to propose the use of such
Schematic representation of the gemcitabine encapsulation process within liposomes by means of the presence of a pH gradient elicited by the co-encapsulation of a 250 mM ammo-nium sulphate solution
Figure 2
Schematic representation of the gemcitabine encapsulation
process within liposomes by means of the presence of a pH
gradient elicited by the co-encapsulation of a 250 mM ammo-
nium sulphate solution.
Cytotoxic effect of gemcitabine-loaded liposomes
Figure 3
Cytotoxic effect of gemcitabine-loaded liposomes. ARO cells
were grown to 50% confluence and treated with increasing
concentrations of either free gemcitabine or gemcitabine-
loaded liposomes for 24 h. Cell mortality was evaluated by
trypan bleu dye exclusion assay (see Methods). The values,
expressed as the ratio over control, represent the mean ±
SEM of at least three different experiments in triplicates.

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preparations at lower (presumably side effect free) doses
in a clinical trial of human thyroid tumours.
Competing interests (medicine)
None declared.
Acknowledgements
This work was supported by a MIUR Cofin 2003 grant to D. Russo, a MIUR
Cofin 2002 grant to M. Fresta and by a grant from Italian Ministry of Health
to S. Filetti.
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