orIGINAL PAPErS - Advances in Clinical and Experimental Medicine

original papers
Adv Clin Exp Med 2010, 19, 4, 481–487
ISSN 1230-025X
© Copyright by Wroclaw Medical University
Alicja Zajdel1, Adam Wilczok1, Małgorzata Latocha2, Zofia Dzierżewicz1
Effect of Polyunsaturated Fatty Acids on Doxorubicin
Cytotoxicity in Glioma Cells in vitro*
Wpływ wielonienasyconych kwasów tłuszczowych na cytotoksyczność
doksorubicyny w komórkach glejaków in vitro
1
2
Department of Biopharmacy, Medical University of Silesia, Sosnowiec, Poland
Department of Cell Biology, Medical University of Silesia, Sosnowiec, Poland
Abstract
Background. In normal and tumour cells, polyunsaturated fatty acids (PUFAs) act as intracellular second messengers, which play a role in signalling, proliferation and cell death. PUFAs have selective tumouricidal action and
may alter sensitivity of tumour cells to radiation and chemotherapy.
Objectives. Investigation of the ability of exogenous linoleic acid (LA, 18:2 n-6), α-linolenic acid (ALA, 18:3 n-3),
arachidonic acid (AA, 20:4 n-6), eicosapentaenoic acid (EPA, 20:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3)
to modulate sensitivity of human glioma cells to doxorubicin.
Material and Methods. The influence of PUFAs (0–100 µM) on the viability of human foreskin fibroblasts (HFF-1),
human glioblastoma (SNB-19), and human gliomas (8-MG-BA, 42-MG-BA) grown in the absence or presence of
doxorubicin was estimated. Viability of the cells was measured after 72 hours of exposure by the WST-1 tetrazolium salt assay.
Results. DHA (25 µM) stimulated growth of fibroblasts, while LA (50 and 100 µM) and AA (100 µM) reduced their
growth. Glioma cell growth was not stimulated by the tested fatty acids. EPA (100 µM) and DHA (100 µM) inhibited viability of 8-MG-BA and 42-MG-BA glioma cells. SNB-19 glioblastoma cells remained resistant to PUFAs.
PUFAs did not modify growth of the fibroblasts exposed to doxorubicin. PUFAs (100 µM), in particular DHA,
enhanced doxorubicin cytotoxicity in all glioma cell lines used. At lower concentrations, EPA (25 µM) and DHA
(25 µM) decreased doxorubicin toxicity in SNB-19 cells. Similar effect was observed for ALA (25 µM) and AA
(25 µM) in 8-MG-BA cells.
Conclusion. To increase doxorubicin antitumour activity in glioma cell lines high concentrations of PUFAs are
required. At low concentrations of PUFAs this activity can be diminished. These facts should be taken into consideration in therapy for patients with brain gliomas (Adv Clin Exp Med 2010, 19, 4, 481–487).
Key words: polyunsaturated fatty acids, doxorubicin, glioma cells.
Streszczenie
Wprowadzenie. Zarówno w komórkach prawidłowych, jak i nowotworowych wielonienasycone kwasy tłuszczowe
(w.n.k.t.) pełnią funkcję wewnątrzkomórkowych, drugorzędowych przekaźników sygnału w procesach proliferacji
oraz śmierci komórki. W.n.k.t. wykazują selektywne działanie przeciwnowotworowe i mogą wpływać na wrażliwość komórek nowotworowych na naświetlanie i chemioterapię.
Cel pracy. Zbadanie zdolności egzogennych kwasów tłuszczowych: linolowego (LA, 18:2 n-6), α-linolenowego
(ALA, 18:3 n-3), arachidonowego (AA, 20:4 n-6), eikozapentaenowego (EPA, 20:5 n-3) i dokozaheksaenowego
(DHA, 20:6 n-3) do modulowania wrażliwości ludzkich komórek glejaków na doksorubicynę.
Materiał i Metody. Do oznaczenia wpływu w.n.k.t. (0–100 µM) na przeżywalność ludzkich komórek fibroblastów
napletka (HFF-1), glejaka wielopostaciowego (SNB-19), ludzkich glejaków (8-MG-BA, 42-MG-BA) hodowanych
w nieobecności i obecności doksorubicyny zastosowano test WST-1. Przeżywalność komórek była mierzona po
72 godzinach ekspozycji.
Wyniki. DHA (25 µM) stymulował wzrost fibroblastów w przeciwieństwie do LA (50 i 100 µM) oraz AA (100 µM),
które hamowały ich wzrost. Badane kwasy tłuszczowe nie stymulowały wzrostu komórek glejaków. EPA (100 µM)
* This work was supported by SUM grant KNW-2-005/10.
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oraz DHA (100 µM) zmniejszały przeżywalność komórek glejaków linii 8-MG-BA i 42-MG-BA. Komórki glejaka
wielopostaciowego SNB-19 nie wykazały wrażliwości na w.n.k.t. W.n.k.t. nie modyfikowały wzrostu fibroblastów
eksponowanych na doksorubicynę. W.n.k.t. (100 µM), w szczególności DHA, zwiększały cytotoksyczność doksorubicyny wobec wszystkich testowanych linii glejaków. W mniejszych stężeniach EPA (25 µM) oraz DHA (25 µM)
zmniejszały cytotoksyczność doksorubicyny w linii komórek SNB-19. Podobne działanie zaobserwowano w przypadku ALA (25 µM) i AA (25 µM) w komórkach 8-MG-BA.
Wnioski. Zwiększenie aktywności przeciwnowotworowej doksorubicyny wobec komórek linii glejaków wymaga
zastosowania w.n.k.t. w dużych stężeniach. W małych stężeniach kwasy te mogą spowodować zmniejszenie przeciwnowotworowej aktywności doksorubicyny. Fakty te powinny być brane pod uwagę w leczeniu pacjentów z glejakami mózgu (Adv Clin Exp Med 2010, 19, 4, 481–487).
Słowa kluczowe: wielonienasycone kwasy tłuszczowe, doksorubicyna, komórki glejaków.
Essential fatty acids, such as linoleic acid (LA,
18:2 n-6) and α-linolenic acid (ALA, 18:3 n-3),
are widely distributed in human food but cannot
be synthesized by mammalian cells. Both LA and
ALA are precursors for the synthesis of longer
chained polyunsaturated fatty acids (PUFAs), crucial for normal cellular function. Thus, γ-linolenic
acid (GLA, 18:3 n-6), dihomo-GLA (DGLA,
20:3 n-6) and arachidonic acid (AA, 20:4 n-6) are
formed from LA, whereas eicosapentaenoic acid
(EPA, 20:5 n-3) and docosahexaenoic acid (DHA,
22:6 n-3) from ALA. PUFAs have been shown to
participate in numerous cellular functions affecting membrane fluidity, membrane enzyme activities and eicosanoid synthesis. Either in normal or
transformed cells, PUFAs act as intracellular second messengers, which participate in signalling,
proliferation and cell death [1, 2].
During the therapy of cancer it is desired that
agents preferentially kill tumour cells without exerting adverse effects on normal cells. In this context, it is interesting to note that PUFAs have selective tumouricidal action in vitro and in vivo [3],
show antiangiogenic action [4, 5] and may increase
sensitivity of tumour cells to radiation and chemotherapy [6, 7]. In addition, PUFAs were reported
to reverse tumour cell drug resistance [8], a major
problem in oncology practice. There is an inverse
relationship between concentrations of lipid peroxides and the rate of cell proliferation. Tumour cells
have low concentrations of lipid peroxides and are
resistant to lipid peroxidation compared to normal
cells [9]. The low rate of lipid peroxidation and levels of lipid peroxides in the tumour cells could be
attributed to their low content of PUFAs. The low
content of PUFAs in the tumour cells is due to the
loss or decreased activity of Δ6 and Δ5 desaturases
[10], enzymes that are essential for the formation
of more highly unsaturated, long-chain fatty acids.
It was shown that local PUFAs deficiencies in tumour cells may be rectified by the supplementation with exogenous PUFAs [11]. The decline in
structural and functional integrity of brain tissue, which is particularly rich in PUFAs (mainly
DHA), appears to correlate with loss in membrane
DHA concentrations. AA, also predominant in
brain tissue, is a major precursor for the synthesis
of eicosanoids, intracellular or extracellular signalling molecules [12]. PUFAs have been reported
to stimulate tumour regression in human glioma
and apoptosis in glioma cells in vitro and in vivo
[3, 5, 13–15].
In the present study we investigated the ability of exogenous LA, ALA, AA, EPA, and DHA
to modulate sensitivity of cultured human glioma
cells to doxorubicin, an anticancer drug commonly
applied in the treatment of malignant gliomas.
Material and Methods
Cell Culture
SNB-19 human glioblastoma cell line, 8-MGBA, and 42-MG-BA human glioma cell lines were
obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). HFF-1 foreskin fibroblasts were purchased from the American Type Culture Collection (ATCC). SNB-19 and
HFF-1 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10%
heat inactivated fetal bovine serum (FBS; PAA The
Cell Culture Company), 100 U/ml penicillin, and
100 µg/ml streptomycin (Sigma). 8-MG-BA and
42-MG-BA cells were grown routinely in Modified
Eagle’s Medium (MEM) supplemented with 10%
heat inactivated fetal bovine serum (FBS; PAA The
Cell Culture Company), 100 U/ml penicillin and
100 µg/ml streptomycin (Sigma). Cells were maintained at 37oC in a humidified atmosphere of 95%
air and 5% CO2.
Cell Exposure to Doxorubicin
and PUFAs
Doxorubicin, linoleic acid (LA, 18:2 n-6),
α-linolenic acid (ALA, 18:3 n-3), arachidonic acid
(AA, 20:4 n-6), eicosapentaenoic acid (EPA, 20:
5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3)
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Cytotoxicity of PUFAs and Doxorubicin in Glioma Cells
were purchased from Sigma. Stock solutions of
doxorubicin were prepared in physiological saline
solution (NaCl 0.9%). The fatty acids were dissolved in 99% ethanol and stored as stock solutions
(100 mM) under nitrogen at –20°C. To achieve the
required concentrations fatty acids and doxorubicin were prepared from stock solutions and diluted with the appropriate growth medium freshly
before each experiment.
Cells were seeded in standard 96-well plates
(5 × 103 cells/well in 100 µl). One day after seeding, the culture medium was removed and replaced by the same medium containing 0.1% ethanol (control), PUFAs (25 µM, 50 µM, 100 µM)
or PUFAs and doxorubicin at IC50 (0.12 µM for
HFF-1 cells and 0.38 µM for glioma cells). Control cells were cultured in the medium containing
the same concentration of ethanol (v/v; 0.1%) as
the experimental cultures. The ethanol solution in
such concentration had no noticeable influence on
the proliferation of the experimental cultures.
Cytotoxicity Assay
Viability of cells was measured after 72 hours
by the tetrazolium salt assay. WST-1 reagent (10 µl,
Roche Diagnostics GmbH) was added to each well.
The plates were gently agitated for 1 min, and the
cells were incubated with the WST-1 reagent for
1 hour in 5% CO2 at 37oC. Metabolically active cells
reduced the dye to purple formazan. Following
incubation, the absorbance at 440 nm was determined using a microplate reader (UVM340, Biogenet). Cell viability was expressed as a percentage
of absorbance measured in the treated wells relative to that in the untreated control wells.
Statistical Analysis
The data obtained from 3 independent series
of experiments were expressed as mean values ±
standard deviations. Statistical significance analysis
based on analysis of variance (ANOVA) followed
by Tukey’s HSD test. The P-value of less than 0.03
was considered significant. Statistical analysis was
performed using Statistica 8 PL software for Windows (StatSoft, Poland).
Results
Effect of PUFAs on Cell Viability
Table 1 shows results of viability tests performed after 72 hours of exposure of human foreskin fibroblasts, which were used for comparison
with tumour cells, and three glioma cell lines to li-
noleic, α-linolenic, arachidonic, eicosapentaenoic,
and docosahexaenoic acid. Results were expressed
as a percentage of viability of PUFA-treated cells
vs. control cells. Fatty acids added to the cultures
in the concentration range 25–100 µM generated
diverse effect in the tested cells. Among the fatty
acids used, only DHA (25 µM) stimulated growth
of fibroblasts (110.7% of control, P = 0.00108),
while LA (50 and 100 µM) and AA (100 µM) reduced their growth by about 10%. In no case glioma cell growth was stimulated by the fatty acids.
EPA (100 µM) and DHA (100 µM) inhibited viability of 8-MG-BA and 42-MG-BA glioma cells.
The effect of DHA was more pronounced. SNB-19
glioblastoma cells remained resistant to PUFAs.
Effect of PUFAs on Doxorubicin
Cytotoxicity
The results of mixed treatment of fibroblasts
and glioma cells with PUFAs in various concentrations and doxorubicin (IC50) added to the cultures simultaneously, are summarised in Table 2.
To allow comparison with the effects of PUFAs
(Table 1), the cell viability of PUFA+ DX-treated
cells was also calculated as a percentage of control. PUFAs did not modify growth of fibroblasts
exposed to doxorubicin. But when LA (100 µM),
ALA (100 µM), AA (100 µM) or DHA (100 µM)
were added to 8-MG‑BA and 42-MG-BA cultures,
statistically significant increase of doxorubicin effect was observed. At lower concentrations of these
acids (50 µM) this effect was not observed and, in
the lowest concentration, ALA (25 µM) and AA
(25 µM) reduced cytostatic activity of doxorubicin
in 8-MG- BA cells. In glioblastoma SNB-19, the addition of ALA, EPA or DHA (100 µM) enhanced
doxorubicin cytotoxicity but EPA (25 µM) and
DHA (25 µM) diminished this effect. Among the
all fatty acids tested DHA (100 µM) increased antitumor activity of doxorubicin in the highest extent.
Discussion
PUFAs are important components of membrane phospholipids. Changes in PUFAs have been
shown to modulate membrane structure, fluidity,
and function. Fatty acids induce modifications in
the cell membrane structure and affect various cellular processes. Several reports showed an increase
of the PUFAs content of tumour cells by altering
the fatty acid composition of the growth medium
in vitro [6, 14, 16] or by feeding PUFAs to laboratory animals implanted with tumours [17]. It was
proved that it might be possible to influence the
lipid composition of cells by controlling the type
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A. Zajdel et al.
Table 1. Cytotoxicity of linoleic acid (LA), α-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA),
and docosahexaenoic acid (DHA) in human foreskin fibroblasts (HFF-1), human glioblastoma (SNB-19) and human gliomas (8-MG-BA, 42-MG-BA) expressed as a percentage of viability of PUFA-treated cells vs. non-treated cells
Tabela 1. Cytotoksyczność kwasu linolowego (LA), α-linolenowego (ALA), arachidonowego (AA), eikozapentaenowego
(EPA) i dokozaheksaenowego (DHA) w komórkach fibroblastów napletka ludzkiego (HFF-1), glejaka wielopostaciowego
(SNB-19), ludzkich glejaków (8-MG-BA, 42-MG-BA) wyrażona jako procent przeżywalności komórek eksponowanych na
w.n.k.t. względem komórek nieeksponowanych
PUFA
(W.n.k.t.)
LA
ALA
AA
EPA
DHA
PUFA concentration
(Stężenie w.n.k.t.)
– µM
Cell viability
(Przeżywalność komórek) – %
HFF-1
SNB-19
8-MG-BA
42-MG-BA
25
96.74 ± 3.33
100.1 ± 4.14
89.27 ± 3.89
98.94 ± 2.34
50
89.93 ± 2.12a
105.8 ± 0.77
88.67 ± 4.16
92.70 ± 1.77
100
83.93 ± 0.21b
102.6 ± 0.52
101.52 ± 39.74
90.14 ± 1.65
25
101.4 ± 1.57
105.3 ± 2.25
95.09 ± 5.81
100.85 ± 6.50
50
95.83 ± 0.73
100.1 ± 4.09
95.33 ± 1.35
99.65 ± 5.60
100
95.11 ± 1.96
100.7 ± 5.12
90.00 ± 1.90
95.05 ± 2.94
25
101.8 ± 3.02
106.5 ± 4.85
103.88 ± 2.53
106.38 ± 1.76
50
98.30 ± 1.27
107.9 ± 0.15
105.33 ± 2.27
102.91 ± 4.12
100
91.70 ± 1.52a
108.2 ± 1.02
110.30 ± 1.94
106.49 ± 2.02
25
106.3 ± 1.91
106.5 ± 1.13
100.30 ± 4.68
106.81 ± 5.31
50
98.52 ± 2.67
104.8 ± 3.82
116.18 ± 16.31
90.92 ± 5.65
100
92.30 ± 2.30
102.8 ± 1.73
79.39 ± 4.66
56.52 ± 4.42b,c
25
110.7 ± 6.85a
101.4 ± 2.98
102.91 ± 6.62
94.36 ±4.85
50
104.1 ± 1.94
103.3 ± 0.79
89.48 ± 4.55
90.80 ± 23.80
100
92.67 ± 3.46
99.68 ± 4.73
22.73 ± 1.40b,c
22.55 ± 2.88b,c
a,c
statistically significant difference in comparison with control; P < 0.03.
statistically significant difference in comparison with control; P < 0.0003.
c
statistically significant difference in comparison with both EPA treated cells and DHA treated cells; P < 0.0003.
a
b
istotna statystycznie różnica w porównaniu z grupą kontrolną; P < 0,03.
istotna statystycznie różnica w porównaniu z grupą kontrolną; P < 0,0003.
c
istotna statystycznie różnica w porównaniu zarówno do komórek traktowanych EPA, jak i traktowanych DHA; P < 0,0003.
a
b
of lipids added to the culture medium. Numerous
studies that have been conducted on various cancer
cells have attempted to determine the antitumoural effects of n-3 and n-6 series fatty acids [11, 18].
Only a few of these studies have been conducted
on human glioma cell lines [6, 14, 15]. PUFAs are
capable of stimulating apoptosis of human glioma
cells [14, 19, 20]. However, marked differences in
the effective doses of PUFAs in various glioma
models have been reported. Moreover, low concentrations of PUFAs and their metabolites may
stimulate tumour proliferation [14, 21].
In this study the comparison of the effects of
LA, ALA, AA, EPA, and DHA on the viability of
fibroblast HFF-1 and glioma SNB-19, 8-MG-BA,
and 42-MG-BA cell lines was made. LA (50 µM,
100 µM) and AA (100 µM) decreased viability of the fibroblasts, DHA (25 µM) stimulated
their growth and the other fatty acids tested did
not show significant effect within the concentration range applied. When the PUFAs were added
to the glioma cell cultures, only EPA (100 µM)
and DHA (100 µM) decreased the growth of
8‑MG‑BA and 42-MG-BA cells, showing selective
tumouricidal action. The observed effect of DHA
was more severe than that of EPA. These results
are similar to those reported for C6 glioma cells,
that GLA, AA, EPA and DHA, selectively kill tumour cells with little or no effect on the survival
of normal cells [14, 19, 22]. In own study, none
of the tested PUFAs modified viability of SNB-19
glioblastoma cells.
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Cytotoxicity of PUFAs and Doxorubicin in Glioma Cells
Table 2. Effect of linoleic acid (LA), α-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and
docosahexaenoic acid (DHA) on the viability of fibroblasts (HFF-1), human glioblastoma (SNB-19) and human gliomas
(8-MG-BA, 42-MG-BA) grown in the presence of doxorubicin (DX). Cell viability changes were expressed as a percentage
of viability of DX treated cells and PUFA + DX treated cells vs. non-treated cells
Tabela 2. Wpływ kwasu linolowego (LA), α-linolenowego (ALA), arachidonowego (AA), eikozapentaenowego (EPA)
i dokozaheksaenowego (DHA) na przeżywalność komórek fibroblastów napletka ludzkiego (HFF-1), glejaka wielopostaciowego (SNB-19) i ludzkich glejaków (8-MG-BA, 42-MG-BA) hodowanych w obecności doksorubicyny (DX). Zmiany
w przeżywalności komórek traktowanych DX oraz w.n.k.t. i DX wyrażono w procentach względem komórek nieeksponowanych na te związki
PUFA
(W.n.k.t.)
PUFA
Concentration
(Stężenie w.n.k.t.)
– µM
Cell viability
(Przeżywalność komórek) – %
HFF-1
SNB-19
8-MG-BA
42-MG-BA
DX*
   0
55.61 ± 3.76
57.48 ± 1.81
44.73 ± 2.76
56.41 ± 5.56
LA + DX
  25
50.58 ± 2.80
54.74 ± 0.79
43.55 ± 1.19
52.37 ± 0.55
  50
43.90 ± 4.34
55.54 ± 0.04
41.53 ± 0.89
48.33 ± 0.95
100
45.24 ± 5.27
56.64 ± 0.14
39.30 ± 1.12a
46.13 ± 0.57a
  25
47.09 ± 6.59
57.69 ± 1.49
53.87 ± 2.37a
59.81 ± 0.58
  50
53.41 ± 2.14
55.07 ± 0.50
43.28 ± 3.18
58.75 ± 0.87
100
50.42 ± 1.77
49.31 ± 0.91a
38.79 ± 0.56a
58.27 ± 1.35
  25
50.42 ± 2.47
60.93 ± 1.00
49.35 ± 1.90
55.38 ± 0.19
  50
52.65 ± 2.72
58.90 ± 2.29
44.35 ± 1.42
51.92 ± 0.49
100
22.43 ± 0.52
54.83 ± 1.54
42.26 ± 0.48
51.60 ± 0.65a
  25
46.77 ± 6.65
62.24 ± 2.29a
48.23 ± 1.05
60.38 ± 1.41
  50
54.23 ± 5.13
55.88 ± 0.47
47.31 ± 1.01
57.18 ± 0.55
100
57.88 ± 2.47
22.83 ± 3.33b,c
47.80 ± 0.68
55.38 ± 1.15
  25
48.89 ± 4.29
62.31 ± 1.66a
48.06 ± 1.61
58.27 ± 2.20
  50
45.98 ± 2.40
58.31 ± 0.38
46.29 ± 0.97
54.94 ± 1.48
100
52.33 ± 6.00
10.55 ± 0.17b,c
15.91 ± 0.79b,c
18.53 ± 1.07b,c
ALA+ DX
AA+ DX
EPA+ DX
DHA+ DX
a
DX* – concentrations were equal to IC50 for each cell line respectively (see Material and Methods).
a
statistically significant difference in comparison with cells treated with DX; P < 0.03.
b
statistically significant difference in comparison with cells treated with DX; P < 0.0003.
c
statistically significant difference in comparison with both EPA + DX treated cells and DHA + DX treated cells; P < 0.0003.
DX* – stężenia były równe wartościom IC50 dla każdej linii komórek (zob. Materiał i metody).
a
istotna statystycznie różnica w porównaniu z komórkami eksponowanymi na DX; P < 0,03.
b
istotna statystycznie różnica w porównaniu z komórkami eksponowanymi na DX; P < 0,0003
c
istotna statystycznie różnica w porównaniu zarówno do komórek traktowanych EPA + DX, jak i DHA + DX; P < 0,0003.
The effects of dietary PUFAs on the prevention and therapy of cancer are still far from clear.
PUFAs are known to induce reactive oxygen species generation and cause lipid peroxidation in tumour cells and lead to altered mitochondrial metabolism, cytochrome c release, caspase activation,
and apoptosis [22]. Both PUFAs and their metabolic products can alter the expression of several
proteins. PUFAs suppress the expression of oncogene ras, inhibit that of Bcl-2 and enhance the activity of p53 [23, 24]. In addition, PUFAs boost the
formation of lipid peroxides due to augmented free
radical generation in tumour cells [16, 19, 25].
One of the major problems of glioma progression in vivo is intense angiogenesis, which has been
related not only to tumour nutrition but also to tumour cell migration along the basement membrane
of the growing blood vessels [26]. In gliomas, the
best characterized proangiogenic factor is vascular
endothelial growth factor (VEGF), whose overexpression is correlated with increasingly malignant
phenotypes [4]. GLA, AA, EPA, and DHA may
486
A. Zajdel et al.
have antiangiogenic action [4, 22]. It was also demonstrated that inhibitors of AA metabolism have
suppressive action on angiogenesis in vivo [27].
PUFAs may interact with anticancer agents
during chemotherapy. Exogenous PUFAs are
readily incorporated into cancer cell membranes.
Biochemical modifications of membrane fatty acids may lead to alteration in drug transport and,
hence, may modulate cell sensitivity to anticancer
drugs. PUFAs incorporated to the tumour cells
may also serve as substrates in reactions, which
generate high amounts of free radicals during anticancer progression and therapy [9, 18, 25].
The authors examined the influence of various PUFAs on doxorubicin induced cell toxicity.
Among the methods available to assess anticancer drug activity, they chose to measure cell mitochondrial activity rather than cell proliferation.
The authors found that in 8-MG-BA cells low concentration of ALA reduced, while high concentration increased anticancer activity of doxorubicin.
Similar effect was observed for EPA and DHA in
SNB-19 cell line. DHA (100 µM) was the most ef-
fective and sensitised all the glioma cell lines tested
to doxorubicin and did not affect fibroblasts. Own
findings agree with the reports that incubation
of human glioblastoma cells A-172 and U-87MG
with non-toxic doses of DHA enhanced cytotoxicity of doxorubicin. DHA enhanced sensitivity
to doxorubicin in the glioblastoma cells was more
pronounced in cells resistant to DHA [7]. In the
present study the authors showed that the effects
of PUFAs on the fibroblasts and gliomas can be
highly diverse. Generally, PUFAs did not influence or slightly inhibited growth of fibroblasts. In
the tumour cells treated with doxorubicin PUFAs,
mostly DHA, enhanced cytotoxicity of this anticancer drug. Very high concentrations of PUFAs
are required to increase doxorubicin antitumour
activity in glioma cells. At low concentrations of
PUFAs this activity can be noticeably decreased.
The alterations of doxorubicin cytotoxicity induced by PUFAs at concentrations, which can result from the dietary intake or supplementation,
should be taken into consideration in therapy of
tumour patients.
References
[1] Eynard AR: Potential of essential fatty acids as natural therapeutic products for human tumors. Nutrition 2003,
19, 386–387.
[2] Benatti P, Peluso G, Nicolai R, Calvani M: Polyunsaturated fatty acids: biochemical, nutritional and epigenetic
properties. J Am Coll Nutr 2004, 23, 281–302.
[3] Das UN: Gamma-linolenic acid therapy of human glioma – a review of in vitro, in vivo, and clinical studies. Med
Sci Monit 2007, 13, 119–131.
[4] Miyake JA, Benadiba M, Colquhoun A: Gamma-linolenic acid inhibits both tumour cell cycle progression and
angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb,
p53 and p27 protein expression. Lipids Health Dis 2009, 8, 8.
[5] Das UN: Tumoricidal and anti-angiogenic actions of gamma-linolenic acid and its derivatives. Curr Pharm
Biotechnol 2006, 7, 457–466.
[6] Vartak S, Robbins ME, Spector AA: Polyunsaturated fatty acids increase the sensitivity of 36B10 rat astrocytoma
cells to radiation-induced cell kill. Lipids 1997, 32, 283–292.
[7] Rudra PK, Krokan HE: Cell specific enhancement of doxorubicin toxicity in human tumor cells by docosahexaenoic acid. Anticancer Res 2001, 21, 29–38.
[8] Das UN, Madhavi N, Sravan Kumar G, Padma M, Sangeetha P: Can tumor cell drug resistance be reversed by
essential fatty acids and their metabolites? Prostaglandins Leukot Essent Fatty Acids 1998, 58, 39–54.
[9] Mahéo K, Vibet S, Steghens JP, Dartigeas C, Lehman M, Bougnoux P, Goré J: Differential sensitization of cancer
cells to doxorubicin by DHA: A role for lipoperoxidation. Free Radic Biol Med 2005, 39, 742–751.
[10] Hansen-Petrik MB, McEntee MF, Johnson BT, Obukowicz G, Masferrer J, Zweifel B, Chiu CH, Whelan J:
Selective inhibition of Δ-6 desaturase impedes intestinal tumorigenesis. Cancer Lett 2002, 175, 157–163.
[11] Berquin IM, Edwards IJ, Chen YQ: Multi-targeted therapy of cancer by omega-3 fatty acids. Cancer Lett 2008,
269, 363–377.
[12] Youdim KA, Martin A, Joseph JA: Essential fatty acids and the brain: possible health implications. Int J Dev
Neurosci 2000, 18, 383–399.
[13] Leaver HA, Wharton SB, Bell HS, Leaver-Yap IM, Whittle IR: Highly unsaturated fatty acid induced tumour
regression in glioma pharmacodynamics and bioavailability of gamma linolenic acid in an implantation glioma
model: effects on tumour biomass, apoptosis and neuronal tissue histology. Prostaglandins Leukot Essent Fatty
Acids 2002, 67, 283–292.
[14] Leaver HA, Bell HS, Rizzo MT, Ironside JW, Gregor A, Wharton SB, Whittle IR: Antitumour and pro-apoptotic
actions of highly unsaturated fatty acids in glioma. Prostaglandins Leukot Essent Fatty Acids 2002, 66, 19–29.
[15] Leaver HA, Williams JR, Smith C, Whittle IR: Intracellular oxidation by human glioma cell populations: effect
of arachidonic acid. Prostaglandins Leukot Essent Fatty Acids 2004, 70, 449–53.
Cytotoxicity of PUFAs and Doxorubicin in Glioma Cells
487
[16] Leonardi F, Attorri L, Benedetto RD, Biase AD, Sanchez M, Tregno FP, Nardini M, Salvati S: Docosahexaenoic
acid supplementation induces dose and time dependent oxidative changes in C6 glioma cells. Free Radic Res 2007,
41, 748–756.
[17] Nasrollahzadeh J, Siassi F, Doosti M, Eshraghian MR, Shokri F, Modarressi MH, Mohammadi-Asl J, Abdi K,
Nikmanesh A, Karimian SM: The influence of feeding linoleic, gamma-linolenic and docosahexaenoic acid rich
oils on rat brain tumor fatty acids composition and fatty acid binding protein 7 mRNA expression. Lipids Health
Dis 2008, 7, 45.
[18] Pardini RS: Nutritional intervention with omega-3 fatty acids enhances tumor response to anti-neoplastic agents.
Chem Biol Interact 2006, 162, 89–105.
[19] Leonardi F, Attorri L, Di Benedetto R, Di Biase A, Sanchez M, Nardini M, Salvati S: Effect of arachidonic,
eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells. Free Radic Res 2005, 8,
865–874.
[20] Williams JR, Leaver HA, Ironside JW, Miller EP, Whittle IR, Gregor A: Apoptosis in human primary brain
tumours: actions of arachidonic acid. Prostaglandins Leukot Essent Fatty Acids 1998, 58, 193–200.
[21] Kokoglu E, Tuter Y, Yazici Z, Sandikci KS, Sonmez H, Ulakoglu EZ, Ozyurt E: Profiles of the fatty acids in the
plasma membrane of human brain tumors. Cancer Biochem Biophys 1998, 16, 301–312.
[22] Spencer L, Mann C, Metcalfe M, Webb M, Pollard C, Spencer D, Berry D, Steward W, Dennison A: The effect
of omega-3 FAs on tumour angiogenesis and their therapeutic potential. Eur J Cancer 2009, 45, 2077–2086.
[23] Ramos KL, Colquhoun A: Protective role of glucose-6-phosphate dehydrogenase activity in the metabolic response
of C6 rat glioma cells to polyunsaturated fatty acid exposure. Glia 2003, 43, 149–166.
[24] Chung FL, Pan J, Choudhury S, Roy R, Hu W, Tang MS: Formation of trans-4-hydroxy-2-nonenal- and other
enal-derived cyclic DNA adducts from omega-3 and omega-6 polyunsaturated fatty acids and their roles in DNA
repair and human p53 gene mutation. Mutat Res 2003, 531, 25–36.
[25] Siddiqui RA, Harvey K, Stillwell W: Anticancer properties of oxidation products of docosahexaenoic acid. Chem
Phys Lipids 2008, 153, 47–56.
[26] Farin A, Suzuki SO, Weiker M, Goldman JE, Bruce JN, Canoll P: Transplanted glioma cells migrate and proliferate on host brain vasculature: a dynamic analysis. Glia 2006, 53, 799–808.
[27] Ito K, Abe T, Tomita M, Morimoto A, Kohno K, Mori T, Ono M, Sugenoya A, Nishihira T, Kuwano M: Antiangiogenic activity of arachidonic acid metabolism inhibitors in angiogensis model systems involving human
microvascular endothelial cells and neovascularization in mice. Int J Cancer 1993, 55, 660–666.
Address for correspondence:
Alicja Zajdel
Department of Biopharmacy
Medical University of Silesia
Narcyzów 1
41-200 Sosnowiec
Poland
E-mail: [email protected]
Tel.: +48 32 364 10 63
Conflict of interest: None declared
Received: 09.03.2010
Revised: 21.07.2010
Accepted: 09.08.2010