Synthesis of doxorubicin α-linolenic acid conjugate and evaluation of its antitumor activity

Article abstract of DOI:10.1021/mp4004139

Doxorubicin (DOX) is a broad-spectrum antitumor drug used in the clinic. However, it can cause serious heart toxicity. To increase the therapeutic index of DOX and to attenuate its toxicity toward normal tissues, we conjugated DOX with either α-linolenic acid (LNA) or palmitic acid (PA) by a hydrazone or an amide bond to produce DOX-hyd-LNA, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA. The cytotoxicity of DOX-hyd-LNA on HepG2, MCF-7, and MDA-231 cells was higher compared to that of DOX, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA. The cytotoxicity of DOX-hyd-LNA on HUVECs was lower than that of DOX. DOX-hyd-LNA released significantly more DOX in pH 5.0 medium than it did in pH 7.4 medium. DOX-hyd-LNA induced more apoptosis in MCF-7 and HepG2 cells than DOX or DOX-ami-LNA. Significantly more DOX was released from DOX-hyd-LNA in both MCF-7 and HepG2 cells compared with DOX-ami-LNA. Compared to free DOX, a biodistribution study showed that DOX-hyd-LNA greatly increased the content of DOX in tumor tissue and decreased the content of DOX in heart tissue after it was intravenously administered. DOX-hyd-LNA improved the survival rate, prolonged the life span, and slowed the growth of the tumor in tumor-bearing nude mice. These results indicate that DOX-hyd-LNA improved the therapeutic index of DOX. Therefore, DOX-hyd-LNA is a potential compound for use as a cancer-targeting therapy.

Full text of DOI:10.1021/mp4004139

Article
pubs.acs.org/molecularpharmaceutics
Synthesis of Doxorubicin α‑Linolenic Acid Conjugate and Evaluation
of Its Antitumor Activity
Chun-hui Liang,†,§,∥ Wei-liang Ye,†,∥ Chun-lai Zhu,† Ren Na,† Ying Cheng,† Han Cui,† Dao-zhou Liu,†
Zhi-fu Yang,*,‡ and Si-yuan Zhou*,†
†Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China
‡Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China
§Department of Pharmacy, Xi’an Children’s Hospital, Xi’an 710002, China
S
* Supporting Information
ABSTRACT: Doxorubicin (DOX) is a broad-spectrum anti-
tumor drug used in the clinic. However, it can cause serious
heart toxicity. To increase the therapeutic index of DOX and to
attenuate its toxicity toward normal tissues, we conjugated DOX
with either α-linolenic acid (LNA) or palmitic acid (PA) by a
hydrazone or an amide bond to produce DOX-hyd-LNA, DOX-
ami-LNA, DOX-hyd-PA, and DOX-ami-PA. The cytotoxicity of
DOX-hyd-LNA on HepG2, MCF-7, and MDA-231 cells was
higher compared to that of DOX, DOX-ami-LNA, DOX-hyd-
PA, and DOX-ami-PA. The cytotoxicity of DOX-hyd-LNA on
HUVECs was lower than that of DOX. DOX-hyd-LNA released
significantly more DOX in pH 5.0 medium than it did in pH 7.4
medium. DOX-hyd-LNA induced more apoptosis in MCF-7 and
HepG2 cells than DOX or DOX-ami-LNA. Significantly more
DOX was released from DOX-hyd-LNA in both MCF-7 and
HepG2 cells compared with DOX-ami-LNA. Compared to free
DOX, a biodistribution study showed that DOX-hyd-LNA
greatly increased the content of DOX in tumor tissue and
decreased the content of DOX in heart tissue after it was
intravenously administered. DOX-hyd-LNA improved the
survival rate, prolonged the life span, and slowed the growth
of the tumor in tumor-bearing nude mice. These results indicate
that DOX-hyd-LNA improved the therapeutic index of DOX.
Therefore, DOX-hyd-LNA is a potential compound for use as a cancer-targeting therapy.
KEYWORDS: doxorubicin, α-linolenic acid, palmitic acid, pH sensitive, tumor targeting
receptor has been used as a target to mediate the uptake of
antitumor drugs by tumor cells.13−15
1. INTRODUCTION
Doxorubicin (DOX) is an important antitumor drug that is
used in the clinic, but it can cause serious adverse effects, such
as cardiomyopathy and congestive heart failure.1−4 Many
methods have been explored to increase the therapeutic index
of DOX, including its use with nanoparticles, liposomes, and
dendrimers.5−10 Additionally, a prodrug strategy has also been
extensively investigated to selectively improve the interaction
between the drug and the cells that it targets.11,12
To design a tumor-targeting drug, it is crucial to understand
the tumor cell microenvironment. Tumor cells are different
from normal cells, as they grow quickly and usually require a
large amount of various nutrients. Therefore, tumor cells highly
express receptors, such as the folate receptor and transferrin
receptor, to facilitate the uptake of nutrients. The folate
Polyunsaturated fatty acids (PUFAs) are essential fatty acids
for human health. Human beings can obtain PUFAs only from
the diet. There are 18−22 carbons and 2−6 unsaturated
carbon−carbon double bonds in a PUFA molecule. PUFAs
have shown significant cytotoxicity toward CFPAC, PANC-1,
and HL-60 leukemia cells, and their in vivo antitumor activities
have been studied with animals and humans.16−21 In addition,
compared with normal cells, PUFAs are more avidly taken up
by tumor cells.22−24 Thus, PUFAs have been used as tumor-
Received: July 17, 2013
Revised: March 21, 2014
Accepted: March 31, 2014
Published: March 31, 2014
© 2014 American Chemical Society
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specific ligands to guide antitumor drugs to recognize tumor
cells selectively.25,26 Taxoprexin was synthesized by connecting
docosahexaenoic acid (DHA, a PUFA) with paclitaxel through
an ester bond. Compared with paclitaxel, taxoprexin greatly
enhanced the therapeutic index and decreased the systemic
toxicity of paclitaxel.27 Taxoprexin has entered phase III clinical
trials.27
The promising antitumor activity of taxoprexin has prompted
more extensive investigation of different PUFA−taxoid
conjugates. Ojima et al. connected second-generation taxoids,
SB-T-1213, with DHA, LNA, and linoleic acid (LA), and their
antitumor activity was evaluated in a drug-resistant (P-
glycoprotein positive) DLD-1 tumor xenograft model. LNA-
SB-T-1213 showed higher antitumor activity than DHA-SB-T-
1213 when given at the same dose. Thus, the antitumor activity
of DHA-SB-T-1213 was greater than LA-SB-T-1213. Paclitaxel,
however, was ineffective. These results implied that LNA could
be utilized for PUFA−antitumor drug conjugates in addition to
DHA.25 Accordingly, Wang et al. conjugated DOX with DHA
through a hydrazone bond, and an in vivo experiment indicated
that compared with free DOX the therapeutic effect of DHA-
DOX was obviously increased in tumor-bearing mice.28
Thereafter, a series of DOX derivatives were synthesized by
connecting doxorubicin-14-hemisuccinate with different sub-
stituent groups, and their cytotoxicity was determined by MTT
assays. The results indicated that the N-tetradecyl amide
derivative showed the greatest cytotoxicity toward tumor cells
of all of the derivatives, and its antitumor activity in vitro was
almost the same as free DOX.29 Furthermore, by using
unsaturated and saturated fatty acid, a series of N-acyl
hydrazone derivatives of DOX (including DOX-hyd-LNA)
were synthesized, and their cytotoxicity was evaluated by MTT
assay. The results indicated that N-acyl hydrazone derivatives
had some merits with regard to their cytotoxicity and ability to
avoid multidrug resistance.30 Although fatty acid derivatives of
DOX have been studied, a deeper and more systematic study
on DOX−fatty acyl conjugates is necessary.
Figure 1. Reaction route for the synthesis of DOX-hyd-LNA: (1)
H2NNHBoc, EDCI, DMF; (2) TFA, CH2Cl2; (3) DOX, TFA,
CH2Cl2.
2.2.1.1. Synthesis of N-Boc-LNA Hydrazine. To synthesize
N-Boc-LNA hydrazine, 278 mg of LNA (1 mmol), 9 mL of
dimethylformamide (DMF), 158.4 mg of H2NNHBoc (1.2
mmol), and 573 mg of EDCI (3 mmol) were added to a flask,
and the mixture was stirred at 25 °C for 12 h. Water (10 mL)
was added into the reaction mixture, which was then extracted
with ethyl acetate. The organic phase was dried with Na2SO4
and evaporated using a vacuum rotary evaporator. N-Boc-LNA
hydrazine was purified via a silica gel column using ethyl
acetate/cyclohexane (v/v 2:1, Rf = 0.46) as the eluant. The
compound was a pale yellow oily liquid. The yield was 372.2
In our previous studies, we conjugated DOX with α-linolenic
acid through an amide bond to improve the uptake of DOX in
cancer cells.31 To study the potential use of LNA in DOX
delivery systems further, we conjugated DOX with LNA or
palmitic acid (PA, a saturated fatty acid) by either a hydrazone
or an amide bond, and their drug-release characteristics and
antitumor activity were investigated in vitro and in vivo.
1
mg (0.95 mmol, 85.3%). The mass spectrum and H NMR
spectrum of N-Boc-LNA hydrazine are shown in Supporting
Information Figure 1. The molecular ion peak ([M − H]−) of
1
N-Boc-LNA hydrazine was 391. The H NMR spectrum of N-
Boc-LNA hydrazine exhibited typical signals at chemical shifts
of δ 5.28−5.40 (6H, m), 2.81 (4H, s), 2.02−2.10 (4H, m),
1.64−1.66 (2H, m), 1.47 (9H, s, Boc), 1.31−1.39 (10H, m),
0.98 (3H, t, CH3, J = 7.4 Hz) ppm.
2. MATERIALS AND METHODS
2.1. Materials. Doxorubicin·HCl (DOX·HCl) was pur-
chased from Hisun Pharmaceutical Co. (Zhejiang province,
China). α-Linolenic acid (LNA), palmitic acid (PA), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), 1-ethyl-(3-(dimethylamino)propyl)-carbodiimide
(EDCI), and N-hydroxysuccinimide (NHS) were purchased
from Sigma-Aldrich (St. Louis, MO). Athymic nude mice
(female, body weight 20−23 g) were purchased from the
Experimental Animal Center of the Fourth Military Medical
University. Mice were fed in a pathogen-free environment with
a 12 h light/dark cycle and were freely supplied with water and
food throughout the experiments. All experiments using mice
were carried out according to protocols that were approved by
the Animal Care and Use Committee of the Fourth Military
Medical University (authorized date: 17/03/2012, no. 12027).
2.2. Methods. 2.2.1. Synthesis of DOX-hyd-LNA. The
synthetic route of DOX-hyd-LNA is shown in Figure 1.
2.2.1.2. Synthesis of LNA Hydrazine. To synthesize of LNA
hydrazine, 274.4 mg of N-Boc-LNA hydrazine (0.7 mmol) was
dissolved in 18 mL of dichloromethane and 6 mL of
trifluoroacetic acid (TFA). The mixture was stirred at 25 °C
for 15 min, the pH of the reaction mixture was then adjusted to
7 using 10% NaHCO3, and 80 mL of dichloromethane was
added. The organic phase was isolated and washed with 80 mL
of water two times. The organic phase was dried with Na2SO4
and evaporated using a vacuum rotary evaporator. LNA
hydrazine was purified via a silica gel column using dichloro-
methane/methanol (v/v 30:1, Rf = 0.29) as the eluant. The
compound was a deep yellow oily liquid. The yield was 219.9
1
mg (0.75 mmol, 80.2%). The mass spectrum and H NMR
spectrum of LNA-NHNH2 are shown in Supporting
Information Figure 2. The molecular ion peak ([M + H]+) of
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1
LNA-NHNH2 was 293. The H NMR spectrum of LNA-
NHNH2 exhibited typical signals at chemical shifts of δ 5.28−
5.43 (6H, m), 2.81 (4H, s), 2.03−2.10 (4H, m), 1.63 (2H, s),
1.24−1.31 (10H, m), 0.97 (3H, t, CH3, J = 7.6 Hz) ppm.
2.2.1.3. Synthesis of the DOX-hyd-LNA Conjugate. To
synthesize the DOX-hyd-LNA conjugate, 70.6 mg of DOX
(0.13 mmol), 52.9 mg of LNA-hydrazine (0.18 mmol), 25 mL
of dichloromethane, and 12 μL of TFA were added to a flask,
the mixture was stirred overnight at 25 °C, and the organic
phase was evaporated in the darkness. Then, DOX-hyd-LNA
was purified via a silica gel column using dichloromethane/
methanol (v/v 5:1, Rf = 0.86) as the eluant. The product was
dried in a vacuum drying chamber. DOX-hyd-LNA was a dark
red solid. The yield was 61.1 mg (0.075 mmol, 49.4%). The
The purity and Log P of DOX-ami-LNA were determined as
described above. The mass spectrum and 1H NMR spectrum of
DOX-ami-LNA have been published previously.31
2.2.3. Synthesis of DOX-hyd-PA and DOX-ami-PA. The
synthetic routes of DOX-hyd-PA and DOX-ami-PA are shown
in Figures 3 and 4, respectively. The synthetic methods for
1
mass spectrum and H NMR spectrum of DOX-hyd-LNA are
shown in Supporting Information Figure 3. The molecular ion
1
peak ([M + H]+) of DOX-hyd-LNA was 819. The H NMR
spectrum of DOX-hyd-LNA exhibited typical signals at
chemical shifts of 5.12−5.60 (9H, m), 3.82−4.21 (7H, m),
3.30−3.53 (3H, m), 2.60−2.78 (6H, m), 2.41−2.58 (5H, m),
1.97−2.06 (5H, m), 1.66−1.90 (3H, m), 1.14−1.66 (14H, m),
0.92 (3H, t, CH3, J = 6.8 Hz) ppm.
The purification of DOX-hyd-LNA (dissolved in DMSO)
was analyzed by a Waters HPLC equipped with a 2996 PDA
detector and 2695 pump (Waters Corporation, USA). A
Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) was used
as the analytical column, 70% acetonitrile and 30% H2O were
used as the mobile phase, and the flow rate was 1 mL/min. The
wavelength of the PDA detector was 230 nm, and the analytical
volume was 20 μL. The column temperature was kept at 25 °C.
Partition coefficients (Log P) of DOX·HCl, DOX, and DOX-
hyd-LNA were measured using the n-octanol/water method.29
The compound concentration in the two phases was detected
by a UV−vis spectrophotometer (Beckman DU-800, USA).
2.2.2. Synthesis of DOX-ami-LNA. The synthetic route of
DOX-ami-LNA is shown in Figure 2: 250 mg of LNA (0.89
Figure 3. Reaction route for the synthesis of DOX-hyd-PA: (1)
H2NNHBoc, EDCI, DMF; (2) TFA, CH2Cl2; (3) DOX, TFA,
CH2Cl2.
Figure 4. Reaction route for the synthesis of DOX-ami-PA: (1) DOX,
Figure 2. Reaction route for the synthesis of DOX-ami-LNA: (1)
NHS, EDCI, TEA, CH2Cl2.
DOX, NHS, EDCI, TEA, CH2Cl2.
DOX-hyd-PA and DOX-ami-PA were as the same as those of
DOX-hyd-LNA and DOX-ami-LNA. The compounds were
dried in a vacuum drying chamber. The yields of DOX-hyd-PA
and DOX-ami-PA were 52.4 and 72.4%, respectively. The
purity and Log P of the compounds were detected as described
above.
mmol), 138 mg of NHS (1.2 mmol), 573 mg of EDCI (3
mmol), 120 μL of triethyl amine (TEA), 234.8 mg of DOX
(0.43 mmol), and 25 mL of dichloromethane were added to a
flask, and the mixture was stirred for 12 h at 25 °C. After that,
the organic phase was evaporated, and the residue was purified
using a silica gel column. The product was dried in a vacuum
drying chamber. The yield was 350.5 mg (0.44 mmol, 72.4%).
1
The mass spectrum and H NMR spectrum of PA-NHNH2,
DOX-hyd-PA, and DOX-ami-PA are shown in Supporting
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Table 1. Mass Spectrum Conditions
dwell time
(s)
capillary voltage
(kV)
cone voltage
(V)
collision energy
compound
precursor
daughter
(eV)
DOX-hyd-LNA
DOX-ami-LNA
DOX
817
803
542
690
416
395
0.2
0.2
0.2
2.5
2.5
2.5
55
48
40
18
22
16
Information Figures 4−6, respectively. The molecular ion peak
centrifuged at 8000g. The supernatant was collected and
analyzed by HPLC−MS/MS.
1
([M + H]+) of PA-NHNH2 was 271. The H NMR spectrum
2.2.7. DOX Release from DOX-hyd-LNA and DOX-ami-LNA
in Medium with Different pH Values. To investigate the DOX-
release characteristics of DOX-hyd-LNA and DOX-ami-LNA in
vitro, sodium phosphate buffer (pH 7.4) and sodium acetate
buffer (pH 5.0 and 6.5) were used as the release media. DOX-
hyd-LNA (2 mg dissolved in 2 mL of water containing 5% (v/
v) PEG400) or DOX-ami-LNA (2 mg dissolved in 2 mL of
water containing 5% (v/v) PEG400) was added into 20 mL of
release medium and cultured in a water bath shaker at 37 °C.
Then, 50 μL of the incubated sample was taken out at
predetermined time intervals, and the same volume of fresh
medium was supplemented into the original mixture. The 20
μL sample was analyzed by HPLC−MS/MS.
2.2.8. HPLC−MS/MS Conditions. A Waters Quattro Premier
LC−MS/MS system (Milford, MA, USA) and MassLynx 4.1
software was used. Briefly, a Waters XTerra C18 column (150 ×
2.1 mm, 5 μm) was used as the analytical column, 80%
acetonitrile and 20% H2O were used as the mobile phase, and
the flow rate was 0.2 mL/min. Multiple reaction monitoring
operated in the negative ion mode was used. The source
temperature and the desolvation temperature were set at 110
and 300 °C, respectively. The desolvation and cone gases were
nitrogen, and its flow rates were 650 and 50 L/h, respectively.
The collision gas was argon, and its flow rate was 0.18 mL/min.
The analytical volume was 20 μL. The mass spectrum
conditions to detect DOX, DOX-ami-LNA, and DOX-hyd-
LNA are shown in Table 1. Under the optimized chromato-
graphic conditions, DOX, DOX-ami-LNA, DOX-hyd-LNA, and
any interference were completely separated. The linear range of
the analytical method was 0.4−300 ng/mL, and the correlation
coefficient was 0.9986.
2.2.9. Apoptosis Analysis. HepG2 cells (or MCF-7 cells)
were grown into 6-well plates at density of 5 × 106 cells/mL for
24 h. Free DOX, DOX-ami-LNA, or DOX-hyd-LNA (dissolved
in cell culture medium containing 5% (v/v) PEG400) was
added, and cells were incubated for 24 h. The medium was
discarded, and the cells were gently rinsed three times with
PBS. Then, the cells were collected in PBS and centrifuged at
1000g for 2 min. After the supernatant was removed, the cells
were resuspended in 0.2 mL of PBS. Lastly, after the cells were
stained with annexin V−FITC and propidium iodide, they were
analyzed with a Becton Dickinson FACScan cytometer.
2.2.10. Uptake of DOX-hyd-LNA and DOX-ami-LNA in
Tumor Cells. HepG2 and MCF-7 cells were grown in 6-well
plates at a density of 1 × 105 cells/mL. After 12 h, the medium
was discarded, and DOX-hyd-LNA or DOX-ami-LNA (dis-
solved in cell culture medium containing 5% (v/v) PEG400)
was added. After the cells were cultured for 30 or 120 min, the
medium was discarded, and the cells were gently rinsed with
PBS three times. Then, cells were collected in 1 mL of
deionized water. The cell lysate was obtained by freezing and
thawing the cells three times. The cell lysate was centrifuged at
8000g, and the supernatant was analyzed by HPLC−-MS/MS.
of PA-NHNH2 exhibited typical signals at chemical shifts δ 6.65
(1H, s), 2.12−2.16 (2H, d,), 1.61−1.65 (3H, t), 1.25−1.28
(31H, m), 0.86−0.89 (3H, t, CH3) ppm. The molecular ion
1
peak ([M + H]+) of DOX-hyd-PA was 797. The H NMR
spectrum of DOX-hyd-PA exhibited typical signals at chemical
shifts of δ 10.01 (1H, s), 8.03 (1H, s), 7.78 (1H, s), 5.53−5.52
(2H, d), 4.68 (2H, d), 3.91−4.09 (3H, m), 3.08−3.46 (6H, m),
2.03−2.60 (7H, m), 1.67−1.98 (8H, m), 1.02−1.42 (29H, m),
0.85−0.88 (6H, m) ppm. The molecular ion peak ([M − H]−)
1
of DOX-ami-PA was 781. The H NMR spectrum of DOX-
ami-PA exhibited typical signals at chemical shifts of δ 13.97
(1H, s), 13.23 (1H, s), 7.78−8.04 (1H, s), 7.26−7.39 (1H, s),
7.26 (1H, s), 5.79−5.81 (1H, s), 5.45−5.55 (1H, s), 5.20−5.32
(2H, d), 4.68−4.83 (2H, d), 4.53−4.58 (1H, s), 3.96−4.28
(5H, m) 3.56−3.66 (1H, s), 2.97−3.02 (2H, d), 1.48−2.52
(14H, m), 1.01−1.42 (34H, m), 0.79−0.96 (4H, m) ppm.
2.2.4. Cell Culture. HUVECs are human umbilical vein
endothelial cells.32−34 MDA-MB-231 cells are a human breast
cancer cell line that does not express the estrogen receptor.35,36
MCF-7 cells are a human breast cancer cell line that expresses
the estrogen receptor normally.36,37 HepG2 cells are a human
hepatoblastoma cell line.38,39 All cell lines were obtained from
the Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences, Shanghai, China. All cells were cultured
in RPMI 1640 medium containing penicillin (100 units/mL),
streptomycin (100 μg/mL), and fetal bovine serum (10%).
2.2.5. Cytotoxicity of DOX Conjugates. The cytotoxicity of
DOX-hyd-LNA, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-
PA was evaluated by MTT assay. All compounds were
dissolved in cell culture medium containing 5% (v/v)
poly(ethylene glycol) 400 (PEG400). The cells were grown
in 96-well plates at a density of 1 × 104 cells/well and incubated
for 12 h. The medium was discarded, fresh drug-containing
medium was added, and cells were incubated for 48 h. The
wells were gently rinsed three times with PBS, fresh medium
containing 5 mg/mL of MTT was added, and cells were
incubated for 4 h. After that, the medium was discarded, and
150 μL of DMSO was added. Lastly, the plate was gently
rocked for 10 min. The absorbance was detected at 570 nm by
a Bio-Rad microplate reader. The cell culture experiments were
performed in quintuplicate.
2.2.6. Stability of DOX-hyd-LNA and DOX-ami-LNA in Rat
Serum. DOX-hyd-LNA (2 mg dissolved in 2 mL of normal
saline containing 5% (v/v) PEG400) or DOX-ami-LNA (2 mg
dissolved in 2 mL of normal saline containing 5% (v/v)
PEG400) was added into 20 mL of fresh rat serum, and the
mixture was cultured in a water bath shaker at 37 °C. Then, 50
μL of the incubated sample was taken out at predetermined
time intervals, and the same volume of fresh serum was
supplemented into the original mixture. One-hundred micro-
liters of acetonitrile was added into the 50 μL drug-containing
serum sample. The mixture was vigorously shaken and
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Table 2. Molecular Properties of DOX Conjugates (n = 5)
molecular
weight
number of oxygens and nitrogens number of -OH and
violations of rule of
number of rotatable
bonds
compound
Log P
atom
-NH
five
DOX·HCl
DOX
−1.45 0.22
0.33 0.08
0.87 0.12
0.93 0.11
1.09 0.16
1.17 0.18
580.0
543.5
781.9
796.1
803.9
818.1
12
12
13
14
13
14
6
6
5
6
5
6
3
3
3
3
3
3
5
5
DOX-ami-PA
DOX-hyd-PA
DOX-ami-LNA
DOX-hyd-LNA
22
22
21
21
Figure 5. Cytotoxicity of DOX, DOX-hyd-LNA, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA against the following cells: HepG2 (A), MCF-7
(B), MDA-MB-231 (C), and HUVEC (D). Cells were incubated with DOX, DOX-hyd-LNA, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA for
48 h. n = 5.
Table 3. IC50 of DOX Conjugates on MCF-7, MDA-MB-231, HepG2, and HUVEC Cells
IC50 (μM)
compound
DOX
HepG2
MCF-7
MDA-MB-231
HUVEC
3.2 0.7
12.3 1.4
7.8 1.9
4.9 0.8
1.3 0.4
3.6 1.3
9.8 2.6
8.8 1.8
4.6 1.5
1.7 0.3
4.3 1.1
13.5 2.2
8.1 1.3
6.6 1.7
5.5 1.8
16.9 3.4
11.1 2.6
19.0 4.9
12.1 3.5
DOX-ami-PA
DOX-hyd-PA
DOX-ami-LNA
abc
, ,
abc
, ,
abc
, ,
a
DOX-hyd-LNA
2.2 0.7
a
b
c
p < 0.05 vs DOX. p < 0.01 vs DOX-ami-LNA. p < 0.01 vs DOX-hyd-PA; n = 5.
The protein content in the cell lysates was determined by the
Bradford method, and the drug content was normalized by the
protein content of the cell lysate. The cell culture experiments
were performed in quintuplicate.
2.2.11.2. In Vivo Antitumor Activity. HepG2 cells were
subcutaneously implanted in the rear right flank of nude mice
(1 × 107 cells/0.1 mL/animal). There were six nude mice in
each group. Treatment was started on the 10th day when the
tumor volume was about 80 mm3. DOX or DOX-hyd-LNA was
dissolved in normal saline containing 5% (v/v) PEG400. Free
DOX (9.2 μmol/kg) or DOX-hyd-LNA (4.6 and 9.2 μmol/kg)
was intravenously injected into tumor-bearing mice by the tail
vein every 7 days for three doses (days 1, 7, and 14). The body
weight of the mice was monitored every 3 days as a sign of
systemic toxicity. The long (L) and short (W) diameter of the
tumor were measured every 3 days with a caliper. The tumor
volume was calculated as LW2/2. On day 42, all surviving mice
were euthanized.
2.2.11. Animal Experiments. 2.2.11.1. Maximum Tolerated
Dose (MTD) Studies. DOX or DOX-hyd-LNA was dissolved in
normal saline containing 5% (v/v) PEG400. DOX or DOX-
hyd-LNA was intravenously administered to healthy BALB/c
mice at doses of 9.2, 18.4, 27.6, 36.8, 46, and 55.2 μmol/kg (n =
4). The survival status and body weight of the mice were
observed daily for 2 weeks. The MTD was defined as the dose
resulting in no more than 15% body weight loss and no death
of the mice for the 2 weeks after the drug was
administered.40−43
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2.2.11.3. Biodistribution. For the biodistribution study in
vivo, tumor-bearing nude mice were treated with free DOX or
DOX-hyd-LNA at a dose of 9.2 μmol/kg. Mice were
euthanized at predetermined time intervals to collect their
organs and tumor tissue. The tissues were rinsed with PBS and
homogenized with water. The homogenate was centrifuged at
8000g for 15 min, and the supernatant was analyzed by HPLC−
MS/MS.
2.2.12. Statistical Analysis. SigmaPlot 8.0 software was used
to analyze the data. Student’s t test was used to evaluate statistic
significance, and p < 0.05 was considered to indicate a
significant difference.
3. RESULTS AND DISCUSSION
3.1. Identification of DOX Conjugates. The typical
HPLC chromatograms of DOX-hyd-LNA, DOX-ami-LNA,
Figure 8. DOX-release characteristics from DOX-hyd-LNA (A) and
DOX-ami-LNA (B) in medium with different pH values. n = 3.
DOX-hyd-PA, and DOX-ami-PA are shown in Supporting
Information Figure 7. The purity of DOX-hyd-LNA, DOX-ami-
LNA, DOX-hyd-PA, and DOX-ami-PA was 98.7, 97.8, 98.2,
and 97.3%, respectively. When LNA-NHNH2 reacted with
DOX to produce DOX-hyd-LNA, it was very important to
control the amount of trifluoroacetic acid in the reaction
mixture because excess trifluoroacetic acid could break the
hydrazone bond between DOX and LNA. To avoid breaking
DOX-hyd-LNA in the silica gel, triethylamine was added in the
eluant.
A molecular description of DOX·HCl, DOX, and its fatty
acid conjugates are presented in Table 2. There were three
parameters that violated rule of five in the DOX conjugates.44
As the length of the fatty acid substituent increase, the water
solubility of the DOX derivative decreased. This was actually an
expected result caused by the increased lipophilicity of the fatty
acid substituent. The partition coefficient (Log P, octanol/
water) was directly correlated to molecular weight, inversely
correlated to the number of -OH and -NH groups (hydrogen-
bond donors), and inversely correlated to water solubility.45
3.2. Cytotoxicity of DOX-hyd-LNA, DOX-ami-LNA,
DOX-hyd-PA, and DOX-ami-PA. The cytotoxicity of free
DOX, DOX-hyd-LNA, DOX-ami-LNA, DOX-hyd-PA, and
DOX-ami-PA toward MCF-7, MDA-MB-231, and HepG2
cells was studied by MTT assay. As shown in Figure 5 and
Table 3, DOX-hyd-LNA showed a higher cytotoxicity toward
tumor cells than DOX, DOX-ami-LNA, DOX-hyd-PA, and
DOX-ami-PA. The cytotoxicity of DOX toward tumor cell was
higher than DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA.
This indicated that modification of the 3′ amino group in DOX
with an unsaturated or saturated fatty acid led to a decrease in
its antitumor activity. It also indicated that the connection of
the saturated fatty acid (PA) with C13 of DOX by a hydrazone
bond did not enhance the cytotoxicity of DOX. Furthermore,
among the three tumor cell lines, DOX-hyd-LNA showed the
highest cytotoxicity against HepG2 cells but the lowest
Figure 6. Effect of exogenous LNA on the viability of HepG2 cells.
HepG2 cells were incubated with 8 μmol/L DOX-hyd-LNA (A) or the
same dose of DOX-hyd-PA (B) for 48 h. *p < 0.05 vs DOX-hyd-LNA.
n = 5.
Figure 7. Stability of DOX-hyd-LNA and DOX-ami-LNA in rat serum.
n = 3.
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Figure 9. Apoptosis of HepG2 cells induced by DOX, DOX-hyd-LNA, and DOX-ami-LNA. DOX, DOX-ami-LNA, or DOX-hyd-LNA was
incubated with HepG2 cells at 37 °C for 24 h.
Figure 10. Apoptosis of MCF-7 cells induced by DOX, DOX-hyd-LNA, and DOX-ami-LNA. DOX, DOX-ami-LNA or DOX-hyd-LNA was
incubated with MCF-7 cells at 37 °C for 24 h.
cytotoxicity toward MDA-MB-231 cells. It was anticipated that
the DOX-hyd-LNA and DOX-ami-LNA would be taken up by
the tumor cells through LNA-mediated endocytosis and
localized in an acidic organelle, such as an endosome or
lysosome, from which DOX would be released from DOX-hyd-
LNA and DOX-ami-LNA and subsequently diffuse into the
nucleus.46,47 The effect of exogenous LNA on the cytotoxicity
of DOX-hyd-LNA is shown in Figure 6. When HepG2 cells
were incubated with DOX-hyd-LNA and different concen-
tration of exogenous free LNA for 48 h, the cytotoxicity of
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Figure 11. Quantitative uptake and DOX-release characteristic of DOX-hyd-LNA and DOX-ami-LNA in HepG2 and MCF-7 cells at 37 °C: (A) 20
μmol/L DOX-hyd-LNA incubated with HepG2 cells, (B) 20 μmol/L DOX-hyd-LNA incubated with MCF-7 cells, (C) 20 μmol/L DOX-ami-LNA
incubated with HepG2 cells, and (D) 20 μmol/L DOX-ami-LNA incubated with MCF-7 cells. n = 5.
DOX-hyd-LNA on HepG2 cells decreased in a dose-dependent
manner. Meanwhile, the cytotoxicity of DOX-hyd-PA on
HepG2 cells was not affected by exogenous LNA. This implied
that LNA plays an important role in the cytotoxicity of DOX-
hyd-LNA toward HepG2 cells.
the hydrazone bond is crucial for DOX conjugates to exert their
efficacy.
3.3. Hydrolysis of DOX-hyd-LNA and DOX-ami-LNA in
Rat Serum. The hydrolytic characteristics of DOX-hyd-LNA
and DOX-ami-LNA in rat serum are shown in Figure 7. The
results indicated that the concentration of DOX-hyd-LNA
decreased 23% in 24 h and that a small amount of DOX was
released during the period from 24 to 48 h. Compared with
DOX-hyd-LNA, DOX-ami-LNA was more stable in rat serum
after 48 h.
3.4. DOX Release from DOX-hyd-LNA and DOX-ami-
LNA in Medium with Different pH Values in Vitro. The
drug-release characteristics of DOX-hyd-LNA and DOX-ami-
LNA in medium with different pH values at 37 °C were
investigated. The results are shown in Figure 8. The time-
dependent chromatograms of the hydrolysis studies are shown
in Supporting Information Figures 8 and 9. On the one hand,
the rate at which DOX was released from DOX-hyd-LNA was
closely related to the pH of the medium. DOX-hyd-LNA
released 81% of the conjugated DOX in pH 5.0 medium in 4 h,
but it released 22% of the conjugated DOX in pH 7.4 medium
in 24 h. The relatively slow DOX release rate of DOX-hyd-LNA
in pH 7.4 medium should ensure the relative stability of DOX-
hyd-LNA in the bloodstream. The faster DOX release rate of
DOX-hyd-LNA in acidic medium mimicked the burst release of
DOX from DOX-hyd-LNA in the endosomal/lysosomal
compartments of the tumor cells.
The cytotoxicity of DOX-hyd-LNA, DOX-ami-LNA, DOX-
hyd-PA, DOX-ami-PA, and free DOX on HUVECs is shown in
Table 3 and Figure 5. The cytotoxicity of DOX-hyd-LNA
toward HUVECs was significantly lower than that of free DOX.
This indicated that DOX-hyd-LNA exhibited selectivity for
tumor cells. Kratz et al. cultured an acid-sensitive transferrin−
DOX conjugate with HUVECs, and the result indicated that
the cytotoxicity of transferrin−DOX conjugate toward
HUVECs was significantly less than that of the free DOX.48
Additionally, Shahin et al. found that p160-decorated micelles
(encapsulated paclitaxel) showed greater cytotoxicity toward
MDA-MB-435 cells than toward HUVECs or MCF10A cells.49
In addition to a small molecule PUFA−DOX conjugate,
Rodrigues et al. synthesized a series of DOX-poly(ethylene
glycol) (PEG) conjugates by an amide or a hydrazone bond in
which the DOX maleimide derivatives were linked with α-
methoxy-PEG-thiopropionic acid amide (MW 20 000 Da), α,ω-
bis-thiopropionic acid amide PEG (MW 20 000 Da), or α-tert-
butoxy-PEG-thiopropionic acid amide (MW 70 000 Da).50 The
hydrazone bond conjugates showed antitumor activity toward
human BXF T24 bladder carcinoma and LXFL 529L lung
cancer cells, but the amide bond conjugates exhibited no
antitumor activity in vitro.50 In our group, DOX was connected
with HOOC-PEG-COOH by a hydrazone bond (PEG-hyd-
DOX) or an amide bond (PEG-ami-DOX) to improve the
therapeutic index of DOX. The results showed that the acid-
sensitive PEG-hyd-DOX conjugate released free DOX in the
acidic environment of the tumor.51 These results implied that
On the other hand, the rate at which DOX was released from
DOX-ami-LNA was independent of the pH of the medium.
DOX-ami-LNA released 5% of conjugated DOX in pH 7.4
medium in 10 h, and 11% of conjugated DOX was released in
pH 5.0 medium in 10 h. These results implied that it is difficult
for DOX-ami-LNA to release DOX in tumor cells. Con-
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Table 4. Statistic Analysis of the Survival of Tumor-Bearing
Mice
median
survival
(days)
maximal
survival
(days)
mean survival
(days)
treatment group
control
p
27
30
26.60 1.63
31.40 1.60
32
36
a
b
b
free DOX
(9.2 μmol/kg)
0.069
0.375
0.007
DOX-hyd-LNA
(4.6 μmol/kg)
34
33.80 2.59
38.80 1.25
42
42
DOX-hyd-LNA
(9.2 μmol/kg)
38
a
b
Compared with control. Compared with free DOX.
MCF-7 cells, DOX-hyd-LNA induced more apoptosis in
HepG2 cells. These data are consistent with the results of
cytotoxicity experiment as well as with the drug-release
characteristics of DOX-hyd-LNA and DOX-ami-LNA in vitro.
3.6. Uptake of DOX-hyd-LNA and DOX-ami-LNA in
MCF-7 and HepG2 Cells. After MCF-7 and HepG2 cells were
incubated with DOX-hyd-LNA or DOX-ami-LNA (20 μmol/
L) for 30 and 120 min, the intracellular amount of DOX, DOX-
hyd-LNA, or DOX-ami-LNA was detected by HPLC−MS/MS.
As shown in Figure 11, both DOX-hyd-LNA and DOX-ami-
LNA were taken up by MCF-7 and HepG2 cells in a time-
dependent manner. Meanwhile, a large amount of DOX was
released from DOX-hyd-LNA in HepG2 and MCF-7 cells, but
a small amount of DOX was released from DOX-ami-LNA in
MCF-7 and HepG2 cells. Compared with MCF-7 cells, a large
amount of DOX-hyd-LNA and DOX-ami-LNA was accumu-
lated in HepG2 cells, and a greater amount of DOX was
released from DOX-hyd-LNA in HepG2 cells. These results
provide an explanation as to why DOX-hyd-LNA exhibited
higher cytotoxicity toward HepG2 cells than MCF-7 cells.
It has been established that PUFAs are taken up much more
readily by tumor cells than by normal cells.22,52 In addition,
PUFAs are able to be inserted quickly into the membrane of
tumor cells, damaging the structure and fluidity of the cell
membrane.53 This implies that PUFAs are able to enhance the
uptake of antitumor drugs by tumor cells,increasing the
sensitivity of chemotherapy drugs.54 A daunomycin−arach-
idonic acid conjugate showed greater cytotoxicity toward
hepatoma cells than free daunomycin. However, the
daunomycin−arachidic acid (a saturated fatty acid) conjugate
exhibited the same antitumor activity as free daunomycin.55
This indicated that arachidonic acid exerts an important role for
the antitumor activity of the daunomycin−arachidonic acid
conjugate. Moreover, when 4′-demethyldeoxypodophyllotoxin
(DDPT) was conjugated with an unsaturated fatty acid by an
ester bond, its antitumor activity in vivo was increased without
any effect on body weight loss.56
Figure 12. Antitumor activity of DOX-hyd-LNA in vivo. Athymic
nude mice xenografted with HepG2 cells were intravenously injected
with different doses of DOX-hyd-LNA (4.6 and 9.2 μmol/kg) and one
dose of DOX (9.2 μmol/kg). Treatment was initiated when the tumor
volume was about 80 mm3. n = 6. Tumor-bearing mice were treated
with normal saline (control), DOX, or DOX-hyd-LNA by tail vein
injection every 7 days (days 1, 7, and 14). (A) Tumor volume changes
in tumor-bearing nude mice, (B) body weight changes in tumor-
bearing nude mice, (C) survival curve of tumor-bearing nude mice.
3.7. MTD of DOX-hyd-LNA. There was no remarkable
reduction in body weight or any other toxic reaction in mice
treated with DOX-hyd-LNA at doses of 9.2−36.8 μmol/kg in
the 2 weeks following its injection. The 46 μmol/kg DOX-hyd-
LNA treatment resulted in body weight reduction (<11%)
during the first week, but the body weight returned to normal
as soon as the experiment ended. The 18.4 μmol/kg free DOX
treatment caused body weight reduction (<14%) without any
death of the mice. The 27.6 μmol/kg free DOX treatment
resulted in a quick reduction in the body weight, and all animals
died in this group within 2 weeks. Therefore, the MTD of
DOX-hyd-LNA and free DOX in BLAB/c mice was, for a single
sequently, DOX-hyd-LNA showed higher cytotoxicity toward
tumor cells than DOX-ami-LNA.
3.5. Cell Apoptosis Induced by DOX-hyd-LNA and
DOX-ami-LNA. DOX-hyd-LNA and DOX-ami-LNA were
incubated with HepG2 and MCF-7 cells for 24 h, and the
apoptotic cells were detected by flow cytometry. As shown in
Figures 9 and 10, DOX-hyd-LNA induced apoptosis in HepG2
and MCF-7 cells in a dose-dependent manner. Compared with
free DOX and DOX-ami-LNA, DOX-hyd-LNA induced
significantly more apoptosis. Additionally, compared with
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Figure 13. Distribution of DOX in tumor-bearing nude mice after intravenous injection of free DOX (A) or DOX-hyd-LNA (B). n = 5.
injection, approximately 46 and 18.4 μmol/kg, respectively.
The MTD value of DOX presented in this article is consistent
with previous literature reports.43,57−60 Doxil is a very stable
liposome of DOX used in the clinic. It has been reported that
the MTD of doxil is almost the same as free DOX in dogs but is
lower than free DOX in mice.61,62
decreased the content of DOX in heart. The level of DOX in
the tumor tissue was 12 μg/g tissue, and this level was sustained
for 24 h. The longer exposure to a high DOX concentration led
to the improvement of the antitumor activity of DOX-hyd-
LNA.
In the human body, fatty acids are transported through the
vascular and lymphatic systems. Fatty acids need to bind plasma
proteins to increase their concentration in vascular and
interstitial compartments because of their poor solubility in
water. Human plasma albumin is the main fatty acid-binding
protein in blood. Plasma albumin possesses high-affinity
binding sites for fatty acids to enhance the concentration of
fatty acids in blood.67 Thus, fatty acids can be used to prolong
the half-life of an antitumor drug and peptide in vivo.68−70
DHA−paclitaxel (taxoprexin) was shown to be stable in
plasma and was extensively bonded with human plasma
albumin in vitro. The plasma protein binding rate of DHA−
3.8. In Vivo Antitumor Activity. Tumor-bearing nude
mice were used to investigate the antitumor activity of DOX-
hyd-LNA in vivo. An equimolar amount of DOX and DOX-
hyd-LNA was administered through the tail vein. The
antitumor activity of DOX-hyd-LNA is shown in Figure 12
and Table 4. A time-related tumor volume increase was
observed in all groups. Although neither DOX nor DOX-hyd-
LNA could completely stop the growth of the tumor, the
tumors in the drug-treated groups showed an obvious growth
retardation in a dose-dependent manner. This result is
consistent with previous observations in the literature.40,63,64
Compared with DOX, DOX-hyd-LNA exhibited a stronger
tumor growth inhibition. The cellular uptake experiment
indicated that a large amount of DOX was released from
DOX-hyd-LNA in HepG2 cells, which exerted an important
role in delaying the growth of the tumor in vivo.
When tumor-bearing nude mice were treated with free DOX
(9.2 μmol/kg), the activity and body weight of the mice were
remarkably reduced. However, no indication of a significant
toxic reaction was discovered in tumor-bearing mice when
treated with an equimolar amount of DOX-hyd-LNA. This
implied that treatment with DOX-hyd-LNA enhances ther-
apeutic efficacy with less systemic toxicity.
INNO-206 is another hydrazone bond-based derivative of
DOX in which doxorubicin is connected with 6-maleimidocap-
roic acid at its C13 keto-position through a hydrazone bond.
INNO-206 can specifically react with cysteine-34 of human
serum albumin to form an albumin-binding DOX prodrug. The
albumin-binding DOX is able to accumulate in tumor tissue by
the enhanced permeability and retention (EPR) effect, and the
DOX can be released in an acidic environment such as tumor
tissue or tumor cell endosomal or lysosomal compartments.65
INNO-206 has entered phase II clinic trials for the treatment of
sarcoma.66
3.9. Drug Biodistribution. After free DOX or DOX-hyd-
LNA was intravenously administered, the biodistribution of
DOX in tumor-bearing nude mice was assessed and is shown in
Figure 13. DOX typically accumulated in the liver, heart, spleen,
lung, kidney, and tumor tissue after free DOX or DOX-hyd-
LNA was administered. Compared with free DOX, treatment
with an equimolar amount of DOX-hyd-LNA greatly increased
the content of DOX in the tumor tissue and significantly
paclitaxel was 99.600
0.057% in human plasma, which
resulted in a small distribution volume and slow systemic
clearance. The plasma protein binding property of DHA−
paclitaxel is favorable for enhancing its antitumor efficacy.70
Pharmacokinetic studies showed that DHA−paclitaxel signifi-
cantly increased the content of paclitaxel in tumor tissue
compared with paclitaxel at an equimolar dose, which was due
to the EPR effect of plasma protein-bound DHA−paclitaxel.
Additionally, DHA−paclitaxel was relatively stable in blood
circulation, and only 0.5% of paclitaxel was released from
DHA−paclitaxel in blood. Thus, the increased accumulation of
paclitaxel in tumor tissue and the relative stability of DHA−
paclitaxel in blood resulted in an enhancement of the
therapeutic efficacy of DHA−paclitaxel in tumor-bearing
mouse model.71 The safety of DHA−paclitaxel has been
demonstrated in metastatic uveal melanoma patients.72,73
The plasma protein binding rate of DOX-hyd-LNA was 98.7
1.1% in rat plasma. Determining whether the increased
distribution of DOX in tumor tissue was caused by the high
plasma protein binding rate of DOX-hyd-LNA still needs
further investigation.
4. CONCLUSIONS
Compared with DOX, DOX-hyd-LNA exhibited improved
antitumor activity with less toxicity in vitro and in vivo. These
results were due to the stability of DOX-hyd-LNA in serum, the
increased distribution of DOX in tumor tissues, and the large
amount of DOX released in tumor cells. Thus, DOX-hyd-LNA
is a compound with great potential, and we expect that it will be
researched further as a cancer-targeting therapy.
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ASSOCIATED CONTENT
* Supporting Information
■
S
MS and 1H NMR data for NBoc-LNA hydrazine, LNA-
NHNH2, DOX-hyd-LNA, PA-NHNH2, DOX-hyd-PA, and
DOX-ami-PA; HPLC chromatograms of DOX, DOX-hyd-
LNA, DOX-ami-LNA, DOX-hyd-PA, and DOX-ami-PA; and
time-dependent chromatograms of hydrolysis of DOX-hyd-
LNA and DOX-ami-LNA in pH 5.0 medium. This material is
available free of charge via the Internet at http://pubs.acs.org.
(18) Hawkins, R. A.; Sangster, K.; Arends, M. J. Apoptotic death of
pancreatic cancer cells induced by polyunsaturated fatty acids varies
with double bond number and involves an oxidative mechanism. J.
Pathol. 1998, 185, 61−70.
(19) Siddiqui, R. A.; Harvey, K. A.; Xu, Z.; Bammerlin, E. M.; Walker,
C.; Altenburg, J. D. Docosahexaenoic acid: A natural powerful adjuvant
that improves efficacy for anticancer treatment with no adverse effects.
BioFactors 2011, 37, 399−412.
AUTHOR INFORMATION
Corresponding Author
*(S.-y.Z.) Tel.: +86 (0)2984776783; Fax: +86 (0)2984779212;
E-mail: zhousy@fmmu.edu.cn.
■
Author Contributions
∥C.-h.L. and W.-l.Y. contributed equally to this work.
(20) Kuan, C. Y.; Walker, T. H.; Luo, P. G.; Chen, C. F. Long-chain
polyunsaturated fatty acids promote paclitaxel cytotoxicity via
inhibition of the MDR1 gene in the human colon cancer Caco-2
cell line. J. Am. Coll. Nutr 2011, 30, 265−273.
(21) Merendino, N.; Costantini, L.; Manzi, L.; Molinari, R.; D’Eliseo,
D.; Velotti, F. Dietary omega-3 polyunsaturated fatty acid DHA: A
potential adjuvant in the treatment of cancer. BioMed Res. Int. 2013,
2013, 310186-1−310186-11.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was sponsored by the Science and Technology
Innovation Project of Shaanxi (2012KTCL03-18) and the
Shaanxi Natural Science Foundation (2013JQ4026).
(22) Sauer, L. A.; Dauchy, R. T.; Blask, D. E. Mechanism for the
antitumor and anticachectic effects of n-3 fatty acids. Cancer Res. 2000,
60, 5289−5295.
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