ANTIPROLIFERATIVE EFFECTS OF HMG-COA REDUCTASE INHIBITORS (STATINS) ARE
SIGNIFICANTLY ENHANCED WHEN USED IN COMBINATION WITH γ-TOCOTRIENOL IN
NEOPLASTIC MAMMARY EPITHELIAL CELLS IN CULTURE
Vikram B. Wali and Paul W. Sylvester
College of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71209-0470
*
3
*
*
2
1
FPP
γ-tocotrienol
GGPP
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2
1
4
3
2
1
0
10
20
50
10
20
50
4
*
3
*
2
*
1
C
0.25 0.5
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6
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4
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C
5
25
50
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*
1
C
100 150 200 300
0.25 0.5
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*
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0
*
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*
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d
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0
C
S
0.25
M
0.25
0.5
0.75
3
T (M)
1
S 0.25+
0.25 0.5
2
0. 75
1
3
T (M)
C
2
L
0.25
M
0.25
0.5
0.75
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T (M)
1
2
L 0.25+
0.25 0.5
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*
*
2
*
*
*
0
C
0.25 0.5
1
2
4
8
Growth of Pravastatin
treated
+SA cells on Day 5
Lovastatin
(M)
4
3
2
1
1
2
4
8
C
10
20
40
80
100
Pravastatin (M)
5
*
*
3
*
*
2
1
0
C
0. 75
1
3
T (M)
*
1
4
1
1
*
Figure 2. Antiproliferative Study. +SA cell growth following
4 days treatment with 0-100µM of various statins. Data points
indicate mean viable cell count + SEM.
Cells per Well (1x10-5)
2
Cells per Well (1x10-5)
Cells per Well (1x10-5)
*
*
2
Mevastatin (M)
4
*
3
0
c5
4
*
0
8
*
Pravastatin(M)
3
4
4
5
5
3
2
0
100 150 200
4
1
5
Growth of +SA cells
on Day 5 after
Mevastatin treatment
Simvastatin
(M)
7
b
5
5
100 150 200
Figure 1. Cytotoxic Study. +SA cell viability following 24hr
treatment with 0-300µM of various statins. Data points indicate
mean viable cells + SEM.
a
6
7
Mevastatin (M)
Cholesterol
6
0
0
C
7
Lethal Dose ofLovastatin
Pravastatin (M)
(24Hrs) for +SA cells
Cells per Well (1x10-5)
Cells per Well (1x10-5)
Mevalonate
*
C
C
10
20 50 (24Hrs)
100 150
200 cells
Lethal Dose
of Mevastatin
for +SA
Simvastatin (M)
5
Reductase
3
0
0
HMGCoA
4
7
Cells per Well (1x10-5)
4
5
Growth of +SA cells on Day 5 after Lovastatin treatment
Cells per well (1x10-5)
5
Cells per Well (1x10-5)
6
Cells per Well (1x10-5)
Statin
6
Cells per Well (1x10-5)
ACoA
Cells per Well (1x10-5)
Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG
CoA) reductase, represent a class of antihyperlipidemic drugs. However,
previous reports have demonstrated that statins also display antiproliferative
and cytotoxic activity against various types of cancers. It is well established
that γ-tocotrienol, a member of the vitamin E family of compounds, also
displays potent anticancer activity. Studies were conducted to determine if
combination therapy of simvastatin, lovastatin, mevastatin, and pravastatin
with γ-tocotrienol resulted in enhanced antiproliferative effects in the highly
malignant +SA mammary epithelial cells grown in culture. Cells were
maintained in serum-free defined media containing EGF (10 ng/mL) and
insulin (10 µg/mL) as co-mitogens. In cytotoxicity studies, cells were treated
with 0-300µM of individual statins, and cell viability was assessed 24 hr later
using the MTT assay. Results showed that none of the statins decrease +SA
viability 24 hr after treatment exposure. In antiproliferative studies, +SA cells
were treated with 0-100µM of individual statins for 4 days. Results showed
that treatment with 2-8µM simvastatin, lovastatin, and mevastatin significantly
inhibited +SA cell growth in a dose-responsive manner, while pravastatin had
no effect on +SA cell growth at any dose tested. In order to achieve plasma
concentrations of 2-8µM of these statins in humans, a treatment dose of 25
mg/kg or higher would be required, and treatment with these dose levels are
associated with severe myotoxicity. However, additional studies showed that
combination treatment with 0.25µM simvastatin, lovastatin or mevastatin, or
10µM pravastatin with sub-effective doses (0.25-2.0µM) of γ-tocotrienol
resulted in significant dose-dependent inhibition in +SA cell proliferation
during the 4 day culture period. In addition, none of the various combinations
of individual statins and γ-tocotrienol were found to be cytotoxic. These
findings suggest that combined treatment with γ-tocotrienol can significantly
enhance the antiproliferative activity of various statins in malignant +SA
mammary epithelial cells. Furthermore, the doses of statins used in these
combination studies are not cytotoxic. These findings suggest that low-dose
treatment of statins used in combination with low-dose γ-tocotrienol therapy
may provide significant health benefits in the prevention and/or treatment of
breast cancer in women, while avoiding myotoxicity associated with high dose
statin treatment. Supported by NIH Grant CA 86833.
Mevalonate Pathway
7
Cells per Well (1x10-5)
Abstract
Growth of +SA cells on Day 5 on Simvastatin treatment
Lethal Dose of Lovastatin (24Hrs) for +SA cells
Lethal Dose of Simvastatin (24Hrs) for +SA cells
2
M
0.25
M
0.25
0.5
0.75
3
T (M)
1
M 0.25+
0.25 0.5
2
C
0. 75
1
3
T (M)
2
P10
M
0.25
0.5
0.75
3
T (M)
1
2
P10+
0.25
0.5
0. 75
1
3
T (M)
2
Figure 2. Antiproliferative Study of the combination. +SA cell growth following 4 days treatment with combination of 0.25µM of simvastatin (a), lovastatin (b) or mevastatin (c), or 10µM pravastatin
(d) and 0.25-2.0µM of γ-tocotrienol. Data points indicate mean viable cell count + SEM. Photomicrographs of +SA cells on day 5 from experiment (a) are shown below.
Materials and Methods
Growth of +SA cells after 24 Hrs of treatment Growth of +SA cells after 24 Hrs of treatment
5
Statistical Analysis: Differences between various treatment groups were
determined by analysis of variance followed by Dunnett’s t-test. Differences
were considered statistically significant at a value of P < 0.05.
Cells per Well (1x10-5)
Measurement of Viable Cell Number: +SA cell count was determined by
o
MTT assay. Briefly, cells were incubated at 37 C for 4hr with media
containing 0.416mg/ml MTT. Then, media was removed and MTT crystals
were dissolved in 1ml isopropanol. The optical density was measured at
570nm and the number of viable cells per well was calculated against a
standard curve prepared by plating various cell concentrations, as determined
by hemocytometer, at the start of each experiment.
Cells per Well (1x10-5)
4
3
2
Sim
T3
0.25M 2M
Sim 0.25 +
0.25
0.5
0.75
3
T (M)
1
2
Sim 0.25 µM +γT³ 1µM
Sim 0.25 µM +γT³ 2µM
Growth of +SA cells after 24 Hrs of treatment
5
5
CONCLUSIONS
4
4
4
1. Statins are not acutely cytotoxic to neoplastic +SA mammary
3
2
1
C
Growth of +SA cells after 24 Hrs of treatment
Sim 0.25 µM +γT³ 0.25µM
5
1
0
γ-Tocotrienol 2 µM
γ-Tocotrienol 1 µM
γ-Tocotrienol 0.25 µM
Simvastatin 0.25 µM
Cells per Well (1x10-5)
Control
Cells per Well (1x10-5)
Cell Culture: +SA, a highly malignant cell line was derived from an
adenocarcinoma that had developed spontaneously in a female BALB/c mouse.
+SA cells were maintained in serum-free defined media consisting of
DMEM/F12 containing 5mg/ml BSA, 10µg/ml transferrin, 100U/ml soybean
trypsin inhibitor, 100U/ml penicillin, 0.1mg/ml streptomycin, 10ng/ml EGF,
and 10µg/ml insulin.
3
2
1
0
C
Lov
T3
0.25M 2M
Lov 0.25 +
0.25
0.5
1
2
2. Simvastatin, lovastatin and mevastatin inhibited +SA cell
growth in a dose-responsive manner, whereas similar doses of
pravastatin had no effect on +SA cell growth.
2
1
0
0.75
T3 (M)
epithelial cells.
3
C
Mev
T3
0.25M 2M
Mev 0.25 +
0.25
0.5
0
0.75
T3 (M)
1
2
C
Prav
10 M
T3
2M
Prav 10 +
0.25
0.5
0.75
T3 (M)
1
2
Figure 1. Cytotoxic Study of the combination. +SA cell viability following 24hr treatment with combination of 0.25µM of
simvastatin, lovastatin or mevastatin, or 10µM pravastatin and 0.25-2.0µM of γ-tocotrienol. Data points indicate mean viable cells +
SEM.
3. Combination of subeffective doses of statins and γ-tocotrienol
resulted in a significant synergistic dose-dependent inhibition
of +SA cell growth.
4. Combined treatment of statins and -tocotrienol may provide
effective anticancer therapy without the adverse side effects
associated with administration of high statin doses.