1. • Ovarian cancer is the fifth most common cancer in
women with an overall 5-year survival rate < 35% [1].
• Statins inhibit HMG-CoA reductase in the
mevalonate pathway which is involved in cholesterol
synthesis (Figure 1).
Figure 1: The Mevalonate Pathway
• Studies have reported the anti-proliferative and pro-
apoptotic effects of atorvastatin, lovastatin,
simvastatin, mevastatin and fluvastatin in ovarian
cancer cells [2-6].
• Anti-cancer effects of statins are likely to result from
suppression of the mevalonate pathway.
− Leads to depletion of downstream products
including farnesyl diphosphate, geranylgeranyl
diphosphate and resulting prenylated proteins
that have roles in cell cycle progression, cell
signalling and membrane integrity.
• Autophagy is a process involved in the degradation
of cellular organelles and proteins during nutrient
starvation and metabolic stress.
• Involves the formation of autophagosomes
containing cellular debris, which fuse with lysosomes
resulting in the degradation of damaged cargo
(Figure 2).
Figure 2: The Autophagic Pathway [7]
• Two geranylgeranylated proteins, Rab5 and Rab7
are involved in autophagy. Rab5 is required for the
formation of autophagosomes whereas Rab7 is
involved in the late stages of autophagosome
maturation.
• Co-treatment of lovastatin and a farnesyl transferase
inhibitor reduced the prenylation of Rab5, which
prevented the membrane localisation and function of
Rab5, thereby inhibiting autophagy in nerve sheath
tumour cells [8].
Concentrations required to inhibit cell growth and
induce apoptosis were similar to those reported in
earlier studies [3-5], but at concentrations significantly
above that obtained with the dose commonly used to
treat hypercholesterolaemia. Simvastatin retained
anti-cancer activity in spheroid models and the
reduction in cellular ATP after simvastatin treatment
indicates the drug is cytotoxic at these concentrations.
These effects were mediated through the mevalonate
pathway and resulted in cell death at least partly
through apoptosis.
The contribution of autophagy to the activity of statins
is complex. Autophagy may allow cancer cells to
survive, so its inhibition by statins may contribute to
the anti-cancer activity that we have observed.
However, in one cell line, simvastatin reduced p62,
suggesting stimulation of autophagy. This may
indicate that statins have effects at different points of
the autophagy pathway.
We found that combining simvastatin with
chemotherapy was modestly antagonistic in all cell
lines. However, the antagonism was profound when
cells were pre-treated with simvastatin. Whilst
antagonism did not occur at simvastatin plasma
concentrations achieved from doses used clinically for
hypercholesterolaemia, much higher concentrations
appear to be necessary for anti-cancer activity. The
antagonism observed with chemotherapy suggests
that statins should be evaluated in clinical trials at high
doses as single-agent therapy.
Conclusion
0
1
2
3
4
- + - + - + - + - + - +
- - + + - - + + - - + +
A2780 Ovcar-5 Ovcar-8
Relativecaspaseactivity
Inhibition of autophagy was supported by a decrease
in the levels of the geranylgeranylated protein
involved in autophagy, Rab7, following simvastatin
treatment (Figure 8).
Figure 8: Rab7 immunofluorescence in Ovcar-8 cells.
Simvastatin in combination with carboplatin or
paclitaxel showed modest antagonism (CI > 1) at high
concentrations, which was most pronounced when
cells were pre-treated with simvastatin (Figure 9).
However, concentrations of simvastatin achieved after
a standard 40 mg dose used to treat
hypercholesterolaemia did not antagonise
chemotherapy.
Figure 9: (Left) Simvastatin and chemotherapy combined at a fixed
ratio based on respective IC50 values; (Right) Ovcar-5 cells treated
with carboplatin or simvastatin for 48 h followed by the reverse
treatment for a further 48 h.
Results
Elizabeth Robinson, Mandrita Nandi, Laurelle Wilkinson, Anthony Curtis, Alan Richardson
Institute for Science and Technology in Medicine, Keele University, Staffordshire
References
1 NICE (2011) The recognition & initial management of ovarian cancer, www.nice.org.uk/guidance/CG122
2 Gauthaman, K. et al. (2007) Reproductive Biomedicine Online, 15 (5): 566-81
3 Liu, H. et al. (2009) Cancer Chemotherapy & Pharmacology, 63 (6): 997-1005
4 Kato, S. et al. (2010) Journal of Cellular & Molecular Medicine, 14 (5): 1180-93
5 Martirosyan, A. et al. (2010) BMC Cancer, 10: 103-15
6 Matsuura, M. et al. (2011) Oncology Reports, 25 (1): 41-7
7 Meléndez, A. and Levine, B. (2009) Autophagy in C. elegans, www.wormbook.org
8 Wojtkowiak, J. W. et al. (2011) Journal of Pharmacology & Experimental Therapeutics, 337 (1): 65-74
Control Simvastatin 3 µM
C
0.00
0.25
0.50
0.75
1.00
1.25
Carboplatin then simvastatin
Simvastatin then carboplatin
-6 -5 -4
Log carboplatin concentration (M)
Cellnumber(absorbance)
Aims
• Evaluate the potential use of statins for the treatment
of ovarian cancer both as single agents and in
combination with carboplatin or paclitaxel.
• Evaluate the mechanism underlying this.
C
0.00
0.25
0.50
0.75
1.00
1.25
Simvastatin
Simvastatin + M
Simvastatin + F
Simvastatin + G
-8 -7 -6 -5
Log simvastatin concentration (M)
Cellnumber(absorbance)
Most statins inhibited the growth of all cell cultures
evaluated (Figure 3). Pravastatin was inactive in all
cell lines (IC50 > 100 µM).
Figure 3: Anti-cancer effects of statins, as illustrated by the IC50
values, in ovarian cancer cell lines.
The activity of simvastatin was retained in ovarian
cancer spheroid models, with IC50 values of 1-15 µM
in several cell lines (Figure 4).
Figure 4: Anti-cancer effects of simvastatin in ovarian cancer
spheroids.
Addition of mevalonate and geranylgeraniol, but not
farnesol, suppressed the activity of simvastatin
(Figure 5).
Figure 5: Dose-response curve for simvastatin alone or with
mevalonate 100 µM (M), farnesol 10 µM (F) or geranylgeraniol 10
µM (G) supplementation in Ovcar-8 cells.
Simvastatin induced apoptosis in ovarian cancer cells,
which was inhibited by mevalonate (Figure 6).
Figure 6: Caspase activity following simvastatin 10 µM treatment with
or without mevalonate 100 µM.
Simvastatin increased LC3-II and p62 levels,
consistent with an inhibition of autophagy (Figure 7).
This was reversed by mevalonate and
geranylgeraniol, suggesting that inhibition of
geranylgeranylation may be the mechanism by which
simvastatin prevents autophagy.
Figure 7: (Left) Rab7, LC3-II and p62 levels following simvastatin 10
µM (+) or DMSO-treated control (–); (Right) Control or statin-treated
Ovcar-8 cells with mevalonate 100 µM (M), farnesol 10 µM (F) or
geranylgeraniol 10 µM (G).
Results
0
5
10
15
20
25
30
35
40
Rosuvastatin Simvastatin Atorvastatin Lovastatin Fluvastatin
IC50(μM)
A2780 Cis A2780 Ovcar-4 Ovcar-5 Ovcar-8 Igrov-1 Skov-3
Introduction
Rab7
LC3-II
p62
GAPDH
Statin – + – + – + – +
A2780 Ovcar-5 Igrov-1 Ovcar-8
– – – – + + + +
– M F G – M F G
HOW CAN STATINS BEST BE EVALUATED TO TREAT OVARIAN CANCER?
Simvastatin
Mevalonate
20 µm 20 µm
1.5 mm
0
5
10
15
20
A2780 Ovcar-5 Ovcar-8 Igrov-1
IC50(μM)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
A2780
cisA2780
Ovcar-4
Ovcar-5
Ovcar-8
Igrov-1
Skov-3
A2780
cisA2780
Ovcar-4
Ovcar-5
Ovcar-8
Igrov-1
Skov-3
Carboplatin Paclitaxel
ConfidenceInterval(CI)