Antineoplastické účinky simvastatínu u experimentálnej rakoviny prsníka
Východiská: Statíny (inhibítory 3-hydroxy-3-metylglutaryl koenzým A reduktázy) predstavujú látky s dobre dokumentovanými terapeutickými a preventívnymi účinkami u kardiovaskulárnych ochorení. V predklinických štúdiách statíny preukázali tumor-supresívne účinky u viacerých typov neoplázií vrátane rakoviny prsníka.
Materiál a metódy: V tejto štúdii sme hodnotili antineoplastický účinok simvastatínu v chemoprevencii N-metyl-N-nitrozoureou – indukovanej mamárnej karcinogenézy u samíc potkanov. Farmakum bolo aplikované v potrave vo dvoch koncentráciách – 18 mg/ kg (SIMVA 18) a 180 mg/ kg (SIMVA 180).
Výsledky: Po dlhodobej aplikácii simvastatínu sme na konci pokusu vyhodnotili základné parametre experimentálnej karcinogenézy. Simvastatín v skupine SIMVA 180 signifikantne znížil frekvenciu nádorov o 80,5 % a incidenciu nádorov o 58,5 % v porovnaní s kontrolou. Simvastatín v tej istej skupine zvierat nesignifikantne znížil priemerný objem nádorov o 23,5 % a nesignifikantne predĺžil latenciu o 14,5 dňa v porovnaní s kontrolnými zvieratami. U simvastatínu podávaného v nižšej dávke sme nepozorovali antineoplastické účinky. Simvastatín v oboch liečených skupinách signifikantne znížil sérové hladiny triacylglycerolov a VLDL-cholesterolu v porovnaní s kontrolnými zvieratami. V porovnaní s kontrolami sme v skupinách SIMVA 18 a SIMVA 180 pozorovali signifikantný nárast príjmu potravy u zvierat. Signifikantné zmeny prírastku telesnej hmotnosti medzi skupinami so simvastatínom a kontrolnou skupinou neboli zistené.
Záver: Táto štúdia je prvou zmienkou o simvastatíne použitom v experimentálnej mamárnej karcinogenéze in vivo.
P. Kubatka 1,2; K. Žihlavniková 1; K. Kajo 3; M. Péč 1,2; N. Stollárová 2; B. Bojková 4; M. Kassayová 4; P. Orendáš 4
Authors place of work:
Department of Medical Biology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic
1; Department of Biology and Ecology, Faculty of Education, Catholic University in Ružomberok, Slovak Republic
2; Department of Pathological Anatomy, Jessenius Faculty of Medicine, Comenius University, Martin, and BB Biocyt, Diagnostic Centre, Ltd., Banská Bystrica, Slovak Republic
3; Department of Animal Physiology, Institute of Biological and Ecological Sciences, Science Faculty, P. J. Šafárik University, Košice, Slovak Republic
Published in the journal:
Klin Onkol 2011; 24(1): 41-45
Backgrounds: Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have proven therapeutic and preventive effects on cardiovascular diseases. Preclinical evidence demonstrates tumor-suppressive effects of statins in several human neoplasias, including breast cancer.
Materials and Methods: In this study, antineoplastic effects of simvastatin in chemoprevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated. The drug was dietary administered at two concentrations – 18 mg/kg (SIMVA 18) and 180 mg/kg (SIMVA 180).
Results: Basic parameters of experimental carcinogenesis after long-term simvastatin treatment in animals were assessed. In the SIMVA 180 group, simvastatin significantly suppressed tumour frequency by 80.5% and tumour incidence by 58.5% in comparison to the controls. Higher dose simvastatin non-significantly decreased the mean tumor volume by 23.5%, as well as non-significantly lengthened the latency period by 14.5 days compared to the control animals. Simvastatin, administered at a lower dose did not change parameters of mammary carcinogenesis in comparison to the control group. Simvastatin in both treated groups significantly decreased serum levels of triacylglycerols and VLDL-cholesterol in comparison to the control animals. Compared to the controls, a significant increase in food intake by the animals was recorded in the SIMVA 18 and SIMVA 180 groups. No significant differences in the final body weight gain between the simvastatin-administered and the control group were found.
Conclusion: This study represents the first report of simvastatin use in experimental mammary carcinogenesis in vivo.
Key words: mammary carcinogenesis – rat – chemoprevention – simvastatin
for non-melanoma skin cancers, breast cancer is the neoplasia with highest
incidence in females all over the world. Chemoprevention is assumed to become
an effective way to combat the above neoplasia. The aim of the chemopreventive
trials is to find an efficient substance that can be administered for
a long period with minimum adverse effects. The statins are highly
effective drugs in lowering cholesterol by inhibiting
3-hydroxy-3-methylglutaryl coenzyme A reductase. The statins have been
shown to decrease the incidence of adverse cardiovascular events, including
death, myocardial infarction, stroke, atrial fibrillation and renal
dysfunction. However, increasing evidence suggests that statins exert
pleiotropic effects in organism, independent of cholesterol reduction.
Recent pre-clinical in vitro studies
have proven direct or indirect effects of statins on regulation mechanisms of
the cell e. g. proliferation,
differentiation and apoptosis. These physiological processes play a key
role in neoplastic transformation; therefore statins is being seriously
discussed in oncology. Statins, through mevalonate, inhibit dolichol-,
farnesyl- and geranylgeranyl pyrophosphate production and block tumor cell
proliferation [1,2]. Lovastatin has been demonstrated to stabilize the cell
cycle kinase inhibitors p21 and p27 and to arrest breast cancer cell lines in
G1 phase of the cell cycle . Cerivastatin has been shown to inhibit Ras- and
Rho-mediated cell growth . Proposed mechanisms for statin-mediated apoptosis
include an upregulation of proapoptotic protein expression (e. g.,
Bax, Bim), combined with decreased antiapoptotic protein expression (e. g.,
Bcl-2) , or activation of caspase-3, caspase-8, and caspase-9 .Angiogenesis
play an important role in the growth of primary tumors and metastasis.
High-dose of cerivastatin decreased tumor vascularisation by 51% in
a murine Lewis lung cancer model .Statins
have been shown to decrease vascular endothelial growth factor production and
to inhibit capillary tube formation . Several lines of evidence suggest that
statins impair the metastatic potential of tumor cells. Statins have been
demonstrated to reduce endothelial leukocyte adhesion molecule E-selectin 
and matrix metalloproteinase-9 expression . Fluvastatin and lovastatin
reduced liver tumorigenesis and liver metastases in pancreatic cancer cells
; atorvastatin decreased melanoma cell metastases .
Also data from experimental studies in
vivo indicated antineoplastic effects of statins in rodent colon  and
hepatal  carcinogenesis. Actual results of our group demonstrated an
apparent antineoplastic effect of dietary administered atorvastatin in the
chemoprevention of rat mammary carcinogenesis [Kubatka et al., unpublished
results]. Epidemiologic studies [13–17] and several human clinical trials have
reported beneficial effects of statins in certain neoplasias [18–20].
Antitumor properties of statins in human
breast cancer have not been tested so far. Original experimental studies are necessary,
which should answer the question about expected tumor suppressive effects of
statins in mammary carcinogenesis. The aim of this study is to evaluate the
chemopreventive potential of simvastatin in rat mammary carcinogenesis. The
adverse effects of the drug after long-term treatment will be assessed.
rats of Sprague-Dawley strain obtained from AnLab (Prague, Czech Republic) aged
31–35 days were used in the experiment. The animals were adapted to standard
vivarium conditions with temperature 23 ± 2 °C, relative humidity 60–70%,
artificial regimen light : dark (12 h : 12 h)
(lights on from 6 a. m., light intensity
150 lux per cage). During the experiment animals drank tap water ad
libitum. The chow containing simvastatin synthesized
by Zentiva (Prague, Czech Republic) was prepared at SSNIFF Spezialdiäten GmbH
(Soest, Germany). Simvastatin was administered in the chow at two
concentrations – 18 mg/ kg
(0.0018%) and 180 mg/ kg
carcinogenesis was induced by N-methyl-N-nitrosourea (Sigma, Deisenhofen,
Germany) administered intraperitoneally in one dose of 50 mg/ kg body weight on
average the 41th postnatal day. Carcinogen was freshly prepared and
dissolved in isotonic saline solution.
simvastatin began 8 days before carcinogen administration and lasted until the
end of the experiment – 17 weeks after N-methyl-N-nitrosourea (NMU) application. Animals were
randomly assigned to one of three experimental groups: 1. control group
without chemoprevention; 2. chemoprevention with simvastatin at
a concentration of 18 mg/ kg in the chow (SIMVA 18); 3. chemoprevention with
simvastatin at a concentration of 180 mg/ kg in the chow (SIMVA 180). Each group consisted of 20
animals. The animals were weekly weighted and since 6th week post
NMU palpated in order to register the presence, number, location and size of
each palpable tumor.
In the last – 17th
week of the experiment, the animals were quickly decapitated, mammary tumors
were excised and tumor size was recorded. Macroscopic changes in selected
organs (liver, kidney, stomach, intestine and lung) were evaluated at autopsy.
Tissue samples of each mammary tumor were fixed in 10% formol and prepared for
histological analysis. The tumors were classified according to the criteria for
the classification of rat mammary tumors . At sacrifice, the blood was collected from
each animal. The selected parameters of serum lipid metabolism were assessed.
The following basic parameters of mammary carcinogenesis were evaluated in each
group: tumor incidence as the percentage representation of tumor-bearing
animals, tumor frequency as the number of tumors per group, latency period
determined by the period from carcinogen administration to the appearance of first
tumor in an animal and average tumor volume. The effect of simvastatin on food,
water intake and final body weight gain was observed. Food and water intake of
animals during 24 hours in 7th and 14th week
administration were found out, overall in 4 measurements (twice in
a mentioned week). The simvastatin doses were calculated in accordance
with the amount of chow consumed.
Tumor incidence was
evaluated by Mann-Whitney test, other parameters by one-way analysis of
variance or Kruskal-Wallis test. Tumor volume was calculated according to:
V = π . (S1)2 . S2/ 12; S1 and
S2 are tumor diameters (S1 < S2).
The experiment was
approved by Ethical Commission of Jessenius Faculty of Medicine of Comenius
University (Protocol No. EK 320/2007) and by State Veterinary and Food
Administration of the Slovak Republic (accreditation No. Ro-2061/08–221). This
work was supported by the Scientific Grant Agency of the Ministry of Education
of the Slovak Republic under contract no. VEGA 1/0029/08.
Apparent tumor-suppressive effects of simvastatin in the
chemoprevention of rat mammary carcinogenesis are summarized in Tab. 1. The
continuous development of tumor incidence and frequency is
presented in Graph 1 and Graph 2, respectively. In experimental group SIMVA
180, simvastatin decreased the incidence by 58.5 % (P = 0.023), frequency
by 80.5 %
= 0.013) and average tumor volume by 23.5 % (P = 0.738), and lengthened the latency by 14.5 days
(P = 0.163) in comparison with the control animals. Chemoprevention with
simvastatin beneficially shifted the rate of malignant to benign lesions in the
group SIMVA 180 (43% : 57%) in comparison with untreated control group (92% :
8%). In comparison with the control group, simvastatin administered at
a lower dose in experimental group SIMVA 18 did not significantly change
the monitored parameters of experimental rat mammary carcinogenesis.
No macroscopic changes
due to simvastatin administration in the selected organs – liver, kidney,
stomach, intestine and lung were observed. With regard to plasma lipid
metabolism, simvastatin in both treated groups significantly decreased the levels
of triacylglycerols (P = 0.041, resp. P < 0.0001 in SIMVA 18, resp. SIMVA
180) and VLDL-cholesterol (P = 0.035, resp. P < 0.0001 in SIMVA 18, resp.
SIMVA 180) in comparison with the controls. The total cholesterol, HDL- and
LDL-cholesterol serum levels were not changed in animals. The evaluation of
final body weight gain did not reveal significant changes in animals with
administered simvastatin compared to control animals. Average daily food intake
per rat in all experimental groups was between 17.7–19.0 g of the chow. Compared
to controls, a significant increase in food intake of animals in the group
SIMVA 18 (P = 0.025) and SIMVA 180 (P = 0.013) were found. Daily average dose of
simvastatin per rat was 0.34 mg in the group SIMVA 18, and 3.42 mg in group SIMVA 180.
This study is the first report
about simvastatin – a 3-hydroxy-3-methylglutaryl coenzyme A reductase
inhibitor, used in experimental rat mammary carcinogenesis. A substantial
chemopreventive effect of simvastatin administered in the concentration of 180 mg/ kg of the diet was
recorded in all evaluated parameters in rat mammary carcinogenesis. In order to
choose the optimal simvastatin doses in this experiment we took into
consideration daily doses of the drug in clinical practice. The lower
concentration of simvastatin – 18 mg/ mg in our experiment was equivalent to daily dose
of the drug (40 mg/day) administered to patients
with hypercholesterolemia. On our previous
experience with atorvastatin [Kubatka et al., unpublished results] we have used
also the 10 times higher concentration of simvastatin in the diet (180 mg/ kg) and this dose has
been shown to be very effective in this experiment.
antitumor effects of atorvastatin administered in the chemoprevention of
NMU-induced rat mammary carcinogenesis in our previous study were observed
[Kubatka et al., unpublished results]. Dietary administered atorvastatin in the
dose of 100 mg/ kg (concentration of 0.01%) significantly decreased
tumor frequency by 80.5% and tumor incidence by 49.5%, and lengthened latency
by 14 days in comparison with control animals. Our study pointed to fact that
antineoplastic effect of atorvastatin in rat mammary carcinogenesis is
independent from its effects on plasma lipid metabolism: atorvastatin in both
concentrations in the diet did not change the serum levels of triacylglycerols,
total cholesterol, and LDL-cholesterol. Narisawa et al in
1,2-dimethylhydrazine-induced colon carcinogenesis in ICR mice, used as
a chemopreventive agent dietary administered simvastatin at
concentrations of 0.01% (100 mg/ kg) and 0.002% (20 mg/ kg) and pravastatin administered in drinking water at
concentrations of 0.01%, 0.001% and 0.005%. Simvastatin and pravastatin (with
exception of pravastatin concentration of 0.001%) significantly reduced tumor
frequency; the tumor incidence was reduced non-significantly by both agents.
Anticarcinogenic effects of statins were proved also in other in vivo
experiments. Pravastatin administered in drinking water has been shown to
reduce the incidence and volume of N-nitrosomorpholine-induced hepatic
neoplastic nodules in Sprague-Dawley rats  and to reduce
N-methyl-N-nitrosourea induced F344 rat colon carcinogenesis . On the other
hand, an actual paper of Lubet et al  reported about dietary administered
atorvastatin and lovastatin either as single agents or in combination with
suboptimal doses of tamoxifen or rexinoid bexarotene in the prevention of NMU –
induced rat mammary carcinogenesis. Atorvastatin alone in this experiment in
high doses of 125 and 500 mg/ kg of chow did not significantly alter incidence and
frequency of mammary tumors. Combining atorvastatin (500 mg/ kg diet) with either of
tamoxifen and bexarotene minimally altered their efficacy. Lovastatin in the
doses of 100 and 400 mg/ kg diet yielded similar results as atorvastatin with
limited oncostatic effects administered alone, without obvious synergy with
tamoxifen or bexarotene . The results of both above mentioned experiments
with atorvastatin and lovastatin of Lubet`s group are in strong contrary with
the apparent antineoplastic effects of atrovastatin or simvastatin observed in
In this experiment, a significant
antineoplastic effect of simvastatin in rat mammary carcinogenesis could be
explained by several mechanisms. Above cited results from preclinical research
suggested that statins have antiproliferative, antiangiogenic and
antimetastatic properties. In addition, data from experimental studies in vitro demonstrated the link between statin application and apoptosis
induction in various human cells [3,4,24]. In order to prove proapoptotic
effects of atorvastatin in our previous study with atorvastatin [Kubatka et
al., unpublished results], the specimens of each mammary tumor from all
experimental groups were evaluated for the mRNA expression of anti-apoptotic
Bcl-2 and pro-apoptotic Bax genes. In this regard, a significant
pro-apoptotic shift of ratio in Bax/Bcl-2 mRNA expression in mammary tumors
after atorvastatin treatment (concentration of 0.01% in the diet) in our
experiment was confirmed.
Although the favourable
effects of statins in the prevention of cardiovascular diseases resulting from
hypercholesterolemia are well established, the increasing evidence suggests,
that these drugs exert pleiotropic effect independent of cholesterol reduction.
Based on favourable results from oncological research, statins may thus
represent a novel clinical approach for cancer risk reduction or maybe
treatment. Several questions are unanswered about the role of statins in cancer
patients. It is unknown, which types of tumors are responsive to statin
therapy. Actual experimental data suggested that statins may be potentially
effective in the treatment of melanoma, leukaemia, brain cancer, hepatocellular
cancer and squamous cell cancer of the head and neck . Further, it is not
known, which statins are most effective in carcinogenesis – hydrophilic statins
(pravastatin, rosuvastatin) or lipophilic statins (atorvastatin, simvastatin,
fluvastatin, lovastatin). Finally, the optimal statin regimens were not defined
yet. Statins administered in combination with other oncostatic substances may
enhance tumor suppressive effects. In order to reduce statin adverse effects
(myopathy, hepatotoxicity, rhabdomyolysis), it is favoured continuous low-dose
drug clinical regimens.
Pleiotropic properties of statins with proven
anticarcinogenic effects in human cells can open a new era in clinical
medicine. The results of this study clearly pointed to simvastatin favourable
effects in experimental rat mammary carcinogenesis and gave the drug
a chance to become a substance with chemopreventive efficacy in various
neoplasias including breast cancer. Our experiment provided a rationale
for the use of the 3-hydroxy-3-methylglutaryl coenzyme A reductase
inhibitor simvastatin in women who require the treatment of
hypercholesterolemia and moreover are high-risk for breast cancer.
This work was supported by the Scientific Grant Agency of the Ministry of
Education of the Slovak Republic under contract no. VEGA
Táto práca bola podporená Vedeckou grantovou agentúrou Ministerstva
školstva Slovenské republiky pod č. VEGA 1/0029/08.
Autoři deklarují, že v souvislosti s předmětem studie nemají žádné komerční
The authors declare they have no potential conflicts of interest
concerning drugs, products, or services used in the study.
Redakční rada potvrzuje, že rukopis práce splnil ICMJE kritéria pro
publikace zasílané do bi omedicínských časopisů.
The Editorial Board declares that the manuscript met the ICMJE “uniform
requirements” for biomedical papers.
Assoc. Prof. RNDr. Peter Kubatka,
Department of Medical Biology
Jessenius Faculty of Medicine
Malá Hora 4
036 01 Martin
Obdrženo/Submitted: 8. 4. 2010
Přijato/Accepted: 16. 8. 2010
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