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Experimental Therapeutics, Preclinical Pharmacology |
ynarczukkowiec-Iskra
witajosa
Departments of Immunology [W. F., I. M., E. Z. B-I., A. C., T. ., T. S., A. G., M. J.] and Histology and Embryology [I. M.], Institute of Biostructure, PL-02-004 Warsaw, Poland, and Department of Pediatric Pneumonology, Allergic Diseases and Hematology, The Medical University Children’s Hospital, PL-01-184 Warsaw, Poland [W. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IFN-
) or chemotherapeutic
drugs (cisplatin). In the present report, we show in three murine tumor
cell lines (Colon-26 cells, v-Ha-ras-transformed NIH-3T3
sarcoma cells, and Lewis lung carcinoma cells) that lovastatin can also
effectively potentiate the cytostatic/cytotoxic activity of
doxorubicin. In three tumor models (Colon-26 cells,
v-Ha-ras-transformed NIH-3T3 sarcoma cells, and Lewis
lung carcinoma cells) in vivo, we have demonstrated
significantly increased sensitivity to the combined treatment with both
lovastatin (15 mg/kg for 10 days) and doxorubicin (3 x 2.5 mg/kg;
cumulative dose, 7.5 mg/kg) as compared with either agent acting alone.
Lovastatin treatment also resulted in a significant reduction of
troponin T release by cardiomyocytes in doxorubicin-treated mice. This
observation is particularly interesting because lovastatin is known to
reduce doxorubicin-induced cardiac injury. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(11)
in murine tumor models. Because lipid-lowering agents have been shown to reduce the cardiotoxic side effects of doxorubicin (14) , we decided to examine how lovastatin influences the antitumor effects of doxorubicin, and whether it will reduce the cardiotoxicity of doxorubicin.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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aw, Poland). Mice were bred in a local
animal facility and kept under standard conditions during the
experimental period. Experiments were performed after formal approval
by the Institutional Ethical Committee for Research on Animals.
Reagents.
Lovastatin in the inactive lactone form was obtained from Merck, Sharp
& Dohme Research Laboratories (Rahway, NJ). It was converted to the
active form by dissolving in ethanol, heating at 50°C in 0.1
N NaOH, and neutralizing with HCl. Distilled water was
added to a final concentration of 8 mg/ml. This stock solution was
stored frozen (-27°C).
Doxorubicin hydrochloride (Adriamycin) was obtained from Farmitalia Carlo Erba (Milan, Italy). The drug was diluted in PBS for in vitro experiments and in 0.9% NaCl for in vivo experiments to obtain the appropriate concentrations.
Cell Lines.
Four types of cells were used in this study: (a) Colon-26
colon adenocarcinoma cells; (b)
LLC3
cells;
(c) v-Ha-ras-transformed NIH-3T3 sarcoma cells
(referred to hereafter as Ras-3T3 cells); and (d)
nontransformed NIH-3T3 cells.
Murine LLC cells (syngeneic to C57BL/6) were provided by Dr. C.
Radzikowski (Institute of Immunology and Experimental Medicine,
Wrocaw, Poland). Murine colon adenocarcinoma cells (Colon-26 cells,
syngeneic to BALB/c) were purchased from the American Type Culture
Collection.
The Ras-3T3 and parental nontransformed NIH-3T3 cell lines were provided by Dr. H. Maruta (Ludwig Institute for Cancer Research, Victoria, Australia). The NIH-3T3 cell line is a nonmalignant murine fibroblast cell line derived from NIH Swiss mouse embryo culture. The Ras-3T3 cells were obtained as described previously (15) . Briefly, normal NIH-3T3 fibroblasts were transfected with the v-Ha-ras oncogene inserted into the mammalian retroviral vector pMV7, leading to tumorigenic Ras-3T3 cell line. Tumorigenicity of transformed cells was assessed in the BALB/c mice model, which indirectly confirmed stability of the transfection.
Cells were maintained in either DMEM or RPMI 1640 (Life Technologies, Inc., Paisley, Scotland) supplemented with antibiotics, 2-mercaptoethanol (50 µM), L-glutamine (2 mM), and 10% FCS (all from Life Technologies, Inc.; culture medium) and passaged every 3–4 days.
MTT Assay.
The cytostatic/cytotoxic effects of lovastatin and/or doxorubicin on
Ras-3T3, NIH-3T3, Colon-26, and LLC cells in vitro were
tested in a standard MTT assay. Portions (100 µl) of Ras-3T3 (4 x 103) and NIH-3T3 (9 x
103) cells as well as Colon-26 (6 x
103) and LLC (5 x
103) cells were dispensed into 96-well microtiter
plates (Corning, Bibby Sterlin, Ltd., Staffordshire, England). The
plates were incubated overnight at 37°C in 5%
CO2, and then serial dilutions of doxorubicin (50
µl; final concentration, 0.025–1 µM) and/or
lovastatin (50 µl; final concentration, 0.1–4
µM) were added in quadruplicate to a final
volume of 200 µl. After an incubation period of 24 h, the medium
was removed, and the cells were washed three times in culture medium.
Serial dilutions of lovastatin (100 µl; final concentration, 0.1–4
µM) were added and supplemented with culture
medium to a final volume of 200 µl. Different incubation periods for
doxorubicin and lovastatin were used to obtain comparable inhibition of
cell proliferation in all experiments, and these concentrations
correspond to those observed in serum of cancer patients
undergoing Phase I trial with lovastatin (12)
.
After an incubation period of 48 h, a standard MTT assay was
performed. Briefly, 25 µl of MTT (Sigma, St. Louis, MO) solution were
added to each well. The plates were centrifuged 4 h later
(350 x g, 10 min), and 200 µl of supernatant were
carefully removed from the wells and replaced with 200 µl of acid
DMSO. Complete solubilization of formazan crystals was achieved by
repeated pipetting of the solution. The plates were read on an ELISA
reader (SLT-Labinstruments Ges. m.b.H., Salzburg, Austria) using
a 550 nm filter. The means and SDs were determined for quadruplicate
samples. The cytostatic/cytotoxic effect of doxorubicin and/or
lovastatin was expressed as the relative viability (percentage of
control) and was calculated as shown below.
 | (1) |
Drug Interaction Analysis.
To examine the interaction between lovastatin and doxorubicin, the
isobolanalysis was used as described by Berenbaum (16)
.
Briefly, inhibition of cell proliferation was determined as described
above. Equieffective concentrations [concentrations of either drug,
alone or in combination, that gave equivalent inhibition of cell growth
as compared with untreated control cells at P 
0.05
(Student’s t test)] were analyzed. The interaction index
for combinations of the two drugs was computed according to the
following equation:
 | (2) |
Synergy occurs when the interaction index is less than 1.0.
Animal Experiments.
On the inoculation day, the tumor cells were harvested from the
cultures and washed, and then 5 x 105 Ras-3T3 or LLC
cells or 1 x 105 Colon-26 cells in 20 µl of medium
were injected s.c. into the footpad of the right hind limb.
Mice were injected with lovastatin and/or doxorubicin in two different regimens: (a) starting at day 5 after inoculation of tumor cells (for Colon-26 tumor); and (b) starting at day 7 after inoculation of tumor cells (for Ras-3T3 and LLC tumors), after the tumor nodules became visible in the footpads.
Local tumor growth was determined by measuring footpad diameter with
calipers every other day, starting with the first day of treatment (day
5 or 7 after inoculation of tumor cells - initial tumor volume). Tumor
volume was estimated by the formula below.
 | (3) |
Relative tumor volume was calculated as follows:
 | (4) |
where % VolX represents mean relative tumor volume on day X, VolX represents mean tumor volume on day X, and VolIni represents mean initial tumor volume.
Treatment and Monitoring.
To investigate the effects of both drugs, tumor-bearing mice were
divided into four groups (6–7 mice/group) and injected with
either: (a) saline; (b) lovastatin (15 mg/kg/day,
i.p.; 10 consecutive days); (c) doxorubicin [2.5 mg/kg,
i.p., in three doses (on the first, fifth, and ninth day of therapy);
or (d) a combination of both drugs (15 mg/kg lovastatin i.p.
for 10 consecutive days + 2.5 mg/kg doxorubicin i.p. on the first,
fifth, and ninth day of therapy).
Tumor growth delay was determined according to the method of Corbett
et al. (17)
and calculated as follows:
 | (5) |
where T represents the median time (in days) required for the treatment group tumors to reach a certain volume (in mm3; different for various types of tumors), and c represents the median time (in days) required for the control group tumors to reach the same size.
Mice were observed daily for survival.
Serum cTnT Levels.
B6D2F1 mice were randomized and divided into four groups (5–6
mice/group), and the therapy protocol was introduced as
presented in Fig. 1
. Blood was drawn from
all mice on days 1, 15 (before the initial dose of doxorubicin), 21
(after two doses of doxorubicin; cumulative dose, 5 mg/kg), and 35
(after six doses of doxorubicin; cumulative dose, 15 mg/kg) of the
experimental cycle. Serum levels of cTnT were measured using a
commercially available cTnT ELISA kit (Enzymun-Test Troponin-T;
Boehringer Mannheim GmbH, Mannheim, Germany) according to
manufacturer’s instructions.
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Statistical Analysis.
Differences between the results of the in vitro tests and
between tumor diameters in the in vivo experiments were
examined and analyzed primarily by Student’s t test and
Mann-Whitney test (two-tailed). The resulting data from our in
vivo studies were also analyzed using the nonparametric ANOVA
(Kruskal-Wallis) test, followed by a Dunn’s multiple comparisons test.
In addition, the data from cTnT studies were analyzed with the
nonparametric repeated measures ANOVA (Friedman) test (all computed
using Instat 2.0; GraphPad Software Inc., San Diego, CA).
P < 0.05 was considered to be statistically
significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Moreover, in all
presented cell lines, lovastatin and doxorubicin demonstrated
potentiated cytostatic/cytotoxic effects in vitro when used
in combination (Fig. 2, A–C)
. Ha-ras-transformed
Ras-3T3 cells were more sensitive to lovastatin than other tumor cells.
|
. Thus, the results of our in
vitro experiments suggest that potentiated cytostatic/cytotoxic
effects of combination therapy with lovastatin and doxorubicin may be
due, at least in part, to the synergistic cytostatic/cytotoxic
interaction of these two drugs acting in Colon-26 tumor cells.
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, Ras-3T3 cells were significantly less
sensitive (P < 0.05 Mann-Whitney test) to its action
as compared with the nontransformed NIH-3T3 cells.
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Lovastatin and Doxorubicin Demonstrate Potentiated Antitumor
Effects When Used in Combination.
To examine whether our in vitro observations could have
potential therapeutic significance, animal studies were performed. A
subthreshold dose of lovastatin in these experiments was established at
15 mg/kg body weight (data not shown). This dose of lovastatin, given
i.p., did not exert any significant antitumor effects against either of
the examined tumors. In the preliminary dose-toxicity study, the
maximum tolerated dose of doxorubicin was established as a total of 7.5
mg/kg body weight (three injections of 2.5 mg/kg doxorubicin; data not
shown). This dose was used in experiments where the combined effects of
lovastatin and doxorubicin were tested. Although doxorubicin when used
alone at this dose did not inhibit tumor growth in two of the three
tumors studied (Colon-26 and LLC tumors), a significant antitumor
effect came to light when lovastatin was included in the therapeutic
regimen (Fig. 5, B and C)
. The resulting significant retardation of Colon-26
adenocarcinoma growth in mice treated with lovastatin combined with
doxorubicin, as compared with each of the remaining groups, was
observed on days 15 (P 0.005, ANOVA), 17
(P < 0.01, ANOVA), and 19, 21, and 25
(P 0.05, ANOVA; Fig. 5B)
. Similar effects
were observed in LLC carcinoma tumors, and beginning on day 11, a
significant retardation of tumor growth was observed in mice treated
with lovastatin in combination with doxorubicin as compared with each
of the remaining groups (days 11–23, P 0.005,
ANOVA; day 25, P 0.05, ANOVA; Fig. 5C)
.
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0.05,
ANOVA), and this effect lasted for 6 days (Fig. 5A)
. No
apparent toxic effects were observed in any of the experimental groups.
To further evaluate the observed antitumor effects, we also assessed
the tumor growth delay. The median tumor growth delay for mice treated
with lovastatin was between 0 and 2 days, and the median tumor growth
delay for mice treated with doxorubicin was between 0 and 4 days (Table 1)
. However, in all of our experiments,
combined treatment with lovastatin and doxorubicin resulted in
significant retardation of tumor growth, and the tumor growth delay was
6 days in three tumor models (Table 1)
. This delay in tumor growth was
higher than a simple addition of the delays produced by any of the
agents used alone, suggesting a synergistic mode of action for this
therapeutic combination.
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Serum cTnT Indicates Protective Activity of Lovastatin on
Doxorubicin-induced Cardiac Injury.
Serum levels of cTnT become elevated in patients with minimal
myocardial cell damage (18)
, making this enzyme an
attractive, noninvasive marker of anthracycline-related cardiotoxicity
both in humans (19)
and in murine experimental models
(20)
. Therefore, in our experiments, we monitored serum
levels of cTnT during combination therapy with lovastatin and
doxorubicin. Pretreatment cTnT level (number of animals, 21) was
0.29 ± 0.03 ng/ml (range, 0.0–0.67 ng/ml). This level remained
relatively unchanged in animals receiving lovastatin or saline solution
for 2 weeks (0.53 ± 0.05 and 0.28 ± 0.06 ng/ml,
respectively; Fig. 6
). As shown in
Fig. 6
, a statistically nonsignificant increase in cTnT level
(3.96 ± 2.74 ng/ml; range, 0.67–14.87 ng/ml) was observed in
mice treated for 1 week with a cumulative dose of 5 mg/kg doxorubicin
(Friedman test, nonparametric repeated measures ANOVA). However,
increasing the cumulative dose of doxorubicin to 15 mg/kg resulted in
an increased cTnT concentration (4.79 ± 3.07 ng/ml; range,
0.88–16.76 ng/ml). This concentration was significantly greater than
that found in mice before the initial dosing of doxorubicin
(P < 0.01, Friedman test, nonparametric repeated
measures ANOVA).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our in vitro studies, lovastatin was used in combination with doxorubicin. This combination was found to be more effective in decreasing the viability of murine Ras-3T3, Colon-26, and LLC tumor cells than each agent alone. Furthermore, in the case of Colon-26 cells, a synergy of these two drugs was revealed. As suggested recently (23) , lovastatin may increase the vulnerability of tumor cells to the action of other chemotherapeutic agents by specifically targeting drug-resistant P-glycoprotein-expressing tumor cells. This observation may be considered as a possible explanation for the increase in cytotoxicity of doxorubicin in combination with lovastatin in our study, although an augmentation of apoptosis induced by both agents cannot be ruled out (13) .
In this study, Ras-3T3 cells were significantly less sensitive to the action of doxorubicin compared with nontransformed NIH-3T3 cells. These results are in accordance with previous reports showing resistance to doxorubicin in tumor cells transformed by ras oncogene (24 , 25) . Lovastatin was able to reverse the resistance of Ras-3T3 cells to doxorubicin. Restoration of doxorubicin sensitivity in ras-transformed tumor cells by lovastatin has been observed previously in osteosarcoma cells (26) . The significance of these observations may be underscored by the high frequency of ras mutations present in human tumors (27) .
We then examined whether the strengthened antitumor effects of
combination treatment with doxorubicin and lovastatin observed in
vitro could have any relevance for in vivo tumor
therapy. In all three tumor models, lovastatin potentiated the
antitumor activity of doxorubicin, resulting in retardation of tumor
growth as compared with both agents used alone and suggesting
synergistic augmentation of antitumor effectiveness in mice treated
with a combination of both drugs. It should be mentioned that
lovastatin has already been shown to increase the antitumor activity of
cisplatin (10)
and tumor necrosis factor
(11)
and to enhance apoptosis induced by cisplatin,
5-fluorouracil (13)
, and sulindac in colon cancer cells
(28)
. In another study, simvastatin, which also belongs to
the statin family, potentiated the cytostatic effects of IFN-ß and
N,N'-bis(2-chlorethyl)-N-nitrosourea
in human glioma cells (29)
.
Our results showing the ability of lovastatin to potentiate the antitumor effects of doxorubicin seem particularly interesting because lovastatin has already been suggested to attenuate but not completely prevent some of the cardiotoxic side effects of doxorubicin (14) . It should be stressed that lovastatin has been used extensively and has well-defined pharmacokinetics at the clinical level, displaying negligible adverse side effects (30) .
There are other drugs that prevent doxorubicin-induced cardiotoxic effects (31, 32, 33, 34) , and some of them seem to be more effective compared with lovastatin as far as their cardioprotective properties are concerned (14) . However, our report is the first to show the ability of a single drug to both potentiate the antitumor activity of doxorubicin and reduce its cardiotoxicity.
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ACKNOWLEDGMENTS |
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bieta Gutowska and Anna
Czerepi
ska for technical assistance. |
FOOTNOTES |
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1 Supported by Grant 6 P207 058 07 from the State
Committee for Scientific Research, Grant 3212/97/GB from The Foundation
of Polish-German Co-operation (Stiftung für Deutsch-Polnische
Zusammenarbeit), and Grants 1W34/S/99 and II-D/83 from The Medical
University of Warsaw (to W. F.). 
2 To whom requests for reprints should be
addressed, at Department of Pediatric Pneumonology, Allergic Diseases
and Hematology, The Medical University Children’s Hospital, ul.
Dziadowska 1/3, PL-01-184 Warsaw, Poland. Phone/Fax:
48-22-632-07-23; E-mail: wfeleszk{at}ib.amwaw.edu.pl
3 The abbreviations used are: LLC, Lewis lung
carcinoma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; cTnT, cardiac troponin T.
Received 10/22/99; revised 2/ 2/00; accepted 2/ 2/00.
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, P 
) lovastatin (15 mg/kg/day, i.p.) and/or (open
arrow) doxorubicin (2.5 mg/kg/dose, i.p.). Logarithmic
(log10) scaling. Data represent the mean relative tumor
volume ± SE. *, P 