In Vitro Cell Cycle Arrest, In Vivo Action on Solid Metastasizing

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0022-3565/99/2891-0559$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 289:559 –564, 1999
Vol. 289, No. 1
Printed in U.S.A.
In Vitro Cell Cycle Arrest, In Vivo Action on Solid Metastasizing
Tumors, and Host Toxicity of the Antimetastatic Drug NAMI-A
and Cisplatin1
A. BERGAMO, R. GAGLIARDI, V. SCARCIA, A. FURLANI, E. ALESSIO, G. MESTRONI, and G. SAVA
Callerio Foundation, Institutes of Biological Research (A.B., R.G., G.S.) and Departments of Biomedical Sciences (V.S., A.F., G.S.) and
Chemical Sciences (E.A., G.M.), University of Trieste, Trieste, Italy
Accepted for publication November 13, 1998
This paper is available online at http://www.jpet.org
Ruthenium complexes originally were synthesized as compounds selectively toxic for solid tumors, because of the selective activation to cytotoxic species into these tissues
(Clarke et al., 1988). A wide series of investigations, performed on sulfoxide-ruthenium complexes, pointed out a
more specific activity of these compounds on solid-tumor
metastases, putting light on the pharmacological possibilities
of such drugs (Sava, 1994; Sava and Bergamo, 1997). The
property that renders ruthenium complexes unique among
anticancer agents is principally the lack of evident direct cell
cytotoxicity at doses that increase lifetime expectancy in
tumor-bearing hosts (Sava et al., 1994, 1995; Capozzi et al.,
1998). Rather than being a limitation, the lack of direct cell
cytotoxicity is the leading aspect of these complexes in that it
indirectly means a low or absent bone marrow or epithelial
toxicity at active dosages (Giraldi et al., 1977; Sava et al.,
1984; Gagliardi et al., 1994).
Although many studies point out the capacity of ruthenium
Received for publication August 18, 1998.
1
This work was done with contributions from Ministero dell’Universitá e
della Ricerca Scientifica e Technologica (60%) and Fondazione CRTrieste and
was developed under the European Economic Community COST D8 Action.
In vitro NAMI-A caused a transient cell cycle arrest of tumor
cells in the premitotic G2/M phase, whereas cisplatin caused a
progressive dose-dependent disruption of cell cycle phases.
Correspondingly, NAMI-A did not modify cell growth, whereas
cisplatin caused a dose-dependent reduction of cell proliferation, as determined by sulforhodamine B test. Thus, NAMI-A,
unlike cisplatin, is a potent agent for the treatment of solid
tumor metastases as well as when these tumor lesions are in an
advanced stage of growth. NAMI-A is endowed with a mechanism of action unrelated to direct tumor cell cytotoxicity, and
such mechanism of action is responsible for a reduced host
toxicity.
complexes to bind to DNA of isolated plasmids or eukaryotic
cells (Clarke and Stubbs, 1996), many others seem to suggest
a certain difficulty of these complexes to penetrate cell membrane, preferring extracellular components as binding sites
(Ghosh et al., 1981; Deinum et al., 1985). These characteristics may contribute to the understanding of the mechanism of
antitumor activity in in vivo systems, where ruthenium interactions may deprive tumor cells of normal cell-cell and
cell-matrix contacts, which are essential for cell growth, division, and metastasis formation (Fox et al., 1995; Schadendorf et al., 1995; Umansky et al., 1996).
The aim of the present investigation, therefore, was to
examine the difference between the effects of NAMI-A [imidazolium trans-imidazole-dimethyl sulfoxide-tetrachlororuthenate, ImH[trans-RuCl4(DMSO)Im]] on tumor cells cultured in vitro and the effects on tumor metastases in vivo, as
determined by a direct count of lung metastatic tumor or
indirectly by measuring the prolongation of lifetime expectancy of tumor-bearing mice. The study was conducted by
comparing the effects of NAMI-A with those of cis-dichlorodiammine platinum(II) (cisplatin), to which the ruthenium
complex is often referred because it contains a heavy metal of
ABBREVIATIONS: NAMI-A, imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate; NAMI, sodium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate; cisplatin, cis-dichlorodiammine platinum(II); SRB, sulphorhodamine B.
559
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ABSTRACT
The effects of NAMI-A (imidazolium trans-imidazoledimethyl
sulfoxide-tetrachlororuthenate) are compared with cisplatin on
tumor cells cultured in vitro at doses of 1 to 100 mM and on
tumor metastases in vivo at maximum tolerated doses. Using
mouse tumors that metastasize to the lungs, NAMI-A given i.p.
for 6 consecutive days at 35 mg/kg/day, was effective independently of the tumor line being treated and of the stage of
metastasis growth. Conversely, cisplatin (2 mg/kg/day for 6
days) was as effective as NAMI-A on MCa mammary carcinoma
and TS/A adenocarcinoma and less effective than NAMI-A on
Lewis lung carcinoma. Cisplatin reduced body weight gain and
spleen weight during treatment and was much more toxic than
NAMI-A on liver sinusoids, kidney tubules, and lung epithelium.
560
Bergamo et al.
group VIII transition metals. In this context, cisplatin represents a drug of particular interest. It is myelosuppressive,
emetic, and nephrotoxic (Tognella, 1990; Rozenweig et al.,
1977); nevertheless, it must be considered a unique agent,
and, since its introduction into clinical trials, it is a drug that
has completely changed the prognosis of some tumors and
significantly ameliorated that of others (AHFS Drug Information 1994, American Society of Hospital Pharmacists, 94,
572). In particular, the work will focus on the relevance of in
vitro cell toxicity, as determined by flow cytometry analysis
of cell cycle, and the correspondent in vivo effects, including
organ toxicity.
Materials and Methods
and buffered with 3 mM tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, 3 mM N,N-bis[2-hydroxyethyl]-2-aminoethane-sulfonic
acid, 3 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid,
and 3 mM Tricine (Sigma Chemical Co.). The cell population doubling time was ca. 24 h. Culture medium was added with penicillinstreptomycin solution (Sigma Chemical Co.) (100 U/ml penicillin G
and 100 mg/ml streptomycin). Cells from confluent monolayers were
removed by 0.05% trypsin solution (Sigma Chemical Co.). Cell viability was determined by trypan blue dye exclusion test.
In Vitro Cytotoxicity Test. Cell cytotoxicity was evaluated
against the KB cell line in 24-well cell culture clusters (Costar)
according to previously described procedures (Alvarez et al., 1997).
Test compounds were dissolved in saline immediately before use,
and the solution was diluted with the growth medium to the desired
concentrations. Cells were incubated with the test compounds at
37°C with 5% CO2 and 100% relative humidity. The cytotoxic effect
was evaluated with the test compound dissolved in PBS. Three
concentrations (1, 10, and 100 mM) of NAMI-A and cisplatin and six
wells per concentration were used. One hour after drug challenge,
unreacted drug was removed and cells were cultured in complete
medium for a further 24, 48, and 72 h. Each experiment was repeated at least twice.
Sulforhodamine B (SRB) Test To Evaluate In Vitro Cytotoxicity. Cell growth was determined by staining with the proteinbinding dye SRB (Sigma Chemical Co.) (Skehan et al., 1990). Briefly,
adherent cell cultures were fixed in situ by the addition of 250 ml of
cold 50% (wt/vol) trichloroacetic acid and were kept for 60 min at 4°C.
The supernatant was then discarded and the plates were washed
three times with deionized and distilled water and dried. SRB solution was added and the cells were allowed to stain for 30 min at room
temperature. Unbound SRB was removed by washing three times
with 1% acetic acid. The plates then were air-dried. Bound stain was
dissolved with unbuffered Tris base (Sigma Chemical Co.), and the
optical density was read at 565 nm on a Perkin-Elmer 550 SE
spectrophotometer. Cytotoxicity was evaluated from the cell-growth
inhibition in the treated cultures versus untreated controls. The
statistical significance of these results was estimated by Student’s t
test (p , .01). IC50, the micromolar concentration of compound at
which cell proliferation was 50% of that observed in control cultures,
was determined by linear regression analysis.
Propidium Iodide Test. Viable cells (1 3 106) of a single cell
suspension, as determined by trypan blue exclusion test, were fixed
in 70% ethanol at 4°C for at least 1 h. Before analysis the ethanol
was removed by centrifugation and cells were washed twice with
PBS. Cells were resuspended in PBS containing 1 mg/ml RNase at
37°C for 30 min and stained further for at least 30 min at room
temperature in the dark with propidium iodide (40 mg/ml) (Sigma
Chemical Co.). Red fluorescence (610 nm) was analyzed, using peak
fluorescence gate to discriminate aggregates. Each analysis consisted of 10,000 events counted. The flow cytometric analyses were
done either at Fondazione Callerio with an EPICS XL-MCL flow
cytometer (Coulter Electronics, Miami, FL) or at the Center for Flow
Cytometry (section of CISP of the University of Trieste) with an
EPICS ELITE ESP cytometer (Coulter Electronics). Cell cycle distribution of the cells was determined by analysis with Multicycle
software (Phoenix Flow Systems).
Histological Examination. Sections for light microscopy were
prepared from paraffin-embedded lungs, which were removed,
washed in water, fixed in 10% formaline, and processed according to
the standard procedure for inclusion and after rehydration (xylene/
alcohol/water), with sections cut at 6 mm. Sections were stained with
Cajal-Gallego mounted in Canada Balsam and were observed with a
Leitz-Orthoplan microscope. Examinations were made on three different slides, each containing three slices for each sample.
Statistical Analysis. Each experiment was subjected to statistical analysis by the Student-Newmann-Keuls ANOVA and by Student’s t test for grouped data. Significance was accepted with p # .05.
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Compounds. NAMI-A was prepared according to a patented procedure (Mestroni et al., 1998). Cisplatin was obtained by Sigma
Chemical Co. (St. Louis, MO). Each compound was administered as
a solution in isotonic saline, in volumes of 10 ml/kg b.wt.
Tumor Lines for In Vivo Test. Lewis lung carcinoma (grown in
C57Bl mice and propagated for experimental purposes in BD2F1
hybrids), MCa mammary carcinoma (grown in CBA mice), and TS/A
adenocarcinoma (grown in Balb/c mice) were used for in vivo testing.
C57Bl, BD2F1, and Balb/c mice were obtained from Harlan Nossan,
and CBA mice were obtained from a locally established breeding
colony grown according to the standard procedure for inbred strains.
The procedure of tumor graft was identical for all tumor lines.
Briefly, for Lewis lung carcinoma and MCa mammary carcinoma,
106 cells of a single cell suspension, prepared from mincing with
scissors the primary tumor masses obtained from donors similarly
implanted 2 weeks before, were injected i.m. into the calf of the left
hind leg of experimental-group mice; for TS/A adenocarcinoma (generously supplied by G. Forni, Consiglio Nazionale delle Ricerche Centro di Immunogenetica ed Oncologia Sperimentale, Torino, Italy),
105 cells were obtained from an in vitro confluent culture of the same
cells. The TS/A cell line was maintained in complete medium consisting of RPMI 1640 medium (Sigma Chemical Co.) supplemented
with 10% fetal bovine serum (HyClone Europe, Holland), 2 mM
L-glutamine (HyClone Europe), and 50 mg/ml gentamycin sulfate
solution (Irvine Scientific, Santa Ana, CA). Cells from confluent
monolayers were removed from flasks by 0.25% trypsin solution
(Sigma Chemical Co.), washed twice, and-pellet diluted with complete medium or PBS.
Primary Tumor Growth and Lung Metastasis Evaluation.
Primary tumor growth was determined by caliper measurements, by
determining two orthogonal axes and calculating tumor weight with
the formula: p/6 3 a2 3 b, were a is the shorter and b is the longer
axis. Lung metastases were counted by carefully examining the
surface of the lungs immediately after killing of the animals by
cervical dislocation. Lungs were dissected into the five lobes, washed
with PBS and examined under a low-power microscope equipped
with a calibrated grid. The weight of each metastasis was calculated
by applying the same formula used for primary tumors, and the sum
of each individual weight gives the total weight of the metastatic
tumor per animal.
Animal Studies. Animal studies were carried out according to
the guidelines enforced in Italy (DDL 116 of February 21, 1992) and
in compliance with the Guide for the Care and Use of Laboratory
Animals (National Institutes of Health, Bethesda, MD).
Tumor Lines for In Vitro Test. An established KB cell line
(ECACC no. 86103004) was cultured according to standard procedure (Craciunescu et al., 1987). Vials of the original line were maintained in liquid N2; from them, cells were obtained, serially subcultured, and used for the experiments reported in the present work.
The KB cell line was maintained in Eagle’s minimum essential
medium (Eagle, 1959) with 1% nonessential amino acids (GIBCO
BRL), supplemented with 10% newborn calf serum (GIBCO BRL),
Vol. 289
1999
Selective Drug Therapy of Metastases
Results
Fig. 1. Effects of NAMI-A and cisplatin on in vitro cell proliferation. KB
cells, seeded on 24-well plastic plates 96 h before, were challenged with
NAMI-A and cisplatin for 1 h at 1, 10, and 100 mM concentration. SRB
test was performed after 24, 48, and 72 h of further cultivation in the
appropriate medium.
cally significant increase of the lifetime expectancy that is
comparable to that caused by cisplatin.
The analysis of histological sections of kidneys of mice
treated with daily doses of 35 mg/kg/day NAMI-A and 2
mg/kg/day cisplatin for 6 consecutive days showed a significant increase of glomeruli that resulted as atrophic or damaged as compared with untreated controls (Table 2). The
compared analysis of this effect, done 24 h after the last dose,
showed a significant difference between cisplatin and
NAMI-A as far as atrophic glomeruli are concerned, whereas
no statistically significant changes were observed on those
identified as damaged, although cisplatin tended to increase
the values more than NAMI-A. When examining the whole
parenchyma of kidney, liver, and lungs (Table 3), cisplatin
always showed effects greater than those of NAMI-A. In
general, the epithelial tissue of these organs appeared edematous and altered compared with untreated controls and also
with mice treated with NAMI-A, either in terms of number of
damages per field of microscopy examination or in terms of
severity of the damage observed. On lungs, cisplatin also
caused an increase of inflammatory sites. Such inflammatory
process, present in the kidney of controls, is virtually negligible in the same organ of mice treated with NAMI-A and
cisplatin; a dramatic reduction of the number and dimensions of lung metastases is observed on these slices as well.
Cisplatin caused an intermediate grade of inflammation of
liver that was more pronounced than that of NAMI-A.
Discussion
Typically, it is expected that a compound such as cisplatin
that shows antitumor action on experimental tumors also
would be cytotoxic against tumor cells in vitro. Therefore, a
compound like NAMI-A, which often is compared with cisplatin because it similarly is based to a heavy metal of the
same group, is totally atypical if it shows the same action as
cisplatin (or even better) in vivo on solid metastasizing tumor
but is virtually devoid of cytotoxicity against tumor cells in
vitro. Data reported in the present investigation show this
discrepancy and show further that NAMI-A is much less toxic
than cisplatin for healthy tissues at equieffective doses. In
vivo doses are optimal for both compounds, considering the
schedule of administration used and the ratio between host
toxicity and antitumor effect (i.e., optimal doses). Furthermore, atomic absorption spectroscopy studies of tissue disposition showed that NAMI-A, at the in vivo dose of 35 mg/kg
given for 6 consecutive days, reaches a 100-mM order concentration in tumor, liver, and lungs (Cocchietto, 1999). Therefore, in vitro comparison of both drugs using a top dose of 100
mM appears to be appropriate.
With the experimental conditions presently adopted, i.e.,
mouse tumors that metastasize to the lungs, NAMI-A resulted as effective independently of the tumor line being
treated, either considering the reduction of the number and
weight of lung tumor or considering the prolongation of lifetime expectancy of tumor-bearing mice. Conversely, cisplatin
was as effective as NAMI-A in mice bearing MCa mammary
carcinoma, which is slightly less effective than NAMI-A in
mice bearing TS/A adenocarcinoma and markedly less effective than NAMI-A in mice bearing Lewis lung carcinoma.
Cisplatin, at the dose and treatment schedule used, was more
toxic than NAMI-A in terms of reduction of body weight gain
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On in vitro cultured KB cells, NAMI-A is virtually devoid of
cytotoxicity up to 100 mM concentration, after 1-h exposure of
confluent cells in PBS, as determined by the SRB test (Fig. 1).
In a dose-dependent way, NAMI-A caused a transient increase of protein content versus controls at 24 h, which is
completely abolished from 48 h onward. In the same experimental conditions, cisplatin caused a significant reduction of
protein content at the maximum dose used; this effect increased from 24 to 72 h after drug exposure.
After DNA analysis of KB cells by flow cytometry, 24 h
after treatment, NAMI-A showed a significant increase of
cells in G2/M phase at 100 mM and 10 mM concentration, and
an increase of cells in S and G2/M phase, with a corresponding reduction of those in G0/G1, at 100 mM concentration.
Such alterations are still evident at 48 h with 100 mM
NAMI-A and are completely abolished at 72 h after drug
exposure (Table 1). Cisplatin caused a marked alteration of
cell cycle distribution of KB cells at 100 mM concentration. A
statistically significant alteration of the distribution of KB
cells within the cell cycle phases also was caused at 10 mM
and 1 mM concentrations; at the lowest dose used, these
alterations were abolished 72 h after drug challenge. Data
reported in Fig. 2 show an example of flow cytometry histograms of DNA distribution of control KB cells and of those
treated with 100 mM NAMI-A and cisplatin. In particular,
data on cisplatin show the complete alteration of DNA distribution that made impossible the calculation of the percentage of cells in the cell cycle phases.
NAMI-A caused a marked reduction of lung metastasis
formation in mice carrying i.m. implants of Lewis lung carcinoma and TS/A adenocarcinoma and significantly increased the postsurgical lifetime expectancy of mice bearing
MCa mammary carcinoma (Fig. 3). The reduction of the
weight of lung metastases on TS/A adenocarcinoma was
equal to that caused by cisplatin, despite the fact that only
cisplatin caused a significant reduction of primary tumor
growth by 32%. When tumor-bearing mice were treated after
surgical removal of primary tumors, the effect of NAMI-A on
Lewis lung carcinoma was markedly greater than that of
cisplatin. In mice bearing MCa mammary carcinoma, a similar postsurgical treatment with NAMI-A caused a statisti-
561
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Bergamo et al.
Vol. 289
TABLE 1
Effects of NAMI-A and cisplatin on cell distribution among cell cycle phases after 24-, 48-, and 72-h drug exposure
KB cells were exposed to drug concentrations for 1 h; then drug was removed and cells were added with fresh medium.
% Cells in Cycle Phase
Drug
Concentration
24 h
G0/G1
mM 0
NAMI-A (1)
NAMI-A
(10)
NAMI-A
(100)
Cisplatin (1)
Cisplatin
(10)
Cisplatin
(100)
S
48 h
G2/M
G0/G1
S
72 h
G2/M
G0/G1
S
G2/M
72.5 6 0.6
70.7 6 0.9
69.5 6 1.1
20.9 6 0.4
21.6 6 0.4
20.4 6 0.8
6.6 6 0.5
7.7 6 0.6
10.2 6 0.4**
70.0 6 2.0
67.9 6 1.0
69.2 6 1.5
22.2 6 1.3
21.3 6 2.0
22.5 6 1.2
7.8 6 0.7
8.5 6 0.7
8.4 6 0.4
72.5 6 0.4
72.2 6 1.8
68.4 6 0.9
19.3 6 0.3
19.4 6 1.0
21.8 6 0.6
8.2 6 0.2
8.4 6 1.2
9.8 6 0.7
39.5 6 2.2**
30.9 6 1.8**
29.6 6 0.8**
61.5 6 1.8**
24.6 6 1.1
13.9 6 0.8*
71.2 6 1.0
20.4 6 0.7
8.4 6 0.4
51.4 6 1.9**
23.6 6 0.6**
33.2 6 1.0**
76.4 6 0.6**
15.5 6 1.1**
0 6 0**
58.1 6 1.2**
16.0 6 1.6**
28.6 6 0.2**
30.7 6 1.5**
13.3 6 1.0*
53.3 6 2.9**
70.6 6 0.8
42.4 6 2.5**
20.6 6 0.7
27.0 6 0.7**
8.8 6 0.3
30.4 6 3.1**
79.4 6 0.5**
18.9 6 0.6
1.7 6 0.6**
ND
ND
ND
ND
ND
ND
ND, not detectable. * p , .05, ** p , .01 versus 0 concentration, Student-Neumann-Keuls ANOVA.
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Fig. 2. Individual flow cytometry histograms of cell cycle distribution of KB cells of untreated controls and of NAMI-A- and cisplatin-treated groups.
KB cells, seeded on six-well plastic plates 96 h before, were challenged with NAMI-A and cisplatin for 1 h at 100 mM concentration. Cell cycles were
obtained at 24, 48, and 72 h after drug challenge.
in the period of drug treatment (up to 211% versus controls)
and in terms of reduction of spleen weight (up to 250%
versus controls). These data, added to those related to the
toxicity for healthy epithelia, stress the more favorable therapeutic properties of NAMI-A versus cisplatin.
Tumor cells, challenged in vitro with NAMI-A, do not show
a significant alteration of cell cycles. NAMI-A causes a mild
and transient arrest of cell cycle in the premitotic phase,
which, besides that reported for KB cells, also was demonstrated with TS/A cells cultured in vitro, with the same doses
1999
Selective Drug Therapy of Metastases
and timings (Fnd Callerio, unpublished data on file). The
correspondent mean increase of total protein content, as determined by the SRB test, should be regarded as a consequence of this effect. This effect is a property of NAMI-A that
is not shared with NAMI (sodium trans-imidazole-dimethyl
sulfoxide-tetrachlororuthenate, Na[trans-RuCl4(DMSO)Im]),
which was totally ineffective in vitro on tumor cells (Sava et
al., 1995; Capozzi et al., 1998), probably associated with the
advantages given by a molecule endowed with better pharmaceutical characteristics (Mestroni et al., 1998). The relevance of these mild effects of NAMI-A on in vitro tumor cells
for the selective action on in vivo metastases is not clear yet.
It could be speculated that NAMI-A has a receptor-mediated
effect, which might explain both its selectivity for tumor
metastases, which behave differently from the primary tumor counterparts, and the reversibility of its in vitro (present
paper) as well as in vivo (Sava et al., 1994) effects. Furthermore, such a mechanism might also help to explain the
effects of NAMI on mRNAs for metalloproteinases and their
inhibitors (Sava et al., 1996), the selection of tumor cell
populations that NAMI caused with vitro-vivo and vivo-vivo
bioassays of treated tumor cells (Sava et al., 1995), and,
particularly, the irrelevance of intracellular migration of
NAMI for its effects on TLX5 lymphoma (Capozzi et al.,
1998). That in vitro treated tumor cells do not show a reduced
rate of growth as compared with in vivo treated lung metastases might be attributed to the cross-talk interactions that
in vivo growing cells have with other healthy cells and with
extracellular matrix constituents that might accentuate
growth arrest or apoptosis (Fox et al., 1995; Schadendorf et
al., 1995, Umanski et al., 1996).
Considering that the main target for antitumor chemotherapy is represented very often by distant metastases, which
are present, although not always diagnosable, at the time of
eradication of the primary lesion and invariably are responsible for the failure of most of the available antitumor therapies (Poste, 1986; Fidler and Balch, 1987), this study presents NAMI-A as a reliable candidate for such work. NAMI-A
is devoid of any link with cisplatin, presently used as a
reference compound, because it is free of direct cytotoxicity
for tumor cells. The lack or low toxicity of NAMI-A for host
tissues, here compared with that exhibited by active doses of
cisplatin, is a support for this new compound, which also
should be regarded as a novel and potent agent for the
treatment of solid tumor metastases when these tumor lesions are already present and in an advanced stage of
growth.
TABLE 2
Histological analysis of glomeruli in mice treated with NAMI-A and cisplatin
Kidneys of three different mice, implanted with MCa mammary carcinoma on day 0 and dosed i.p. with NAMI-A or cisplatin daily on days 14 –19, were removed from animals
24 h after last dose, fixed with formalin, and examined with light microscopy. For each animal, three different glasses, each mounted with three different slices, were read
completely in a single blind procedure.
Atrophic Glomeruli
Treatment Group (dose)
Altered Glomeruli
Number/total
counted
% of total, mean
6 S.E.
Number/total
counted
% of total, mean
6 S.E.
15/3259
233/4485
278/4484
0.46 6 0.06a,b
5.27 6 0.36a,c
6.31 6 0.71b,c
66/3259
1207/4485
1594/4484
2.04 6 0.29a,b
27.20 6 3.40a
35.54 6 1.61b
Controls
NAMI-A (35 mg/kg)
Cisplatin (2 mg/kg)
Means with the same letter differ statistically;
a,b
p , .01 and c p , .05, Student-Newmann-Keuls ANOVA.
TABLE 3
Histological analysis of kidney, liver, and lung parenchyma after treatment with NAMI-A and cisplatin
Kidneys, livers, and lung of three different mice, implanted with MCa mammary carcinoma on day 0 and dosed i.p. with NAMI-A or cisplatin daily on days 14 –19, were
removed from animals 24 h after last dose, fixed with formalin, and examined with light microscopy. For each animal, three different glasses, each mounted with four different
slices, were read completely in a single blind procedure.
Treatment Group (dose)
NAMI-A (35 mg/kg)
Cisplatin (2 mg/kg)
Kidney
Swelling of
proximal tubules
and edema
Liver
Swelling of hepatic
sinusoids and
damage of lobular
architecture
Liver
Compression of
alveoli and
increased thickness
of epithelium
111
1111
11
1111
1
111
1, Focal; 11, moderate; 111, diffuse; 1111, marked and generalized.
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Fig. 3. Effects of NAMI-A and cisplatin on the reduction of lung metastasis and on the increase of survival time versus controls. Groups of seven
mice, implanted i.m. on day 0, underwent surgical ablation of primary
tumor on days 11 (Lewis lung carcinoma, A), 13 (MCa mammary carcinoma, B), and 15 (TS/A adenocarcinoma, C). NAMI-A (35 mg/kg/day) or 2
mg/kg/day cisplatin was given i.p. on days 12–17 (Lewis lung carcinoma,
A), 14 –19 (MCa mammary carcinoma, B), and 9 –14 (TS/A adenocarcinoma, C). The effects of drug treatment were determined as an increase
of the lifetime expectancy (MCa mammary carcinoma, B) or by measuring
the weight of the metastatic tumor in the lungs after killing the mice on
day 23 (Lewis lung carcinoma, A) or day 30 (TS/A adenocarcinoma, C).
*p , .05; **p , .01, statistically different from controls using the Student-Newmann-Keuls test.
563
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Bergamo et al.
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