In Vivo Antitumor Activity of Bis(4,7-dimethyl-1,10-phenanthroline) Sulfatooxovanadium(IV) METVAN VO(SO4)(Me2-Phen)2
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2124 Vol. 7, 2124 –2133, July 2001 Clinical Cancer Research
In Vivo Antitumor Activity of Bis(4,7-dimethyl-1,10-phenanthroline)
Sulfatooxovanadium(IV) {METVAN [VO(SO4)(Me2-Phen)2]}
Rama Krishna Narla, Chun-Lin Chen, after tumor cell inoculation than those of the vehicle-treated
Yanhong Dong, and Fatih M. Uckun1 control SCID mice (174 ⴞ 29 mm3 versus 487 ⴞ 82 mm3;
P ⴝ 0.002). The favorable in vivo pharmacodynamic fea-
Parker Hughes Cancer Center, Departments of Oncology [R. K. N.,
F. M. U.], Pharmaceutical Sciences [C-L. C.], and Chemistry [Y. D.], tures and antitumor activity of METVAN warrants further
and Drug Discovery Program [R. K. N., C-L. C., Y. D., F. M. U.], development of this novel oxovanadium compound as a
Parker Hughes Institute, St. Paul, Minnesota 55113 potential new anticancer agent.
INTRODUCTION
ABSTRACT
The use of vanadium compounds and vanadium salts for
The compound bis(4,7-dimethyl-1,10-phenanthroline)
clinical applications received renewed interest in the late 1970s
sulfatooxovanadium(IV) {METVAN [VO(SO 4 )(Me 2 -
and early 1980s because of the discovery that vanadate(V)
Phen)2]}, exhibits potent cytotoxicity against human cancer
solutions produced insulin-like effects in rat diaphragms and
cells at low micromolar concentrations. At concentrations
isolated adipocytes in vitro (1–3). Subsequently, the adminis-
>1 M, METVAN treatment was associated with a nearly
tration of vanadate solutions to diabetic rats was shown to lower
complete loss of the adhesive, migratory, and invasive prop-
blood glucose levels (4). Despite a long history of vanadium
erties of the treated tumor cell populations. METVAN did
compounds being used in the treatment of human diseases,
not cause acute or subacute toxicity in mice at dose levels
however, only a small number of organometallic compounds
ranging from 12.5 mg/kg to 100 mg/kg. Therapeutic plasma
containing vanadium have been tested for antitumor activity
concentrations >5 M were rapidly achieved and main-
(5–11); a few of these were shown to possess anticancer activity
tained in mice for at least 24 h after i.p. bolus injection of a
in vitro as well as in vivo (6, 8, 12–14).
single 10 mg/kg nontoxic dose of METVAN. At this dose
Vanadium can be found in both anionic and cationic forms
level, the maximum plasma METVAN concentration was
with oxidation states ranging from ⫺1 to ⫹5 (15–18). Vana-
37.0 M, which was achieved with a tmax of 21.4 min. Plasma
dium complexes with oxidation states ⫹4 (IV) and ⫹5 (V) have
samples (diluted 1:16) from METVAN-treated mice killed
been shown to modulate cellular redox potential, regulate enzy-
85% of human breast cancer cells in vitro. METVAN was
matic phosphorylation, and exert various other effects in mul-
slowly eliminated with an apparent plasma t1/2 of 17.5 h and
tiple biological systems (19 –25). In addition to the ability of
systemic clearance of 42.1 ml/h/kg. In accordance with its
vanadium to assume various oxidation states, its coordination
potent in vitro activity and favorable in vivo pharmacokinet-
chemistry also plays a key role in its interactions with various
ics, METVAN exhibited significant antitumor activity and
biomolecules. As part of a systematic effort aimed at developing
delayed tumor progression in CB.17 severe combined im-
compounds with anticancer activity, we recently identified
munodeficient (SCID) mouse xenograft models of human
METVAN, {bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxo-
glioblastoma and breast cancer. In these experiments,
vanadium(IV) [VO(SO4)(Me2-Phen)2]} as an active apoptosis-
METVAN was administered in daily injections of a single
inducing agent with potent in vitro antitumor activity (26 –28).
nontoxic 10 mg/kg i.p. dose on 5 consecutive days per week
At nanomolar to low micromolar concentrations, METVAN
for 4 consecutive weeks beginning the day after the s.c.
induced apoptosis in leukemic cell lines, multiple myeloma cell
inoculation of U87 glioblastoma or MDA-MB-231 breast
lines, and solid tumor cell lines derived from breast cancer,
cancer cells. At 40 days after the inoculation of tumor cells,
glioblastoma, and testicular cancer patients (26 –28). Apoptosis
the U87 tumor xenografts in the vehicle-treated control
induced by treatment with METVAN is mediated by the gen-
SCID mice were much larger than those of the mice treated
eration of reactive oxygen species, depletion of glutathione, and
with METVAN (4560 ⴞ 654 mm3 versus 1688 ⴞ 571 mm3;
depolarization of mitochondrial membranes (29). Furthermore,
P ⴝ 0.003). Similarly, the MDA-MB-231 tumors in SCID
METVAN inhibited the constitutive expression of MMP2-9
mice treated with METVAN were much smaller 40 days
protein and its gelatinolytic activity in HL-60 acute myeloid
leukemia cells and MMP-2 as well as of MMP-9 gelatinolytic
activities in leukemic cells from acute lymphoblastic leukemia,
Received 2/14/01; revised 3/28/01; accepted 3/30/01.
The costs of publication of this article were defrayed in part by the
2
payment of page charges. This article must therefore be hereby marked The abbreviations used are: MMP, matrix metalloproteinase; SCID,
advertisement in accordance with 18 U.S.C. Section 1734 solely to severe combined immunodeficient; IR, infrared; LC, liquid chromatog-
indicate this fact. raphy; MS, mass spectrometry; ES, electron spin; MTT, 3-(4,5-dimeth-
1
To whom requests for reprints should be addressed, at Parker Hughes ylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ECM, extracellular
Institute, 2665 Long Lake Road, Suite 330, St. Paul, MN 55113. Phone: matrix; AUC, area under the concentration-time curve; TdT, terminal
(651) 697-9228; FAX: (651) 697-1042; E-mail: fatih_uckun@ deoxynucleotidyltransferase; TUNEL, TdT-mediated dUTP nick-end
mercury.ih.org. labeling.
Downloaded from clincancerres.aacrjournals.org on January 15, 2021. © 2001 American Association for Cancer
Research.
Clinical Cancer Research 2125
acute myeloid leukemia, and chronic myeloid leukemia patients
(30). We now report that concentrations of METVAN, highly
cytotoxic to human glioblastoma and breast cancer cells in vitro,
can be achieved in vivo after i.p. administration of a single
nontoxic dose. METVAN showed favorable pharmacokinetics
in mice and exhibited significant in vivo antitumor activity in
SCID mouse xenograft models of human glioblastoma and
breast cancer.
MATERIALS AND METHODS
Preparation and Characterization of METVAN. The
coordination compound METVAN, bis(4,7-dimethyl-1,10-phen-
anthroline) sulfatooxovanadium(IV) [VO(SO4)(Me2-Phen)2],
was synthesized as described previously in detail (26, 27).
METVAN was characterized by IR, LC-MS, and elemental
analysis. The results were as follows: (a) IR (KBr pellet),
IR (KBr pellet): (cm⫺1) 3421, 3066, 2924, 1624, 1608, 1578,
1524, 1423, 1383, 1297, 1237, 1157, 1030, 972, 930, 868, 729,
696, 663, 650, 590, 557, 545, 529, 485, 464; (b) LC-MS (ES),
LC conditions: eluant, 30:70 v/v mixture of acetonitrile and an
aqueous solution of 0.1% acetic acid; flow rate, 0.5 ml/min;
column, 250 ⫻ 4 mm LiChrospher 100 RP-18 (5 m); injection
volume, 10 l; detection, 300 nm; and LC-MS (ES), MS con-
ditions (positive ion mode): fragmentor voltage, 50 V; drying
gas flow rate, 10 liter/min; drying gas temperature, 350°C;
nebulizer pressure, 25 p.s.i.; retention time, 7.84 min; ES-MS,
Calculated for {M ⫹ H}⫹ 580.1 {M ⫽ [VO(SO4)(Me2-
Phen)2]}, C28H24N4O5SV}; found, 580.1; and (c) elemental Fig. 1 Cytotoxic activity of METVAN against human glioblastoma
and breast cancer cells. Human glioblastoma (A, U87, U118, U138,
analysis: calculated for [VO(SO4)(Me2-Phen)2]䡠2H2O U373, and T98) and breast cancer (B, BT-20, MDA-MB-231, MDA-MB-
(C28H28N4O7SV), C, 54.63; H, 4.59; and N, 9.10; found: C, 361, and MCF-7) cells were incubated with increasing concentrations
54.79; H, 4.50; N, 9.26. (0.1–250 M) of METVAN for 48 h (A and B) or 16 h (C) in 96-well
Cell Lines. Human brain tumor cell lines U87 MG, U118 plates. Cell survival was determined by MTT assays. Data points, the
mean (⫾ SE) values from three independent experiments. The mean (⫾
MG, U138, U373 MG, and T98G, and breast cancer cell lines
SE) IC50 values for 48-h incubation were 2.1 ⫾ 0.6 M (U87), 0.82 ⫾
BT-20, MDA-MB-231, MDA-MB-361, and MCF-7, were ob- 0.1 M (U118), 2.7 ⫾ 0.4 M (U138), 2.0 ⫾ 0.4 M (U373), 3.9 ⫾ 0.5
tained from American Type Culture Collection (Rockville, MD) M (T98), 1.6 ⫾ 0.7 M (BT-20), 0.5 ⫾ 0.1 M (MDA-MB-231), 1.9 ⫾
and were maintained as continuous cell lines in DMEM (U87, 0.5 M (MDA-MB-361), and 2.3 ⫾ 0.4 M (MCF-7). The mean (⫾ SE)
U118, U138, U373, T98, BT-20) or in Leibovitz’s L-15 medium IC50 values for 16 h incubation were 16.3 ⫾ 1.8 M (U87) and 11.1 ⫾
1.8 M (MDA-MB-231).
(MDA-MB-231 and MDA-MB-361). All of the media were
supplemented with 10% FCS, 4 mM glutamine, 100 units/ml
penicillin G, and 100 mg/ml streptomycin sulfate. All of the
tissue culture reagents were obtained from Life Technologies, reader (Labsystems). The percentage survival and the IC50
Inc. Inc. (Gaithersburg, MD). values were calculated using Graphpad Prism v2.0 (Graphpad
Cytotoxicity MTT Assays. The cytotoxicity of Software, Inc., San Diego, CA).
METVAN against human cancer cell lines was analyzed Adhesion Assays. In vitro adhesion assays were used to
using the MTT assay (Boehringer Mannheim Corp., Indian- evaluate the effects of METVAN on the adhesive properties of
apolis, IN) as described previously (27). Briefly, exponen- U87 and MDA-MB-231 cells as described previously (31). The
tially growing tumor cells were seeded into a 96-well plate at plates for the adhesion assays were precoated with the ECM
a density of 2.5 ⫻ 104 cells/well. METVAN was dissolved in proteins laminin, fibronectin, vitronectin, and type IV collagen
0.1% DMSO in PBS and then added to each well to yield (each at a final concentration of 1 g/ml in PBS) overnight at
final concentrations ranging from 0.1 to 250 M. After incu- 4°C and dried. Exponentially-growing cells were incubated with
bation at 37°C for 48 or 16 h, 10 l of MTT (0.5 mg/ml final METVAN at concentrations ranging from 0.5 M to 25 M for
concentration) were added to each well. The plates were then 16 h in a humidified 5% CO2 atmosphere. The cells were then
incubated at 37°C for an additional 4 h to allow MTT to form detached from the flasks with 0.05% trypsin (Life Technologies,
formazan crystals by reacting with metabolically active cells. Inc.), resuspended in medium, incubated at 37°C for 2 h to allow
The formazan crystals were solubilized overnight at 37°C in them to recover from the trypsinization stress, and examined for
a solution containing 10% SDS in 0.01 M HCl. The absorb- their ability to adhere to the plates precoated with ECM proteins.
ance at 540 nm (a reference wavelength of 690 nm was used) Cells were centrifuged, washed twice with serum-free medium,
of the solution in each well was measured using a microplate counted, and resuspended in serum-free medium at a final
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2126 In Vivo Antitumor Activity of METVAN
Fig. 2 METVAN induces apoptosis
in glioblastoma and breast cancer
cells. U87 glioblastoma (A, A⬘) and
MDA-MB-231 breast cancer (B, B⬘)
cells were incubated with 10 M of
METVAN for 24 h, fixed, permeabi-
lized and visualized for DNA degra-
dation in a TUNEL assay using
dUTP-labeling. Red fluorescence,
nuclei stained with propidium iodide.
Green or yellow (i.e., superimposed
red and green) fluorescence, apop-
totic nuclei containing fragmented
DNA. When compared with controls,
treated with 0.1% DMSO (A, B), sev-
eral of the cells incubated with
METVAN (A⬘, B⬘) exhibited apo-
ptotic nuclei.
concentration of 2.5 ⫻ 105 cells/ml. The cell suspension was per filter were counted to determine the invasive fraction. The
added to each well in aliquots of 100 l, and the cells were invasive fractions of cells treated with METVAN were com-
allowed to adhere for 1 h at 37°C in a humidified 5% CO2 pared with those of DMSO-treated control cells and the percent-
atmosphere. The nonadherent cells were removed by gently age inhibition of invasiveness was determined.
washing the cells with PBS. The adherent fraction was quanti- Migration Assay. Migration of brain tumor cells was
tated using MTT assays as described previously (27, 32). monitored using U373 glioblastoma cell spheroids, as described
In Vitro Invasion Assays. The in vitro invasiveness of previously (31). Glioblastoma cell spheroids were cultured in
cancer cells treated with METVAN was assayed using Matrigel- 100-mm2 tissue culture plates precoated with 0.75% agar pre-
coated Costar 24-well transwell cell culture chambers (Boyden pared in MEM supplemented with 10% fetal bovine serum. The
chambers) with 8.0-m-pore polycarbonate filter inserts as de- cells (5 ⫻ 106 cells) were suspended in the medium, seeded onto
scribed previously (31, 33, 34). Exponentially-growing U87 and agar-coated plates, and cultured for 5–7 days at 37°C. Spheroids
MDA-MB-231 cells were incubated with METVAN at various with a diameter of 200 – 400 m were selected for use in
concentrations ranging from 0.5 M to 25 M overnight. The additional experiments. For the migration experiments, the se-
cells were trypsinized, washed twice with serum-free medium lected spheroids were incubated for 2 h at 37°C in serum-free
containing BSA, counted and resuspended in a serum-free me- medium containing METVAN in concentrations ranging from 1
dium at 1 ⫻ 105 cells/ml. The cell suspension (0.5 ml aliquots) M to 25 M. The METVAN-treated and vehicle-control
was added to the Matrigel-coated and rehydrated filter inserts. (DMSO, 0.1%) spheroids were transferred onto fibronectin-
The inserts were then placed in 24-well plates containing 750 l coated coverslips (Becton Dickinson, Bedford, MA) and placed
of NIH fibroblast-conditioned medium (31, 34) as a chemoat- in 6-well plates containing the same concentrations of
tractant and were incubated at 37°C for 48 h. The filter inserts METVAN or DMSO in serum-free medium. Spheroids were
were then removed, the medium was decanted, and the cells on then kept in a humidified 5% CO2 incubator at 37°C for 48 h. A
the top of the filter that did not migrate were scraped off with a total of four to six spheroids were used for each concentration.
cotton-tipped applicator. The invasive cells that migrated to the After the 48-h incubation period, the spheroids were fixed and
lower side of the filter were fixed, stained with Hema-3 solution, stained with Hema-3 solutions and mounted onto glass slides.
and counted under a light microscope. Five to 10 random fields The distance of tumor cell migration from the spheroid was
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Research.Clinical Cancer Research 2127
measured using an ocular micrometer and a transmitted light
microscope.
Mice. The animal protocols used in this study were ap-
proved by the Parker Hughes Institute Institutional Animal Care
and Use Committee (IACUC), and all of the animal care pro-
cedures conformed to the Guide for the Care and Use of Lab-
oratory Animals (National Research Council, National Acad-
emy Press, Washington DC 1996). Female CB.17 SCID and
CD-1 mice were obtained from Taconic (Germantown, NY) and
housed in a specific-pathogen-free room located in a secure
indoor facility with controlled temperature, humidity, and noise
levels. Mice were housed in microisolater cages and fed with
autoclaved rodent chow. Water was also autoclaved and sup-
plemented with trimethoprim/sulfomethoxazol 3 days a week.
Toxicity Studies in Mice. The toxicity profile of
METVAN in CD-1 mice was examined, as reported previously
for other new agents (35–38). Female CD-1 mice (7 weeks old;
average weight, 24.5 g) were used and monitored daily for
lethargy, cleanliness, and morbidity. At the time of death, nec-
ropsies were performed, and the toxic effects of METVAN
administration were assessed. For histopathological studies, tis-
sues were fixed in 10% neutral buffered formalin, dehydrated,
and embedded in paraffin by routine methods. Glass slides with
affixed 6-m tissue sections were prepared and stained with
H&E. Female CD-1 mice were given an i.p. bolus injection of
METVAN in 0.2 ml of PBS supplemented with 10% DMSO, or
Fig. 3 Effect of METVAN on U87 and MDA-MB-231 cell adhesion to
0.2 ml of PBS supplemented with 10% DMSO alone (control ECM proteins. The U87 glioblastoma (A) and MDA-MB-231 breast
mice). No sedation or anesthesia was used throughout the treat- cancer (B) cells were preincubated with various concentrations of
ment period. Mice were monitored daily for mortality for de- METVAN and processed for adhesion assays. METVAN inhibited
termination of the day-30 LD50 values. Blood samples were adhesion of the U87 and MDA-MB-231 cells to ECM proteins. Data
points, the mean (⫾SE) values from three independent experiments.
collected prior to and after METVAN administration for a
complete blood cell count (CBC) with differential and platelets,
serum samples for total protein, albumin, bilirubin/ALT, alka-
line phosphatase, creatinine, CPK, and electrolytes. Mice sur- Pharmacokinetic Studies in Mice. Female CD-1 mice
viving until the end of the 30-days monitoring were killed. and (6 – 8 weeks old) from Charles River (Wilmington, MA) were
the tissues were immediately collected from randomly selected housed in a controlled environment (12-h light/12-h dark pho-
mice and preserved in 10% neutral buffered formalin. Standard toperiod, 22 ⫾ 1°C, 60 ⫾ 10% relative humidity). The mice
tissues collected for histological evaluation included: bone, bone were allowed free access to autoclaved pellet food and tap water
marrow, brain, cecum, heart, kidney, large intestine, liver, lung, throughout the study. The animal studies are approved by the
lymph node, ovary, pancreas, skeletal muscle, skin, small intes- Parker Hughes Institute Animal Care and Use Committee, and
tine, spleen, stomach, thymus, thyroid gland, urinary bladder, all animal care procedures conformed to the Guide for the Care
and uterus (as available). and Use of Laboratory Animals (National Research Council,
SCID Mouse Xenograft Models of Human Glioblas- National Academy Press, Washington DC 1996). A solution
toma and Breast Cancer. The right and left hind legs of the (100 l) of METVAN (dissolved in 5% DMSO in PBS) was
SCID mice were inoculated s.c. with 1 ⫻ 106 U87 human administered i.p. to mice at a dose of 10 mg/kg. This volume of
glioblastoma or 1 ⫻ 106 MDA-MB-231 breast cancer cells in DMSO is well tolerated by mice when administrated by rapid
0.1 ml of PBS. Mice were treated with i.p.-administered injec- i.v. or extravascular injection (40, 41). Blood samples (⬃500
tions of METVAN (10 mg/kg in 5% DMSO in PBS; n ⫽ 10) or l) were obtained from the ocular venous plexus by retro-orbital
vehicle alone (5% DMSO in PBS; n ⫽ 10). Injections were venipuncture at 0, 2, 5, 10, 15, 30, 45 min, and 1, 2, 4, 6, and
given once daily, 5 days per week, for 4 consecutive weeks 24 h after the i.p. injection. All of the collected blood samples
beginning the day after inoculation of the tumor cells. Mice were heparinized and centrifuged at 7000 ⫻ g for 5 min to
were monitored daily for health status as well as tumor growth separate the plasma fraction from the whole blood. The plasma
and were killed if they became moribund or developed tumors samples were then processed immediately, using the procedure
that impeded their ability to attain food or water, or at the end described below for determining vanadium concentrations.
of the six-week observation period. Tumors were measured Determination of Plasma METVAN Levels by Induc-
using Vernier calipers three times per week. Tumor volumes tively Coupled Plasma Atomic Emission Spectroscopy. All
were calculated according to the following formula, as described of the glassware was kept in 10% nitric acid for at least 48 h and
previously (32, 39): tumor volume ⫽ (width)2 (length)/2. was subsequently washed with distilled and Millipore water
Downloaded from clincancerres.aacrjournals.org on January 15, 2021. © 2001 American Association for Cancer
Research.2128 In Vivo Antitumor Activity of METVAN
Fig. 4 Effects of METVAN
on glioblastoma cell migration
from spheroids. U373 glioblas-
toma spheroids with diameters
of 200 –356 m were treated
for 2 h with 1 M (B), 2 M (C)
or 5 M (D) METVAN in 0.1%
DMSO or with 0.1% DMSO
alone (A). The control cells mi-
grated 745 ⫾ 29 m from the
spheroids, whereas treatment of
the spheroids with 1 M (B), 2
M (C) and 5M (D) METVAN
resulted in migration distances
of only 311.1 ⫾ 54.4 m,
214.9 ⫾ 25.7 m and 32.6 ⫾
8.4 m (corresponding to 58.3
⫾ 7.3%, 71.2 ⫾ 3.4%, and 95.6
⫾ 5.8% inhibition of tumor cell
migration), respectively.
before use. Standard solutions containing 0 –200 g/liter of
vanadium were prepared by diluting 100 l of plasma samples
containing various concentrations of METVAN with 3.7% HCl
to 5 ml. The plasma samples from mice (100 l) were diluted
with 3.7% HCl to 5 ml and were analyzed by inductively
coupled plasma atomic emission (ICP) spectroscopy carried out
in the Research Analytical Laboratory at University of Minne-
sota using a Perkin-Elmer Optima 3000DV spectrometer. The
analytical wavelength was 292.396 nm with plasma gas flow of
15 liters/min, auxiliary gas flow of 0.5 liter/min and nebulizer
gas flow of 0.75 liter/min. The output power was 1350 W. The
reporting limit for vanadium was 0.001 g/ml. (Reporting limit
is based on the concept of the lowest quantitatively determinable
concentration (LQDC). Precision at the LQDC is approximately
⫾10% and analytical results are quantitative.).
Pharmacokinetic Analysis. Pharmacokinetic modeling
and parameter calculations were carried out using the WinNon-
lin Professional Version 3.0 (Pharsight, Inc., Mountain, CA)
pharmacokinetics software (35, 36, 42, 43). An appropriate
model was chosen on the basis of the lowest sum of weighted
squared residuals, the lowest Schwartz criterion (SC), the lowest
Akaike’s information criterion (AIC) value, the lowest SEs of
the fitted parameters, and the dispersion of the residuals. The
elimination t1/2 life was estimated by linear regression analysis
of the terminal phase of the plasma concentration-time profile.
The AUC was calculated according to the linear trapezoidal rule Fig. 5 Anti-invasive activity of METVAN against U87 glioblastoma
between the first sampling time (0 h) and the last sampling time and MDA-MB-231 breast cancer cells. Cells were incubated for 24 h
plus C/k, where C is the concentration of the last sampling and with METVAN in concentrations ranging from 0.5 to 10 M. The cells
k is the elimination rate constant. The systemic clearance (CL) were then trypsinized and processed for invasion assays using Matrigel
matrix-coated Boyden chambers. Data points, the mean (⫾SE) values
was determined by dividing the dose by the AUC. from 3 independent experiments. The mean (⫾ SE) IC50 values were
In Situ Detection of Apoptosis. Apoptosis was detected 0.78 ⫾ 0.4 M and 0.61 ⫾ 0.3 M for the U87 and MDA-MB-231 cells,
using an in situ cell death detection kit (Boehringer Mannheim respectively.
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Research.Clinical Cancer Research 2129
Table 1 Laboratory studies of METVAN toxicity in mice
METVAN dose level (mg/kg)
Vehicle
Laboratory test (n ⫽ 20) 12.5 (n ⫽ 20) 25 (n ⫽ 20) 50 (n ⫽ 20) 100 (n ⫽ 20)
Hematology
WBCa (⫻ 109/liter) 8.7 ⫾ 0.9 9.6 ⫾ 0.7 9.2 ⫾ 0.8 9.9 ⫾ 0.9 10.6 ⫾ 0.9
Hgb (g/dl) 14.0 ⫾ 0.2 13.8 ⫾ 0.2 14.1 ⫾ 0.2 13.7 ⫾ 0.1 13.2 ⫾ 0.2
9
Plt. ct. (⫻ 10 /liter) 488 ⫾ 65 400 ⫾ 59 452 ⫾ 65 392 ⫾ 71 530 ⫾ 101
Electrolytes and kidney function
K (mmol/liter) 3.6 ⫾ 0.1 3.7 ⫾ 0.1 4.0 ⫾ 0.3 3.3 ⫾ 0.1 3.3 ⫾ 0.1
Ca (mg/dl) 8.7 ⫾ 0.1 8.7 ⫾ 0.1 8.8 ⫾ 0.2 8.9 ⫾ 0.1 9.4 ⫾ 0.1
PO4 (mg/dl) 6.6 ⫾ 0.1 6.1 ⫾ 0.2 6.3 ⫾ 0.2 6.9 ⫾ 0.2 6.6 ⫾ 0.2
Creat. (mg/dl) 0.4 ⫾ 0.0 0.3 ⫾ 0.0 0.4 ⫾ 0.0 0.3 ⫾ 0.0 0.4 ⫾ 0.0
Metabolism and liver function
Alb. (g/dl) 1.6 ⫾ 0.0 1.6 ⫾ 0.0 1.5 ⫾ 0.0 1.5 ⫾ 0.0 1.5 ⫾ 0.0
Total protein (g/dl) 4.9 ⫾ 0.1 5.0 ⫾ 0.1 4.8 ⫾ 0.1 4.6 ⫾ 0.1 4.6 ⫾ 0.1
ALT (IU/liter) 71 ⫾ 3.2 40 ⫾ 6 65 ⫾ 15 49 ⫾ 5 38 ⫾ 5
Alk. Ptse. (IU/liter) 99 ⫾ 6 98 ⫾ 7 123 ⫾ 11 106 ⫾ 6 106 ⫾ 5
Bili. (mg/dl) 0.4 ⫾ 0.0 0.4 ⫾ 0.0 0.5 ⫾ 0.1 0.4 ⫾ 0.0 0.5 ⫾ 0.0
LD-L (IU/liter) 536 ⫾ 77 709 ⫾ 101 666 ⫾ 83 554 ⫾ 41 598 ⫾ 74
Cardiac enzymes
CPK (IU/liter) 581 ⫾ 188 682 ⫾ 222 363 ⫾ 65 396 ⫾ 85 482 ⫾ 163
a
WBC, white blood count; Hgb, hemoglobin; Plt. ct., platelet count; K, potassium; Ca, calcium, PO4, phosphorous; Creat., creatinine; Alb.,
albumin; Bili., bilirubin; LD-L, lactate dehydrogenase-liver derived; ALT, alanine aminotransferase; Alk. Ptse., alkaline phosphatase; CPK, creatine
phosphokinase.
Corp., Indianapolis, IN) as described earlier (27, 44). Cells were mine whether METVAN is capable of impairing the adhesion of
incubated with either METVAN in 0.1% DMSO or 1:16-diluted cancer cells to ECM proteins, cells were incubated for 16 h with
plasma samples from METVAN-treated mice for 48 h at 37°C, noncytotoxic concentrations of METVAN (the cytotoxic IC50
and were fixed, permeabilized, incubated with the reaction mix- values for 16 h incubation were 16.3 ⫾ 1.8 M for U87 and
ture containing TdT- and FITC-conjugated dUTP, and counter- 11.1 ⫾ 1.8 M for MDA-MB-231 (see Fig. 1C) and the integrin-
stained with propidium iodide. Cells were transferred to slides mediated cancer cell adhesion was examined. As shown in Fig.
and viewed with a confocal laser scanning microscope (Bio-Rad 3, pretreatment of cells with noncytotoxic concentrations of
MRC 1024) mounted on a Nikon Eclipse E800 series upright METVAN inhibited the adhesion of U87 glioblastoma and
microscope as reported previously (27, 44). MDA-MB-231 breast cancer cells to laminin-, fibronectin-,
vitronectin-, and collagen-coated plates. The inhibition of glio-
RESULTS AND DISCUSSION blastoma cell adhesion was concentration-dependent with aver-
In Vitro Activity of METVAN against Glioblastoma and age IC50 values of 1.4 ⫾ 0.1 M, 1.3 ⫾ 0.1 M, 1.2 ⫾ 0.1 M,
Breast Cancer Cells. The cytotoxic activity of METVAN and 1.2 ⫾ 0.2 M for laminin, fibronectin, vitronectin and
against brain tumor and breast cancer cells was first confirmed collagen, respectively. The corresponding IC50 values for the
using MTT assays. As shown in Fig. 1, METVAN exhibited MDA-MB-231 breast cancer cells were 1.0 ⫾ 0.1 M, 1.0 ⫾ 0.0
potent cytotoxicity against each of the five brain tumor and four M, 1.1 ⫾ 0.1 M, and 1.3 ⫾ 0.1 M, respectively.
breast cancer cell lines with nanomolar or low micromolar IC50 Dissociation and migration are the initial steps for infiltra-
values. The METVAN-induced cell death was confirmed to be tion of tumor cells into the surrounding tissue (53–55). To
apoptotic using the TUNEL of exposed 3⬘-OH termini of DNA determine whether METVAN is capable of preventing the mi-
with dUTP-FITC. As shown in the confocal laser scanning gration of tumor cells, U373 glioblastoma multicellular sphe-
microscopy images in Fig. 2, METVAN-treated U87 glioblas- roids were used. The spheroids were prepared by plating the
toma and MDA-MB-231 breast cancer cells, examined for cells over agar plates as described above. Spheroids measuring
dUTP-FITC incorporation (green fluorescence) and propidium 259.2 ⫾ 41.9 m in diameter were incubated with METVAN or
iodide counterstaining (red fluorescence), exhibited many ap- with DMSO on fibronectin-coated coverslips. As shown in Fig.
optotic yellow nuclei (superimposed green and red fluores- 4, U373 glioblastoma cells incubated with DMSO rapidly mi-
cence) at 24 h after treatment. grated to a distance 745 ⫾ 29 m away from the spheroid.
During the multistep process of tissue invasion, tumor cells Treatment of the spheroids with METVAN inhibited tumor-cell
initially adhere to the ECM proteins via cell surface integrin migration in a concentration-dependent fashion with an IC50
receptors and then gain migratory capacity to enter the sur- value of ⬍1 M. Treatment with 1 M, 2 M, and 5 M
rounding tissue. ECM proteins such as laminin, fibronectin, METVAN resulted in 58.3 ⫾ 7.3% (migration distance of
vitronectin and type IV collagen are thought to play an impor- 311.1 ⫾ 54.4 m), 71.2 ⫾ 3.4% (migration distance of 214.9 ⫾
tant role in tumor cell attachment and migration because these 25.7 m), and 95.6 ⫾ 5.8% (migration distance of 32.6 ⫾ 8.4
proteins have been found in the basal lamina that promote the m) inhibition of tumor cell migration from the spheroids,
adhesion and invasion of tumor cells in situ (45–52). To deter- respectively (Fig. 4).
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Research.2130 In Vivo Antitumor Activity of METVAN
Fig. 6 Pharmacokinetic features of
METVAN in mice. The measured plasma
concentration of vanadium (E) as a function
of time in mice after i.p. injection of 10
mg/kg METVAN (five mice per time point).
A, plasma concentration-time curve. B, com-
puter-fitted pharmacokinetic parameter val-
ues. The pharmacokinetic parameters in
CD-1 mice (n ⫽ 5 mice per time point) are
presented as the average values estimated
from composite plasma concentration-time
curves of pooled data; in parentheses,
mean ⫾ SE values; CL, systemic clearance
from plasma; Cmax, measured maximum
plasma concentration; t ⁄ , terminal elimina-
12
tion half-life; tmax, the time required to reach
the maximum plasma drug concentration af-
ter i.p. administration; Vc, central volume of
distribution. aApparent Vc and systemic CL
without correction for bioavailability (F).
Tumor invasion of the basement membrane is a crucial step injections of METVAN at dose levels of 12.5 mg/kg (n ⫽ 40),
in the complex multistage process that leads to metastatic 25 mg/kg (n ⫽ 40), or 50 mg/kg (n ⫽ 40). Twenty mice from
spread. Tumor cells cross the basement membrane as they each group were electively killed on days 7 and 30 after the
initially invade the lymphatic or vascular beds during dissemi- administration of METVAN. METVAN was not toxic to mice at
nation and as they penetrate their target tissues (56 –59). The these dose levels. None of the 120 mice treated with i.p. bolus
Matrigel matrix, an artificial basement membrane composed of injections of METVAN in this dose range experienced side
growth factors and several ECM components such as collagens, effects or died of toxicity (data not shown). No evidence of
laminin, and proteoglycans, was used to examine the effect of acute toxicity was evident from laboratory tests performed on
METVAN on tumor cell invasion. To determine whether the blood samples from mice killed on day 7 (Table 1). In
METVAN impairs cancer cell invasion through Matrigel, the particular, there was (a) no neutropenia, anemia, or thrombocy-
cells were incubated with various noncytotoxic concentrations topenia suggestive of a hematological toxicity, (b) no increase in
of the compound, and then the invasion was examined using serum creatinine levels or an electrolyte imbalance suggestive of
MDA-MB-231 and U87 cells (Fig. 5). METVAN inhibited the renal toxicity, (c) no elevation of liver enzymes or total bilirubin
invasion of tumor cells through Matrigel matrix in a concentra- levels suggestive of hepatic toxicity, and (d) no CPK elevation
tion-dependent fashion with mean IC50 values of 0.78 ⫾ 0.4 M suggestive of cardiac toxicity (Table 1). Similarly, no laboratory
and 0.6 ⫾ 0.3 M for U87 and MDA-MB-231 cells, respec- abnormalities suggestive of subacute toxicity were evident from
tively. analysis of blood samples obtained from mice killed on day 30.
Taken together, these findings show that treatment with No histopathological lesions were found in the organs of
METVAN at concentrations ⬎1 M is associated with a nearly METVAN-treated mice that were electively killed at day 7 or
complete loss of the adhesive, migratory, and invasive proper- day 30.
ties of the treated tumor cell populations. We next sought to determine whether cytotoxic concentra-
In Vivo Pharmacokinetic Features of METVAN after tions of METVAN are achievable in vivo at nontoxic dose
i.p. Administration. We first evaluated the toxicity of levels. To this end, a single nontoxic 10 mg/kg bolus dose of
METVAN in mice to identify nontoxic dose levels for pharma- METVAN was administered to mice i.p., and the plasma vana-
codynamic studies. Mice were treated with single i.p. bolus dium concentrations were determined at various time points
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Research.Clinical Cancer Research 2131
Fig. 7 Anticancer activity of plasma
samples from METVAN-treated mice.
Plasma samples, obtained from vehicle-
treated (A, B) and METVAN-treated (A⬘,
B⬘) mice 30 min after administration of the
drug, were diluted with PBS 1:16 and then
tested for their cytotoxic activity against
MDA-MB-231 human breast cancer (A,
A⬘) and NALM-6 human leukemic (B, B⬘)
cells in vitro. After a 48-h incubation, cells
were examined for apoptotic changes us-
ing standard TUNEL/apoptosis assays, as
described in “Materials and Methods.”
after the injection using inductively coupled plasma atomic MDA-MB-231 breast cancer cells. METVAN significantly de-
emission (ICP) spectroscopy. A two-compartment, first-order layed the onset of tumor growth and inhibited visible tumor
pharmacokinetic model was fit to the pharmacokinetic data (Fig. progression when administered i.p. in single daily injections (10
6, A and B). The computer-fitted pharmacokinetic parameter mg/kg/dose) given 5 days per week for 4 weeks beginning the
values are shown in Fig. 6B. Therapeutic plasma concentrations day after s.c. inoculation of the tumor cells (Fig. 8). All of the
ⱖ5M, which are highly cytotoxic against human cancer cells, control mice developed measurable tumors within 7 days,
were rapidly achieved and maintained in mice for at least 24 h whereas 40% of the mice with U87 xenografts treated with
after i.p. bolus injection of a single 10-mg/kg nontoxic dose of METVAN remained alive and free of detectable tumors for ⬎16
METVAN. At this dose level, the maximum plasma METVAN days. At 40 days after the inoculation of tumor cells, the average
concentration (Cmax) was 37.0 M, which was achieved with a size of the U87 tumor xenografts in vehicle-treated control
tmax of 21.4 min. Notably, 1:16-diluted plasma samples obtained SCID mice was 4560 ⫾ 654 mm3, whereas the average size of
from METVAN-treated mice 30 min after the administration of the U87 tumors in METVAN-treated SCID mice was only
the drug killed 85% of MDA-MB-231 human breast cancer cells 1688 ⫾ 571 mm3 (P ⫽ 0.003; Fig. 8A). Similarly, the average
and ⬎99% of NALM-6 human leukemia cells in vitro (Fig. 7). size of MDA-MB-231 tumors in vehicle-treated control SCID
METVAN was slowly eliminated with an apparent plasma mice at 40 days after tumor cell inoculation was 487 ⫾ 82 mm3,
half-life of 17.5 h and systemic clearance of 42.1 ml/h/kg, which whereas the average size of MDA-MB-231 tumors in
is much slower than the blood flow to liver (5400 ml/h/kg) or METVAN-treated SCID mice was 174 ⫾ 29 mm3 (P ⫽ 0.002;
kidney (3900 ml/h/kg). The apparent volume distribution of Fig. 8B). Ninety % of the control mice developed measurable
METVAN at central compartment was 276.5 ml/kg [⬃5-fold tumors within 7 days, whereas 50% of the mice treated with
greater than the plasma volume (50 ml/kg) but less than the total METVAN remained alive and free of detectable tumors for ⬎19
body water (725 ml/kg); Ref. 60], indicating that METVAN days.
might have limited extravascular distribution after i.p. adminis- In summary, in accordance with its potent in vitro activity
tration. and favorable in vivo pharmacokinetics, METVAN exhibited
In Vivo Antitumor Activity of METVAN in SCID significant antitumor activity and delayed tumor progression in
Mouse Xenograft Models of Human Glioblastoma and CB.17 SCID mouse xenograft models of human glioblastoma
Breast Cancer. CB.17 SCID mice developed rapidly growing and breast cancer. The favorable in vivo pharmacodynamic
tumors after s.c. inoculation of 1 ⫻ 106 U87 glioblastoma or features and antitumor activity of METVAN warrants further
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Research.2132 In Vivo Antitumor Activity of METVAN
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Research.In Vivo Antitumor Activity of
Bis(4,7-dimethyl-1,10-phenanthroline) Sulfatooxovanadium(IV)
{METVAN [VO(SO4)(Me2-Phen)2]}
Rama Krishna Narla, Chun-Lin Chen, Yanhong Dong, et al.
Clin Cancer Res 2001;7:2124-2133.
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