Download Antimelanoma Effect of 4-S-Cysteaminylcatechol, an Activated Form

[CANCER RESEARCH 55, 2603-2607,
June 15, 19M5]
Antimelanoma Effect of 4-S-Cysteaminylcatechol,
4-5- Cy steaminylphenol1
an Activated Form of
Shigeki Inoue, Katsutoshi Hasegawa, Shosuke Ito,2 Hiroyuki Ozeki, Francisco Solano, Celia Jimenez-Cervantes,
Kazumasa Wakamatsu,
and Keisuke Fujita
Schon/ of Health Sciences ¡S.I., S. I.. H. O., K. W.] and Institute for Comprehensive Medical Sciences [K. H., K. F.], Fujita Health University, Timmke. Aichi 470-11, Japan,
tinti Department of Biochemistry ariti Molecular Biology. School of Medicine, University of Murcia, 30100 Murcia. Spain IF. S., C 7-C/
ABSTRACT
Rational chemotherapy of malignant melanoma
taking advantage of the presence of melanogenic
cells. 4-S-Cysteaminylphenol
(4-S-CAP) has been
cytotoxicity and antimelanoma effect. Although
could be
enzymes
evaluated
4-5-CAP
developed by
in melanoma
for melanois selectively
toxic to pigmented melanoma cells, it is not potent enough when applied
as a single agent. To increase the efficacy of 4-S-CAP, we synthesized
4-S-cysteaminylcatechol
(4-S-CAC), an activated form of 4-S-CAP, and
compared its biochemical properties and antimelanoma effects with those
of the isomers 3-S-cysteaminylcatechol
(3-S-CAC) and 2-S-cysteaminylhydroquinone (2-S-CAH). 4-S-CAC was found to be a better substrate for
melanoma tyrosinase than was i.-3,4-dihydroxyphenylalanine,
the natural
catecholic substrate. 3-S-CAC was a poor substrate, whereas 2-S-CAH
was not a substrate. 4-S-CAC was the most cytotoxic to three lines of
melanoma cells in vitro, followed by 2-S-CAH and 3-S-CAC. When ap
plied i.p. for 9 days at a dose of 100 mg/kg, 4-S-CAC-HCI, increased by
46-52% the life span of C57BL/6 mice inoculated i.p. with B16 melanoma;
this effect was comparable to that of a 50 mg/kg dose of 5-(3,3-dimethyltriazenyl)-l//-imidazole-4-carboxamide.
3-S-CAC was marginally effec
tive, whereas 2-S-CAH was toxic to the host. This systemic toxicity of
2-S-CAH reflected its susceptibility to autoxidation. Growth of B16 mel
anoma cells inoculated s.c. was significantly inhibited by i.p. administra
tion of 4-S-CAC-HCI (200 mg/kg) for 5 days (P < 0.05). These results
suggest that 4-S-CAC is a potent antimelanoma agent, the effect of which
is mostly mediated through tyrosinase oxidation.
INTRODUCTION
Melanogenesis is a biochemical property unique to melanoma
cells among cancers. In normal and malignant cells, the specific
enzyme tyrosinase catalyzes the oxidative conversion of the com
mon amino acid L-tyrosine to melanin pigments (reviewed in Ref.
1). The first steps are the hydroxylation of L-tyrosine to form
L-dopa'1 and the subsequent oxidation of L-dopa to i.-dopaquinone.
L-Dopaquinone, an o-quinone, is highly reactive and immediately
undergoes cyclization followed by oxidation to form a more stable
intermediate, t.-dopachrome. The rearrangement of dopachrome
leads to 5,6-dihydroxyindole
and 5,6-dihydroxyindole-2-carboxylic acid. These dihydroxyindoles are oxidized to form indolequinones, which are polymerized to form ultimately the brown-black
pigment, eumelanin. It has recently been shown that TRP1, another
enzyme unique to pigmented cells, also catalyzes oxidation of
5,6-dihydroxyindole-2-carboxylic
acid to the indolequinone,
whereas tyrosinase oxidizes 5,6-dihydroxyindole
but not its carboxylic acid (2).
Received 11/2/94; accepted 4/11/95.
The costs of publication of this article were defrayed in part hy the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
IX U.S.C. Section 1734 solely to indicate this fact.
1This study was supported in part by Grants-in-Aid for Cancer Research from the
Ministry of Health and Welfare of Japan.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: L-dopa, u-3,4-dihydroxyphenylalaninc;
TRP1, tyrosinase-related protcin-1; CAP, cysteaminylphenol; MAO, monoamine oxidase; CAC, cysteaminylcatechol; CAH, cysteaminylhydroquinone; DTIC, 5-(3,3-dimethyl-triazenyl)-l//imidazole-4-carboxamide; MBTH. 3-methyl-2-benzothiazoline hydrazone; NMR, nuclear
magnetic resonance; %ILS, percentage increase in life span.
Hochstein and Cohen (3) suggested as early as 1963 that reactive
quinoid intermediates generated during melanogenesis could be toxic
to the cell in which they are produced, i.e., the melanocyte. In 1969,
Riley (4, 5) introduced 4-methoxyphenol (4-hydroxyanisole) as a
melanocytotoxic compound. Subsequently, Wick, Byers, and Frei (6)
and Wick (7) showed that L-dopa and related catechols (o-diphenols)
are selectively toxic to melanoma cells. Since then, many groups have
attempted to develop chemotherapeutic agents that exhibit selective
toxicity to melanoma cells through oxidative conversion to quinoid
intermediates (reviewed in Refs. 8 and 9). We have synthesized a
number of phenolic and catecholic melanin precursors and evaluated
their cytotoxicity in vitro and in vivo (10-12). Among the new
compounds tested, 4-5-CAP appeared to be the most potentially
promising antimelanoma agent (Fig. 1). 4-5-CAP is a good substrate
for tyrosinase, and the oxidized form binds to thiol enzymes (13). In
vitro experiments have shown that it is incorporated and becomes
cytotoxic to melanoma cells (14, 15) and that it inhibits DNA syn
thesis in melanoma cells (16); these phenomena are correlated to the
degree of pigmentation. The inhibition of DNA synthesis is correlated
to inhibition of thymidylate synthase, a thiol enzyme essential for
DNA synthesis and, eventually, cell survival (17). Furthermore, sev
eral in vivo studies have shown that 4-5-CAP accumulates in the eye
and the tumor of a B16 melanoma-bearing mouse (14), inhibits
growth of B16 mouse melanoma (12, 16, 18) and a human melanoma
xenograft (19), and increases the life span of B16 melanoma-bearing
mice (16, 18). The phenol also destroys melanocytes in hair follicles of
black mice and reduces the number of B16F10 colonies in lungs (20).
However, 4-5-CAP is not potent enough to pursue further chemo
therapeutic trials. Reasons for this limitation may be related to the
strong hypotensive effect of 4-5-CAP (21) and its conversion to the
aldehyde form through oxidation by plasma MAO (22); these prop
erties result in a low LD5(, of 430 mg/kg (16). To increase the efficacy
of 4-5-CAP, we newly synthesized 4-5-CAC, the catechol derivative
of 4-5-CAP, and evaluated its biochemical properties and antimela
noma effects in vitro and in vivo. These properties were compared
with those of 3-5-CAC and 2-5-CAH, the two isomers of 4-5-CAC. It
is well known that the oxidation of L-dopa, a catechol, proceeds
approximately two orders of magnitude faster than the hydroxylation
of L-tyrosine, a phenol (23). Therefore, it was expected that, in
melanoma cells, the oxidation of 4-5-CAC would proceed much faster
than the hydroxylation (and hence oxidation) of 4-5-CAP, and thus,
4-5-CAC may exhibit a much higher antimelanoma effect than
4-5-CAP.
MATERIALS
AND METHODS
Chemicals
4-S-CAP was prepared as described by Padgette et al. (21). l.-dopa, DTIC,
MBTH, and mushroom lyrosinase (5600 units/mg) were obtained from Sigma
Chemical Co. (St. Louis, MO). All other chemicals were of analytical grade.
Melanoma tyrosinase and TRP1 were obtained from B16 mouse melanomas.
These enzymes were purified according to the procedure published elsewhere
(2, 24). The purity of the preparations was checked by SDS-PAGE and
subsequent Western blots using specific antibodies «PEP1 (anti-TRPI) and
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ANTIMELANOMA
EFFECT OF 4-S-CYSTEAMINYLCATECHOL
2-S-CAH-HC1. A mixture of 11 g (100 mmol) of hydroquinone and 5.63
y :i
SCHpCHjNH?
4-S-CAP
[i:i
g (25 mmol) of cystamine dihydrochloride in 250 ml of 47% HBr was refluxed
for 3 h. The brown solution was evaporated to dryness in a rotary evaporator.
The workup, as described for 4-S-CAC-HCI, gave a pale yellow oil of
2-S-CAH-HC1 (1.41 g; 25.5% yield), which could not be crystallized but was
CH2CH2NH2
ii
SCH2CH2NH2
4-S-CAC
3-S-CAC
found to be homogeneous as judged by HPLC: UV (0.1 M HC1) Amax265 and
310 nm (absorbance ratio, 3.2:1). 'H and 13C NMR spectra were consistent
2-S-CAH
with the structure of 2-S-CAH.
3-S-CAC-HC1. To a solution of 1.1 g (10 mmol) of pyrocatechol in 100 ml
Fig. 1. Structures of 4-S-CAP and Ihe cysteaminyldiphenols. Note that 4-S-CAC is the
catechol («-diphenol) derivative of 4-S-CAP and that 3-S-CAC and 2-S-CAH are the two
possible isomers of 4-S-CAC.
«PEP7 (anti-tyrosinase). One enzymatic unit is defined as the amount of
enzyme that catalyzes the oxidation of 1 /xmol of L-dopa/min at 30°Cin both
cases.
HPLC
The HPLC system consisted of a Jasco PU-980 intelligent liquid Chromato
graph with a Jasco 851-AS intelligent sampler (JASCO, Tokyo, Japan), a Jasco
840-EC electrochemical detector, and a 4.6 x 150 mm (internal diameter) Cls
reverse-phase column packed with Jasco Catecholpack (particle size, 7 jj,m).
The mobile phase was 0.1 M potassium phosphate buffer (pH 2.1) containing
1 mM EDTA:methanol, 92:8 (v/v). The column was maintained at 30°C,and
of methanol containing 1 ml of 98% formic acid, were added 6 g of anhydrous
sodium sulfate and 4.7 g (20 mmol) of silver oxide at room temperature. The
mixture was vigorously stirred for 5 min and filtered through a layer of sodium
sulfate, and the filtered mass was washed with methanol. The combined,
wine-red filtrate was added dropwise over 10 min to a stirred solution of 1.13
g (10 mmol) of cysteamine hydrochloride in 20 ml of water and 20 ml of
methanol at room temperature. Five ml of 6% sulfurous acid were added to the
solution, and the mixture was concentrated to approximately 25 ml. The
residue was extracted with ethyl acetate (3 x 60 ml) to remove byproducts. The
aqueous layer was evaporated, and the residue was chromatographed on a
Dowex 50W-X2 column (3.6 X 10 cm). Elution with 3 MHC1 and evaporation
of fractions containing 3-S-CAC left the HC1 salt of 3-S-CAC. Crystallization
from ethanol yielded 1.45 g (65.4%) of 3-S-CAC-HC1 as colorless needles:
m.p. 141-144°C (dec.); UV (0.1 M HC1) Amax265 nm (e 5600) and 310 nm
(3200). JH and 13C NMR spectra were consistent with the structure of
3-S-CAC.
the flow rate was 0.7 ml/min. The diphenols were detected at 500 mV versus
a mercury/mercury chloride reference electrode.
Biochemical Studies
Melanoma Cell Lines
Tyrosinase Oxidation and Autoxidation of Cysteaminyldiphenols.
Re
action mixtures contained 0.1 mM 4-S-CAC, 3-S-CAC, or 2-S-CAH in 1 ml of
The origin of human melanoma cell lines HMV-II (pigmented) and HMV-I
(nonpigmented) was described previously (22). These cells were cultured as
monolayers at 37°Cin Ham's F-10 medium (with glutamine; from GIBCO
BRL, Grand Island, NY) supplemented with 15% PCS, (100 units/ml) peni
cillin, and (100 /ng/ml) streptomycin in a 5% CO2 atmosphere. The human
melanoma cell line MM418 (highly pigmented) was obtained from Dr. P. G.
Parsons (Queensland Institute of Medical Research, Brisbane, Australia). The
cells were cultured in RPMI 1640 (GIBCO BRL) supplemented with 5% PCS,
penicillin, and streptomycin.
Synthesis of Cysteaminyldiphenols
4-S-CAC-HCI and 2-5-CAH-HC1 were prepared and purified in an essen
tially similar manner as that described for the preparation of 4-S-cysteinylcatechol (11). 3-S-CAC-HC1 was prepared by the addition reaction of cysteamine
to o-benzoquinone (11).
4-S-CAC-HCI. A mixture of 33 g (300 mmol) of pyrocatechol and 16.9 g
50 mM sodium phosphate buffer (pH 6.8). For tyrosinase oxidation, 5 jug of
mushroom tyrosinase were added, and the oxidation was carried out at 37°C.
At 5, 10, 20, and 30 min, a 10-yu.laliquot of the reaction mixture was removed
and mixed with 90 /j.1 of 0.4 M HC1O4. The concentrations of the substrate
remaining were measured by HPLC (see above). Autoxidation was carried out
similarly but without tyrosinase. The results were averages for two separate
experiments, with each experiment giving a similar result.
Determination of Kinetic Constants for Melanoma Tyrosinase and
TRP1. Reaction mixtures (1 ml total volume) were prepared by adding
different concentrations of a substrate ranging from 0.2 to 1 mM in media
consisting of 10 mM sodium phosphate buffer (pH 6.8), 0.1 mM EDTA, and 3
mM MBTH. After the addition of 2 milliunits (10 /xg) of mouse melanoma
tyrosinase or 1.4 milliunits (126 jug) of TRP1, the reactions were followed
spectrophotometrically at 500 nm and 37°C,and the formation of the conju
gates between MBTH and o-quinones formed from 4-S-CAC or L-dopa was
recorded (25). The solution of 4-S-CAC was freshly prepared in each exper
(75 mmol) of cystamine dihydrochloride in 500 ml of 47% HBr was refluxed
for 2 h. The reddish brown solution was evaporated to dryness in a rotary
evaporator. The residue was dissolved in 100 ml of 3 M HC1 and extracted with
ethyl acetate (3 x 100 ml) to remove unreacted pyrocatechol and byproducts.
The aqueous layer was evaporated, and the residue was chromatographed on a
Dowex 50W-X2 column (3.6 x 15 cm; equilibrated with 3 MHC1). The column
iment to avoid autoxidation. The reaction was followed for 5 min against
appropriate controls containing the same chemicals but lacking the enzyme.
The Km and Vmalldeterminations were repeated twice and had good reproducibility. Oxidation of 4-S-CAP was performed in the presence of 20 /XMof
L-dopa as the cofactor for the tyrosine hydroxylase activity of the enzyme.
was eluted with 3 M HC1, and fractions of 20 ml were collected and analyzed
by UV spectrophotometry. Evaporation of fractions 36-85 left the HC1 salts of
4-S-CAC and 3-S-CAC; the 4-S isomer was the major one. A concentrated
In Vitro Cytotoxicity
solution of the mixture in ethanol was left in a freezer. Colorless needles of
4-S-CAC-HCI were precipitated and filtered and washed with acetone. 4-SCAC-HC1 (5.07 g; 30.5% yield) was obtained with an isomerie purity of 97%,
as judged by HPLC. Pure 4-S-CAC-HCI, free of 3-S-CAC, could be obtained
by processing fractions appearing later (71-85) from the Dowex 50W-X2
column: m.p. 142-145°C (dec.); UV (0.1 M HC1) Amax265 nm (e 5600) and
310 nm (3200); 'H NMR (D2O) 5 2.95 (4H, s, -S-CH2-CH2-NH2), 6.72 (IH,
d, J = 8.1 Hz, C6H), 6.77 (IH, dd, J = 2.0, 8.1 Hz, C5H), 6.86 (IH, d, J = 2.0
Hz, C3H); and 13C NMR (D2O) 6 32.4 (-S-CHj-), 38.2 (-CH2-NH2), 116.8
(C3), 120.0 (C6), 122.8 (C5), 125.4 (C4), 144.49 (C2), and 144.52 (C1).
Assay
Exponentially growing melanoma cells were harvested, inoculated at a
concentration of 2 X IO5 cells/dish into 60-mm Falcon Petri dishes, and
allowed to attach for 24 h prior to exposure to the cysteaminyldiphenols. The
cells were incubated continuously with either a regular medium or a medium
containing one of the chemicals at concentrations of 10, 30, 100, and 300 JUM.
After 48 h of incubation, cells were harvested by trypsinization with 0.25%
trypsin-EDTA and counted in a Sysmex F-610 Micro Cell Counter (Kobe,
Japan). The IC50s were calculated on the basis of three separate experiments,
with each experiment being performed in duplicate. In addition, cytotoxicity of
4-S-CAC was assayed in a serum-free medium. The experiments were per
formed in a similar manner as described above, but BIO-RICH 1 serum-free
medium (Flow, McLean, VA) was used.
In Vivo Cytotoxicity
Calculated:
Found:
C43.34,
C43.21,
H5.46,
H5.61,
Cl, 15.99,
Cl, 15.86,
N6.32,
N6.32,
S 14.46
S14.42
Assay
Pigmented B16 melanoma has been maintained by following the National
Cancer Institute protocol (26). On day 0, 5-week-old C57BL/6 x DBA/2 fl
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ANTIMELANOMA
EFFECT OF 4-S-CYSTEAMINYLCATK
(BDF,) mice, weighing 20-21 g, were given i.p. inoculations of 5 X 10'' B16
Table 1 Kinetic constants of 4-S-CAC, L-dopa, and 4-S-CAP for melanoma ttrosinase
ami TRPI"
melanoma cells. Treatment of mice, 10 animals/group, was begun on day 1 and
continued daily for 9 days. The chemicals, dissolved in 1 ml of 0.9% NaCI,
were given i.p. once a day. Control animals received i.p. injections of 1 ml of
0.9% NaCI. The effect of the chemicals was expressed in the %ILS.
In Vivo Growth
Inhibition
B16 melanoma cells (IX
Tyrosinasc
Substrate
4-S-CAC
L-dopa
4-S-CAP*
of B16 Melanoma
IO6 cells) were inoculated s.c. at the axillary
"Tyrosinase
region of 25 C57BL/6 mice (5-week-old mice; 18-20 g), and treatment was
initiated on day 8, when tumors reached an estimated volume of approximately
100 mm\ The 25 mice were divided into 5 groups of 5 mice each: control; 300
mg/kg 4-S-CAP; 100 mg/kg 4-5-CAC-HCI; 2(K) mg/kg 4-5-CAC-HCI; and 50
mg/kg DTIC. The chemicals, dissolved in 1 ml of 0.9% NaCI, were given i.p.
once a day for 5 consecutive days. Control animals received 0.9% NaCI.
Tumor diameters were measured with calipers on alternate days, and tumor
volumes were calculated by the formula: long axis X (short axis)2 X 1/2.
3-S-CAC
o^\^_75SO25vX.^*
—¿
2-S-CAH
755025^ü~~0~o^-.0
0
10
20
Vma»/tfm (nmol/min/mg)
(mM)
Vm„/Km
on melanoma cells in vitro"
content
cells)PTCA*
(ng/10"
CellMM418
4-S-CAC
(mM)
101
0.40
253
13.6
2.27
6.0
0.51
87
171
19.6
l.W
10.3
6.4
0.32
20
and TRPI were purified from B16 mouse melanomas (2, 24). The
Table 2 Cylotoxicity of i:\steaminyUiphenols
RESULTS
a—oV.—¿
(nmol/min/mg)
TRPI
reaction mixtures consisted of 0.2 to 1 mM substrate in 10 mM of phosphate buffer (pH 6.8)
containing 0.1 mM EDTA and 3 mM MBTH. The oxidation was started by adding 2
milliunits of tyrosinase or 1.4 milliunits of TRPI and was followed spectrophotometrically
at 500 nm and 37°C;the formation of the conjugate between MBTH and u-quinone
formed from 4-5-CAC or t.-dopa was recorded (25).
h Oxidation was carried out in the presence of 20 fiM L-dopa as a cofactor.
' The hydroxylation of 4-S-CAP by TRPI was too slow to be measured accurately.
Growth rates were expressed in log Vt/Vo, where Vt is the tumor volume at
each measurement, and Vo is the tumor volume on day 8.
Synthesis of Cysteaminyldiphenols. The three isomers of cysteaminyldiphenols, 4-5-CAC, 3-5-CAC, and 2-5-CAH, were newly
synthesized using standard procedures. They were prepared in gram
scale, and 4-5-CAC and 3-5-CAC were obtained as crystals.
Cysteaminyldiphenols
as Substrates for Tyrosinase. The Cys
teaminyldiphenols were examined as substrates for tyrosinase and for
autoxidation. Fig. 2 shows that 4-5-CAC was rapidly oxidized by
mushroom tyrosinase, whereas 3-5-CAC was oxidized very slowly.
Although detailed kinetic experiments were not performed, 4-5-CAC
was oxidized at least 5-fold faster than 3-5-CAC. 2-5-CAH did not act
as a substrate for tyrosinase, as expected from the /7-diphenol struc
ture. By autoxidation, 2-5-CAH was oxidized most rapidly, followed
by 4-S-CAC and 3-5-CAC; the difference between the latter two was
small.
Because 4-5-CAC was found to be a good substrate for mushroom
tyrosinase, we next examined the efficacy of this catechol, in com
parison with L-dopa, as a substrate for tyrosinase and TRP1 isolated
from B16 mouse melanoma (2, 24). Oxidation of the catechols by
these melanogenic enzymes was monitored spectrophotometrically at
500 nm following the formation of dark pink conjugates between
MBTH and o-quinones (25). 4-5-CAC had a lower Km value and a
higher Vmax value than L-dopa, the catecholic natural substrate for
tyrosinase (Table 1). The specificity constant VmaJKm was 1.5 times
higher for 4-5-CAC than for L-dopa, indicating that 4-5-CAC is a
better substrate for tyrosinase than is L-dopa. At concentrations higher
than 0.5 mM, 4-5-CAC became inhibitory to tyrosinase. 4-5-CAP was
HOI
AHP*130
+ 2 (6.4 ±0.6)
1460
±27
±ft
HMV-II
W
169
27 ±4 (5.5 ±0.4)
39 ±1
92 ±2
HMV-IMelanin <1.0
63+ 1
10IC5l4-S-CAC21
15 ±1(8.1 ±0.7)3U*M)3-S-CAC157
108 ±142-S-CAH70
"Two hundred thousand cells were plated in 60-mm Petri dishes in RPMI 1640-5%
PCS (MM418) or Ham's F-10-15% PCS (HMV-II and HMV-I). Twenty-four h later, the
medium was aspirated and replaced with the same medium containing 10. 30, 100, and
300 UMof the compound. After 48 h of incubation, cells were harvested by trypsini/ation
and counted. The 1C,,, values were calculated on the basis of three separate experiments,
with each experiment being performed in duplicate. Data are expressed as means ±SE.
' PTCA, pyrrole-2,3,5-tricarboxylic
acid; AHP, aminohydroxyphenylalaninc.
PTCA
and AHP are specific indicators of eumelanin and pheomelanin, respectively. They were
determined by using our HPLC method (32, 33).
The values in parentheses were obtained by culturing in a serum-free medium.
Twenty-four h after incubation as described in footnote a, the medium was replaced with
BIO-RICH serum-free medium containing 2.5, 5, 10, and 20 /¿M
of 4-S-CAC. After 48 h
of incubation, cells were harvested and counted.
not oxidized by tyrosinase unless L-dopa was added as a cofactor. In
its presence, 4-5-CAP was slowly oxidized by tyrosinase, but the rate
was approximately 16-fold lower than that for 4-5-CAC.
TRPI was less effective than tyrosinase in oxidizing these cat
echols. Compared with L-dopa, 4-5-CAC was a slightly poorer sub
strate for TRPI. Inhibition of TRPI activity was not observed, even at
a 1 mM concentration of 4-5-CAC.
In Vitro Cytotoxicity of Cysteaminyldiphenols.
The in vitro cytotoxicity of Cysteaminyldiphenols was assessed against three lines of
melanoma cells, a highly pigmented MM418, a moderately pigmented
HMV-II, and a nonpigmented HMV-I (Table 2). The Cysteaminyldi
phenols were highly toxic to the melanoma cells; the IC5l,s were in the
10-100 JAM range. 4-5-CAC was the most toxic, followed by
2-5-CAH and 3-5-CAC. The toxicity of 4-5-CAC was not affected
by the difference in ability to synthesize melanin.
We have shown previously that in vitro cytotoxicity of 4-5-CAP is
mostly mediated through its conversion to the aldehyde formed by the
action of plasma MAO present in the PCS (22). Therefore, we
examined the cytotoxicity of 4-5-CAC in a serum-free medium. The
results, shown in Table 1, indicate that the cytotoxicity did not
diminish, but rather increased, in the serum-free medium.
In Vivo Cytotoxicity of Cysteaminyldiphenols.
4-5-CAC-HCI
.75-5025"SÕT^
30
0
10
20
30
0
10
20
30
Time (min)
Fig. 2. Tyrosinasc oxidation and autoxidation of Cysteaminyldiphenols. Cysteaminyl
diphenols (0.1 mM) were oxidized at pH 6.8 either by 5 Mg/ml mushroom tyrosinase (•)
or by air (O). Concentrations of the substrate remaining were measured by HPLC. The
data arc averages tor two determinations. 2-S-CAH was rapidly converted to an interme
diate within a few minutes even when tyrosinase was not added; the figure depicts the
decrease of this intermediate. Although the structure of this intermediate is unknown, one
possibility is that it is the cyclized, dihydrobenzothiazinc derivative, such as that formed
in the course of phcomelanogenesis (1).
was found to be highly cytotoxic to B16 melanoma cells inoculated
i.p. to BDF, mice, with the %ILS values being 46 and 49% at doses
of 100 and 200 mg/kg, respectively (Table 3, Experiment 1). The
effects were comparable to a 50-mg/kg dose of DTIC, which is the
single most effective chemotherapeutic agent clinically used against
malignant melanoma (27). The catechol was not toxic to the host, as
judged by the body weight change. The two isomers of 4-5-CAC, i.e.,
3-5-CAC and 2-5-CAH, were not effective against B16 melanoma;
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ANTIMELANOMA
EFFECT OF 4-S-CYSTEAMINYLCATECHOL
200 mg/kg dose did not give signs of systemic toxicity. In addition,
we did not observe depigmentation of growing hair, such as was
frequently seen when 4-S-CAP was administered to mice (12, 20).
weight change
A major limitation of 4-S-CAP is the strong hypotensive effect (21).
%ILS"
(g)c46^49P42'-516471.11.30.6K).31.11.51.21.4+
It appears that this hypotensive effect of 4-S-CAP was abolished by its
conversion to the catecholic structure of 4-S-CAC. MAO plays a
major role in promoting the cytotoxicity of 4-S-CAP in vitro through
its conversion to the aldehyde and the formation of H2O2 (22, 28).
However, MAO does not seem to play such a role in 4-S-CAC,
because 4-S-CAC exhibited a higher cytotoxicity in a serum-free
medium than in a serum-containing medium (Table 2). It seems that
the oxidative metabolism proceeds much faster than the MAO-
Table 3 Effects of cysteaminyldiphenols on the life span of mice who received
¡.p.B16 melanoma"
(day)Compound
Survival time
(mg/kg]Experiment Dose
1Control4-S-CAC-HC14-S-CAC-HC1DTICExperiment
2Control3-S-CAOHC13-S-CAC-HC1DTICExperiment
3Control2-S-CAH-HC12-S-CAH-HC1Experiment
dependent degradation.
Although 4-S-CAC is as potent as DTIC when applied i.p. to
-1.9+
i.p.-transplanted B16 melanoma, the catechol is not as potent as DTIC
4Control4-S-CAOHC14-S-CAOHC14-S-CAPDTIC010020050050100500100200010020030050Median28.541.542.540.521.520.525.031.531.527.024.527.041.045.031.040.0Range23-3721-4436-4631^4519-4319-4522when applied i.p. to the same melanoma transplanted s.c. This sug
1.052'
+1.0bT1
gests that some of the 4-S-CAC administered may be metabolized
1.2-15
+0.3-22
+1.215
+0.948f
+1.2
" On day 0, 5-week-old BDF, mice (10/group), 20-21 g, were given i.p. inoculations
of 5 X IO6 B16 melanoma cells. Compounds were administered i.p. daily for 9 days
starting on day
* Percentage
' Difference
'' Significant
' Significant
1.
of increase in life span of treated versus control animals.
in body weight between days 5 and 1.
at P < 0.005 from the control using the log-rank test (Cox-Mantel test).
at P < 0.001 from the control using the log-rank test (Cox-Mantel test).
3-S-CAC was marginally effective, whereas 2-S-CAH was toxic to the
host.
Because 4-S-CAC-HC1 was found to be effective against B16
melanoma, we then compared its antimelanoma potential with 4-SCAP. The results show that the catechol 4-S-CAC was much more
effective in prolonging the life span of B16 melanoma-bearing mice
than was the parent phenol 4-S-CAP (Table 3, Experiment 4).
during circulation before it reaches s.c. transplanted tumor. Possible
metabolic pathways include conversion to the aldehyde by MAO,
O-methylation by catechol O-methyltransferase, and nonspecific ox
idation to the o-quinone form. To overcome this problem, we plan to
administer 4-S-CAC more frequently in future in vivo experiments.
Another approach is to protect either the o-dihydroxyl group by
acetylation or phosphorylation (29) or the amino group by N-acetylation. The latter approach has been evaluated for 4-S-CAP in the form
of yV-acetyl-4-S-CAP, which showed some improvements in efficacy
as an antimelanoma agent (30). Furthermore, combination therapy
with buthionine sulfoximine was shown to enhance the antimelanoma
activity of A'-acetyl-4-S-CAP through the depletion of tissue glutathione (30). The same approach may also be applied to enhance the
therapeutic efficacy of 4-S-CAC. In connection with the significance
of tissue glutathione level, it should be noted that mouse melanoma
In Vivo Growth Inhibition of Melanoma. We examined the ef
ficacy of 4-S-CAC in inhibiting the growth of B16 melanoma inoc
ulated s.c. to C57BL/6 mice (Fig.3). At 14 days after tumor inocula
tion, the growth was inhibited by 67% in mice treated with a 200
mg/kg dose of 4-S-CAC-HC1 (P < 0.05); this effect was slightly lower
J3
O
than that exhibited by a 50 mg/kg dose of DTIC. The antimelanoma
effect of 4-S-CAP at a dose of 300 mg/kg was comparable to that of
4-S-CAC-HC1 at a dose of 100 mg/kg, although their effects were not
Cont
CAP 300
CAC 100
CAC200
DTIC 50
k_
O
statistically significant.
DISCUSSION
(0
0)
O)
In this study, we have shown that 4-S-CAC is a better substrate for
tyrosinase than is L-dopa, and the two isomers are either a poor
substrate or not a substrate at all. TRP1, a second oxidase unique to
melanocytes, can oxidize 4-S-CAC as well, although at a much slower
rate. 2-S-CAH was the most susceptible to autoxidation. These bio
chemical properties fit well with the in vivo cytotoxicity of the
cysteaminyldiphenols to B16 mouse melanoma. 4-S-CAC is as potent
as DTIC in increasing the life span of B16 melanoma-bearing mice,
whereas 3-S-CAC is marginally effective, and 2-S-CAH is toxic to the
host. The strong melanocytotoxicity of 4-S-CAC in vitro appears to
correspond to its ability to be oxidized by tyrosinase and TRP1. These
results suggest that the antimelanoma effect of 4-S-CAC is dependent
on tyrosinase activity, and the systemic toxicity of 2-S-CAH is related
to its susceptibility to autoxidation.
It is argued that catechols may not be suitable for antimelanoma
agents in view of their susceptibility to autoxidation and systemic
toxicity. However, mice tolerated a single i.p. administration of
400-600 mg/kg 4-S-CAC-HC1, and repeated administration at a
co
u
O)
8
10
12
14
16
18
Days after tumor inoculation
Fig. 3. Effect of 4-5-CAC on growth rate of B16 melanoma in vivo. Arrows at the
bottom, times of injection. Treatment was initiated on day 8 after tumor inoculation. •¿,
control; O, 300 mg/kg 4-S-CAP; •¿100 mg/kg 4-S-CAC-HC1; D, 200 mg/kg 4-SCAC-HC1; A, 50 mg/kg DTIC; 5 mice/group. Growth rates were expressed in log Vt/Vo,
where Vt is the tumor volume at each measurement and Vo is the tumor volume on day
8. *, P < 0.05 from the control with the use of the Mann-Whitney nonparametric U test.
4-S-CAP (300 mg/kg) and 4-S-CAC-HC1 (100 mg/kg) did not show a significant differ
ence from the control on any measurement.
2606
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1995 American Association for Cancer Research.
ANTIMELANOMA
EFFECT OF 4-S-CYSTEAMINYLCATECHOL
cells become more sensitive to oxidative lysis through the glutathione
depletion (31).
In conclusion, the o-diphenol 4-5-CAC is a more potent antimelanoma
agent than its parent phenol 4-S-CAP. The tyrosinase (and TRP1)dependent cytotoxicity is suggested to be the major mechanism of antimelanoma action of 4-5-CAC. The oxidized o-quinone form of 4-5-CAC
may bind essential thiol enzymes such as thymidylate synthase (13,17).
However, not all of the present results fit this hypothesis; we did not
observe a correlation between in vitro cytotoxicity and cellular melanin
content (Table 2). It is possible that, in the in vitro experiments, 4-S-CAC
might be oxidized by free iron present in the medium, and the resultant
o-quinone and/or active oxygen species could exert cytotoxicity. Addi
tional studies are certainly necessary to clarify the mechanism(s) of
cytotoxicity of this potentially promising antimelanoma agent and to
augment the cytotoxic effect of 4-5-CAC against melanoma.
ACKNOWLEDGMENTS
We thank Dr. P. G. Parsons for critical reading of the manuscript and for
donating the MM418 melanoma cell line.
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Antimelanoma Effect of 4-S-Cysteaminylcatechol, an Activated
Form of 4- S-Cysteaminylphenol
Shigeki Inoue, Katsutoshi Hasegawa, Shosuke Ito, et al.
Cancer Res 1995;55:2603-2607.
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