C.R.C.-B.A.C.R.-A.I.C.R. International Workshop
Melanogenesis : its chemistry as a therapeutic strategy in melanoma
E.J. Land
CR.C. Department of Biophysical Chemistry, Paterson Institute
for Cancer Research, Christie Hospital and Holt Radium
Institute, Manchester M20 9BX, U.K.
This Workshop brought together 60 scientists from 13 countries
investigating mechanisms of melanogenesis and rational approaches towards using the
melanogenic pathway as a means of targeting the cytotoxic therapy of malignant melanoma.
The organisers were : Prof. D.G. Harnden and Dr E.J. Land (Paterson Institute for Cancer
Research, Manchester), Prof. P.A. Riley (University College and Middlesex School of
Medicine, London), Dr N. Thatcher (Christie Hospital, Manchester) and Prof T.G. Truscott
(University of Keele).
The unique biochemical characteristic of melanocytes is the propensity to produce melanin.
The meeting was convened specifically to examine recent advances in knowledge of
melanogenesis and the possibility that there might be some way of exploiting the
melanin-forming property of malignant melanocytes as a means treating melanoma. An
improved understanding of the chemistry of melanogenesis might lead to the ability to
manipulate such chemistry which could lead to a treatment of melanoma based on subverting
the chemistry of melanogenesis.
The problem of melanoma was put in perspective by Prof. Rona MacKie (Glasgow, U.K.) who
discussed the epidemiology and pathogenesls of melanoma and emphasized the
importance of early detection, with the prognosis worsening rapidly for increasingly thick
lesions due to disseminated disease. Epidemiological and case-controlled studies strongly
implicate ultra-violet radiation, in particular its B component (280-315 nm), as a major
aetiological factor in cutaneous melanoma. Those at greatest risk appear to be
white-skinned subjects who have an indoor occupation but who indulge in intense sun
exposure for short periods of time, e.g. on vacation. Additional risk factors include fair
complexion and large numbers of benign melanocytic naevi. The unsatisfactory nature of current
therapy for disseminated melanoma was reviewed by Dr N. Thatcher (Manchester, U.K.)
who gave an overview of the metastatic behaviour of melanoma and the relatively poor
results of treatment with a range of cytotoxic agents, immunotherapy, and adjuvant
treatments. Although in most clinical series there were a proportion of responses, the
efficacy of current treatments for metastasizing melanoma is currently highly
unsatisfactory and new strategies are urgently needed.
The classical Raper-Mason scheme for melanogenesis postulates a pathway involving,
successively, tyrosine, dopa, dopaquinone, dopachrome, dihydroxyindoles, indolequinones...
leading to eumelanin, or phaeomelanin with the added involvement of cysteine. Enzymes
are essential to some reactions in this process. Dr F. Solano (Murcia, Spain) described
how the regulation of mammalian eumelanogenesis is mainly carried out not only by the
well-known enzyme tyrosinase, but also by the more recently discovered enzyme dopachrome
tautomerase. This enzyme is able to catalyze dopachrome tautomerisation into
5,6-dihydroxyindole-2-carboxylic acid (DHICA), thus preventing dopachrome decarboxylation
taking place in the spontaneous rearrangement of dopachrome into 5,6-dihydroxyindole (DHI)
at neutral pH. As o-diphenols, both DHI and DHICA are possible substrates of
tyrosinase leading to the corresponding o-quinones. The relative concentrations of
these dihydroxyindoles. and hence their o-quinones and the composition of the
resulting eumelanin polymer, depends crucially upon the activity of both enzymes. This
controlled polymerisation may be a natural mechanism to protect melanocytes against the
known cytotoxicity of the decarboxylated indoles, which are more reactive than their
2-carboxylated counterparts. The possibility that peroxidase could also be involved
in later stages of the biosynthesis of eumelanins was discussed by Prof G. Prota (Naples,
Italy). Although peroxidase cannot convert the monophenol tyrosine to the diphenol, dopa,
and hence lead to melanin, evidence was presented showing that peroxidase is much more
effective than tyrosinase in catalysing the oxidative conversion of DHI and DHICA to
melanin pigments.
The stimulation of tyrosinase in melanoma cells by adrenoceptor agonists and by
catecholic compounds was described by Prof H. Rorsman (Lund, Sweden), the aim being to try
to develop ways of influencing the metabolism of catechols. An increase in tyrosinase
activity could thus lead to the enhanced production of cytotoxic quinones. The stimulating
and cytotoxic effects of isoprenaline, theophylline, terbutaline and DOPAC on IGR1
melanoma cells was discussed in terms of their abilities to penetrate the cells, modify
cell proliferation and generate active oxygen species, including H2O2.
Active oxygen species appear to increase tyrosinase activity in IGR1 cells. Moreover, both
the activation of tyrosinase and the cytotoxic effects of catechols were found to be
eliminated by catalase.
The melanocytes of normal humans and melanoma patients also contain the enzyme calechol-O-methyl
transferase (COMT). This enzyme methylates the indolic melanin precursors DHI and
DHICA. Dr S. Pavel (Amsterdam, The Netherlands) described the detection of such indoles in
media from human melanoma cell cultures.
Most of these possess a methoxy group in positions 5 or 6 which prevents their oxidation
to simple o-quinones. The methylation, which can be considered as a protective
mechanism in melanocytes against intrinsically generated toxic o-hydroxy, products,
is accomplished by intracellularly localised COMT.
Several contributions dealt with detailed aspects of the chemistry of early stages,
melanogenesis and related processes. An overview of quinone reactivity with respect
to melanin formation was provided by Dr J.M. Bruce (Manchester, U.K.). Although much of
the chemistry of the melanogenesis pathway up to the dihydroxyindoles DHI and DHICA is
fairly well understood, the subsequent stages, even those leading to species with
comparatively low molecular weight, are not. The problem is compounded by the complexity
of the oxidation of DHI (and DHICA), which can, in principle, lead not only to the
corresponding ortho-quinone, but also to both a quinone-imine and a
quinone-methide, all three of which may be in tautomeric equilibrium. Home- and
hetero-coupling reactions involving one or more of these tautomers, leading in the first
place to a multiplicity of isomeric dimers, could be important in the polymerisation
ultimately resulting in melanin.
Quinones and quinone-methides are also involved in the molecular mechanisms for cuticular
sclerotization as described by Prof M. Sugumaran (Boston, USA). The exoskeleton of
insects and other arthropods are hardened to protect their soft bodies by a process called
sclerotization. During hardening, soluble structural proteins and chitin fibres are
rendered insoluble by reaction with reactive species derived from enzymatic activation of
catecholamines, such as N-acetyldopamine and
N-Ò -alanyldopamine. Based on the reactive species formed,
two different mechanisms have been identified to acount for sclerotization reactions. They
are quinone tanning and quinone-methide sclerotization. Although initially these two
mechanisms were considered to be dependent of each other, the recent discovery of two new
enzymes, quinone isomerase and quinone methide isomerase, led to a unification of these
mechanisms.
The initial steps involved in sclerotization closely resemble the initial reactions
observed during melanization. Both processes involve the initial enzymic oxidation of
catechols to quinones, and quinone tautomerisation to cluinone methides. The introduction
of a double bond into the side-chain and the oxidation of the side-chain desaturated
catecholamine parallels the aromatisation of dopachrome to dihydroxyindole, and the
oxidation of dihydroxyindole to the corresponding quinone.
Details of the chemistry of the transition : dopachrome --> dihydroxyindole were
described by Prof H. Wyler (Lausanne, Switzerland). Four protonated forms of dopachrome
were identified, the 6-OH group deprotonating with a pK of 0.8, the carboxy group with a
pK of 3.1 and the nitrogen with a pK of 9.1. At neutral pH, the only product of dopachrome
decay is dihyroxyindole, whereas at high pH (> 10) it is exclusively the anion of
dihydroxyindole-2-carboxylic acid.
The maturity of the technique of pulse racliolysis is such that one can now be
confident enough to tackle problems as complex as the process of melanogenesis. Although one-electron
oxidatinn of DHI and DHICA is not thought to be a part of the melanogenic pathway, the
rapid disproportionation of such radicals is an excellent means of preparing high
concentrations of the subsequent metastable intermediates, e.g. indolequinones, which are
thought to be important components of the melanogenic pathway. Dr P. O'Neill (Chiltonl
U.K.) described the one-electron oxidation of a series of hydroxy- and methoxy-indoles
using pulse radiolysis. One-electron oxidation of dihydroxyindole in the pH range 5-10
yields the corresponding oxygen-centred indole semiquinone radical (pK 6.8). With
hydroxylated mono-methoxyindoles, the corresponding methoxyindoloxyl radical is formed.
Further methylation of the hydroxy substituents, as in dimethoxyindole, results in the
stabilisation of the corresponding cation which, depending on the pH, deprotonates at N(1)
to yield the nitrogen-centred indolyl radical, with a pK of 6.0.
With the exception of dihydroxyindole, the radicals were all found to decay bimolecularly
to yield semi-permanent products which eventually decayed unimolecularly. In the
case of dihydroxyindole, using very low pulse doses the radical was found to decay
unimolecularly. The latter decay was assigned to a reaction of the radical with the parent
dihydroxyindole, rate constant ~ 106 M-1
s-1. This could be a component of the route for melanin polymerisation.
Prof T.G. Truscott (Keele, U.K.) described a closely similar study of the species
resulting from pulse radiolytic one-electron oxidation of dihydroxvindoles and their
methoxylated metabolites. Whereas there was good agreement with the results reported by
the previous speaker on the initially formed one-electron oxidised radicals, differences
were apparent in the nature of the reactions of some of the radicals and subsequent
metastable intermediates. A possible scheme for polymerisation to melanin was
proposed involving a reaction of a quinone-methide with water to produce a
trihydroxyindole, which may itself then react successively with the methide to produce
dimers, then trimers, etc.
Prof B. Kalyanaraman (Milwaukee, USA) described the application of the technique of electron
spin resonance spectroscopy to the identification and characterisation of free
radicals derived from melanin precursors. Complexed with diamagnetic ions, especially Mg²+
and ZnL²+, catechol(amine) semiquinones live 104 times as long as
in the absence of metal, allowing their protonation-deprotonation reactions to be readily
studied. The addition reactions of quinones with nucleophiles e.g. amino acids (proline
and methionine), peptides and proteins were also studied via the radicals derived from
such adducts.
It was the discovery some 20 years ago that tyrosinase is capable of oxidising substrates
that are structural analogues of tyrosine which suggested the possibility of using
the melanogenic pathway as a targeting strategy for melanoma chemotherapy. Prof P.A. Riley
(London, U.K.) described a series of investigations on the mechanism of action of
substituted phenols of which the lead compound is the well-known depigmenting agent
4-hydroxyanisole (4HA) which is oxidised to the corresponding orthoquinone. The mechanism
of the cytotoxlcity of the orthoquinone probably depends predominantly on the formation of
adducts with important cellular thiol-containing proteins. The possibility that the action
depends on the generation of active oxygen species by redox cycling has been excluded and
there is little evidence that semiquinones are involved in the toxic process (see below).
4HA also exhibits direct toxic actions, including inhibition of ribonucleotide reductase,
inhibition of mitochondrial electron transport, and other effects on cell physiology.
Separation of these direct actions from the tyrosinase-dependent cytotoxicity would serve
to amplify the therapeutic index against melanogenic cells, and the results of tests on a
series of derivatives of 4HA including a range of oxy-ethers and thio-ethers of differing
chain length were presented. In discussion, Dr K. Schwabe (Berlin, Germany) reported that
his group had synthesized over 100 analogues of 4HA and shown that the propyl oxy-ethers
were therapeutically effective against tumour volume in melanoma-bearing animals, but had
shown no effect on survival of the animals employed in these tests.
The results of studies on the mechanism of cytotoxicity and analogues were
described by Dr E.J. Land (Manchester, U.K.). Using pulse radiolysis, semiquinones were
generated from the corresponding hydroquinones chemically synthesized by Mr C.J. Cooksey.
The semiquinones neither reacted with oxygen nor with trans-butanoic acid, a
water-soluble model for unsaturated fatty acids. Consequently, it appears unlikely that
redox cycling or the initiation of lipid peroxidation via semiquinones comprise the
cytotoxic mechanism. The 3,4-quinones which form rapidly by disproportionation of the
corresponding semiquinone were found to react rapidly with several thiols and with
ascorbic acid. Nucleophilic addition of protein thiols to the quinone is thus a more
probable mediator of cytotoxicity. 4-(n-propoxy)phenol, which possesses five times the in
vitro tyrosinase-dependent cytotoxicity towards rat cells compared with 4HA, was found
to behave almost identically as far as the reactivity of semiquinones and quinones was
concerned.
A closely similar approach to targeted cytotoxicity was described by Prof K. Jimbow
(Edmonton, Canada) who showed that 4-S-cysteaminylphenol and some analogues are
selectively incorporated into murine melanoma tissue and into actively melanising hair
follicles with selective destruction of melanocytes resulting in depigmentation of
black hair follicles in mice, N-acetyl-4-S-cysteaminylphenol being the most potent inducer
of selective destruction of follicular melanocytes. This compound may prove to be a useful
anti-tumour agent judged by the data on subcutaneously inoculated B16 F10 melanoma cells
in mice.
Dr P.G. Parsons (Queensland, Australia) reported that despite many promising in vitro
demonstrations of antimelanoma activity by redox active agents such as catechols,
and by drugs such as buthionine sulphoximine which depress cellular defences against
oxygen radicals, there remain many problems regarding the successful application of this
approach to melanomas in vivo. It may be possible to overcome the lack of potency
and selectivity of such agents by using combinations of drugs. Such combination therapy,
together with measures for minimising mutations rates, may also combat phenotypic
instability leading to the development of drug resistance.
Dr B.S. Larsson (Uppsala, Sweden) described an approach towards the therapy of melanoma
using aminothiol compounds which are selectively incorporated into
newly-synthesized melanin by reacting with orthoquinones generated during melanogenesis.
Various compounds of this class, including thiourea and 2-thiouracil, have been shown by
autoradiography to be specifically taken up into melanising tissue including murine
melanoma. Radioiodinated 5-iodo-2-uracil has been used in patients for melanoma screening
and pilot studies on treatment with 35S-thioracil have been carried out on
melanoma-bearing mice. However, the radiation doses required for substantial effects using
this radionuclide are extremely high, making therapeutic clinical application hazardous.
As an alternative, boron neutron capture therapy has been attempted, using boronated
thioureas which are selectively incorporated into newly-synthesized melanin and the 10B
can then be activated to undergo nuclear fission by irradiatin of tumours with thermal
neutrons from an external source.
Dr A.J. Winder (Oxford, U.K.) reported on the effects of L-tyrosine phosphate and
cytochalasin D on induction of pigmentation in cells, with a view to the possibility of
developing differenciation therapy for melanoma. Both agents produced a small
increase in the amount of tyrosinase messenger RNA, although the amount observed was
insufficient to account for the increase in enzyme activity.
The relationship between abnormal melanosome structure and cytotoxic phenomena was
discussed by Dr J. Borovansky (Prague, Czechoslovakia). Electron microscope investigations
have confirmed the presence of abnormal and incomplete melanosomes in human melanomas from
epidermal and mucosal sites, in melanoma metastases and in the B16 mouse melanoma. For
example, 90% of melanosomes in cutaneous melanomas had membrane defects. Evidence that
this leads to significant leakage of reactive melanin precursors was furnished by raised
free radical-mediated lipid peroxidation in the liver of B16 melanoma-bearing mice.
Prof T. Sarna (Krakow, Poland) discussed thephotoactivation of melanin and the possible
generation of cytotoxic products resulting from it. Direct photo-oxidation of melanin was
accompanied by oxygen consumption and the production of hydrogen peroxide. This process
was shown to be strongly wavelength- and pH-dependent and was also influenced by the
presence of metal ions. Photo-oxidation of melanin in the presence of photosensitizers was
also reported, and the possibility of covalent binding of photosensitizers to melanin as
an approach to photodynamic therapy was discussed. Dr A.R. Young (London, U.K.) discussed
the apparently paradoxical photoprotection of skin to UV-induced damage by prior
exposure to photosensitizers such as psoralens. Tans induced by 5-methoxypsoralen (5-MOP)
contained in a UVB sun-screen with solar stimulating radiation (SsR) were found to reduce
unscheduled DNA svnthesis following minimal erythema doses of SSR. Despite the fact that
5-MOP is weak photocarcinogen, judicious use of 5-MOP-containing sun-screens may offer
benefits with regard to protection against various forms of light-induced skin cancer
which could outweigh the risk involved. This may be of importance in protecting future
generations from the risk of developing malignant melanoma.
The Workshop generated much detailed discussion which it is hoped will be reflected in the
stimulation of new initiatives towards the introduction of a successful treatment for
disseminated melanoma. Clearly one potentially powerful strategy is targeted cytotoxicity
utilising the melanogenic pathway to activate an otherwise innocuous pro-drug.
Acknowledgements
As well as the Cancer Research Campaign, the British Association for Cancer
Research and the Association for International Cancer Research, the following are thanked
for providing financial support : Amgen Ltd., Asta Pharma AG, Beckman
Instruments Inc., Boehringer Ingelheim, Bristol-Myers Pharmaceuticals, Ciba Geigy
Pharmaceuticals, Farillon Ltd., Institut de Recherches Internationales Servier,
Laboratoires Pharmaceutiques Bergaderm SA, Lilly Industries Ltd., I'Oréal, Sterling Drug
Inc., US Air Force Office of Aerospace Research and Development, and Windsor
Phamaceuticals Ltd.