Estrogens and qrowth of malignant melanoma

Myra K. Jacobsohn and Gert M. Jacobsohn

Department of Biological Chemistry, Hahnemann University,
Philadelphia, PA 19102, USA

Melanin, the brown-black polymer in pigment cells, is being recognized increasingly as a non-specific binding agent for various organic substances. This binding is in lieu of, or in addition to, receptor protein binding which has a relatively higher specificity than the melanin. We have found that melanin is capable of binding estrogens; 2-hydroxyestradiol, a metabolite of estradiol produced in the liver, brain and uterus, binds to a somewhat higher extent than estradiol (Jacobsohn et al. 1988). Melanin which is being actively synthesized from dopa binds more estradiol than preformed melanin (Jacobsohn et al. 1988). The binding is not a unique phenomenon. A number of aromatic substances have the ability to bind to melanin and this phenomenon has been used to explain the localization of drugs at their site of action or the concentration of toxins in specific locales where they may exercise a damaging effect. The binding may also remove harmful substance from causing damage elsewhere. The successful treatment of tinnitus by lidocaine injection was thought by Lyttkens (1986) to be due to the drug accumulating on melanin of the inner ear. He found lidocaine, bupivacaine and chlorpromazine, a phenothiazine derivarive, to associate with melanin-containing structures of the eye and of the inner ear (Lyttkens et al, 1979). Melanin of the eye, hair and skin of pigmented mice can take up serotoni, dopamine, epinephrin, and norepinephrin (Lindquist, 1973). Selegiline and other amphetamines, chloroquine, dopamine, norepinephrin, and the morphine derivative MPTP, can bind to melanin granules (Bathory et al,1987; Stepien et al, 1987; Lindquist 1973; Lindquist et al, 1987). Debing et al (1988) reported the binding of 15 aromatic drugs to calf eye melanosomes and to synthetic dopa-melanin.
The binding of estrogens to melanin of melanoma could be responsible for the favorable effect associated with estrogens during the later course of development of the malignancy in women.
Women with melanoma, especially during the child-bearing age, have a better survival rate than men. The effect may be due to oxidative reactions of estradiol leading to formation of ortho-quinones which, by acting as melanocytotoxic agents, could provide the host with a mechanism for defense. Precedence exists for the action of quinones as cytotoxic intermediaries. A specific strategy for treatment of melanoma is based upon tyrosinase activation of pro-drugs with selectivity conferred by the enzyme (Wick 1983; Pezzuto et al, 1988). Quinones and semiquinones are generated and it is assumed that semiquinones act biologically via generation of superoxide (Pezzuto et al, 1988). The cytotoxicity of 4-hydroxyanisole to malignant melanoma was proposed to be due to its oxidation to the ortho-quinone and subsequent reverse dismutation to the semiquinone, but it is uncertain whether the quinone or the semiquinone is the actual cytotoxic product (Riley 1985; Nilges et al, 1984). In studies on malignant cells in culture, estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol were found to be cytotoxic to dividing MCF-7 and HeLa cells (Seegers et al, 1989). On exposure af these cells to high concentrations ( 1 x 10-6 M) of the steroids, they showed increased mitosis with formation of abnormal and fragmented polar bodies and disoriented microtubule arrangements (Seegers et al. 1989).
Tyrosinase, the enzyme responsible for melanin formation by way of successive oxidations of tyrosine and its products, has the ability to oxidize estrogens (Jellinck et al, 1971; Jellinck et al, 1963; Jacobsohn et al, 1988; Jacobsohn et al, 1984). Mushroom tyrosinase has been used for some time in the laboratory preparation of 2-hydroxyestradiol from estradiol (Jellinck et al, 1971; Hersey et al, 1981), and we  found that the enzyme can oxidize the hydroxylated product in stoichiometric relationship to molecular oxygen in presence of catechol (Jacobsohn et al, 1984). The enzyme used in this work originated from mushrooms but it is likely that the mammalian enzyme can oxidize estrogens as well. Incubations of 2,4,6,7-3H-estradiol with tvrosinase isolated from B-16 melanoma cells yielded 3H-water (Ewaskiewicz and Jacobsohn, in prepn.) by methodology analogous to the Pomerantz assay for tyrosinase activity (Pomerantz, 1969). Incubations of [14C ] - estradiol or [14C]-2-hydroxyestradiol, together with DOPA and tyrosinase from the B16 mouse melanoma cell line, produced melanin with the estrogen label firmly attached to the pigment (Jacobsohn et al, 1988). The steroids were attached to melanin in such a way that they were resistant to extraction with organic solvents such as ethyl ether or hot methanol. This was taken as evidence that the unextractable portion of steroids had been oxidized and bound to melanin in covalent linkage; similar experiments with the mushroom enzyme have already shown that the 2-hydroxy estrogen can be oxidized and incorporated into melanin (Jacobsohn et al, 1988; Jacobsohn et al, 1984). An alternate but remote possibility is that the estrogens are bound to inner spaces within the pigmented polymer where the solvent cannot reach them. In more recent experiments, it was possible to isolate and identify the 2,3- orthoquinone of estradiol from incubation mixtures of the fungal enzyme and 2-hydroxyestradiol (Jacobsohn et al. 1989; Jacobsohn et al, 1990). Incubations of the quinone with murine tyrosinase and dopa have shown that the quinone can be incorporated into melanin, perhaps by additional enzyme-catalyzed steps or through reverse dismutation with dopa-melanin intermediates (Jacobsohn et al, 1990). It should be noted that melanin itself, in the absence of tyrosinase, may function as an oxidant for estrogens, because exposure of melanin to estradiol or 2-hydroxyestradiol can cause physiologically significant amounts of steroid to be retained by the pigment (Jacobsohn et al, 1988; Jacobsohn et al,1990).
Estrogens are thought to be co-carcinogens because they are able to exacerbate the activity of true carcinogens but do not appear to initiate growth of a malignancy by themselves. After the growth of melanoma cells has been triggered by external events, estrogens may intensify the malignancy by binding to melanin of affected cells. This may explain the often-noted higher incidence of melanoma in women compared to men. As the disease progresses and increased amounts of estrogens are concentrated in melanoma cells, their conversion to cytotoxic oxidation products may present a more favourable outcome. We hope that this hypothesis will stimulate additional research on the relationship of estrogens to growth and development of pigment celIs.

References
Bathory G. Szuts T, Magyar K: Pol J Pharmacol Pharm, 39, 195-201,1987
Debing I, Ijzeman AP, Vauquelin G:Molec Pharmacol, 33, 470-476, 1988
Hersey RM, Williams KIH, Weisz J: Endocrinology, 109, 1912-1920, 1981
Jacobsohn GM, Jacobsohn MK: Arch Biochem Biophys, 232, 189-196,1984
Jacobsohn GM, Chiartas PL, HearingVJ, Jacobsohn MK: Biochim Biophys Acta, 966. 222-230, 1988
Jacobsohn GM, Jacobsohn MK. Enu B. Byler DM: The Endocrine Society.
71st Annual Meeting, 1403. 1989
Jacobsohn MK, Dobre VC, Branam C, Jacobsohn GM: J Steroid Biochem, 31, 377-385, 1988
Jacobsohn MK, Byler DM, Jacobsohn GM: submitted
Jellinck PH, Irwin L: Biochim Biophys Acta. 78, 778-780, 1963
Jellinck PH, Brown BJ: Steroids, 17, 133-140, 1971
Lindquist NG: Acta Radiol, Suppl. 325, 325, 67-82, 1973
Lindquist NG, Larsson BS, Lyden-Sokolowski A: Pigment Cell Res. 1, 133-136, 1987
Lyttkens L, Larsson BS, Stahle J, Englesson F: Adv Oto-rhino-laryng, 25, 17-25, 1979
Lyttkens L: Scand Audiol. Suppl., 26. 27-31, 1986
Nilges MJ, Swartz HM, Riley PA: J Biol Chem. 259. 2446-2451. 1984
Pezzuto JM. Shieh HL, Shaughnessyy E, Beattie CW: Sem Oncol, 150, 578-588, 1988
Pomerantz SH: Science, 164. 838-839, 1969
Riley PA: Phil Trans R Soc Lond B, 311. 679-689, 1985
Seegers JC, Aveling ML, Van Aswegen CH, Cross M, Koch F, Joubert WS: J Steroid Biochem, 32, 797-809, 1989
Stepien K, Porebska M, Wilczok T: Studia Biophys, 122, 165-174, 1987.
Wick MM, J Invest Dermatol, 80 (suppl), 61s-62s, 1983