SUPPRESSION OF PIGMENTATION IN MOUSE MELANOMA CELLS BY 5-BROMODEOXYURIDINE

Low concentrations (1–3 µg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B559) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C3471), derived by long-term culture of B559 cells in 1 µg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B559 cells during a 7-day period of growth with BrdU (3 µg/ml). Assays of tyrosinase activity showed that activity was reduced after 1 day and decreased progressively, approaching zero by 7 days. A quantitatively minor part of this reduction was directly attributable to the appearance of a dialyzable inhibitor of tyrosinase activity. Acrylamide gel electrophoresis revealed two bands of activity corresponding in Rx values to the T1 and T2 forms of soluble tyrosinase. Both were progressively reduced during growth with BrdU but one form (T1) was consistently affected earlier than the other (T2). Ultrastructural-cytochemical studies also showed an early effect on the localization of tyrosinase reaction product. At day 3, reaction product was no longer present in Golgi saccules and Golgi-associated smooth surfaced tubules, but was still seen within premelanosomes, compound melanosomes, and occasional Golgi-associated vesicles. By 7 days tyrosinase reaction product was usually not demonstrable. The number of premelanosomes was progressively decreased during growth with BrdU. Premelanosomes became concentrated in the juxtanuclear region and at day 3 many were contained within abnormally large and numerous compound melanosomes. Premelanosomes and compound melanosomes were rarely seen at 7 days, by which time the cultures were nearly amelanotic. The coordinated suppression of melanogenesis by BrdU may provide a useful model in which to study the normal regulation of this process.

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Novel Chemically Modified Curcumin (CMC) Derivatives Inhibit Tyrosinase Activity and Melanin Synthesis in B16F10 Mouse Melanoma Cells

Biomolecules ◽

10.3390/biom11050674 ◽

2021 ◽

Vol 11 (5) ◽

pp. 674

Author(s):

Shilpi Goenka ◽

Francis Johnson ◽

Sanford R. Simon

Keyword(s):

Enzyme Activity ◽

Parent Compound ◽

Radical Scavenging ◽

Melanoma Cells ◽

Tyrosinase Activity ◽

Partial Recovery ◽

Tyrosinase Inhibitor ◽

Pyrocatechol Violet ◽

Mouse Melanoma ◽

Chemically Modified

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.

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