RS-4-(4-Hydroxyphenyl)-2-butanol (rhododendrol, RD) was reported to induce leukoderma of the skin. To explore the mechanism underlying that effect, we previously showed that oxidation of RD with mushroom tyrosinase produces RD-quinone, which is converted to secondary quinone products, and we suggested that those quinones are cytotoxic because they bind to cellular proteins and produce reactive oxygen species. We then confirmed that human tyrosinase can oxidize both enantiomers of RD. In this study, we examined the metabolism of RD in B16F1 melanoma cells in vitro. Using 4-amino-3-hydroxy-n-butylbenzene as a specific indicator, we detected moderate levels of RD-pheomelanin in B16F1 cells exposed to 0.3 to 0.5 mM RD for 72 h. We also confirmed the covalent binding of RD-quinone to non-protein thiols and proteins through cysteinyl residues. The covalent binding of RD-quinone to proteins was 20- to 30-fold greater than dopaquinone. These results suggest that the tyrosinase-induced metabolism of RD causes melanocyte toxicity.
RS-4-(4-Hydroxyphenyl)-2-butanol (rhododendrol, RD), a skin-whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD-pheomelanin and RD-quinone bound to non-protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5- to 3-h exposure, due to oxidation to cystine. This pro-oxidant activity was then examined using synthetic melanins. Indeed, we find that RD-eumelanin exerts a pro-oxidant activity as potent as Dopa-pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD-eumelanin with a concomitant production of H O . We propose that RD-eumelanin induces cytotoxicity through its potent pro-oxidant activity.
These results suggest that RD exerts the cytotoxicity in melanocytic cells through its oxidative metabolites and that ROS plays a role in RD-mediated cytotoxicity.
Melanogenesis substrate, N-propionyl-4-S-cysteaminylphenol (NPr-CAP) is specifically taken up by melanoma cells and inhibits their growth by producing cytotxic free radicals. By taking advantage of this unique chemical agent, we have established melanomatargeting intracellular hyperthermia by conjugating NPrCAP with magnetite nanoparticles (NPrCAP/M) upon exposure to an alternating magnetic field (AMF). This treatment causes cytotoxic reaction as well as heat shock responses, leading to elicitation of antitumor immune response, which was proved by tumor rechallenge test and CTL induction. We found the level of heat shock protein 72 (Hsp72) to be increased in the cell lysate and culture supernatant after intracellular hyperthermia. Melanoma-specific CD8 + T-cell response to dendritic cells loaded with hyperthermiatreated tumor lysate was enhanced when compared with non-treated tumor lysate. When heat shock protein, particularly Hsp72, was immuno-depleted from hyperthermia-treated tumor cell lysate, specific CD8 + T-cell response was abolished. Thus, it is suggested that antitumor immune response induced by hyperthermia using NPrCAP/M is derived from the release of HSP-peptide complex from degraded tumor cells. Therefore, this chemothermo-immuno (CTI)-therapy might be effective not only for primary melanoma but also for distant metastasis because of induction of systemic antimelanoma immune responses. (Cancer Sci 2010; 101: 1939-1946 M elanoma has been increasing in incidence leading to a rise in morbidity and mortality in recent decades. Metastatic melanoma is extremely difficult to cure and continues to have a poor prognosis. Only 12% with metastatic melanoma survive for 5 years.(1) The reason for this poor prognosis is the lack of effective conventional therapies. Various types of therapies such as immunotherapy, chemotherapy, and biologic therapy have been studied in melanoma management. However, a very modest effect was recorded in advanced malignant melanoma. Therefore, there is an emerging need for innovative therapies for the control of advanced melanoma.It has been reported that the intracellular hyperthermia using magnetic nanoparticles is effective for treating certain types of cancer in not only primary but also metastatic lesions.(2-8) Incorporated magnetic nanoparticles generate heat within the cells after exposure to the alternating magnetic field (AMF) due to hysteresis loss or relaxational loss.(9,10) One of us has shown that hyperthermic treatment using magnetite cationic liposomes (MCLs), which are cationic liposomes containing 10-nm magnetite nanoparticles, induces antitumor immunity by enhancement of heat shock protein (HSP) expression. (3,(11)(12)(13) We previously proposed that cross-presentation of extracellular HSP-peptide complex released from hyperthermia-induced necrotic tumor cells is the mechanism for inducing antitumor immunity.(7) In this paper, we present evidence that tumor-derived HSP-peptide complex is responsible for the hyperthermia-mediated antitumor immunity.In addition, ex...
To study the molecular mechanisms responsible for inducing the spatial proliferation of malignant melanomas (MM), three-dimension (3D) spheroids were produced from several MM cell lines including SK-mel-24, MM418, A375, WM266-4, and SM2-1, and their 3D architectures and cellular metabolisms were evaluated by phase-contrast microscopy and Seahorse bio-analyzer, respectively. Several transformed horizontal configurations were observed within most of these 3D spheroids, and the degree of their deformity was increased in the order: WM266-4, SM2-1, A375, MM418, and SK-mel-24. An increased maximal respiration and a decreased glycolytic capacity were observed within the lesser deformed two MM cell lines, WM266-4 and SM2-1, as compared with the most deformed ones. Among these MM cell lines, two distinct cell lines, WM266-4 and SK-mel-24, whose 3D appearances were the closest and farthest, respectively, from being horizontally circular-shaped, were subjected to RNA sequence analyses. Bioinformatic analyses of the differentially expressed genes (DEGs) identified KRAS and SOX2 as potential master regulatory genes for inducing these diverse 3D configurations between WM266-4 and SK-mel-24. The knockdown of both factors altered the morphological and functional characteristics of the SK-mel-24 cells, and in fact, their horizontal deformity was significantly reduced. A qPCR analysis indicated that the levels of several oncogenic signaling related factors, including KRAS and SOX2, PCG1α, extracellular matrixes (ECMs), and ZO1 had fluctuated among the five MM cell lines. In addition, and quite interestingly, the dabrafenib and trametinib resistant A375 (A375DT) cells formed globe shaped 3D spheroids and showed different profiles in cellular metabolism while the mRNA expression of these molecules that were tested as above were different compared with A375 cells. These current findings suggest that 3D spheroid configuration has the potential for serving as an indicator of the pathophysiological activities associated with MM.
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