Tyrosinase (TYR) is a key enzyme for melanin production. We previously showed that hinokitiol, a naturally occurring seven-membered ring terpenoid, potently inhibits human TYR activity. Interestingly, hinokitiol was recently reported to decrease expression of TYR and microphthalmia-associated transcription factor (MITF), which is a main transcription factor of the TYR gene, in murine melanoma cells. However, the mechanisms by which hinokitiol decreases the intracellular levels of TYR and MITF have not been fully elucidated. Here, we investigated the underlying mechanisms of the decreases using cultured human melanoma cells. As a result, hinokitiol treatment decreased TYR protein level in a time- and dose-dependent manner in G361 human melanoma cells, while MITF protein level was decreased only at higher concentrations after 3 days treatment. Notably, the mRNA levels of TYR and MITF were slightly increased by hinokitiol treatment. Therefore, we focused on the degradation of TYR and MITF in endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Importantly, co-treatment of ERAD inhibitor with hinokitiol restored the protein levels of TYR and MITF to approximately 30% and 20% of total those in untreated control cells, respectively. Hinokitiol affected the ER homeostasis as well as degradation of TYR and MITF in two human melanoma cell lines, G361 and HT-144, but the changes of ER-stress markers under the hinokitiol treatment were different in the two human melanoma cell lines. Taken together, these observations indicate that hinokitiol may induce ER stress and trigger the degradation of unfolded newly synthesizing TYR and MITF via the ERAD pathway.

酪氨酸酶 (TYR) 是黑色素生成的关键酶。我们之前表明，一种天然存在的七元环萜类化合物 hinokitiol 可有效抑制人类 TYR 活性。有趣的是，最近报道了扁柏醇降低 TYR 和小眼相关转录因子 (MITF) 的表达，MITF 是TYR的主要转录因子基因，在小鼠黑色素瘤细胞中。然而，桧醇降低细胞内 TYR 和 MITF 水平的机制尚未完全阐明。在这里，我们使用培养的人黑色素瘤细胞研究了减少的潜在机制。结果，桧醇处理以时间和剂量依赖性方式降低了 G361 人黑色素瘤细胞中的 TYR 蛋白水平，而 MITF 蛋白水平仅在处理 3 天后的较高浓度下降低。值得注意的是， TYR和MITF的 mRNA 水平通过桧醇处理略有增加。因此，我们专注于内质网（ER）相关蛋白降解（ERAD）途径中TYR和MITF的降解。重要的是，ERAD 抑制剂与桧醇的共同处理将 TYR 和 MITF 的蛋白质水平分别恢复到未处理对照细胞中总蛋白质水平的约 30% 和 20%。桧醇影响两种人黑色素瘤细胞系 G361 和 HT-144 的 ER 稳态以及 TYR 和 MITF 的降解，但在两种人黑色素瘤细胞系中，桧醇处理下 ER 应激标志物的变化是不同的。总之，这些观察结果表明，桧醇可能会诱导 ER 应激，并通过 ERAD 途径触发未折叠的新合成 TYR 和 MITF 的降解。