ERK and p MAPK signaling pathways play critical role
ERK and p38 MAPK signaling pathways play critical role in genesis and metastasis of melanoma (Tang et al., 2018). >50% of melanoma Cytarabine (Abildgaard and Guldberg, 2015) show abnormal activity of BRAF-RAS-RAF to activate MEK signal that drives the growth-promoting extracellular signal-regulated kinase (ERK) pathway (Burotto et al., 2014; Paluncic et al., 2016). Thus, compounds inhibiting the kinase activities of BRAF and MEK have yielded promising clinical results, and some of them have progressed to the clinical usage for the treatment of melanoma (Flaherty et al., 2012; Herrero et al., 2015; Montagut and Settleman, 2009). However, whether hinokitiol may act through the similar way to inhibit ERK pathway is not known.
Hinokitiol has been reported to inhibit EGFR phosphorylation and ERK pathway, offering a possible mechanism by which hinokitiol suppressed proliferation in H1975 cells (a gefitinib resistant lung adenocarcinoma cell) (Li et al., 2014). This notion is supported by several investigations; hinokitiol treatment of mouse melanoma (B16-F10 cells) concentration-dependently inhibited ERK1/2, p38 MAPK, and JNK1/2 phosphorylation (Huang et al., 2015a). Accordingly, hinokitiol can inhibit ERK in B16-F10 cells, but, the mechanism of hinokitiol-induced dose-dependent inhibition of ERK pathway is not fully understood.
Dual-specificity phosphatase 6 (DUSP6) is also known as MAP kinase phosphatase-3 (MKP-3); it can specifically induce ERK dephosphorylation leading to inhibition of ERK activity in mammalian cell. Expression of MKP-3 is regulated by ERK signaling pathway while MKP-3 by itself can be a regulator of negative feedback in ERK activity. MKP-3 is a tumor suppressor in many cancer cells such as pancreatic cancer, non-small cell lung cancer, ovarian cancer and human melanoma cell line (Ahmad et al., 2018; Díaz-García et al., 2015; Furukawa et al., 2003), and at least part of tumor-suppressive activity of MKP-3 is from its ability to inactivate ERK (Ahmad et al., 2018). Previous study has indicated that hinokitiol suppressed B16 melanoma growth by inhibiting MAPK signaling pathway (Huang et al., 2015b), however the exact mechanism in hinokitiol-induced inhibition of ERK activity and tumor growth in B16 melanoma remains unclear. Thus, this study investigated the role of MKP-3 in inhibition of ERK signaling pathway to suppress cell growth in B16 melanoma treated with hinokitiol.
Materials and methods
Discussion We investigated the anticancer mechanism of hinokitiol treatment in metastatic B16-F10 melanoma cells. The hinokitiol (5–20 μM) treatment significantly inhibited colony formation and cell viability in a time and concentration-dependent manner (Fig. 1), exhibited apoptotic features (Fig. 1B, D), and decreased survivin protein levels with an increased suvivin ubiquitination (Fig. 2, Fig. 3). Pretreatment with proteosome inhibitors effectively prevented hinokitiol-reduced survivin expression, implying an involvement of ubiquitin/proteosome pathway (Fig. 4). Hinokitiol rapidly induced ERK phosphorylation followed by a sustained dephosphorylation, which was accompanied by an increase in expression of tumor suppressor MKP-3 (Fig. 6A). Inhibition of hinokitiol-induced ERK activation by MEK inhibitor U0126 completely blocked expression of MKP-3 (Fig. 7B). More importantly, inhibition of MKP-3 activity by NSC 95397 significantly inhibited hinokitiol-induced ERK dephosphorylation, ubiquitination, and downregulation of survivin (Fig. 6). These results suggested that hinokitiol treatment inhibited growth of B16-F10 melanoma through down regulated survivin by activating ERK/MKP-3/proteosome pathway. Hinokitiol at low concentrations appears to have good anticancer effects. Its concentration at 5–10 μM exhibits marked anticancer effect on adenocarcinoma A549 cell through suppression of MMPs and induction of antioxidant enzymes and apoptosis (Jayakumar et al., 2018); 5 μM inhibits significantly melanogenesis of melanocytes (Choi et al., 2006); and 1–5 μM inhibits invasion capability of melanoma B16-F10 cells via increasing factor matrix metalloproteinase (MMP)-2 and MMP-9 along with an increase of intracellular antioxidant enzymes (Huang et al., 2015a). In addition, hinokitiol exhibits divergent biological effects in different concentration ranges by modulating different signaling pathways, such as hinokitiol inhibits melanogenesis and induces expression of autophagic marker in B16 melanoma by inhibiting AKT/mTOR pathway at 1–5 nM range (Tsao et al., 2016), however it induce apoptotic cell death in colon cancer by increasing expression of p21 (a Cdk inhibitor) at 1–10 μM range(Lee et al., 2013). Consistent with aforementioned studies, we demonstrated that low dose of hinokitiol at 5–20 μM induced concentration-dependent anti-cancer effects in melanoma B16-F10 cells (Fig. 1). In addition, our study is the first that demonstrated the anticancer effect of hinokitiol is mediated through downregulated survivin by activating ERK/MKP-3/proteosome pathway.