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  • br Acknowledgement br Introduction Malignant melanoma is


    Introduction Malignant melanoma is an aggressive and highly metastatic cancer. The incidence of malignant melanoma is increasing all over the world, and the population of patients with advanced melanoma is growing [1], [2]. Malignant melanoma is often resistant to conventional chemotherapy, and there is little chance of long-term survival for patients suffering from its metastasis. Only less than 15% of stage IV patients respond to the standard therapy with dacarbazine [3]. Therefore, novel chemotherapeutic agents are urgently required. Glycogen synthase kinase-3 (GSK-3) regulates a variety of cell biological processes, such as proliferation, differentiation, migration, and survival, through a number of signaling pathways. GSK-3 is also known to play important roles in tumor growth and development [4], [5]. GSK-3 phosphorylates and induces degradation of the major proto-oncogenes cyclin D1 and c-Myc [6], [7], which play key roles in the initiation and progression of the G1 phase in the Altiratinib [8], [9]. Moreover, GSK-3 is a negative regulator of the canonical Wnt signaling pathway, the activation of which can lead to cancer development [10], [11], [12], [13]. Therefore, GSK-3 could be a target of new anti-cancer drugs. Differentiation-inducing factors (DIFs) were identified in Dictyostelium discoideum as morphogens required for stalk cell differentiation [14], [15]. In the DIF family, DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was the first identified. However, the activity of DIFs is not limited to Dictyostelium: DIFs have been shown to strongly inhibit the proliferation of various mammalian cells [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29]. Previously, we reported that DIFs accelerated the degradation of cyclin D1 and c-Myc through the activation of GSK-3β, resulting in cell cycle arrest at the G0/G1 phase in various human cells. Moreover, we demonstrated that DIFs inhibited the expression levels of transcription factor 7-like 2 (TCF7L2) and β-catenin, leading to the suppression of the canonical Wnt signaling pathway [20], [21], [22], [23], [24], [25], [26], [27], [28]. However, whether DIFs has an anti-proliferative effect on malignant melanoma has not been elucidated.
    Materials and methods
    Discussion In the present study, we demonstrated that DIF-1 inhibited cell proliferation by a GSK-3-dependent mechanism and suppressed cell migration and invasion by a GSK-3-independent mechanism in malignant melanoma cells. Moreover, oral administration of DIF-1 inhibited lung tumor formation of malignant melanoma in vivo. DIF-1 suppressed melanoma cell proliferation by a GSK-3-dependent mechanism, as it did in several other tumor cell lines (HeLa, SaOS-1, HCT-116, DLD-1, A2058 cells, and others). We previously reported that the growth arrest induced by DIF-1 was reversible because the cell proliferation was immediately and completely recovered when DIF-1 was removed from the culture medium [40]. Interestingly, murine melanoma B16BL6 cells were most sensitive to DIF-1 among the cell lines we examined. It has been reported that , which inhibits the complex of cyclin-dependent kinase 4 and cyclin D [41], is absent in B16BL6 cells [42]. Therefore, cyclin D1 probably works as a more powerful cell cycle engine in B16BL6 cells than in other cell lines, so the proliferation of B16BL6 cells is more dependent on cyclin D1. This could be one reason why B16BL6 cell line was most sensitive to DIF-1. Another reason could be that DIF-1 arrested the cell cycle not only in the G0/G1 phase, but also in the G2/M phase in B16BL6 cells (Fig. 1), although DIF-1 arrests the cell cycle only in the G0/G1 phase in A2058 cells and other cell lines (Fig. 9) [21], [26], [28]. Reduction of MMP-2 expression induced by DIF-1 might be related to the suppression of cell migration and invasion. MMP-2, which facilitates migration and invasion of cancer cells [34], [35], [36], [37], is a canonical Wnt signaling pathway target gene product [38]. Transcription activity of the canonical Wnt signaling pathway was suppressed by DIF-1 via the reduction of TCF7L2 and β-catenin protein expression levels in B16Bl6 cells. Therefore, we first hypothesized that the reduction of MMP-2 level could be due to the suppression of the canonical Wnt signaling pathway. However, as shown in Fig. 6, SB216763 did not have a significant effect on the DIF-1-induced reduction of TCF7L2 and MMP-2 protein levels, although it attenuated the DIF-1-induced reduction of β-catenin expression. This result was in line with the inability of the GSK-3 inhibitors SB216763 and LiCl to prevent inhibition of cell migration and invasion by DIF-1 (Fig. 5). These results suggested that DIF-1 exhibited GSK-3-dependent and GSK-3-independent effects in B16BL6 cells and that TCF7L2 might be a more critical regulator of MMP-2 expression level.