Morusinol extracted from Morus alba induces cell cycle arrest and apoptosis via inhibition of DNA damage response in melanoma by CHK1 degradation through the ubiquitin-proteasome pathway.

BACKGROUD: Flavonoids have a variety of biological activities, such as anti-inflammation, anti-tumor, anti-thrombosis and so on. Morusinol, as a novel isoprene flavonoid extracted from Morus alba root barks, has the effects of anti-arterial thrombosis and anti-inflammatory in previous studies. However, the anti-cancer mechanism of morusinol remains unclear. PURPOSE: In present study, we mainly studied the anti-tumor effect of morusinol and its mode of action in melanoma. METHODS: The anti-cancer effect of morusinol on melanoma were evaluated by using the MTT, EdU, plate clone formation and soft agar assay. Flow cytometry was used for detecting cell cycle and apoptosis. The É£-H2AX immunofluorescence and the alkaline comet assay were used to detect DNA damage and the Western blotting analysis was used to investigate the expressions of DNA-damage related proteins. Ubiquitination and turnover of CHK1 were also detected by using the immunoprecipitation assay. The cell line-derived xenograft (CDX) mouse models were used in vivo to evaluate the effect of morusinol on tumorigenicity. RESULTS: We demonstrated that morusinol not only had the ability to inhibit cell proliferation, but also induced cell cycle arrest at G0/G1 phase, caspase-dependent apoptosis and DNA damage in human melanoma cells. In addition, morusinol effectively inhibited the growth of melanoma xenografts in vivo. More strikingly, CHK1, which played an important role in maintaining the integrity of cell cycle, genomic stability and cell viability, was down-regulated in a dose- and time-dependent manner after morusinol treatment. Further research showed that CHK1 was degraded by the ubiquitin-proteasome pathway. Whereafter, morusinol-induced cell cycle arrest, apoptosis and DNA damage were partially salvaged by overexpressing CHK1 in melanoma cell lines. Herein, further experiments demonstrated that morusinol increased the sensitivity of dacarbazine (DTIC) to chemotherapy for melanoma in vitro and in vivo. CONCLUSION: Morusinol induces CHK1 degradation through the ubiquitin-proteasome pathway, thereby inducing cell cycle arrest, apoptosis and DNA damage response in melanoma. Our study firstly provided a theoretical basis for morusinol to be a candidate drug for clinical treatment of cancer, such as melanoma, alone or combinated with dacarbazine.

CSN6 promotes melanoma proliferation and metastasis by controlling the UBR5-mediated ubiquitination and degradation of CDK9.

As a critical subunit of the constitutive photomorphogenesis 9 (COP9) signalosome (CSN), CSN6 is upregulated in some human cancers and plays critical roles in tumorigenesis and progression, but its biological functions and molecular mechanisms in melanoma remain unknown. Our study showed that CSN6 expression was upregulated in melanoma patients and cells, and correlated with poor survival in melanoma patients. In melanoma cells, CSN6 knockdown remarkably inhibited cell proliferation, tumorigenicity, migration, and invasion, whereas CSN6 recovery rescued the proliferative and metastatic abilities. Notably, we identified that CSN6 stabilized CDK9 expression by reducing CDK9 ubiquitination levels, thereby activating CDK9-mediated signaling pathways. In addition, our study described a novel CSN6-interacting E3 ligase UBR5, which was negatively regulated by CSN6 and could regulate the ubiquitination and degradation of CDK9 in melanoma cells. Furthermore, in CSN6-knockdown melanoma cells, UBR5 knockdown abrogated the effects caused by CSN6 silencing, suggesting that CSN6 activates the UBR5/CDK9 pathway to promote melanoma cell proliferation and metastasis. Thus, this study illustrates the mechanism by which the CSN6-UBR5-CDK9 axis promotes melanoma development, and demonstrate that CSN6 may be a potential biomarker and anticancer target in melanoma.

ZC3H15 Correlates with a Poor Prognosis and Tumor Progression in Melanoma.

Zinc figure CCCH-type containing 15 (ZC3H15), also called developmentally regulated GTP-binding protein 1 (DRG1) family regulatory protein 1 (DFRP1), is a zinc finger containing protein. Despite playing a role in cellular signaling, it is found overexpressed in acute myeloid leukemia and also an independent prognostic marker in hepatocellular carcinoma patients. However, the biological effect of ZC3H15 in malignant melanoma (MM) remains unexplored. The expression of ZC3H15 in patients was analyzed using the R2: Genomics Analysis and Visualization Platform database. Immunohistochemical analysis, western blot, and qRT-PCR were used to detect ZC3H15 expression in melanoma tissues and cell lines. MTT, BrdU, flow cytometry assay, transwell, and western blot were performed to explore the proliferation, cell cycle, invasion, and migration of melanoma cells. We undertaken colony formation assay in vitro and tumor xenograft in vivo to detect the tumorigenicity of melanoma cells. In the present study, ZC3H15 was demonstrated highly expressed in melanoma tissues and cells. Elevated ZC3H15 impairs the survival of melanoma patients. Meanwhile, attenuation of ZC3H15 in melanoma cells inhibited cell proliferation and induced cycle arrest at G0/G1 phase. Consistently, the expression of cell cycle-related proteins cyclin dependent kinase 4 (CDK4), CDK6, and cyclin D1 (CCND1) was decreased while p21 was upregulated. Furthermore, we found the migration and invasion abilities were inhibited in ZC3H15-knockdown melanoma cells. In addition, downregulation of ZC3H15 resulted in inhibition of colony formation abilities in vitro and tumorigenesis in vivo. ZC3H15 promotes proliferation, migration/invasion, and tumorigenicity of melanoma cells. As a promising biomarker and therapeutic target in MM, ZC3H15 is worthy of further exploration.