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Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone), a novel quinone derivative, has been shown to modulate cellular redox balance. However, effect of this compound on melanoma remains unclear. This study examined the in vitro or in vivo anti-tumor, apoptosis, and anti-metastasis activities of CoQ0 (0-20 μM) through inhibition of Wnt/β-catenin signaling pathway. CoQ0 exhibits a significant cytotoxic effect on melanoma cell lines (B16F10, B16F1, and A2058), while causing little toxicity toward normal (HaCaT) cells. The suppression of β-catenin was seen with CoQ0 administration accompanied by a decrease in the expression of Wnt/β-catenin transcriptional target c-myc, cyclin D1, and survivin through GSK3β-independent pathway. We found that CoQ0 treatment caused G1 cell-cycle arrest by reducing the levels of cyclin E and CDK4. Furthermore, CoQ0 treatment induced apoptosis through caspase-9/-3 activation, PARP degradation, Bcl-2/Bax dysregulation, and p53 expression. Notably, non- or sub-cytotoxic concentrations of CoQ0 markedly inhibited migration and invasion, accompanied by the down-regulation of MMP-2 and -9, and up-regulation of TIMP-1 and -2 expressions in highly metastatic B16F10 cells. Furthermore, the in vivo study results revealed that CoQ0 treatment inhibited the tumor growth in B16F10 xenografted nude mice. Histological analysis and western blotting confirmed that CoQ0 significantly decreased the xenografted tumor progression as demonstrated by induction of apoptosis, suppression of β-catenin, and inhibition of cell cycle-, apoptotic-, and metastatic-regulatory proteins. The data suggest that CoQ0 unveils a novel mechanism by down-regulating Wnt/β-catenin pathways and could be used as a potential lead compound for melanoma chemotherapy., Competing Interests: The authors declare no conflicts of interest.

Simple Summary: Melanoma is the most dangerous type of skin cancer that develops from the pigment-producing cells known as melanocytes. One of the primary causes of melanoma is ultraviolet light (UV) exposure in those with low levels of the skin pigment melanin. Flavokawain B (FKB) is a naturally occurring chalcone, which is known to possess anti-proliferative pharmacological activity on various cancer cells. However, the effect of FKB on the anti-melanoma pharmacological role has not been investigated. Therefore, in this study, we explored the anti-melanoma properties of FKB on human melanoma cells and the cell death mechanisms that were mediated through the induction of reactive oxygen species (ROS) were investigated via in vitro and in vivo approaches. Our study results support promising application prospects of FKB in the treatment of human melanoma cancer. Melanoma is the most prevalent type of skin cancer with high mortality rates. This study demonstrates the in vitro and in vivo anticancer properties of chalcone flavokawain B (FKB) induced ROS-mediated apoptosis and autophagy in human melanoma (human epithelial melanoma cell line A375 and/or human skin lymph node derived melanoma cell line A2058) cells. Cell viability was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression patterns of various apoptosis, autophagy-associated proteins were determined by Western blot methods. Annexin V was detected by flow cytometry, whereas acidic vesicular organelles (AVOs) and intracellular ROS levels were measured by fluorescence microscopy. The in vivo anticancer properties of FKB were evaluated by xenografting the A375 cells into nude mice. The results convey that FKB inhibited cell viability, B-Raf proto-oncogene, serine/threonine kinase (BRAF)/extracellular signal-regulated kinase (ERK) expression in human melanoma cells. Caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage pathway, and Bcl2 associated X (Bax)/B-cell lymphoma 2 (Bcl-2) dysregulation were involved in the execution of apoptosis. Moreover, FKB-induced autophagy was observed through increased microtubule-associated protein 1A/1B-light chain 3B (LC3-II) accumulation and AVOs formation, which was also associated with an increase in sequestosome 1 (SQSTM1/p62), decreased protein kinase B (AKT)/mammalian target of rapamycin (mTOR) expressions, and dysregulated Beclin-1/Bcl-2 levels. Autophagy inhibitors [3-methyladenine (3-MA)/chloroquine (CQ)] and LC3 silencing suppressed FKB-induced apoptosis by decreasing caspase-3 in melanoma cells. The antioxidant N-acetylcysteine (NAC) diminished FKB-induced apoptotic and autophagic cell death. However, the inhibition of apoptosis decreased FKB-induced autophagy (LC3-I/II). The in vivo study confirmed that FKB inhibited melanoma growth in A375-xenografted nude mice. This study concluded that FKB is critically associated with the execution and generation of ROS-modulated apoptotic and autophagic cell death of melanoma cells. FKB also repressed tumor growth in xenografted nude mice. Therefore, flavokawain B might be a potential anti-tumor agent in human melanoma treatment. [ABSTRACT FROM AUTHOR]