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// Xiao-Yu Chen 1 , De-Fang Li 2 , Ji-Chun Han 2 , Bo Wang 3 , Zheng-Ping Dong 2 , Li-Na Yu 2 , Zhao-Hai Pan 2 , Chuan-Jun Qu 2 , Ying Chen 2 , Shi-Guo Sun 1 , Qiu-Sheng Zheng 2 1 College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, China 2 Binzhou Medical University, Yantai, Shandong, 264003, China 3 Key Laboratory of Xinjiang Endemic Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832002, China Correspondence to: Qiu-Sheng Zheng, email: zqsyt@sohu.com Shi-Guo Sun, email: sunsg@nwsuaf.edu.cn Keywords: reprogramming, melanoma, cachexia, isoliquiritigenin, mTORC2-AKT-GSK3β signaling Received: December 08, 2016 Accepted: March 15, 2017 Published: March 29, 2017 ABSTRACT Isoliquiritigenin (ISL), a member of the flavonoids, is known to have anti-tumor activity in vitro and in vivo . The effect of ISL on reprogramming in cancer cells, however, remains elusive. In this study, we investigated the effect of ISL on reprogramming in human melanoma A375 cells. ISL (15 μg/ml) significantly inhibited A375 cell proliferation, anchorage independent cell proliferation and G2/M cell cycle arrest after ISL exposure for 24 h. However, there were no significant changes in apoptosis rate. Terminal differentiation indicators (melanin content, melanogenesis mRNA expression, tyrosinase (TYR) activity) were all up-regulated by ISL treatment. In ISL-treated cells, glucose uptake, lactate levels and mRNA expression levels of GLUT1 and HK2 were significantly decreased, and accompanied by an increase in O 2 consumption rate (OCR) and adenosine triphosphate (ATP) deficiency. Protein expression levels of mTORC2-AKT-GSK3β signaling pathway components (mTOR, p-mTOR, RICTOR, p-AKT, p-GSK3β) decreased significantly after ISL treatment. Co-treatment of ISL and the mTOR-specific inhibitor Ku-0063794 had a synergistic effect on the inhibition of proliferation, and increased melanin content and TYR activity. Glucose uptake and lactate levels decreased more significantly than treatment with ISL alone. These findings indicate that ISL induced reprogramming in A375 melanoma cells by activating mTORC2-AKT-GSK3β signaling.

The aim of the present study was to determine the effects of Licochalcone D (LD) on the apoptosis and migration and invasion in human melanoma A375 cells. Cell proliferation was determined by sulforhodamine B assay. Apoptosis was assessed by Hoechst 33258 and Annexin V-FITC/PI staining and JC-1 assay. Total intracellular reactive oxygen species (ROS) was examined by DCFH-DA. Wound healing and Transwell assays were used to detect migration and invasion of the cells. The activities of matrix metalloproteinase (MMP-2 and MMP-9) were assessed via gelatin zymography. Tumor growth in vivo was evaluated in C57BL/6 mice. RT-PCR, qPCR, ELISA and western blot analysis were utilized to measure the mRNA and protein levels. Our results showed that LD inhibited the proliferation of A375 and SK-MEL-5 cells in a concentration-dependent manner. After treatment with LD, A375 cells displayed obvious apoptotic characteristics, and the number of apoptotic cells was significantly increased. Pro-apoptotic protein Bax, caspase-9 and caspase-3 were upregulated, while anti-apoptotic protein Bcl-2 was downregulated in the LD-treated cells. Meanwhile, LD induced the loss of mitochondrial membrane potential (ΔΨm) and increased the level of ROS. ROS production was inhibited by the co-treatment of LD and free radical scavenger N-acetyl-cysteine (NAC). Furthermore, LD also blocked A375 cell migration and invasion in vitro which was associated with the downregulation of MMP-9 and MMP-2. Finally, intragastric administration of LD suppressed tumor growth in the mouse xenograft model of murine melanoma B16F0 cells. These results suggest that LD may be a potential drug for human melanoma treatment by inhibiting proliferation, inducing apoptosis via the mitochondrial pathway and blocking cell migration and invasion.