Advanced Bio Convergence Center (ABCC), Pohang Technopark Foundation, Pohang, Gyeongbuk 37668, Korea.
Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk 38453, Korea.
Int J Mol Sci. 2021 Apr 15;22(8):4096. doi: 10.3390/ijms22084096.
Melanoma is known to aggressively metastasize and is one of the prominent causes of skin cancer mortality. This study was designed to assess the molecular mechanism of decursinol angelate (DA) against murine melanoma cell line (B16F10 cells). Treatment of DA resulted in growth inhibition and cell cycle arrest at G0/G1 ( < 0.001) phase, evaluated through immunoblotting. Moreover, autophagy-related proteins such as ATG-5 ( < 0.0001), ATG-7 ( < 0.0001), beclin-1 ( < 0.0001) and transition of LC3-I to LC3-II ( < 0.0001) were markedly decreased, indicating autophagosome inhibition. Additionally, DA treatment triggered apoptotic events which were corroborated by the occurrence of distorted nuclei, elevated reactive oxygen species (ROS) levels and reduction in the mitochondrial membrane potential. Subsequently, there was an increase in the expression of pro-apoptotic protein Bax in a dose-dependent manner, with the corresponding downregulation of Bcl-2 expression and cytochrome C expression following 24 h DA treatment in A375.SM and B16F10 cells. We substantiated our results for apoptotic occurrence through flow cytometry in B16F10 cells. Furthermore, we treated B16F10 cells with N-acetyl-L-cysteine (NAC). NAC treatment upregulated ATG-5 ( < 0.0001), beclin-1 ( < 0.0001) and LC3-I to LC3-II ( < 0.0001) conversion, which was inhibited in the DA treatment group. We also noticed a systematic upregulation of important markers for progression of G1 cell phase such as CDK-2 ( < 0.029), CDK-4 ( < 0.036), cyclin D1 ( < 0.0003) and cyclin E ( < 0.020) upon NAC treatment. In addition, we also observed a significant fold reduction ( < 0.05) in ROS fluorescent intensity and the expression of Bax ( < 0.0001), cytochrome C ( < 0.0001), cleaved caspase-9 ( > 0.010) and cleaved caspase-3 ( < 0.0001). NAC treatment was able to ameliorate DA-induced apoptosis and cell cycle arrest to support our finding. Our in vivo xenograft model also revealed similar findings, such as downregulation of CDK-2 ( < 0.0001) and CDK-4 ( < 0.0142) and upregulation of Bax ( < 0.0001), cytochrome C ( < 0.0001), cleaved caspase 3 ( < 0.0001) and cleaved caspase 9 ( < 0.0001). In summary, our study revealed that DA is an effective treatment against B16F10 melanoma cells and xenograft mice model.
黑色素瘤具有侵袭性转移的特点，是皮肤癌死亡的主要原因之一。本研究旨在评估当归酰基桃叶珊瑚苷（DA）对小鼠黑色素瘤细胞系（B16F10 细胞）的分子机制。通过免疫印迹法评估，DA 处理导致细胞生长抑制和细胞周期停滞在 G0/G1 期（<0.001）。此外，自噬相关蛋白如 ATG-5（<0.0001）、ATG-7（<0.0001）、beclin-1（<0.0001）和 LC3-I 向 LC3-II 的转化（<0.0001）明显减少，表明自噬体抑制。此外，DA 处理引发了凋亡事件，这通过扭曲的细胞核、升高的活性氧（ROS）水平和线粒体膜电位的降低得到证实。随后，在 A375.SM 和 B16F10 细胞中，随着时间的推移，促凋亡蛋白 Bax 的表达呈剂量依赖性增加，同时在 24 小时 DA 处理后，Bcl-2 表达和细胞色素 C 表达下调。我们通过 B16F10 细胞中的流式细胞术证实了凋亡的发生。此外，我们用 N-乙酰-L-半胱氨酸（NAC）处理 B16F10 细胞。NAC 处理上调了 ATG-5（<0.0001）、beclin-1（<0.0001）和 LC3-I 向 LC3-II 的转化，而在 DA 处理组中，这种转化受到抑制。我们还注意到，在 NAC 处理后，G1 细胞周期进展的重要标志物如 CDK-2（<0.029）、CDK-4（<0.036）、cyclin D1（<0.0003）和 cyclin E（<0.020）的表达呈系统上调。此外，我们还观察到 ROS 荧光强度和 Bax（<0.0001）、细胞色素 C（<0.0001）、裂解的 caspase-9（>0.010）和裂解的 caspase-3（<0.0001）的表达显著降低（<0.05）。NAC 处理能够改善 DA 诱导的细胞凋亡和细胞周期阻滞，支持我们的发现。我们的体内异种移植模型也揭示了类似的发现，例如 CDK-2（<0.0001）和 CDK-4（<0.0142）的下调以及 Bax（<0.0001）、细胞色素 C（<0.0001）、裂解的 caspase-3（<0.0001）和裂解的 caspase-9（<0.0001）的上调。总之，我们的研究表明，DA 是一种有效的治疗 B16F10 黑色素瘤细胞和异种移植小鼠模型的方法。
