Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis

Abstract

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 (p < 0.001) phase, evaluated through immunoblotting. Moreover, autophagy-related proteins such as ATG-5 (p < 0.0001), ATG-7 (p < 0.0001), beclin-1 (p < 0.0001) and transition of LC3-I to LC3-II (p < 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 (p < 0.0001), beclin-1 (p < 0.0001) and LC3-I to LC3-II (p < 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 (p < 0.029), CDK-4 (p < 0.036), cyclin D1 (p < 0.0003) and cyclin E (p < 0.020) upon NAC treatment. In addition, we also observed a significant fold reduction (p < 0.05) in ROS fluorescent intensity and the expression of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase-9 (p > 0.010) and cleaved caspase-3 (p < 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 (p < 0.0001) and CDK-4 (p < 0.0142) and upregulation of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase 3 (p < 0.0001) and cleaved caspase 9 (p < 0.0001). In summary, our study revealed that DA is an effective treatment against B16F10 melanoma cells and xenograft mice model.

A comprehensive review of antitumor properties of Angelica species and their antitumor‐responsible constituents and the underlying molecular mechanisms involved in tumor inhibition

“…Decursinol angelate exerted growth inhibitory effects against murine melanoma B16F10 cells and xenograft mice model by arresting cell cycle at G0/G1 via suppression and downregulation of cyclin D1 and cyclin‐dependent kinases such as CDK2, CDK4 and CDK5 and upregulation of p21 that is a CDK inhibitor. Besides, DA treatment caused inhibition of autophagosome formation (expression of ATG‐5, ATG‐7, beclin‐1, and conversion of LC3‐I to LC3‐II were markedly decreased) and induced apoptosis via Bcl2 downregulation, Bax upregulation, activation of caspases (caspase‐9, caspase‐3), the release of cytochrome C, and generation of ROS that ultimately caused oxidative burst in B16F10 cells (Chang et al, 2021).…”

Role of ROS‑mediated autophagy in melanoma (Review)

“…Autophagy as a protective role to ameliorate ROS-induced apoptosis. Several studies have reported that various anticancer agents are employed to treat melanoma through ROS production and ROS-mediated apoptosis (77,78). Kalantuboside B, a natural bufadienolide derivative, has been demonstrated to enhance intracellular ROS levels, which induce apoptosis and autophagy; moreover, apoptosis was revealed to be potentiated by the autophagy inhibitors chloroquine and 3-methyladenine in A2058 melanoma cells and xenografts, suggesting that autophagy plays a protective role in melanoma (9).…”

“…A possible explanation is that the functional effects of ROS-induced autophagy on cancer cell death are dependent on the type of oxidative stress, as well as the quantity and location of the ROS produced ( 92 ). It should be noted that anticancer drug-induced ROS also inhibit cytoprotective autophagy to promote apoptosis, and that blocking autophagy fails to affect ROS generation ( 78 , 89 ), suggesting that ROS serve as messengers to modulate autophagy and thus determine the cell fate. In this context, further clarifying the role of anticancer drug-induced ROS and the resultant autophagy is essential for melanoma treatment.…”

Mitochondria-endoplasmic reticulum contacts in sepsis-induced myocardial dysfunction

“…Beclin-1 is a highly conserved autophagy regulator protein in eukaryotic cells, which is mainly responsible for inducing the nucleation of autophagosomes during autophagy. Overexpression of Beclin-1 enhances autophagy and reduces apoptosis ( Chang et al, 2021 ). It has been found that Beclin-1 relocates to MERCs and interacts with PINK1 to promote the formation of MERCs and autophagosome precursor in mitophagy and starvation-induced autophagy ( Gelmetti et al, 2017 ).…”