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(B) DA was treated to B16F10 cells for 24 h and cell cycle protein levels were detected, such as cyclin D1, CDK2, 5, 4, cyclin E, and p21. Densitometry analysis of the respective proteins was evaluated by Image J software, and results were normalized with -actin. Figure Graph: Figure 2 (A) Clonogenic assay and quantification of B16F10 cells cultured in the presence and absence of DA over 7 days, followed by crystal violet staining. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Alpinia oxyphylla is a widely used medicinal herb for diarrhea, gastralgia, tumors, hypertention, and cerebrovascular disorders. Here, we evaluated the chondroprotective effect of A. oxyphylla dried fruit ethanol extract (AOE) against cartilage degradation in rabbit articular cartilage explants. Treatment of interleukin‐1α (IL‐1α) and plasminogen increased degraded collagen release in culture supernatants, but pretreatment of AOE (50, 100, 200 µg/ml) inhibited the collagen release in dose‐dependent manner. To examine the mechanism of action of AOE on chondroprotection, the level of matrix metalloproteinases‐3 (MMP‐3), matrix metalloproteinases‐13 (MMP‐13), tissue inhibitor of metalloprotease‐1 (TIMP‐1), and inflammatory mediators like prostaglandin E2 (PGE2) and nitric oxide (NO) was evaluated. AOE inhibited upregulation of MMP‐3 and MMP‐13 and downregulation of TIMP‐1 and also reduced increase of PGE2 and NO level induced by exposure of IL‐1α and plasminogen. These results indicate that AOE show chondroprotective effect through inhibiting collagen degradation via regulating MMPs, TIMP‐1, and inflammatory mediators. Practical applications: Osteoarthritis (OA) is a one of the most common chronic disorders in elderly persons. Because the regenerative power of joint articular cartilage is very low, treatment of OA is difficult to expect complete recovery. Therefore, there is a need to develop a therapeutic agent that can safely and effectively inhibit the cartilage destruction. For the first time, we exhibited the inhibitory effect of AOE on collagen degradation through regulating MMPs and TIMP‐1 in articular cartilage explants. These findings support AOE could be used as herbal therapeutic application for protecting articular cartilage to prevent OA. [ABSTRACT FROM AUTHOR]