ACSL4 accelerates osteosarcoma progression via modulating TGF-ß/Smad2 signaling pathway.

Osteosarcoma (OS) is a malignant bone sarcoma arising from mesenchymal stem cells. The biological role of Acyl-CoA synthetase long-chain family member 4 (ACSL4), recently identified as an oncogene in numerous tumor types, remains largely unclear in OS. In this study, we investigated the expression of ACSL4 in OS tissues using immunohistochemistry staining (IHC) staining of a human tissue microarray and in OS cells by qPCR assay. Our findings revealed a significant up-regulation of ACSL4 in both OS tissues and cells. To further understand its biological effects, we conducted a series of loss-of-function experiments using ACSL4-depleted MNNG/HOS and U-2OS cell lines, focusing on OS cell proliferation, migration, and apoptosis in vitro. Our results demonstrated that ACSL4 knockdown remarkably suppressed OS cell proliferation, arrested cells in the G2 phase, induced cell apoptosis, and inhibited cell migration. Additionally, a subcutaneous xenograft mice model was established to validate the in vivo impact of ACSL4, revealing ACSL4 silencing impaired tumor growth in the OS xenograft mice. Additionally, we discovered that ACSL4 could regulate the phosphorylation level of Smad2 through cooperative interactions, and treatment with a TGF-ß inhibitor weakened the promoting effects of ACSL4 overexpression. In short, ACSL4 regulated OS progression by modulating TGF-ß/Smad2 signaling pathway. These findings underscore ACSL4 as a promising therapeutic target for OS patients and contribute novel insights into the pathogenesis of OS.

[Research of epigallocatechin gallate in delaying chondrocyte senescence].

Objective: To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods: The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1ß group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 µmol/L EGCG+10 ng/mL IL-1ß group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1ß group (group B), EGCG+10 ng/mL IL-1ß group (group C), and EGCG+10 ng/mL IL-1ß+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by ß-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results: The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1ß group significantly decreased ( P<0.05). Compared with the 10 ng/mL IL-1ß group, the cell activity of EGCG+10 ng/mL IL-1ß groups increased, and the 50.0, 100.0, and 200.0 µmol/L EGCG significantly promoted the activity of chondrocytes ( P<0.05). The 100.0 µmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant ( P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend ( P<0.05). Conclusion: EGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.