Over the last two or three decades, the pace of development of treatments for osteosarcoma tends has been slow. Novel effective therapies for osteosarcoma are still lacking. Previously, we reported that tumor-suppressing STF cDNA 3 (TSSC3) functions as an imprinted tumor suppressor gene in osteosarcoma; however, the underlying mechanism by which TSSC3 suppresses the tumorigenesis and metastasis remain unclear. We investigated the dynamic expression patterns of TSSC3 and autophagy-related proteins (autophagy related 5 (ATG5) and P62) in 33 human benign bone tumors and 58 osteosarcoma tissues using immunohistochemistry. We further investigated the correlations between TSSC3 and autophagy in osteosarcoma using western blotting and transmission electronic microscopy. CCK-8, Edu, and clone formation assays; wound healing and Transwell assays; PCR; immunohistochemistry; immunofluorescence; and western blotting were used to investigated the responses in TSSC3-overexpressing osteosarcoma cell lines, and in xenografts and metastasis in vivo models, with or without autophagy deficiency caused by chloroquine or ATG5 silencing. We found that ATG5 expression correlated positively with TSSC3 expression in human osteosarcoma tissues. We demonstrated that TSSC3 was an independent prognostic marker for overall survival in osteosarcoma, and positive ATG5 expression associated with positive TSSC3 expression suggested a favorable prognosis for patients. Then, we showed that TSSC3 overexpression enhanced autophagy via inactivating the Src-mediated PI3K/Akt/mTOR pathway in osteosarcoma. Further results suggested autophagy contributed to TSSC3-induced suppression of tumorigenesis and metastasis in osteosarcoma in vitro and in vivo models. Our findings highlighted, for the first time, the importance of autophagy as an underlying mechanism in TSSC3-induced antitumor effects in osteosarcoma. We also revealed that TSSC3-associated positive ATG5 expression might be a potential predictor of favorable prognosis in patients with osteosarcoma.

The nephrotoxicity of myoglobin released during rhabdomyolysis (RM) is the main cause of RM-induced acute kidney injury (AKI), leading to iron overload in renal tubular cells. Ferroptosis is characterized by iron overload and iron-dependent accumulation of lipid peroxidation. ACSL4 is a sensitive regulator and important contributor to ferroptosis which promotes lipid peroxidation and consequently results in acute tubular necrosis. The role of ferroptosis and ACSL4 in RM-induced AKI remains unclear. Therefore, we conducted this study, aiming to evaluate the potential role of ferroptosis in the progression of RM-induced AKI and determine whether lutein could act as a potential pharmaceutical against ferroptosis in renal tubules by suppressing ACSL4 expression. The real-time PCR, western blot, TUNEL staining, immunohistochemistry and transmission electron microscopy were used to evaluate the renal function, lipid peroxidation changes, histological changes and mitochondrial morphology changes in this model. The KEGG pathway analysis suggested that ferroptosis pathway played a significant role in RM-induced AKI. ACSL4 was upregulated in the RM-induced AKI mice model and tubule-specific knockout of ACSL4 markedly reduced ferroptosis as well as attenuated the functional, pathological and mitochondrial damage in this model. Furthermore, lutein improves renal function by combining with ACSL4 to reduce the occurrence of ferroptosis. Tubule-specific knockout of ACSL4 confers protection against RM-induced AKI by inhibiting ferroptosis. Lutein has been shown to prevent ferroptosis in renal tubules by suppressing the expression of ACSL4, suggesting its potential as a pharmaceutical agent to alleviate RM-induced AKI.