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.

Background: No prominent advancements in osteosarcoma (OS) treatment have been made in the past 20 years. Although photodynamic therapy (PDT) is an emerging technique for cancer therapy, the lack of targeted photosensitizers for OS treatment severely limits its applications. Results: In this study, we constructed a potential theranostic nanoplatform by using (poly (lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) encapsulating IR780 into the shell (PLGA-IR780 NPs), which were further camouflaged with human OS cell membranes from the HOS cell line (MH-PLGA-IR780 NPs). These constructed NPs showed the capacity for homologous targeting with excellent photoacoustic (PA)/fluorescence (FL) imaging ability. Benefitting from their homologous targeting capacity, MH-PLGA-IR780 NPs obviously promoted cell endocytosis in vitro and tumor accumulation in vivo, which could further improve PDT performance under near-infrared (NIR) irradiation. In addition, to their homologous targeting and PA/FL dual-mode imaging ability, MH-PLGA-IR780 NPs had advantages in penetrating deeper into tumor tissues and in real-time dynamic distribution monitoring in vivo, which laid a foundation for further clinical applications in OS. Moreover, we demonstrated that PDT guided by the constructed NPs could significantly induce HOS cells apoptosis and ferroptosis via excessive accumulation of reactive oxygen species (ROS), and further determined that the potential anticancer molecular mechanism of apoptosis was triggered by the release of cytochrome c-activated mitochondrial apoptosis (endogenous apoptosis), and that ferroptosis caused the activation of nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and the inactivation of glutathione peroxidase 4 (GPX4), synergistically leading to excessive accumulation of Lipid-ROS and Lipid peroxides (LPOs). Concurrently, MH-PLGA-IR780 NPs-guided PDT also showed an obvious inhibitory effect on tumor growth in vivo. Conclusion: These results suggest that this homologous targeting-based theranostic nanoplatform provides an effective method to improve PDT performance in OS and contributes a new and promising approach for OS therapy. Graphical

The combination of phytochemicals with chemotherapy drugs is an emerging new strategy for cancer therapy to increase antitumor responses. The present study investigates the effect of the combination of capsaicin (CAP) with cisplatin (DDP) and the potential underlying anticancer mechanisms in osteosarcoma (OS) cells in vitro and in vivo. Cell viability assays and isobolographic analyses demonstrated that the combination of CAP and DDP showed synergistic cytotoxic effects on OS cells. We chose relatively low concentrations of CAP (100 μM) and DDP (16.7 μM) for subsequent experiments. Generally, the combination of CAP and DDP had significant effects on apoptosis induction, cell cycle arrest and cell invasion inhibition in OS cells compared with the individual-treatment groups and the control group. Moreover, cotreatment with CAP and DDP triggered prosurvival autophagy through reactive oxygen species (ROS)/JNK and p-AKT/mTOR signaling in OS cells. The combination regimen of CAP and DDP also inhibited tumor growth in an OS xenograft model. These results suggest that the combination of CAP and DDP has strong inhibitory effects on OS cells and identify CAP as a promising agent for supplementing standard chemotherapy and possible future targeted therapy in OS.

Capsaicin, a pungent molecular compound present in many hot peppers, exerts anticancer activities against various human cancer cell lines by inducing apoptosis. However, the effects of capsaicin on human osteosarcoma (OS) as well as the related mechanisms remain to be fully elucidated. In the present study, the anticancer effects of capsaicin on 3 human OS cell lines (MG63, 143B and HOS) were investigated. Various concentrations of capsaicin (50-300 µM) effectively decreased cell viability in all 3 OS cell lines in a dose-dependent manner. Notably, capsaicin-induced apoptosis was observed when OS cells were treated with relatively high concentrations of capsaicin (starting at 250 µM). In addition, the mitochondrial apoptotic pathway was involved in the capsaicin-induced apoptosis in the OS cells. Meanwhile, our results also indicated that at relatively low concentrations (e.g., 100 µM), capsaicin could inhibit the proliferation, decrease the colony forming ability and induce G0/G1 phase cell cycle arrest of OS cells in a dose-dependent manner. Moreover, our results revealed that the anticancer effects induced by capsaicin on OS cell lines involved multiple MAPK signaling pathways as indicated by inactivation of the ERK1/2 and p38 pathways and activation of the JNK pathway. Furthermore, the results of animal experiments showed that capsaicin inhibited tumor growth in a xenograft model of human OS. In conclusion, these results indicate that capsaicin may exert therapeutic benefits as an adjunct to current cancer therapies but not as an independent anticancer agent.

Background: Notch signaling abnormalities are associated with the development of various tumors, including hematopoietic and epithelium-derived tumors. However, the role of Notch signaling in tumors originating from mesenchymal cells is unclear. The effect of Notch3 expression on the prognosis of osteosarcoma and its role and mechanism in osteosarcoma cells have never been reported. Materials and methods: In this study, we performed a clinicopathological analysis of 70 cases of osteosarcoma, with primary focus on survival. Osteosarcoma cell lines MTH and U2OS were used. After knockdown of Notch3 by lentiviral transfection and siRNA, the cell cycle, cell viability, and wound healing capacity were assessed. Subsequently, the Transwell assay was performed, and the expression levels of hairy and enhancer of split-1 (Hes1) and matrix metalloproteinase 7 (MMP7) were detected by RT-PCR and Western blot assay. The expression of MMP7 was also detected after knockdown of Hes1. Animal experiments were performed by injecting the cell lines MTH of Notch3 knockdown into mice tail veins and comparing the development of lung metastasis with the control group. Results: Comparison of survival curves showed that Notch3 expression significantly impacts patient survival. Additionally, multivariate analysis revealed that Notch3 is an independent prognostic factor for osteosarcoma. In in vivo experiments, osteosarcoma-associated pulmonary metastasis in nude mice was reduced after Notch3 silencing. The expression of downstream effector molecule, Hes1, and that of the invasion and metastasis-associated proteolytic enzyme, MMP7, were reduced, and MMP7 was further decreased by Hes1 knockdown in in vitro experiments. Conclusion: Notch3 is a prognostic factor for osteosarcoma and might regulate its invasion and metastasis through the downstream target gene Hes1 and effector MMP7.

Tumor cells must resist anoikis to metastasize. There is a key role of angiogenesis in the growth and metastasis of tumors. However, the relationship between anoikis resistance and angiogenesis has not been well explored in human osteosarcoma. In the present study, we reported the higher expression of vascular endothelial growth factor‑A (VEGF‑A) in osteosarcoma cells that were resistant to anoikis than in parental osteosarcoma cells, promoting the proliferation, tube formation, and migration of human umbilical vein endothelial cells (HUVECs). Src, JNK (Jun amino‑terminal kinase) and ERK (extracellular signal‑regulated kinase) signaling pathway phosphorylation was activated in anoikis‑resistant cells; Src inhibitor reduced the expression of VEGF‑A and angiogenesis and inhibited JNK and ERK pathway activity. Overexpression of phosphorylated (p)‑Src and VEGF‑A was positively correlated to the metastatic potential in human osteosarcoma tissues, as quantified by immunohistochemistry. In addition, p‑Src expression was directly correlated with VEGF‑A expression and microvessel density in vivo. Our findings revealed that anoikis resistance in osteosarcoma cells increased the expression of VEGF‑A and angiogenesis through the Src/JNK/ERK signaling pathways. Thus, Src may be a potential therapeutic alternative in osteosarcoma angiogenesis and metastasis.