3-5-bis(2-fluorobenzylidene)piperidin-4-one and Bone-Neoplasms

EF24, a synthetic analogue of curcumin, was developed as an anti-tumor compound to induce apoptosis, inhibit proliferation and metastasis in various cancers. However, whether EF24 induces ferroptosis in osteosarcoma cells or not, and its underlying mechanism remains largely elusive.. After EF24 combining with or without other compounds treatments, mRNA expression profiles were proceeded by RNA sequencing. Cytotoxicity was measured by cell counting kit-8 assay. Cell death was quantified by flow cytometer. Gene expression was quantified by real-time PCR. Protein level was detected by western blot. Malonydialdehyde (MDA) level was measured by lipid peroxidation MDA assay kit. Reactive oxygen species (ROS) level was measured by ROS Assay Kit. Ferric ion was measured by Iron Assay kit.. EF24 significantly induced cell death in osteosarcoma cell lines, and this effect was significantly reversed by ferrostatin-1, but not Z-VAD(Ome)-FMK, MRT68921 or necrosulfonamide. EF24 significantly increased MDA level, ROS level and intracellular ferric ion level, these effects were significantly attenuated by ferrostatin-1. EF24 upregulated HMOX1 expression in a dose dependent manner, overexpression of HMOX1 facilitated EF24 to induce ferroptosis in osteosarcoma cell lines. HMOX1 knockdown attenuated EF24-induced cytotoxicity and attenuated EF24-induced inhibition of Glutathione Peroxidase 4 (GPX4) expression.. Our results showed that EF24 upregulated HMOX1 to suppress GPX4 expression to induce ferroptosis by increasing MDA level, ROS level and intracellular ferric ion level. Thus, EF24 might serve as a potential agent for the treatment of HMOX1-positive osteosarcoma patients.

Topics: Antineoplastic Agents; Benzylidene Compounds; Bone Neoplasms; Cell Line, Tumor; Ferroptosis; Gene Expression Regulation, Neoplastic; Heme Oxygenase-1; Humans; Iron; Malondialdehyde; Osteosarcoma; Phospholipid Hydroperoxide Glutathione Peroxidase; Piperidones; Reactive Oxygen Species; Signal Transduction

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Benzylidene Compounds; BH3 Interacting Domain Death Agonist Protein; Bone Neoplasms; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Curcumin; Enzyme Activation; Fas-Associated Death Domain Protein; Humans; Osteosarcoma; Piperidones; Poly(ADP-ribose) Polymerases; Tumor Suppressor Protein p53