Synergistic antitumor efficacy of gemcitabine and cisplatin to induce ferroptosis in pancreatic ductal adenocarcinoma via Sp1-SAT1-polyamine metabolism pathway.

PURPOSE: The combination of cisplatin and gemcitabine-based chemotherapy has been recommended as a preferred regimen for pancreatic ductal adenocarcinoma (PDAC) patients with germline-based mutations. However, the underlying mechanism remains poorly elucidated. Therefore, our study aimed to explore the mechanistic basis of the cell-killing activity of gemcitabine plus cisplatin and identify potential therapeutic targets. METHODS: First, we explored the synergistic cytotoxic effects of gemcitabine and cisplatin on PDAC through in vitro and in vivo experiments. Then, we investigated ferroptosis-related biomarkers, to assess the impact of the combination therapy on ferroptosis. Using bioinformatics methods, we identified SAT1 as a potential key mediator of ferroptosis induced by gemcitabine and cisplatin. We tested the polyamine levels in PDAC cells by LC-MS after overexpressed or knocked down SAT1, and explored the role of polyamines in ferroptosis using exogenous supplementation. Finally, we explored the regulatory effect of Sp1 on SAT1 through ChIP-qPCR and dual-luciferase reporter assay. RESULTS: Gemcitabine plus cisplatin enhanced cell death and induced ferroptosis in PDAC. This combination upregulated SAT1 transcription by inhibiting Sp1. SAT1 activation promoted the catabolism of spermine and spermidine, leading to iron accumulation and lipid peroxide generation, ultimately resulting in ferroptosis. CONCLUSIONS: In summary, our findings suggested the gemcitabine and cisplatin combination therapy induced ferroptosis in a GSH-independent manner in PDAC. The combined treatment inhibited Sp1 and upregulated SAT1 transcription, leading to the breakdown of spermine and spermidine. Therefore, targeting SAT1-induced polyamine metabolism may represent a promising therapeutic strategy for PDAC.

Anti-Osteoporotic Effects of Kukoamine B Isolated from Lycii Radicis Cortex Extract on Osteoblast and Osteoclast Cells and Ovariectomized Osteoporosis Model Mice.

Osteoporosis is an abnormal bone remodeling condition characterized by decreased bone density, which leads to high risks of fracture. Previous study has demonstrated that Lycii Radicis Cortex (LRC) extract inhibits bone loss in ovariectomized (OVX) mice by enhancing osteoblast differentiation. A bioactive compound, kukoamine B (KB), was identified from fractionation of an LRC extract as a candidate component responsible for an anti-osteoporotic effect. This study investigated the anti-osteoporotic effects of KB using in vitro and in vivo osteoporosis models. KB treatment significantly increased the osteoblastic differentiation and mineralized nodule formation of osteoblastic MC3T3-E1 cells, while it significantly decreased the osteoclast differentiation of primary-cultured monocytes derived from mouse bone marrow. The effects of KB on osteoblastic and osteoclastic differentiations under more physiological conditions were also examined. In the co-culture of MC3T3-E1 cells and monocytes, KB promoted osteoblast differentiation but did not affect osteoclast differentiation. In vivo experiments revealed that KB significantly inhibited OVX-induced bone mineral density loss and restored the impaired bone structural properties in osteoporosis model mice. These results suggest that KB may be a potential therapeutic candidate for the treatment of osteoporosis.

Trodusquemine enhances Aß42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes.

Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-ß peptide (Aß42) are key pathogenic agents in Alzheimer's disease (AD). To investigate the relationship between Aß42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aß42-induced toxicity. In addition to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small molecule to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.

Nitric oxide rescues thalidomide mediated teratogenicity.

Thalidomide, a sedative drug given to pregnant women, unfortunately caused limb deformities in thousands of babies. Recently the drug was revived because of its therapeutic potential; however the search is still ongoing for an antidote against thalidomide induced limb deformities. In the current study we found that nitric oxide (NO) rescues thalidomide affected chick (Gallus gallus) and zebrafish (Danio rerio) embryos. This study confirms that NO reduced the number of thalidomide mediated limb deformities by 94% and 80% in chick and zebrafish embryos respectively. NO prevents limb deformities by promoting angiogenesis, reducing oxidative stress and inactivating caspase-3 dependent apoptosis. We conclude that NO secures angiogenesis in the thalidomide treated embryos to protect them from deformities.

Anti-tumoral effect of native and immobilized bovine serum amine oxidase in a mouse melanoma model.

Bovine serum amine oxidase (BSAO) oxidatively deaminates polyamines containing primary amine groups, spermidine and spermine, to form the cytotoxic products hydrogen peroxide and aldehyde(s). Polyamines are present at elevated levels in many tumor tissues. The aims of the study were to evaluate the anti-tumoral activities of native and immobilized BSAO in mouse melanoma and also to determine the mechanism of tumor cell death. C57BL mice received a subcutaneous injection of B16 melanoma cells to induce formation of tumors, prior to antitumor treatments with native and immobilized BSAO. The enzyme was immobilized in a poly(ethylene glycol) (PEG) biocompatible matrix. Antitumor treatments consisted of a single injection of enzyme into the tumor. When immobilized BSAO (2.5mU) was injected into the tumor, there was a marked decrease of 70% of the tumor growth. This was compared with a decrease of only 32% of tumor size when the same amount of native BSAO was administered. The type of cell death was analysed in tumors that were treated with native or immobilized BSAO. When tumors were treated with immobilized BSAO, there was induction of a high level of apoptosis (around 70%), compared to less than 10% with the native enzyme. Apoptotic cell death was assessed by nuclear chromatin condensation using Hoechst staining and labelling of externalized phosphatidylserine using Annexin V. However, native BSAO, probably due to a burst of cytotoxic products, induced a high level of necrosis of about 40%, compared to less than 10% with immobilized BSAO. In conclusion, the advantage is that immobilized BSAO can act by allowing the slow release of cytotoxic products, which induces tumor cell death by apoptosis rather than necrosis.

Inhibition of arterial thrombosis by polyamines in a canine coronary artery injury model.

Polyamines are polycations present in all living organisms and have been shown to play an important role in various physiological functions. Previous studies have shown that various amines including polyamines inhibit platelet activation. Among the amines tested tetra-amine, spermine is the potent inhibitor of platelet aggregation. In spite of vast literature on the anti-aggregatory effect of amines, there are no definitive studies testing their efficacy in an in vivo thrombosis model. In the present study, we investigated if polyamines could inhibit in-vivo thrombosis. A partially occlusive thrombus was generated by application of electric current in canine coronary artery. In control animals, the artery was completely in 76+/-14 min after the current was discontinued. When 40 mg/kg (1.44 mM) spermine was given immediately after stopping the current blood flow remained patent for >240 min. At equimolar concentration, triamine, spermidine and diamine putrescine are also equally effective in preventing thrombus development. The anti thrombic effect of polyamines was not associated with increased bleeding tendency, as judged by the amount of blood adsorbed by a gauge pad placed in a surgical incision extending to the muscle tissue and by a standard template bleeding. These results indicate that apart from inhibiting in-vitro platelet aggregation polyamines can also inhibit in-vivo thrombus formation.

Inhibition of platelet aggregation by putrescine, spermidine, and spermine in hypercholesterolemic rabbits.

BACKGROUND: Hypercholesterolemia causes alterations in platelet function. Platelet hyperaggregation is considered a predisposing factor for atherosclerosis. In this paper, the antiaggregating effect of the polyamines putrescine, spermidine, and spermine was studied on platelets of normal and hypercholesterolemic rabbits. METHODS: New Zealand rabbits were fed with a cholesterol-enriched diet for 10 weeks. Lipids and glucose were determined in serum. The assays of platelet aggregation were carried out using platelet-rich plasma (PRP) obtained from both control and cholesterol-fed rabbits. We used 2.5 micromol /mL ADP and 2 microg/mL collagen as inductors of platelet aggregation. In addition, arginase activity and L-arginine content were determined in PRP. RESULTS: Serum total cholesterol and LDL-cholesterol concentrations were increased from 26.3 +/- 8.1 to 1,485.0 +/- 26.8 mg/dL and from 15.9 +/- 5.9 to 1,383.8 +/- 58.9 mg/dL, respectively, whereas triglyceride concentration increased from 88.3 +/- 35.6 to 411.0 +/- 154.5 mg/dL upon cholesterol feeding. Seventy-five percent of platelet aggregation inhibition was observed with 10 microM of polyamines in PRP of normal rabbits. Spermine inhibited platelet aggregation by 54% in PRP of hypercholesterolemic rabbits when ADP was used as agonist. The order of polyamine action was spermine > spermidine > putrescine. In addition, we found that platelet arginase activity and L-arginine content were unaltered upon hypercholesterolemia. CONCLUSIONS: These results show that the polyamines putrescine, spermidine, and spermine have antagonist action in platelet aggregation and suggest a key role of polyamines in platelet aggregation under normal and hypercholesterolemic conditions.