Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells.

OBJECTIVE: Research has shown that celastrol can effectively treat a variety of diseases, yet when passing a certain dosage threshold, celastrol becomes toxic, causing complications such as liver and kidney damage and erythrocytopenia, among others. With this dichotomy in mind, it is extremely important to find ways to preserve celastrol's efficacy while reducing or preventing its toxicity. METHODS: In this study, insulin-resistant HepG2 (IR-HepG2) cells were prepared using palmitic acid and used for in vitro experiments. IR-HepG2 cells were treated with celastrol alone or in combination with N-acetylcysteine (NAC) or ferrostatin-1 (Fer-1) for 12, 24 or 48 h, at a range of doses. Cell counting kit-8 assay, Western blotting, quantitative reverse transcription-polymerase chain reaction, glucose consumption assessment, and flow cytometry were performed to measure celastrol's cytotoxicity and whether the cell death was linked to ferroptosis. RESULTS: Celastrol treatment increased lipid oxidation and decreased expression of anti-ferroptosis proteins in IR-HepG2 cells. Celastrol downregulated glutathione peroxidase 4 (GPX4) mRNA. Molecular docking models predicted that solute carrier family 7 member 11 (SLC7A11) and GPX4 were covalently bound by celastrol. Importantly, we found for the first time that the application of ferroptosis inhibitors (especially NAC) was able to reduce celastrol's toxicity while preserving its ability to improve insulin sensitivity in IR-HepG2 cells. CONCLUSION: One potential mechanism of celastrol's cytotoxicity is the induction of ferroptosis, which can be alleviated by treatment with ferroptosis inhibitors. These findings provide a new strategy to block celastrol's toxicity while preserving its therapeutic effects. Please cite this article as: Liu JJ, Zhang X, Qi MM, Chi YB, Cai BL, Peng B, Zhang DH. Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells. J Integr Med. 2024; 22(3): 286-294.

ADAMTS-13-regulated nuclear factor E2-related factor 2 signaling inhibits ferroptosis to ameliorate cisplatin-induced acute kidney injuy.

ADAMTS-13 plays an important role in acute kidney injury (AKI), but the mechanism of cisplatin (CP) induced AKI remains unclear. Ferroptosis is increased in CP-induced AKI, and ADAMTS13 levels are associated with ferritin expression. In this article, we will explore the relationship between the three. After CP induction, mice were given 0.1 and 0.3 nmol/kg ADAMTS-13, and then serum creatinine (Scr) and blood urea nitrogen (BUN) were detected by the kits. The pathological changes of renal tissue were observed by staining with HE and PAS staining, and Western blot detected the expressions of KIM1 and NGAL in renal tissu. Perl's staining detected iron deposition in renal tissues, the kits detected iron levels, and western blot detected the expression of ferroptosis related proteins. Then the mechanism was further explored by adding ferroptosis inhibitors Ferrostatin 1 (Fer-1) and iron supplements Fe. The expression of Nrf2 pathway related proteins were detected by Western blot. We found that ADAMTS13 alleviated CP-induced ferroptosis in AKI mice with renal function impairment and tubular damage. Fer-1partially reversed CP-induced AKI, and Fe exacerbated this effect. ADAMTS13 alleviated CP-induced inflammatory response and oxidative stress in AKI mice, during which the Nrf2 signaling pathway was abnormal. Overall, ADAMTS-13-regulated Nrf2 signaling inhibits ferroptosis to ameliorate CP-induced AKI.

Hematopoietic protection and mechanisms of ferrostatin-1 on hematopoietic acute radiation syndrome of mice.

PURPOSE: Baicalein (an anti-ferroptosis drug) was recently reported to synergistically improve the survival rate of mice following a high dose of total body irradiation with anti-apoptosis and anti-necroptosis drugs. At the same time, our group has demonstrated that ferrostatin-1, a ferroptosis inhibitor, improves the survival rate of a mouse model of hematopoietic acute radiation syndrome to 60% for 150 days (p < .001). These phenomena suggest that ferroptosis inhibition can mitigate radiation damage. In this study, we continued to study the mechanisms by which ferrostatin-1 alleviated radiation-induced ferroptosis and subsequent hematopoietic acute radiation syndrome. MATERIALS AND METHODS: Male ICR mice (8-10 weeks old) were exposed to doses of 0, 8, or 10 Gy irradiated from a 137Cs source. Ferrostatin-1 was intraperitoneally injected into mice 72 h post-irradiation. Bone marrow mononuclear cells (BMMCs) and peripheral blood cells were counted. The changes in iron-related parameters, lipid metabolic enzymes, lipid peroxidation repair molecules (glutathione peroxidase 4, glutathione, and coenzyme Q10), and inflammatory factors (TNF-α, IL-6, and IL-1ß) were evaluated using biochemical or antibody techniques. RESULTS: Ferrostatin-1 increased the number of red and white blood cells, lymphocytes, and monocytes in the peripheral blood after total body irradiation in mice by mitigating the ferroptosis of BMMCs. Total body irradiation induced ferroptosis in BMMCs by increasing the iron and lipid peroxidation levels and depleting the acyl-CoA synthetase long-chain family member 4 (ASCL4), lipoxygenase 15, glutathione peroxidase 4, and glutathione levels. Ferroptotic BMMCs did not release TNF-α, IL-6, or IL-1ß at the early stage of radiation exposure. Ferrostatin-1 mitigated the lipid peroxidation of radiation-induced ferroptosis by attenuating increases in levels of hemosiderin and liable iron pool and decreases in levels of ASCL4 and glutathione peroxidase 4. CONCLUSIONS: The onset of total body irradiation-induced ferroptosis in BMMCs involved changes in iron, lipid metabolic enzymes, and anti-lipid peroxidation molecules. Ferrostatin-1 could be a potential radiation mitigation agent by acting on these targets.

Integrated stress response inhibition provides sex-dependent protection against noise-induced cochlear synaptopathy.

Noise-induced hearing loss (NIHL) is a common health concern with significant social, psychological, and cognitive implications. Moderate levels of acoustic overstimulation associated with tinnitus and impaired speech perception cause cochlear synaptopathy, characterized physiologically by reduction in wave I of the suprathreshold auditory brainstem response (ABR) and reduced number of synapses between sensory hair cells and auditory neurons. The unfolded protein response (UPR), an endoplasmic reticulum stress response pathway, has been implicated in the pathogenesis and treatment of NIHL as well as neurodegeneration and synaptic damage in the brain. In this study, we used the small molecule UPR modulator Integrated Stress Response InhiBitor (ISRIB) to treat noise-induced cochlear synaptopathy in a mouse model. Mice pretreated with ISRIB prior to noise-exposure were protected against noise-induced synapse loss. Male, but not female, mice also exhibited ISRIB-mediated protection against noise-induced suprathreshold ABR wave-I amplitude reduction. Female mice had higher baseline wave-I amplitudes but greater sensitivity to noise-induced wave-I reduction. Our results suggest that the UPR is implicated in noise-induced cochlear synaptopathy, and can be targeted for treatment.

Ferroptosis driven by radical oxidation of n-6 polyunsaturated fatty acids mediates acetaminophen-induced acute liver failure.

Acetaminophen (APAP) overdose is a common cause of drug-induced acute liver failure. Although hepatocyte cell death is considered to be the critical event in APAP-induced hepatotoxicity, the underlying mechanism remains unclear. Ferroptosis is a newly discovered type of cell death that is caused by a loss of cellular redox homeostasis. As glutathione (GSH) depletion triggers APAP-induced hepatotoxicity, we investigated the role of ferroptosis in a murine model of APAP-induced acute liver failure. APAP-induced hepatotoxicity (evaluated in terms of ALT, AST, and the histopathological score), lipid peroxidation (4-HNE and MDA), and upregulation of the ferroptosis maker PTGS2 mRNA were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1). Fer-1 treatment also completely prevented mortality induced by high-dose APAP. Similarly, APAP-induced hepatotoxicity and lipid peroxidation were prevented by the iron chelator deferoxamine. Using mass spectrometry, we found that lipid peroxides derived from n-6 fatty acids, mainly arachidonic acid, were elevated by APAP, and that auto-oxidation is the predominant mechanism of APAP-derived lipid oxidation. APAP-induced hepatotoxicity was also prevented by genetic inhibition of acyl-CoA synthetase long-chain family member 4 or α-tocopherol supplementation. We found that ferroptosis is responsible for APAP-induced hepatocyte cell death. Our findings provide new insights into the mechanism of APAP-induced hepatotoxicity and suggest that ferroptosis is a potential therapeutic target for APAP-induced acute liver failure.

Preclinical evaluation of EPHX2 inhibition as a novel treatment for inflammatory bowel disease.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by cytochrome P450 epoxygenation of arachidonic acid, which are metabolized by EPHX2 (epoxide hydrolase 2, alias soluble epoxide hydrolase or sEH). EETs have pleiotropic effects, including anti-inflammatory activity. Using a Connectivity Map (CMAP) approach, we identified an inverse-correlation between an exemplar EPHX2 inhibitor (EPHX2i) compound response and an inflammatory bowel disease patient-derived signature. To validate the gene-disease link, we tested a pre-clinical tool EPHX2i (GSK1910364) in a mouse disease model, where it showed improved outcomes comparable to or better than the positive control Cyclosporin A. Up-regulation of cytoprotective genes and down-regulation of proinflammatory cytokine production were observed in colon samples obtained from EPHX2i-treated mice. Follow-up immunohistochemistry analysis verified the presence of EPHX2 protein in infiltrated immune cells from Crohn's patient tissue biopsies. We further demonstrated that GSK2256294, a clinical EPHX2i, reduced the production of IL2, IL12p70, IL10 and TNFα in both ulcerative colitis and Crohn's disease patient-derived explant cultures. Interestingly, GSK2256294 reduced IL4 and IFNγ in ulcerative colitis, and IL1ß in Crohn's disease specifically, suggesting potential differential effects of GSK2256294 in these two diseases. Taken together, these findings suggest a novel therapeutic use of EPHX2 inhibition for IBD.

UVA and UVB Photoprotective Capabilities of Topical Formulations Containing Mycosporine-like Amino Acids (MAAs) through Different Biological Effective Protection Factors (BEPFs).

The safety and stability of synthetic UV-filters and the procedures for evaluating the photoprotective capability of commercial sunscreens are under continuous review. The influence of pH and temperature stressors on the stability of certain Mycosporine-like amino acids (MAAs) isolated at high purity levels was examined. MAAs were highly stable at room temperature during 24 h at pH 4.5⁻8.5. At 50 °C, MAAs showed instability at pH 10.5 while at 85 °C, progressive disappearances were observed for MAAs through the studied pH range. In alkaline conditions, their degradation was much faster. Mycosporine-serinol and porphyra-334 (+shinorine) were the most stable MAAs under the conditions tested. They were included in four cosmetically stable topical sunscreens, of which the Sun Protection Factor (SPF) and other Biological Effective Protection Factors (BEPFs) were calculated. The formulation containing these MAAs showed similar SPF and UVB-BEPFs values as those of the reference sunscreen, composed of synthetic UV absorbing filters in similar percentages, while UVA-BEPFs values were slightly lower. Current in vitro data strongly suggest that MAAs, as natural and safe UV-absorbing and antioxidant compounds, have high potential for protection against the diverse harmful effects of solar UV radiation. In addition, novel complementary in vitro tests for evaluation of commercial sunscreens efficacy are proposed.

Anti-adult T­cell leukemia/lymphoma activity of cerdulatinib, a dual SYK/JAK kinase inhibitor.

Adult T­cell leukemia/lymphoma (ATLL) constitutes an aggressive malignancy caused by human T­cell leukemia virus type 1 (HTLV­1) that is resistant to available chemotherapeutics. The constitutive activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is an important feature of ATLL, and spleen tyrosine kinase (SYK) is overexpressed in HTLV­1-transformed T­cell lines. In this study, we evaluated the effects of SYK- (PRT060318) or JAK- (JAK inhibitor 1) selective inhibitors and the dual SYK/JAK inhibitor, cerdulatinib, on the viability of HTLV­1-transformed and ATLL-derived T­cell lines. Cell proliferation, viability, cell cycle, apoptosis and intracellular signaling cascades were analyzed by the water-soluble tetrazolium-8 assay, flow cytometry and western blot analysis. HTLV­1-infected T­cell lines were sensitive to both SYK-selective and pan-JAK inhibitors, whereas cerdulatinib more potently suppressed cell proliferation and reduced cell viability than either of these agents alone. By contrast, the cytotoxic effects of cerdulatinib on uninfected T­cell lines and peripheral blood mononuclear cells from a healthy donor were less pronounced. Cerdulatinib induced cell cycle arrest in the G2/M phase, which was associated with a decreased cyclin-dependent kinase 1 and cyclin B1, and an increased p21 and p27 expression. Hoechst staining revealed chromatin condensation and nuclear fragmentation in the cells treated with cerdulatinib, and an increased fraction of apoptotic APO2.7-stained cells was detected by flow cytometry. This corresponded to the activation of caspase-8, -9 and -3, and decreased levels of the anti-apoptotic factors, Bcl-xL, survivin, X-linked inhibitor of apoptosis (XIAP) and c­FLIP. The cerdulatinib-induced decrease in cell viability was partly reversed by the caspase inhibitor, z­VAD­FMK. These anti-ATLL effects were associated with the suppression of SYK and JAK/STAT signaling, along with that of the downstream factors, AKT, ERK, activator protein­1 and nuclear factor-κB. Finally, oral dosing with cerdulatinib lowered the tumor burden in a murine model of ATLL. Thus, our findings indicate that the simultaneous inhibition of therapeutically relevant targets, such as SYK and JAK is a more effective approach than single-agent therapy for the treatment of ATLL.

Cell growth potential drives ferroptosis susceptibility in rhabdomyosarcoma and myoblast cell lines.

PURPOSE: Ferroptosis is a programmed form of iron-dependent cell death caused by lipid hydroperoxide accumulation, which can be prevented by glutathione peroxidase 4 (GPx4) activity. Here we investigated the effects of ferroptosis inducers called erastin and RSL3, which act by glutathione depletion and GPx4 inactivation, respectively, on muscle-derived cell lines of embryonal and alveolar rhabdomyosarcoma (RMS), and mouse normal skeletal C2C12 myoblasts. METHODS: Myogenic lines were exposed to stepwise increasing concentrations of ferroptosis inducers either alone or in combination with iron supplementation, iron chelating agents (bathophenanthrolinedisulfonic acid, BPS), antioxidant molecules (glutathione, N-acetylcysteine), lipid peroxidation inhibitors (ferrostatin-1), and chemotherapeutic agents (doxorubicin and actinomycin D). Drug susceptibility was quantified by measuring cell viability, proliferation and differentiation via neutral red assay, crystal violet assay and Giemsa staining, respectively. The detection of lipid hydroperoxide and protein levels was performed by immunofluorescence and Western blot analysis, respectively. RESULTS: Erastin and RSL3 increased lipid hydroperoxide levels preferentially in the embryonal U57810 and myoblast C2C12 lines, leading to ferroptosis that was accentuated by iron supplementation or prevented by co-treatment with BPS, glutathione, N-acetylcysteine and ferrostatin-1. The inhibition of extracellular regulated kinases (ERK) pathway prevented ferroptosis in U57810 and C2C12 cells, whereas its increased activation in the embryonal RD cells mediated by caveolin-1 (Cav-1) overexpression led to augmented ferroptosis susceptibility. Finally, we observed the combination of erastin or RSL3 with chemotherapeutic doxorubicin and actinomycin D agents to be effective in increasing cell death in all RMS lines. CONCLUSIONS: Erastin and RSL3 trigger ferroptosis in highly proliferating myogenic lines through a ERK pathway-dependent fashion.

Pseudolaric acid B triggers ferroptosis in glioma cells via activation of Nox4 and inhibition of xCT.

Ferroptosis is a form of programmed cell death decided by iron-dependent lipid peroxidation, but its role in glioma cell death remains unclear. In this study, we found Pseudolaric acid B (PAB) inhibited the viabilities of glioma cells in vitro and in vivo, which was accompanied by abnormal increases of intracellular ferrous iron, H2O2 and lipid peroxidation, as well as depletion of GSH and cysteine. In vitro studies revealed that the lipid peroxidation and the cell death caused by PAB were both inhibited by iron chelator deferoxamine, but exacerbated by supplement of ferric ammonium citrate. Inhibition of lipid peroxidation with ferrostatin-1 or GSH rescued PAB-induced cell death. Morphologically, the cells treated with PAB presented intact membrane, shrunken mitochondria with increased membrane density, and normal-sized nucleus without chromatin condensation. Mechanistically, PAB improved intracellular iron by upregulation of transferrin receptor. The increased iron activated Nox4, which resulted in overproduction of H2O2 and lipid peroxides. Moreover, PAB depleted intracellular GSH via p53-mediated xCT pathway, which further exacerbated accumulation of H2O2 and lipid peroxides. Thus, PAB triggers ferroptosis in glioma cells and is a potential medicine for glioma treatment.

Heme oxygenase-1 mitigates ferroptosis in renal proximal tubule cells.

Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI because of its antiapoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1-/- mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of antioxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared with vehicle-treated controls. HO-1-/- cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1-/- PTCs compared with HO-1+/+ cells. Cotreatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-l-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1-/- PTCs. These results demonstrate an important antiferroptotic role of HO-1 in renal epithelial cells.

Neuropharmacological characterization of the new psychoactive substance methoxetamine.

The use of new psychoactive substances (NPS) is steadily increasing. One commonly used NPS is methoxetamine (MXE), a ketamine analogue. Several adverse effects have been reported following MXE exposure, while only limited data are available on its neuropharmacological modes of action. We investigated the effects of MXE and ketamine on several endpoints using multiple in vitro models. These included rat primary cortical cells, human SH-SY5Y cells, human induced pluripotent stem cell (hiPSC)-derived iCell® Neurons, DopaNeurons and astrocyte co-cultures, and human embryonic kidney (HEK293) cells. We investigated effects on several neurotransmitter receptors using single cell intracellular calcium [Ca2+]i imaging, effects on neuronal activity using micro-electrode array (MEA) recordings and effects on human monoamine transporters using a fluorescence-based plate reader assay. In rat primary cortical cells, 10 µM MXE increased the glutamate-evoked increase in [Ca2+]i, whereas 10 µM ketamine was without effect. MXE and ketamine did not affect voltage-gated calcium channels (VGCCs), but inhibited spontaneous neuronal activity (IC50 0.5 µM and 1.2 µM respectively). In human SH-SY5Y cells, 10 µM MXE slightly inhibited the K+- and acetylcholine-evoked increase in [Ca2+]i. In hiPSC-derived iCell®(Dopa)Neurons, only the ATP-evoked increase in [Ca2+]i was slightly reduced. Additionally, MXE inhibited spontaneous neuronal activity (IC50 between 10 and 100 µM). Finally, MXE potently inhibits uptake via monoamine transporters (DAT, NET and SERT), with IC50 values in the low micromolar range (33, 20, 2 µM respectively). Our combined in vitro data provide an urgently needed first insight into the multiple modes of action of MXE. The use of different models and different (neuronal) endpoints can be complementary in pharmacological profiling. Rapid in vitro screening methods as those presented here, could be of utmost importance for gaining a first mechanistic insight to aid the risk assessment of emerging NPS.

PRT062607 Achieves Complete Inhibition of the Spleen Tyrosine Kinase at Tolerated Exposures Following Oral Dosing in Healthy Volunteers.

The spleen tyrosine kinase (SYK) regulates immune cell activation in response to engagement of a variety of receptors, making it an intriguing target for the treatment of inflammatory and autoimmune disorders as well as certain B-cell malignancies. We have previously reported on the discovery and preclinical characterization of PRT062607, a potent and highly selective inhibitor of SYK that exhibits robust anti-inflammatory activity in a variety of animal models. Here we present data from our first human studies aimed at characterizing the pharmacokinetics (PK), pharmacodynamics (PD), and safety of PRT062607 in healthy volunteers following single and multiple oral administrations. PRT062607 demonstrated a favorable PK profile and the ability to completely inhibit SYK activity in multiple whole-blood assays. The PD half-life in the more sensitive assays was approximately 24 hours and returned to predose levels by 72 hours. Selectivity for SYK was observed at all dose levels tested. Analysis of the PK/PD relationship indicated an IC50 of 324 nM for inhibition of B-cell antigen receptor-mediated B-cell activation and 205 nM for inhibition of FcεRI-mediated basophil degranulation. PRT062607 was safe and well tolerated across the entire range of doses. Clinical PK/PD was related to in vivo anti-inflammatory activity of PRT062607 in the rat collagen-induced arthritis model, which predicts that therapeutic concentrations may be safely achieved in humans for the treatment of autoimmune disease. PRT062607 has a desirable PK profile and is capable of safely, potently, and selectively suppressing SYK kinase function in humans following once-daily oral dosing.

[COMPARISON OF CHRONIC ANTICONVULSANT ACTIVITY AND SAFETY OF IEM-2062, SODIUM VALPROATE AND ME-MANTINE IN THE PENTYLENETETRAZOL KINDLING MODEL IN RATS].

IEM-2062 [1-(6-aminohexylamino)-1-phenylcyclohexyl dihydrochloride], causing a combined block NMDA and AMPA receptors, after chronic oral administration in doses, respectively, 0.3 and 3 mg/kg, induce maximal anticonvulsant effect in the pentylenetetrazol kindling rats because decrease the number of completely kindling rats by 100 %, and also decrease in 2.5-3.3 times the average severity of clonic-tonic kindling seizures. IEM-2062 causes significant anticon- 299 vulsant effects in the widest range of doses, 1-48 mg/kg, which is 24-22 times more than that of memantine (12-20 mg/kg) and sodium valproate (100-200 mg/kg). Sodium valproate and memantine cause significant disturbances of locomotor activity in the «open field¼ test in doses causing maximal anticonvulsant effect in the kindling rats. At the same time IEM-2062 cause disturbance of locomotor activity only in very high dose of 92 mg/kg, which exceeds in 30.7 times the dose causing the maximum anticonvulsive effect in the kindling rats. Thus, IEM-2062 reduces the severity of kindling seizures in 1.7-1.9 times stronger than sodium valproate and memantine and also by 30.7 times is safer than sodium valproate and memantine.

The novel ketamine analog methoxetamine produces dissociative-like behavioral effects in rodents.

RATIONALE: Methoxetamine (MXE) is a ketamine analog sold online that has been subject to widespread abuse for its dissociative and hallucinogenic effects. Previous studies have shown that MXE has high affinity for the phencyclidine (PCP) binding site located within the channel pore of the NMDA receptor (NMDAR), but little is known about its behavioral effects. Dissociative anesthetics such as ketamine and PCP produce a characteristic behavioral profile in rats that includes locomotor hyperactivity and disruption of prepulse inhibition (PPI) of acoustic startle. METHODS: The goal of the present investigation was to determine whether MXE produces PCP-like effects in Sprague-Dawley rats using the PPI paradigm and the behavioral pattern monitor (BPM), which enables analyses of patterns of locomotor activity and investigatory behavior. PPI studies were conducted with several other uncompetitive NMDAR antagonists that produce dissociative effects in humans, including PCP, the S-(+) and R-(-) isomers of ketamine, and N-allylnormetazocine (NANM; SKF-10,047). RESULTS: MXE disrupted PPI when administered at 3 and 10 mg/kg SC. The rank order of potency of MXE and the other test compounds in the PPI paradigm (PCP > MXE > S-(+)-ketamine > NANM > R-(-)-ketamine) parallels their affinities for the PCP binding site reported in the literature. When tested in the BPM, 10 mg/kg MXE induced locomotor hyperactivity, reduced the number of rearings, increased the roughness of locomotor paths, and produced perseverative patterns of locomotion. Administration of PCP (2.25 and 6.75 mg/kg, SC) produced a similar profile of effects in the BPM. CONCLUSIONS: These results indicate that MXE produces a behavioral profile similar to that of other psychotomimetic uncompetitive NMDAR antagonists. Our findings support the classification of MXE as a dissociative drug and suggest that it likely has effects and abuse potential similar to that of PCP and ketamine.

Artemisinin derivatives induce iron-dependent cell death (ferroptosis) in tumor cells.

BACKGROUND: Apoptosis and other forms of cell death have been intensively investigated in the past years to explain the mode of action of synthetic anticancer drugs and natural products. Recently, a new form of cell death emerged, which was termed ferroptosis, because it depends on intracellular iron. Here, the role of genes involved in iron metabolism and homeostasis for the cytotoxicity of ten artemisinin derivatives have been systematically investigated. MATERIAL AND METHODS: Log10IC50 values of 10 artemisinin derivatives (artesunate, artemether, arteether, artenimol, artemisitene, arteanuin B, another monomeric artemisinin derivative and three artemisinin dimer molecules) were correlated to the microarray-based mRNA expression of 30 iron-related genes in 60 cell lines of the National Cancer Institute (NCI, USA) as determined in 218 different microarray hybridization experiments. The effect of desferoxamine and ferrostatin-1 on the cytotoxicity of artenimol of CCRF-CEM cells was determined by resazurin assays. The mRNA expression of TFRC was exemplarily validated by immunohistochemical detection of transferrin receptor protein expression. RESULTS: The mRNA expression of 20 genes represented by 59 different cDNA clones significantly correlated to the log10IC50 values for the artemisinins, including genes encoding transferrin (TF), transferrin receptors 1 and 2 (TFRC, TFR2), cerulopasmin (CP), lactoferrin (LTF) and others. The ferroptosis inhibitor ferrostatin-1 and the iron chelator deferoxamine led to a significantly reduced cytotoxicity of artenimol, indicating ferroptosis as cell death mode. CONCLUSION: The numerous iron-related genes, whose expression correlated with the response to artemisinin derivatives speak in factor for the relevance of iron for the cytotoxic activity of these compounds. Treatment with ferroptosis-inducing agents such as artemisinin derivatives represents an attractive strategy for cancer therapy. Pre-therapeutic determination of iron-related genes may indicate tumor sensitivity to artemisinins. Ferroptosis induced by artemisinin-type drugs deserve further investigation for individualized tumor therapy.

Inhibition of Collagenase by Mycosporine-like Amino Acids from Marine Sources.

Matrix metalloproteinases play an important role in extracellular matrix remodeling. Excessive activity of these enzymes can be induced by UV light and leads to skin damage, a process known as photoaging. In this study, we investigated the collagenase inhibition potential of mycosporine-like amino acids, compounds that have been isolated from marine organisms and are known photoprotectants against UV-A and UV-B. For this purpose, the commonly used collagenase assay was optimized and for the first time validated in terms of relationships between enzyme-substrate concentrations, temperature, incubation time, and enzyme stability. Three compounds were isolated from the marine red algae Porphyra sp. and Palmaria palmata, and evaluated for their inhibitory properties against Chlostridium histolyticum collagenase. A dose-dependent, but very moderate, inhibition was observed for all substances and IC50 values of 104.0 µM for shinorine, 105.9 µM for porphyra, and 158.9 µM for palythine were determined. Additionally, computer-aided docking models suggested that the mycosporine-like amino acids binding to the active site of the enzyme is a competitive inhibition.

Selective spleen tyrosine kinase inhibition delays autoimmune arthritis in mice.

Spleen tyrosine kinase (SYK) has an important role in immunoreceptor signaling, and SYK inhibition has accordingly attenuated immune-mediated injury in several models. Therefore, the present study examined the effect of SYK inhibition with the selective spleen tyrosine kinase inhibitor P505-15 in experimental rheumatoid arthritis (RA) using a murine model of collagen-induced arthritis (CIA). Treatment with the selective SYK inhibitor P505-15, a small molecule kinase inhibitor selective for SYK, led to a reduction in arthritis score and attenuated histological damage. P505-15 reduced cartilage destruction and macrophage infiltration in CIA mice. In addition, P505-15-treated mice showed lower circulating levels of type II-collagen immunoglobulin (Ig)G1 and IgG2 and pro-inflammatory cytokines. Importantly, P505-15 treatment markedly reduced the interleukin 1ß-stimulated inflammatory response in human RA synovial cells. Given these encouraging results, a key function for SYK in the development of RA was identified, highlighting that SYK may be a potential therapeutic target for human RA.

Modulation of viability and apoptosis of UVB-exposed human keratinocyte HaCaT cells by aqueous methanol extract of laver (Porphyra yezoensis).

We investigated the effect of 80% methanol extract of laver (Porphyra yezoensis) on the UVB-exposed HaCaT cells, human keratinocytes. The laver extract showed absorbance spectrum characteristic of porphyra-334 or shinorine, major mycosporine-like amino acids (MAAs) in red algae, and contained phenolic compounds. UVB exposure decreased cell viability and increased apoptotic cell fractions, and it also decreased the ratio of reduced (GSH) to oxidized glutathione (GSSG) and the total glutathione content. Post-treatment with the laver extract significantly increased the net viability and also the apoptotic cell fractions of UVB-exposed cells. The extract caused increase in GSH/GSSG ratio, yet it exacerbated the decrease in glutathione content in the UVB-exposed cells. These effects of the laver extract were also manifested in the sham-exposed cells, suggesting that those effects might be general phenomena caused by the laver extract. The extract treatment enhanced the UVB-induced phosphorylation of JNK and ERK, affecting more the latter. Our results suggest that the post-treatment with laver extract may protect UVB-exposed skin cells not only by increasing overall cell proliferation but also by enhancing apoptosis of damaged cells, via activating JNK and ERK signaling pathways, in which modulation of the content and redox status of glutathione may take significant parts.

From PCP to MXE: a comprehensive review of the non-medical use of dissociative drugs.

PCP or phencyclidine was discovered in 1956 and soon became a popular street drug. Dissociatives including PCP, ketamine, and dextromethorphan have been used non-medically for their mind-altering effects for over 60 years. Many of these compounds have also been used clinically and in legitimate research. At least 14 derivatives of PCP were sold for non-medical and illict use from the late 1960s until the 1990s. With the advent of the Internet, the drug market underwent a dramatic evolution. While initially gray-market chemical vendors offering dextromethorphan and ketamine thrived, most recently the market has shifted to legal high and online-based research chemical vendors. Starting with the first dissociative research chemical, 4-MeO-PCP in 2008, the dissociative research chemical market has rapidly evolved and currently comprises at least 12 dissociatives, almost half of which were unknown in the scientific literature prior to their introduction. Several of these, including methoxetamine, have reached widespread use internationally. A historical account of non-medical use of over 30 dissociative compounds was compiled from a diverse collection of sources. The first complete portrait of this underground market is presented along with the relevant legal, technological, and scientific developments which have driven its evolution.