Piperazine ring toxicity in three novel anti-breast cancer drugs: an in silico and in vitro metabolic bioactivation approach using olaparib as a case study.

The metabolic activation of small-molecule drugs into electrophilic reactive metabolites is widely recognized as an indicator of idiosyncratic adverse drug reactions (IADRs). Three novel anti-breast cancer drugs containing piperazine rings, ribociclib (Kisqali®, RCB), abemaciclib (Verzenio®, ABC), and olaparib (Lynparza®, OLP), were selected to study the effect of different chemical environment on the piperazine ring activation using in silico and in vitro metabolic experiments. ABC and RCB were previously studied and we noticed the piperazine ring in ABC could be strongly bioactivated generating more reactive intermediates than piperazine ring in RCB. OLP was further used as a case study to show the power of in silico software to predict the piperazine ring activation that was approved using in vitro experiments. Initially, predictions of susceptible sites in the metabolism and reactivity pathways were performed using the StarDrop P450 model and XenoSite reactivity tool, respectively. Later, in vitro OLP metabolites were characterized based on rat liver microsomes (RLMs) using KCN (trapping agent) using LC-MS/MS. The main goal of the current study was to answer the question of whether the presence of a piperazine ring in the chemical structure should be always considered a structural alert. Piperazine ring in RBC and ABC was bioactivated through a metabolic sequence that involves the hydroxylation of α-carbon to the tertiary nitrogen atoms of the piperazine ring. In the case of OLP, no cyano adduct was formed due to the presence of two carbonyl groups attached to the two nitrogen atoms of the piperazine ring (neutral amide groups). From the results, piperazine ring in certain cases should not be considered as a structural alert as in the case of OLP due to the presence of two electron withdrawing group that stops the proposed toxicity. Also blocking the bioactive center (α-carbon) using steric hindrances such as methyl group, also the isosteric replacement of α-carbon hydrogen with fluoro atom can aid in reducing the toxic side effects of ABC and RCB. These experiments were done in vitro through incubation with RLMs in the presence of KCN. Also, the results are supported by data generated from in silico software. In the future, drug discovery studies using this concept could be undertaken, allowing for the development of new drugs with reasonable safety profiles. Overall, in vitro RLMs incubations and in silico experiments were able to predict successfully that the piperazine ring should not always be considered a structural alert.

Toxicological and pharmacological characterization of novel cinnamylpiperazine synthetic opioids in humans and in vitro including 2-methyl AP-237 and AP-238.

The recent scheduling actions of fentanyl-related substances in both the United States and China have sparked the emergence and proliferation of other generations of "legal" opioids that are structurally distinct from fentanyl, including the recently emerged class of cinnamylpiperazines. In contrast to fentanyl, which contains a piperidine core and a phenethyl moiety, the primary structural components of cinnamylpiperazines are the piperazine core and a cinnamyl moiety. This manuscript reports on the toxicological profile for antemortem and postmortem cases where a cinnamylpiperazine was detected. Samples were quantitatively confirmed using liquid chromatography tandem mass spectrometry. The cases were received between February 2020 and April 2021. Concentrations of 2-methyl AP-237 from four postmortem cases ranged from 820 to 5800 ng/mL, and concentrations of AP-238 from two postmortem cases were 87 and 120 ng/mL. µ-Opioid receptor (MOR) activation potential for 2-methyl AP-237, AP-237, para-methyl AP-237, and AP-238 were studied using a ßarr2 recruitment assay. Efficacies (Emax, relative to hydromorphone) and potencies (EC50) were derived and of the compounds tested AP-238 was the most potent compound in the panel with an EC50 of 248 nM. 2-Methyl AP-237 was found to be the most efficacious drug (Emax = 125%) of the tested cinnamylpiperazines; however, it had substantially less efficacy than fentanyl. The in vitro MOR activation potential of the studied cinnamylpiperazines was lower than that of fentanyl and other novel synthetic opioids (NSOs), in line with the relatively higher concentrations observed in postmortem toxicology samples-an important observational link between in vitro pharmacology and in vivo toxicology.

Real-Life Impact of Drug Toxicity on Dolutegravir Tolerability: Clinical Practice Data from a Multicenter Italian Cohort.

Dolutegravir (DTG) is currently one of the most used Integrase inhibitors (INI) in antiretroviral therapies (ARV) in both naïve and experienced people living with HIV (PLWHIV). We analyzed a multicenter cohort of PLWHIV, both naïve and experienced, starting an ARV including DTG. We enrolled 3775 PLWHIV: 2763 (73.2%) were males, with a median age of 50 years. During 9890.7 PYFU, we observed 930 discontinuations (9.4 per 100 PYFU). Estimated probabilities of maintaining DTG at three and five years were 75.1% and 67.2%, respectively. Treatment-naïve pts showed a lower probability of maintaining DTG at three and five years compared to treatment-experienced PLWHIV (log-rank p < 0.001). At a multivariate analysis, a longer time of virological suppression (aHR 0.994, p < 0.001) and having experienced a previous virological failure (aHR 0.788, p = 0.016) resulted protective against DTG discontinuation. Most discontinuations (84.0%) happened within the first 12 months of DTG initiation, in particular, 92.2% of discontinuations due to neuropsychiatric toxicity were observed in the first year. Our data confirm the overall good tolerability of DTG in clinical practice, with a low rate of discontinuations. CNS toxicity resulted the main reason for DTG discontinuation, with most related interruptions happening in the first year from DTG introduction.

Facile green approach towards the synthesis of some phenyl piperazine based dithiocarbamates as potent hemolytic and thrombolytic agents.

The facile and efficient protocol for the synthesis of N-phenyl piperazine based di-thio-carbamates has been reported under neat conditions. A library of novel piperazine based di-thio-carbamates (3a-h) in excellent yields has been prepared. Solvent free, catalyst free and easy work up conditions make this protocol an attractive synthetic protocol to achieve novel biologically active di-thio-carbamates. The synthesized molecules have been characterized by FT-IR, 1HNMR and 13CNMR spectroscopic techniques. The pharmacological aspects of these derivatives have been evaluated via hemolysis and thrombolysis. All the target molecules (3a-h) exhibit mild to medium potential as hemolytic and thrombolytic agents. Among the synthesized derivatives, compound 3c showed least cytotoxicity and better thrombolytic potential.

Metabolism is not a Major Contributor to the Toxicity of Piperaquine, a Long-acting Antimalarial Agent in Artemisinin-based Combination Therapy.

BACKGROUND: Hepatocellular damage has been reported for the antimalarial piperaquine (PQ) in the clinic after cumulative doses. OBJECTIVES: The role of metabolism in PQ toxicity was evaluated, and the mechanism mediating PQ hepatotoxicity was investigated. METHODS: The toxicity of PQ and its major metabolite (PQ N-oxide; M1) in mice was evaluated in terms of serum biochemical parameters. The role of metabolism in PQ toxicity was investigated in mice pretreated with an inhibitor of CYP450 (ABT) and/or FMO enzyme (MMI). The dose-dependent pharmacokinetics of PQ and M1 were studied in mice. Histopathological examination was performed to reveal the mechanism mediating PQ hepatotoxicity. RESULTS: Serum biochemical levels (ALT and BUN) increased significantly (P < 0.05) in mice after three-day oral doses of PQ (> 200 mg/kg/day), indicating hepatotoxicity and nephrotoxicity of PQ at a high dose. Weaker toxicity was observed for M1. Pretreatment with ABT and/or MMI did not increase PQ toxicity. PQ and M1 showed linear pharmacokinetics in mice after a single oral dose, and multiple oral doses led to their cumulative exposures. Histopathological examination showed that a high dose of PQ (> 200 mg/kg/day for three days) could induce hepatocyte apoptosis. The mRNA levels of targets in NF-κB and p53 pathways could be up-regulated by 2-30-fold in mice by PQ or M1. CONCLUSION: PQ metabolism led to detoxification of PQ, but there was a low possibility of altered toxicity induced by metabolism inhibition. The hepatotoxicity of PQ and its N-oxidation metabolite was partly mediated by NF-κB inflammatory pathway and p53 apoptosis pathway.

Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death.

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.

Designer exosomes for targeted and efficient ferroptosis induction in cancer via chemo-photodynamic therapy.

Background: Efficient and specific induction of cell death in liver cancer is urgently needed. In this study, we aimed to design an exosome-based platform to deliver ferroptosis inducer (Erastin, Er) and photosensitizer (Rose Bengal, RB) into tumor tissues with high specificity. Methods: Exosome donor cells (HEK293T) were transfected with control or CD47-overexpressing plasmid. Exosomes were isolated and loaded with Er and RB via sonication method. Hepa1-6 cell xenograft C57BL/6 model was injected with control and engineered exosomes via tail vein. In vivo distribution of the injected exosomes was analyzed via tracking the fluorescence labeled exosomes. Photodynamic therapy was conducted by 532 nm laser irradiation. The therapeutic effects on hepatocellular carcinoma and toxic side-effects were systemically analyzed. Results: CD47 was efficiently loaded on the exosomes from the donor cells when CD47 was forced expressed by transfection. CD47 surface functionalization (ExosCD47) made the exosomes effectively escape the phagocytosis of mononuclear phagocyte system (MPS), and thus increased the distribution in tumor tissues. Erastin and RB could be effectively encapsulated into exosomes after sonication, and the drug-loaded exosomes (Er/RB@ExosCD47) strongly induced ferroptosis both in vitro and in vivo in tumor cells after irradiation of 532 nm laser. Moreover, compared with the control exosomes (Er/RB@ExosCtrl), Er/RB@ExosCD47 displayed much lower toxicity in liver. Conclusion: The engineered exosomes composed of CD47, Erastin, and Rose Bengal, induce obvious ferroptosis in hepatocellular carcinoma (HCC) with minimized toxicity in liver and kidney. The proposed exosomes would provide a promising strategy to treat types of malignant tumors.

Dolutegravir Inhibition of Matrix Metalloproteinases Affects Mouse Neurodevelopment.

Dolutegravir (DTG) is a first-line antiretroviral drug (ARV) used in combination therapy for the treatment of human immunodeficiency virus type-1 (HIV-1) infection. The drug is effective, safe, and well tolerated. Nonetheless, concerns have recently emerged for its usage in pregnant women or those of child-bearing age. Notably, DTG-based ARV regimens have been linked to birth defects seen as a consequence of periconceptional usages. To this end, uncovering an underlying mechanism for DTG-associated adverse fetal development outcomes has gained clinical and basic research interest. We now report that DTG inhibits matrix metalloproteinases (MMPs) activities that could affect fetal neurodevelopment. DTG is a broad-spectrum MMPs inhibitor and binds to Zn++ at the enzyme's catalytic domain. Studies performed in pregnant mice show that DTG readily reaches the fetal central nervous system during gestation and inhibits MMP activity. Postnatal screenings of brain health in mice pups identified neuroinflammation and neuronal impairment. These abnormalities persist as a consequence of in utero DTG exposure. We conclude that DTG inhibition of MMPs activities during gestation has the potential to affect prenatal and postnatal neurodevelopment.

Maculosin, a non-toxic antioxidant compound isolated from Streptomyces sp. KTM18.

CONTEXT: Streptomyces species are prolific sources of bioactive secondary metabolites known especially for their antimicrobial and anticancer activities. OBJECTIVE: This study sought to isolate and characterize antioxidant molecules biosynthesized by Streptomyces sp. KTM18. The antioxidant potential of an isolated compound and its toxicity were accessed. MATERIALS AND METHODS: The compound was purified using bioassay-guided chromatography techniques. Nuclear magnetic resonance (NMR) experiments were carried out for structure elucidation. The antioxidant potential of the isolated compound was determined using DPPH free radical scavenging assay. The toxicity of the isolated compound was measured using a brine shrimp lethality (BSL) assay. RESULTS: Ethyl acetate extract of Streptomyces sp. KTM18 showed more than 90% inhibition of DPPH free radical at 50 µg/mL of the test concentration. These data were the strongest among 13 Streptomyces isolates (KTM12-KTM24). The active molecule was isolated and characterized as maculosin (molecular formula, C14H16N2O3 as determined by the [M + H]+ peak at 261.1259). The DPPH free radical scavenging activity of pure maculosin was higher (IC50, 2.16 ± 0.05 µg/mL) than that of commercial butylated hydroxyanisole (BHA) (IC50, 4.8 ± 0.05 µg/mL). No toxicity was observed for maculosin (LD50, <128 µg/mL) in brine shrimp lethality assay (BSLA) up to the compound's antioxidant activity (IC50) concentration range. The commercial standard, berberine chloride, showed toxicity in BSLA with an LD50 value of 8.63 ± 0.15 µg/mL. CONCLUSIONS: Maculosin may be a leading drug candidate in various cosmetic and therapeutic applications owing to its strong antioxidant and non-toxic properties.

Correlation between Protonation of Tailor-Made Polypiperazines and Endosomal Escape for Cytosolic Protein Delivery.

Responsive polymers, which become protonated at decreasing pH, are considered a milestone in the development of synthetic cell entry vectors. Exact correlations between their properties and their ability to escape the endosome, however, often remain elusive due to hydrophobic interactions or limitations in the design of water-soluble materials with suitable basicity. Here, we present a series of well-defined, hydrophilic polypiperazines, where systematic variation of the amino moiety facilitates an unprecedented fine-tuning of the basicity or pKa value within the physiologically relevant range (pH 6-7.4). Coincubation of HEK 293T cells with various probes, including small fluorophores or functioning proteins, revealed a rapid increase of endosomal release for polymers with pKa values above 6.5 or 7 in serum-free or serum-containing media, respectively. Similarly, cytotoxic effects became severe at increased pKa values (>7). Although the window for effective transport appears narrow, the discovered correlations offer a principal guideline for the design of effective polymers for endosomal escape.

Prognostic nomogram for progression-free survival in patients with BRCA mutations and platinum-sensitive recurrent ovarian cancer on maintenance olaparib therapy following response to chemotherapy.

BACKGROUND: The impact of maintenance therapy with PARP inhibitors (PARPi) on progression-free survival (PFS) in patients with BRCA mutations and platinum-sensitive recurrent ovarian cancer (PSROC) varies widely. Individual prognostic factors do not reliably distinguish patients who progress early from those who have durable benefit. We developed and validated a prognostic nomogram to predict PFS in these patients. METHODS: The nomogram was developed using data from a training patient cohort with BRCA mutations and high-grade serous PSROC on the placebo arm of two maintenance therapy trials, Study 19 and SOLO2/ENGOT-ov21. We performed multivariable Cox regression analysis based on pre-treatment characteristics to develop a nomogram that predicts PFS. We assessed the discrimination and validation of the nomogram in independent validation patient cohorts treated with maintenance olaparib. RESULTS: The nomogram includes four PFS predictors: CA-125 at randomisation, platinum-free interval, presence of measurable disease and number of prior lines of platinum therapy. In the training (placebo) cohort (internal validation C-index 0.64), median PFS in the model-predicted good, intermediate and poor-risk groups was: 7.7 (95% CI 5.3-11.3), 5.4 (4.8-5.8) and 2.9 (2.8-4.4) months, respectively. In the validation (olaparib) cohort (C-index 0.71), median PFS in the model-predicted good, intermediate and poor-risk groups was: not reached, 16.6 (13.1-22.4) and 8.3 (7.1-10.8) months, respectively. The nomogram showed good calibration in the validation cohort (calibration plot). CONCLUSIONS: This nomogram can be used to predict PFS and counsel patients with BRCA mutations and PSROC prior to maintenance olaparib and for stratification of patients in trials of maintenance therapies.

Nonclinical Safety Profile of Sotorasib, a KRASG12C-Specific Covalent Inhibitor for the Treatment of KRAS p.G12C-Mutated Cancer.

Sotorasib is a first-in-class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. A comprehensive nonclinical safety assessment package, including secondary/safety pharmacology and toxicology studies, was conducted to support the marketing application for sotorasib. Sotorasib was negative in a battery of genotoxicity assays and negative in an in vitro phototoxicity assay. Based on in vitro assays, sotorasib had no off-target effects against various receptors, enzymes (including numerous kinases), ion channels, or transporters. Consistent with the tumor-specific target distribution (ie, KRASG12C), there were no primary pharmacology-related on-target effects identified. The kidney was identified as a target organ in the rat but not the dog. Renal toxicity in the rat was characterized by tubular degeneration and necrosis restricted to a specific region suggesting that the toxicity was attributed to the local formation of a putative toxic reactive metabolite. In the 3-month dog study, adaptive changes of hepatocellular hypertrophy due to drug metabolizing enzyme induction were observed in the liver that was associated with secondary effects in the pituitary and thyroid gland. Sotorasib was not teratogenic and had no direct effect on embryo-fetal development in the rat or rabbit. Human, dog, and rat circulating metabolites, M24, M10, and M18, raised no clinically relevant safety concerns based on the general toxicology studies, primary/secondary pharmacology screening, an in vitro human ether-à-go-go-related gene assay, or mutagenicity assessment. Overall, the results of the nonclinical safety program support a high benefit/risk ratio of sotorasib for the treatment of patients with KRAS p.G12C-mutated tumors.

Mercapturate pathway metabolites of sotorasib, a covalent inhibitor of KRASG12C, are associated with renal toxicity in the Sprague Dawley rat.

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and ß-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.

ADMETlab 2.0: an integrated online platform for accurate and comprehensive predictions of ADMET properties.

Because undesirable pharmacokinetics and toxicity of candidate compounds are the main reasons for the failure of drug development, it has been widely recognized that absorption, distribution, metabolism, excretion and toxicity (ADMET) should be evaluated as early as possible. In silico ADMET evaluation models have been developed as an additional tool to assist medicinal chemists in the design and optimization of leads. Here, we announced the release of ADMETlab 2.0, a completely redesigned version of the widely used AMDETlab web server for the predictions of pharmacokinetics and toxicity properties of chemicals, of which the supported ADMET-related endpoints are approximately twice the number of the endpoints in the previous version, including 17 physicochemical properties, 13 medicinal chemistry properties, 23 ADME properties, 27 toxicity endpoints and 8 toxicophore rules (751 substructures). A multi-task graph attention framework was employed to develop the robust and accurate models in ADMETlab 2.0. The batch computation module was provided in response to numerous requests from users, and the representation of the results was further optimized. The ADMETlab 2.0 server is freely available, without registration, at https://admetmesh.scbdd.com/.

Evaluation of a Low-Toxicity PARP Inhibitor as a Neuroprotective Agent for Parkinson's Disease.

Repurposing PARP-1 inhibitors (PARPi) for non-oncological applications offers an attractive therapeutic strategy for pathological conditions characterized by PARP-1 hyperactivity. In the context of Parkinson's disease (PD), PARP-1 hyperactivity has been linked to neuronal death and disease progression. From a therapy perspective, the evaluation of PARPi as neuroprotective agents may offer a new therapeutic alternative for neurodegenerative disorders. An ideal PARPi needs to inhibit PARP-1 hyperactivity while also limiting downstream DNA damage and cellular toxicity-an effect that is attractive in cancer but far from ideal in neurological disease applications. Consequently, in this study, we set out to evaluate the neuroprotective properties of a previously reported low-toxicity PARPi (10e) using in vitro neuronal models of PD. 10e is a structural analogue of FDA-approved PARPi olaparib, with high PARP-1 affinity and selectivity. Our studies revealed that 10e protects neuronal cells from oxidative stress and DNA damage. In addition, 10e exhibits neuroprotective properties against α-synuclein pre-formed fibrils (αSyn PFF) mediated effects, including reduction in the levels of phosphorylated αSyn and protection against abnormal changes in NAD+ levels. Our in vitro studies with 10e provide support for repurposing high-affinity and low-toxicity PARPi for neurological applications and lay the groundwork for long-term therapeutic studies in animal models of PD.

Clinical feasibility of oral low-dose etoposide and sobuzoxane for conventional chemotherapy-intolerant lymphoma patients.

Introduction: In Japan, etoposide or sobuzoxane, a type of topoisomerase II inhibitor, is orally administered in patients with lymphoma who cannot tolerate conventional combination chemotherapy. However, the related clinical data remain to be fully summarized.Areas covered: We evaluate the efficacy and toxicity of etoposide and sobuzoxane.Expert opinion: Previous studies on etoposide or sobuzoxane monotherapy, including those among patients who could not tolerate conventional chemotherapy, suggested a favorable overall response rate (ORR) with moderate gastrointestinal or liver/renal toxicity. As for adult T-cell leukemia/lymphoma, a clinical trial with a limited sample size exhibited an ORR of >70%. Remarkably, the percentage of patients with a poor performance status was high among those receiving etoposide/sobuzoxane. Given a lack of randomized studies, etoposide/sobuzoxane might be a therapeutic option for lymphoma in a palliative setting. In the future, prospective trials with a homologous treatment schedule are warranted, in which the association between clinical efficacy and characteristics of lymphomas, such as specific gene alterations, should be elucidated.

Synthesis of artemisinin-piperazine-furan ether hybrids and evaluation of in vitro cytotoxic activity.

For the first time, eight novel artemisinin-piperazine-furane ether hybrids (5a-h) were efficiently synthesized and investigated for their in vitro cytotoxic activity against some human cancer and benign cells. The absolute configuration of hybrid 5c was determined by X-ray crystallographic analysis. Hybrids 5a-h exhibited more pronounced growth-inhibiting action on hepatocarcinoma cell lines than their parent dihydroartemisinin (DHA) and the reference cytosine arabinoside (ARA). The hybrid 5a showed the best cytotoxic activity against human hepatocarcinoma cells SMMC-7721 (IC50 = 0.26 ± 0.03 µM) after 24 h. Furthermore, hybrid 5a also showed good cytotoxic activity against human breast cancer cells MCF-7 and low cytotoxicity against human breast benign cells MCF-10A in vitro. We found the cytotoxicity of hybrid 5a did not change when tumour cells absorb iron sulfate (FeSO4); thus, we conclude the anti-tumour mechanism induced by iron ions (Fe2+) is unclear.

Cytoglobin promotes sensitivity to ferroptosis by regulating p53-YAP1 axis in colon cancer cells.

Ferroptosis is an iron-dependent mode of non-apoptotic cell death characterized by accumulation of lipid reactive oxygen species (ROS). As a regulator of ROS, cytoglobin (CYGB) plays an important role in oxygen homeostasis and acts as a tumour suppressor. However, the mechanism by which CYGB regulates cell death is largely unknown. Here, we show that CYGB overexpression increased ROS accumulation and disrupted mitochondrial function as determined by the oxygen consumption rate and membrane potential. Importantly, ferroptotic features with accumulated lipid ROS and malondialdehyde were observed in CYGB-overexpressing colorectal cancer cells. Moreover, CYGB significantly increased the sensitivity of cancer cells to RSL3- and erastin-induced ferroptotic cell death. Mechanically, both YAP1 and p53 were significantly increased based on the RNA sequencing. The knock-down of YAP1 alleviated production of lipid ROS and sensitivity to ferroptosis in CYGB overexpressed cells. Furthermore, YAP1 was identified to be inhibited by p53 knock-down. Finally, high expression level of CYGB had the close correlation with key genes YAP1 and ACSL4 in ferroptosis pathway in colon cancer based on analysis from TCGA data. Collectively, our results demonstrated a novel tumour suppressor role of CYGB through p53-YAP1 axis in regulating ferroptosis and suggested a potential therapeutic approach for colon cancer.

TNFα enhances trovafloxacin-induced in vitro hepatotoxicity by inhibiting protective autophagy.

Trovafloxacin (TVX) is associated with idiosyncratic drug-induced liver injury (iDILI) and inflammation-mediated hepatotoxicity. However, the inflammatory stress-regulated mechanisms in iDILI remain unclear. Herein, we elucidated the novel role of tumor-necrosis factor alpha (TNFα), an inflammatory stress factor, in TVX-induced in vitro hepatotoxicity and synergistic toxicity. TVX specifically induced synergistic toxicity in HepG2 cells with TNFα, which inhibits autophagy. TVX-treated HepG2 cells induced protective autophagy by inhibiting the expression of mTOR signaling proteins, while ATG5 knockdown in HepG2 cells, responsible for the impairment of autophagy, enhanced TVX-induced toxicity due to the increase in cytochrome C release and JNK pathway activation. Interestingly, the expression of mTOR signal proteins, which were suppressed by TVX, disrupted the negative feedback of the PI3K/AKT pathway and TNFα rebounded p70S6K phosphorylation. Co-treatment with TVX and TNFα inhibited protective autophagy by maintaining p70S6K activity, which enhanced TVX-induced cytotoxicity. Phosphorylation of p70S6K was inhibited by siRNA knockdown and rapamycin to restore TNFα-inhibited autophagy, which prevented the synergistic effect on TVX-induced cytotoxicity. These results indicate that TVX activates protective autophagy in HepG2 cells exposed to toxicity and an imbalance in negative feedback regulation of autophagy by TNFα synergistically enhanced the toxicity. The finding from this study may contribute to a better understanding of the mechanisms underlying iDILI associated with inflammatory stress.

Cardiohemodynamic and Arrhythmogenic Effects of the Anti-Atrial Fibrillatory Compound Vanoxerine in Halothane-Anesthetized Dogs.

While vanoxerine (GBR-12909) is a synaptosomal dopamine uptake inhibitor, it also suppresses IKr, INa and ICa,L in vitro. Based on these profiles on ionic currents, vanoxerine has been developed as a candidate compound for treating atrial fibrillation. To investigate electropharmacological profiles, vanoxerine dihydrochloride was intravenously administered at 0.03 and 0.3 mg/kg to halothane-anesthetized dogs (n = 4), possibly providing subtherapeutic and therapeutic concentrations, respectively. The low dose increased the heart rate and cardiac output, whereas it prolonged the ventricular refractoriness. The high dose decreased the heart rate but increased the total peripheral vascular resistance, whereas it delayed the ventricular repolarization and increased the atrial refractoriness in addition to further enhancing the ventricular refractoriness. The extent of increase in the refractoriness in the atrium was 0.8 times of that in the ventricle. The high dose also prolonged the early and late repolarization periods of the ventricle as well as the terminal repolarization period. Meanwhile, no significant change was detected in the mean blood pressure, ventricular contraction, preload to the left ventricle, or the intra-atrial, intra-ventricular or atrioventricular conductions. The high dose can be considered to inhibit IKr, but it may not suppress INa or ICa in the in situ heart, partly explaining its poor atrial selectivity for increasing refractoriness. The prolongation of early repolarization period may reflect enhancement of net inward current, providing potential risk for intracellular Ca2+ overload. Thus, vanoxerine may provide both trigger and substrate toward torsade de pointes, which would make the drug less promising as an anti-atrial fibrillatory drug.