Novel 9-Methylanthracene Derivatives as p53 Activators for the Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme, a highly aggressive and lethal brain tumor, is a substantial clinical challenge and a focus of increasing concern globally. Hematological toxicity and drug resistance of first-line drugs underscore the necessity for new anti-glioma drug development. Here, 43 anthracenyl skeleton compounds as p53 activator XI-011 analogs were designed, synthesized, and evaluated for their cytotoxic effects. Five compounds (13d, 13e, 14a, 14b, and 14n) exhibited good anti-glioma activity against U87 cells, with IC50 values lower than 2 µM. Notably, 13e showed the best anti-glioma activity, with an IC50 value up to 0.53 µM, providing a promising lead compound for new anti-glioma drug development. Mechanistic analyses showed that 13e suppressed the MDM4 protein expression, upregulated the p53 protein level, and induced cell cycle arrest at G2/M phase and apoptosis based on Western blot and flow cytometry assays.

C-Jun N-Terminal Kinase Post-Translational Regulation of Pain-Related Acid-Sensing Ion Channels 1b and 3.

Neuronal proton-gated acid-sensing ion channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathologic diseases. Such conditions often involve in parallel the activation of various signaling pathways such as mitogen activated protein kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal kinase (JNK), as a new post-translational positive regulator of ASICs in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit-dependent manner, targeting both rodent and human ASIC1b and ASIC3 subunits (except mouse ASIC3). The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost on mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNFα. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory-neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.SIGNIFICANCE STATEMENT ASICs are a class of excitatory cation channels critical for the detection of tissue acidosis, which is a hallmark of several painful diseases. Previous work in sensory neurons has shown that ASICs containing the ASIC3 or the ASIC1b subunit are important players in different pain models. We combine here functional and pharmacological in vitro and in vivo approaches to demonstrate that the MAP Kinase JNK is a potent post-translational positive regulator, probably via direct phosphorylation, of rodent and human ASIC1b- and ASIC3-containing channels. This JNK-dependent, fast post-translational mechanism of regulation of sensory-neuron-expressed ASICs may contribute to peripheral sensitization and pain hypersensitivity. These data also identify pain-related channels as direct downstream effectors of JNK in nociceptors.

A phase II study of bisantrene in patients with relapsed/refractory acute myeloid leukemia.

OBJECTIVES: To determine the current role of bisantrene, an anthracene with anthracycline-like activity which was shown in earlier studies to be effective therapy in relapsed/refractory acute myeloid leukemia with no discernible cardiotoxicity, in the treatment of patients with R/R AML. METHODS: This phase 2, single-center study (NCT03820908) enrolled adult R/R AML to receive bisantrene (250 mg/m2 daily for 7 days) which was administered via an intravenous infusion over 2 hours on days 1-7. Disease assessment included routine blood work and bone marrow studies. RESULTS: In all, 10 patients were enrolled with a median of 3 lines of prior therapy including seven patients who had relapsed following allogeneic stem cell transplantation. The most frequently reported grade ≥3 treatment-attributed hematologic AE was thrombocytopenia, whereas the most frequently reported grade ≥3 treatment-attributed non-hematologic AE was mucositis. Of the 10 patients, one (10%) achieved a complete remission and three patients achieved a partial remission resulting in an overall response rate of 40%. Next-generation sequencing of patient samples identified a wide array of mutations associated with activated signaling, splicing, and epigenetic modification. CONCLUSIONS: In view of the observed low toxicity, a follow-up study combining bisantrene with complementary anti-leukemic therapy is planned.

The combination therapy with esomeprazole and flupenthixol/melitracen in symptom improvement of erosive gastritis complicated with negative feelings compared with Esomeprazole alone.

The purpose of this study was to evaluate the co-prescription efficacy of esomeprazole and flupenthixol/melitracen relative to that of solitary esomeprazole on erosive gastritis complicated with negative feelings. 140 erosive gastritis patients complicated with negative feelings enrolled in the present study. Seventy cases in the control group took esomeprazole, and 70 cases in the observation group received esomeprazole plus flupenthixol/Melitracen, both for 4 weeks. We gastroscopically checked the clinical symptoms, mucosal erosion, PGE2 and MDA levels in gastric mucosa, anxiety, depression, and recurrence before and after treatment in the groups. After treatment, the observation group had lower scores of clinical symptoms, mucosal erosions, Hamilton Depression Rating Scale (HAMD), and Hamilton Depression Rating Scale (HAMA) than the control group (p<0.05); as well, the observation group showed higher PGE2 and lower MDA levels than the control group (p<0.05); during six months of follow-up (100% follow-up rate), 16 and 34 recurrent cases occurred, respectively, in the observation and control groups (p<0.05).  Co-prescription of esomeprazole and flupenthixol/melitracen improved the clinical symptoms and mucosal erosions, relieved negative feelings and reduced the recurrence rate. The efficacy of the co-prescription is higher than that of the solitary prescription.

Inhibition of c-Jun N-Terminal Kinase Protects Against Brain Damage and Improves Learning and Memory After Traumatic Brain Injury in Adult Mice.

Traumatic brain injury (TBI) is a global risk factor that leads to long-term cognitive impairments. To date, the disease remains without effective therapeutics because of the multifactorial nature of the disease. Here, we demonstrated that activation of the c-Jun N-terminal kinase (JNK) is involved in multiple pathological features of TBI. Therefore, we investigated the disease-modifying therapeutic potential of JNK-specific inhibitor (SP600125) in TBI mice. Treating 2 different models of TBI mice with SP600125 for 7 days dramatically inhibited activated JNK, resulting in marked reductions of amyloid precursor protein (APP) expression level and in amyloid beta production and hyperphosphorylated tau and regulation of the abnormal expression of secretases. Furthermore, SP600125 strongly inhibited inflammatory responses, blood-brain barrier breakdown, apoptotic neurodegeneration, and synaptic protein loss, regulated prosurvival processes and improved motor function and behavioral outcomes in TBI mice. More interestingly, we found that SP600125 treatment ameliorated amyloidogenic APP processing and promoted the nonamyloidogenic pathway in TBI mouse brains. Our findings strongly suggest that active JNK is critically involved in disease development after TBI and that inhibition of JNK with SP600125 is highly efficient for slowing disease progression by reducing multiple pathological features in TBI mouse brains and regulating cognitive dysfunction.

Barbaloin Attenuates Mucosal Damage in Experimental Models of Rat Colitis by Regulating Inflammation and the AMPK Signaling Pathway.

BACKGROUND Barbaloin is one of the main medicinal ingredients of aloe vera, which displays various anti-inflammatory and anti-apoptosis properties in several inflammatory and fibrotic diseases. Our study evaluated its efficacy against dextran sulfate sodium (DSS)-induced colitis in rats. MATERIAL AND METHODS Ulcerative colitis (UC) rat models were established in vivo, and after barbaloin treatment, body weight and inflammation index were measured. Additionally, the signaling mechanism by which barbaloin protects against UC was investigated using LPS-infected Caco-2 cells. RESULTS Barbaloin could significantly reverse UC-induced weight loss and colon injury. Further, it could effectively increase the mRNA expression of IL-4 and IL-10 in colon tissues, while decreasing the expression of IFN-γ, IL-6, IL-1ß, and TNF-alpha. Furthermore, it significantly enhanced UC-inhibited atresia band 1 (ZO-1), occludin, and E-cadherin, and was also found to activate the AMPK signaling pathway. Additionally, si-RAN-induced knockdown, and overexpression assay showed that barbaloin could inhibit the UC-enhanced MLCK signaling pathway by activating the AMPK signaling pathway. CONCLUSIONS Barbaloin can effectively inhibit inflammation and reverse epithelial barrier function to protect against UC, possibly via activation of the AMPK signaling pathway.

The Matrine Derivate MASM Inhibits Recruitment of Gr1hi Monocyte and Alleviates Liver Injury.

BACKGROUNDS: (6aS, 10S, 11aR, 11bR, 11cS)-10-methylaminododecahydro-3a, 7a-diaza-benzo (de) anthracene-8-thione (MASM), a novel derivative of matrine, exhibits better anti-inflammatory activity. This study was designed to evaluate the protective effect of MASM on acute and chronic liver injuries and explore the possible mechanisms. METHODS: Acute and chronic liver injury models were established by the CCl4 intraperitoneal injection and the protective effect of MASM was assessed by biochemical and histological examination. The infiltration of different monocyte subsets into the liver was characterized and analyzed by flow cytometry. The in vitro effect of MASM on liver nonparenchymal cells was evaluated by real-time PCR and transwell chemotaxis assays. RESULTS: Administration of MASM markedly attenuated acute liver injury and liver fibrosis induced by CCl4 injection. Meanwhile, the infiltrations of Gr1hi monocytes in injured livers and induced hepatic expression of monocyte chemoattractant protein-1 (MCP-1) were greatly inhibited. Cellular experiments demonstrated that MASM not only decreased the expression of MCP-1 but also inhibited its chemotactic activity. CONCLUSIONS: This study demonstrates that the protective effect of MASM on liver injury could be contributed to the suppression of Gr1hi monocyte infiltration to the liver and the inhibition of MCP-1 production and activity. These findings provide new insights into the protective role of MASM in liver injury.

Synergetic and Antagonistic Molecular Effects Mediated by the Feedback Loop of p53 and JNK between Saikosaponin D and SP600125 on Lung Cancer A549 Cells.

We jointly analyzed the changes in cell cycle arrest and distribution, the accumulation of subphase cells, apoptosis, and proliferation in A549 cells treated with Saikosaponin D (Ssd) and JNK inhibitor SP600125 alone or in combination. Our results indicated that cell cycle arrest at G0/G1, S, and G2/M phases was coupled with the accumulation of subG1, subS, and subG2 cells, corresponding to early apoptosis, DNA endoreplication, and later inhibitory proliferation, respectively. Analyzing the expression of 18 cell cycle regulatory genes and JNK and phosphorylated JNK (pJNK) levels revealed an enhancement in these factors by Ssd. Additional SP600125 weakened or eliminated the Ssd-induced increase of these factors except that p53/p21 and Rassfia levels were further improved. Ingenuity Pathway Analysis (IPA) of the interactions of these factors revealed a negative synergistic effect on apoptosis while a positive synergistic effect on proliferative inhibition of the two drugs: (1) Ssd induced apoptosis via the activation of two axes, TGFα-JNK-p53 and TGFα-Rassfia-Mst1. By eliminating the Ssd-induced increase of JNK/pJNK, additional SP600125 weakened the Ssd-induced apoptotic axis of TGFα-JNK-p53 and simultaneously abolished Ssd-induced apoptosis; (2) Ssd inhibited proliferation by the activation of two axes, TGFß-p53/p21/p27/p15/p16 and TGFα-Rassfia-cyclin D1. By improving the Ssd-induced increase of p53/p21 and Rassfia, additional SP600125 enhanced the two axes of Ssd-induced inhibitory proliferation. Analyzing JNK/pJNK, p53, phospho-p53, and TNF-α levels revealed an opposite association of JNK/pJNK with p53 while consistent with phospho-p53 and TNF-α, which supported the proposals that JNK/pJNK negatively regulated p53 level, while it mediated p53 phosphorylation to transcriptionally activate TNF-α expression of apoptotic gene and trigger apoptosis. With the multiple roles, JNK/pJNK forms a synergetic and antagonistic feedback loop with phospho-p53/p53. Within the feedback loop, (1) Ssd-induced apoptosis depended on JNK/pJNK activities mediating phospho-p53 that activated TNF-α expression; (2) by weakening the negative regulation of JNK/pJNK in p53, SP600125 enhanced p53 level and the Ssd-induced inhibitory proliferation axes of TGFß-p53/p21/p27/p15/p16. The results indicated the central coordinating roles of the feedback loop in the synergistic and antagonistic effects of the two drugs in A549 cells and provided a rationale for the combination of Ssd with SP600125 in the treatment of lung cancer.

Chronic intermittent hypobaric hypoxia provides vascular protection in the aorta of the 2-kidney, 1-clip rat model of hypertension.

Many studies have demonstrated that chronic intermittent hypobaric hypoxia (CIHH) can reduce blood pressure in spontaneously hypertensive rats and renovascular hypertensive (RVH) rats in which endothelial dysfunction is determined as a critical factor. However, whether CIHH can regulate vasodilation of the aorta in RVH rats remains unknown. The purpose of this study was to investigate the effect of CIHH on impaired relaxation of the aorta in the 2-kidney, 1-clip (2K1C) RVH rat model. The results showed CIHH improved the impaired endothelium-dependent relaxation in the 2K1C rat aorta. The endothelial dysfunction was prevented by the p38 antagonist SB203580, but not by the ERK1/2 antagonist PD98059 or JNK antagonist SP600125. Furthermore, the expression of p-eNOS, HIF-1α, and HIF-2α increased while that of p-p38 and BMP-4 decreased in CIHH-treated aortas from 2K1C rats. Finally, the p-eNOS expression was upregulated and the p-p38 expression was downregulated by pre-incubation of SB203580 or the BMP-4 antagonist Noggin with the aorta. CIHH ameliorated the impairment of endothelium-dependent relaxation through upregulating the expression of p-eNOS, which may be mediated by the inhibition of BMP-4/p-p38 MAPK, and upregulating the expression of HIFs in the 2K1C rat aorta.

[Effect analysis on Deanxit combined with Shuganjieyu capsule in the treatment of refractory gastroesophageal reflux disease].

Objective: To investigate the curative effect of Deanxit combined with Shuganjieyu capsule on the treatment of refractory gastroesophageal reflux disease. Methods: A total of 125 patients with refractory gastroesophageal reflux disease who had failed in standard lansolazole capsule treatment in the Department of Gastroenterology, First People's Hospital of Neijiang were selected. According to the symptom scores and mood scores of gastroesophageal reflux disease, patients were randomly and double-blindly divided into five groups. Group A(Lansoprazole Capsules + Mosapride Citrate + Deanxit), B(Lansoprazole Capsules + Mosapride Citrate + Shuganjieyu capsule), C(Lansoprazole Capsules + Mosapride Citrate+ Deanxit + Shuganjieyu capsule) and D(Deanxit) groups are study groups, the other was control group (Lansoprazole Capsules + Mosapride Citrate). The scores of symptoms and mood were compared after 4 weeks and 8 weeks of treatment. Results: The clinical symptoms score, HAMA and HAMD scores were significantly lower in the all study groups in comparison to the control group after 4 weeks and 8 weeks therapy. The clinical symptoms score, HAMA and HAMD scores in group C were significantly lower than those in group A and B (P<0.05), while the difference between group A and B was not statistically significant(P>0.05). The HAMA and HAMD scores of group D were significantly higher than those of group A, B, C and control group, and the differences were statistically significant (P<0.05). The total effective rate of study groups were significantly higher than those of the control group, and the difference was statistically significant (P<0.05). The total effective rate of group C was significantly higher than that of group A and B (χ(2)=6.47, P<0.05). The total effective rate of group A at the end of the 8th week was significantly higher than that of group B (χ(2)=6.52, P<0.05). The total effective rate of group D at the end of the 4th and 8th week was significantly lower than those of the group A, B, C and control group, the difference was statistically significant (χ(2)=5.85, P<0.05). Conclusions: Deanxit combined with Shuganjieyu capsule is significantly effective in treatment of refractory gastroesophageal reflux disease, which can effectively improve the total treatment efficiency, reduce the symptom scores and mood scores of gastroesophageal reflux disease patients.

Psychopharmacological Effects of JNK Inhibitor in Posthypoxic Encephalopathy and Mechanisms of Their Development.

Psychopharmacological effects of JNK inhibitor were studied using a mouse model of posthypoxic encephalopathy. The preparation exhibited a pronounced cerebroprotective effect manifested in normalization of orientation and exploratory behavior and conditioned responses in posthypoxic mice. These effects were accompanied by marked elevation of neural stem cell content in the paraventricular region of the brain.

Augmentation of M-type (KCNQ) potassium channels as a novel strategy to reduce stroke-induced brain injury.

Cerebral ischemic stroke is a worldwide cause of mortality/morbidity and thus an important focus of research to decrease the severity of brain injury. Therapeutic options for acute stroke are still limited. In neurons throughout the brain, "M-type" K(+) currents, underlain by KCNQ subunits 2-5, play dominant roles in control over excitability, and are thus implicated in myriad neurological and psychiatric disorders. Although KCNQ channel openers, such as retigabine, have emerged as anti-epilepsy drugs, their effects on ischemic injury remain unknown. Here, we investigated the protective effects of M-channel openers on stroke-induced brain injury in mouse photothrombotic and middle cerebral artery occlusion (MCAo) models. Both photothrombosis and MCAo led to rapid, predictable, and consistently sized necrotic brain lesions, inflammatory responses, and behavioral deficits. Administration of three distinct M-channel openers at 0-6 h after ischemic injury significantly decreased brain infarct size and inflammation, and prevented neurological dysfunction, although they were more effective when administered 0-3 h poststroke. Thus, we show beneficial effects against stroke-induced brain injury and neuronal death through pharmacological regulation of ion channels that control neuronal excitability.

Inhibition of KV7 Channels Protects the Rat Heart against Myocardial Ischemia and Reperfusion Injury.

The voltage-gated KV7 (KCNQ) potassium channels are activated by ischemia and involved in hypoxic vasodilatation. We investigated the effect of KV7 channel modulation on cardiac ischemia and reperfusion injury and its interaction with cardioprotection by ischemic preconditioning (IPC). Reverse-transcription polymerase chain reaction revealed expression of KV7.1, KV7.4, and KV7.5 in the left anterior descending rat coronary artery and all KV7 subtypes (KV7.1-KV7.5) in the left and right ventricles of the heart. Isolated hearts were subjected to no-flow global ischemia and reperfusion with and without IPC. Infarct size was quantified by 2,3,5-triphenyltetrazolium chloride staining. Two blockers of KV7 channels, XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] (10 µM) and linopirdine (10 µM), reduced infarct size and exerted additive infarct reduction to IPC. An opener of KV7 channels, flupirtine (10 µM) abolished infarct size reduction by IPC. Hemodynamics were measured using a catheter inserted in the left ventricle and postischemic left ventricular recovery improved in accordance with reduction of infarct size and deteriorated with increased infarct size. XE991 (10 µM) reduced coronary flow in the reperfusion phase and inhibited vasodilatation in isolated small branches of the left anterior descending coronary artery during both simulated ischemia and reoxygenation. KV7 channels are expressed in rat coronary arteries and myocardium. Inhibition of KV7 channels exerts cardioprotection and opening of KV7 channels abrogates cardioprotection by IPC. Although safety issues should be further addressed, our findings suggest a potential role for KV7 blockers in the treatment of ischemia-reperfusion injury.

Inhibition of c-Jun N-terminal kinase activation reverses Alzheimer disease phenotypes in APPswe/PS1dE9 mice.

OBJECTIVE: Growing evidence indicates that the activation of c-Jun N-terminal kinase (JNK) is implicated in the multiple major pathological features of Alzheimer disease (AD). However, whether specific inhibition of JNK activation could prevent disease progression in adult transgenic AD models at moderate stage remains unknown. Here we first investigated the potential disease-modifying therapeutic effect of systemic administration of SP600125, a small-molecule JNK-specific inhibitor, in middle-aged APPswe/PS1dE9 mice. METHODS: Using behavioral, histological, and biochemical methods, outcomes of SP600125 treatment on neuropathology and cognitive deficits were studied in APPswe/PS1dE9 mice. RESULTS: Compared with vehicle-treated APPswe/PS1dE9 mice, chronic treatment of SP600125 for 12 weeks potently inhibited JNK activation, which resulted in a marked improvement of behavioral measures of cognitive deficits and a dramatic reduction in amyloid plaque burden, ß-amyloid production, tau hyperphosphorylation, inflammatory responses, and synaptic loss in these transgenic animals. In particular, we found that SP600125 treatment strongly promoted nonamyloidogenic amyloid precursor protein (APP) processing and inhibited amyloidogenic APP processing via regulating APP-cleavage secretase expression (ie, ADAM10, BACE1, and PS1) in APPswe/PS1dE9 mice. INTERPRETATION: Our findings demonstrate that chronic SP600125 treatment is powerfully effective in slowing down disease progression by markedly reducing multiple pathological features and ameliorating cognitive deficits associated with AD. This study highlights the concept that active JNK actually contributes to the development of the disease, and provides critical preclinical evidence that specific inhibition of JNK activation by SP600125 treatment may be a novel promising disease-modifying therapeutic strategy for the treatment of AD.

Inhibition of c-Jun N-terminal kinase signaling suppresses skin flap apoptosis in a rat ischemia and/or reperfusion model.

BACKGROUND: The goals of this study were to validate the role of c-Jun N-terminal kinase (JNK) activation in skin flap apoptosis in a rat model of abdomen skin ischemia and/or reperfusion (IR) and to compare the protective effect of SP600125 and hydrogen-rich saline in skin IR injury. METHODS: Male Sprague-Dawley rats were divided into five groups: one sham surgery group and four surgery groups. Before undergoing 3 h of IR management, the surgery groups were treated with normal saline (IR), dimethyl sulfoxide, SP600125 (SP), and hydrogen-rich saline (H). On the third postoperative day, blood perfusion of the flap was measured using Laser Doppler flowmeters. Hematoxylin and eosin staining was used to observe morphologic changes. Early apoptosis was observed using TdT-mediated dUTP-X nick end-labeling staining. pASK-1, pJNK, Bcl-2, and Bax were examined by immunodetection. Caspase-3 activity was also measured 24 h after reperfusion. RESULTS: Compared to the IR group and the dimethyl sulfoxide group, the SP group and the H group had larger skin flap survival area, more blood perfusion and lower levels of caspase-3 activity. The SP and the H groups had high expression levels of Bcl-2 and low expression levels of pASK-1 and pJNK. Bax was significantly decreased in the SP group. In addition, cell apoptosis was decreased in both the sham surgery and the H groups. CONCLUSIONS: IR-induced JNK phosphorylation was reduced by SP600125, indicating that JNK mediates the apoptosis pathways in rat skin. In the SP and the H groups, the apoptotic factors measured showed similar expression levels, indicating that JNK inhibition during IR may be associated with H-mediated protection against skin IR apoptosis.

The Role of Platelet-Derived Growth Factor Receptor in Early Brain Injury Following Subarachnoid Hemorrhage.

OBJECTIVE: This study aims to observe encephaledema and cell apoptosis in rats following subarachnoid hemorrhage (SAH) and to explore the mechanism of platelet-derived growth factor receptor (PDGFR) in the development of early brain injury (EBI). METHODS: Adult and male Sprague-Dawley rats were randomly divided into 4 groups: sham operation, SAH, SAH + imatinib, and SAH + platelet-derived growth factor-BB (PDGF-BB). SAH model was established using intravascular silk puncture of the internal carotid artery crotch. The SAH + imatinib group was treated with intraperitoneal injection of imatinib 1 hour before establishing the model. The SAH + PDGF-BB group was administered with intracerebroventricular injection of PDGF-BB 1 hour before establishing the model. The mortality, encephaledema, and nerve functional scoring were observed after 24 hours in all groups. The expression of caspase-3 in hippocampus was tested by reverse transcription polymerase chain reaction and Western blotting. RESULTS: Mortality and encephaledema were the highest in the SAH + PDGF-BB group, which were alleviated when the rats were injected with imatinib (P < .01). CONCLUSION: PDGFR may participate in the pathogenesis of EBI following SAH. The antagonist of PDGFR, imatinib, can reduce brain damage to some degree.

The role of the c-Jun N-terminal kinases 1/2 and receptor-interacting protein kinase 3 in furosemide-induced liver injury.

1. The mechanisms of furosemide (FS) hepatotoxicity were explored in mice. Specifically, C57Bl/6 J mice were treated with 500 mg FS/kg bodyweight, and c-Jun N-terminal kinase (JNK) activation and receptor-interacting protein kinase 3 (RIP3) expression were measured by western blotting. Co-treatment with FS and the JNK inhibitor SP600125 was also performed, and FS-induced liver injury was compared in wild-type and RIP3 knockout (KO) mice. 2. JNK phosphorylation and RIP3 expression were increased in livers from the FS-treated mice as early as 6 h after treatment and persisted until at least 24 h. JNK phosphorylation was also observed in primary mouse hepatocytes and human HepaRG cells treated with FS. 3. Phosphorylated JNK translocated into mitochondria in livers, but no evidence of mitochondrial damage was observed. 4. SP600125-treated mice, SP600125 co-treated primary mouse hepatocytes and RIP3 KO mice were not protected against FS hepatotoxicity. These data show that, although JNK activation and RIP3 expression are induced by FS, neither contributes to the liver injury.

Blockade of the JNK signalling as a rational therapeutic approach to modulate the early and late steps of the inflammatory cascade in polymicrobial sepsis.

Cecal ligation and puncture (CLP) is an experimental polymicrobial sepsis induced systemic inflammation that leads to acute organ failure. Aim of our study was to evaluate the effects of SP600125, a specific c-Jun NH2-terminal kinase (JNK) inhibitor, to modulate the early and late steps of the inflammatory cascade in a murine model of CLP-induced sepsis. CB57BL/6J mice were subjected to CLP or sham operation. Animals were randomized to receive either SP600125 (15 mg/kg) or its vehicle intraperitoneally 1 hour after surgery and repeat treatment every 24 hours. To evaluate survival, a group of animals was monitored every 24 hours for 120 hours. Two other animals were sacrificed 4 or 18 hours after surgical procedures; lung and liver samples were collected for biomolecular and histopathologic analysis. The expression of p-JNK, p-ERK, TNF-α, HMGB-1, NF-κB, Ras, Rho, Caspase 3, Bcl-2, and Bax was evaluated in lung and liver samples; SP600125 improved survival, reduced CLP induced activation of JNK, NF-κB, TNF-α, and HMGB-1, inhibited proapoptotic pathway, preserved Bcl-2 expression, and reduced histologic damage in both lung and liver of septic mice. SP600125 protects against CLP induced sepsis by blocking JNK signalling; therefore, it can be considered a therapeutic approach in human sepsis.

XI-011 enhances cisplatin-induced apoptosis by functional restoration of p53 in head and neck cancer.

Head and neck cancer (HNC), one of the most common cancers worldwide, frequently involves mutation of the TP53 gene and dysregulation of the p53 pathway. Overexpression of MDM2 or MDM4 inactivates the tumor-suppressive function of p53. Restoration of p53 function that counteracts these p53 repressors can lead to in vivo tumor regression. Therefore, the present study assessed the ability of the small molecule p53 activator XI-011 (NSC146109) to induce apoptosis in HNC by restoring p53 function. We tested the effects of XI-011 treatment in HNC cell lines, either individually or in combination with cisplatin and assessed growth suppression, cell cycle arrest, and apoptosis. The drug effects on in vivo growth of HNC cells were examined in mice xenograft model. XI-011 exerted the highest growth suppression in tumor cells that overexpress MDM4, in which p53 is degraded. XI-011 treatment downregulated MDM4 mRNA and protein levels, and upregulated expression of proapoptotic genes and promoted apoptosis, in a dose-dependent manner. The apoptotic response was blocked by inhibition of p53 or expression of MDM4, demonstrating that the effects of XI-011 depend on p53 and MDM4. In combination treatments, XI-011 acted synergistically with cisplatin to inhibit growth of HNC cells in vitro and in vivo. MDM4 inhibition and functional restoration of p53 by XI-011 effectively enhanced cisplatin-induced cytotoxicity in HNC cells, an activity that suggests a promising strategy for treating HNC.

Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase.

UNLABELLED: Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300mg/kg) caused severe liver injury in male mice but 69-77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4h, and 2.5 and 3.3 fold higher in the total liver at 4h and 6h, respectively. This faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4h post-APAP, it was 3.1 fold lower at 6h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17ß-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. CONCLUSION: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17ß-estradiol partially protects male mice, it does not affect hepatic GSH recovery.