Discovery of novel paeonol-based derivatives against skin inflammation in vitro and in vivo.

T-LAK-cell-originated protein kinase (TOPK), a novel member of the mitogen-activated protein kinase family, is considered an effective therapeutic target for skin inflammation. In this study, a series (A - D) of paeonol derivatives was designed and synthesised using a fragment growing approach, and their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells were tested. Among them, compound B12 yielded the best results (IC50 = 2.14 µM) with low toxicity (IC50 > 50 µM). Preliminary mechanistic studies indicated that this compound could inhibit the TOPK-p38/JNK signalling pathway and phosphorylate downstream related proteins. A murine psoriasis-like skin inflammation model was used to determine its therapeutic effect.

Functional Roles of JNK and p38 MAPK Signaling in Nasopharyngeal Carcinoma.

c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) family members integrate signals that affect proliferation, differentiation, survival, and migration in a cell context- and cell type-specific way. JNK and p38 MAPK activities are found upregulated in nasopharyngeal carcinoma (NPC). Studies have shown that activation of JNK and p38 MAPK signaling can promote NPC oncogenesis by mechanisms within the cancer cells and interactions with the tumor microenvironment. They regulate multiple transcription activities and contribute to tumor-promoting processes, ranging from cell proliferation to apoptosis, inflammation, metastasis, and angiogenesis. Current literature suggests that JNK and p38 MAPK activation may exert pro-tumorigenic functions in NPC, though the underlying mechanisms are not well documented and have yet to be fully explored. Here, we aim to provide a narrative review of JNK and p38 MAPK pathways in human cancers with a primary focus on NPC. We also discuss the potential therapeutic agents that could be used to target JNK and p38 MAPK signaling in NPC, along with perspectives for future works. We aim to inspire future studies further delineating JNK and p38 MAPK signaling in NPC oncogenesis which might offer important insights for better strategies in diagnosis, prognosis, and treatment decision-making in NPC patients.

CC-90001, a c-Jun N-terminal kinase (JNK) inhibitor, in patients with pulmonary fibrosis: design of a phase 2, randomised, placebo-controlled trial.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a progressive and often fatal interstitial lung disease (ILD); other ILDs have a progressive, fibrotic phenotype (PF-ILD). Antifibrotic agents can slow but not stop disease progression in patients with IPF or PF-ILD. c-Jun N-terminal kinases (JNKs) are stress-activated protein kinases implicated in the underlying mechanisms of fibrosis, including epithelial cell death, inflammation and polarisation of profibrotic macrophages, fibroblast activation and collagen production. CC-90001, an orally administered (PO), one time per day, JNK inhibitor, is being evaluated in IPF and PF-ILD. METHODS AND ANALYSIS: This is a phase 2, randomised, double-blind, placebo-controlled study evaluating efficacy and safety of CC-90001 in patients with IPF (main study) and patients with PF-ILD (substudy). Both include an 8-week screening period, a 24-week treatment period, up to an 80-week active-treatment extension and a 4-week post-treatment follow-up. Patients with IPF (n=165) will be randomised 1:1:1 to receive 200 mg or 400 mg CC-90001 or placebo administered PO one time per day; up to 25 patients/arm will be permitted concomitant pirfenidone use. Forty-five patients in the PF-ILD substudy will be randomised 2:1 to receive 400 mg CC-90001 or placebo. The primary endpoint is change in per cent predicted forced vital capacity from baseline to Week 24 in patients with IPF. ETHICS AND DISSEMINATION: This study will be conducted in accordance with Good Clinical Practice guidelines, Declaration of Helsinki principles and local ethical and legal requirements. Results will be reported in a peer-reviewed publication. TRIAL REGISTRATION NUMBER: NCT03142191.

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns.

Chemical splicing modulators that bind to the spliceosome have provided an attractive avenue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. However, the biological effect of proteins containing translated intron sequences remains unclear. Here, we identify a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) via genome-wide ribosome profiling and bio-orthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through c-Jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

Pirfenidone attenuates acetaminophen-induced liver injury via suppressing c-Jun N-terminal kinase phosphorylation.

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in Western countries. Pirfenidone (PFD), an orally bioavailable pyridone derivative, is clinically used for idiopathic pulmonary fibrosis treatment and has antifibrotic, anti-inflammatory, and antioxidant effects. Here we examined the PFD effect on APAP-induced liver injury. In a murine model, APAP caused serum alanine aminotransferase elevation attenuated by PFD treatment. We performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and vital propidium iodide (PI) stainings simultaneously. APAP induced TUNEL-positive/PI-negative necrosis around the central vein and subsequent TUNEL-negative/PI-positive oncotic necrosis with hemorrhage and caused the upregulation of hypercoagulation- and hypoxia-associated gene expressions. PFD treatment suppressed these findings. Western blotting revealed PFD suppressed APAP-induced c-Jun N-terminal kinase (JNK) phosphorylation despite no effect on JNK phosphatase expressions. In conclusion, simultaneous TUNEL and vital PI staining is useful for discriminating APAP-induced necrosis from typical oncotic necrosis. Our results indicated that PFD attenuated APAP-induced liver injury by suppressing TUNEL-positive necrosis by directly blocking JNK phosphorylation. PFD is promising as a new option to prevent APAP-induced liver injury.

Proliferation maintains the undifferentiated status of stem cells: The role of the planarian cell cycle regulator Cdh1.

The coincidence of cell cycle exit and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since they may possess a simple cell cycle regulation in which Cdh1 is one of the factors responsible for exiting the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the knockdown of cdh1 caused pronounced accumulation of the stem cells, the progenitor and differentiated cells were decreased. Further analyses indicated that the stem cells with cdh1 knockdown did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle exit. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation.

Role of JNK in the Regulation of Xenobiotic Metabolizing Function of Hepatocytes.

We studied the role of JNK in the regulation of the metabolism of xenobiotic venlafaxine by liver cells under in vitro conditions. The inhibitory role of this protein kinase in the biotransformation of this psychotropic agent by hepatocytes was demonstrated. JNK inhibitor added to the liver homogenate containing antidepressant enhanced and accelerated the formation of the only pharmacologically active venlafaxine metabolite O-desmethylvenlafaxine in the cell suspension. The results show the promise of studying modifiers of activity of intracellular signaling molecules (in particular, mitogen-activated protein kinases) to develop a fundamentally new approach to control the transformation of xenobiotics and to create a new class of pharmaceutical, target regulators of drugs metabolism.

Discovery of the c-Jun N-Terminal Kinase Inhibitor CC-90001.

As a result of emerging biological data suggesting that within the c-Jun N-terminal kinase (JNK) family, JNK1 and not JNK2 or JNK3 may be primarily responsible for fibrosis pathology, we sought to identify JNK inhibitors with an increased JNK1 bias relative to our previous clinical compound tanzisertib (CC-930). This manuscript reports the synthesis and structure-activity relationship (SAR) studies for a novel series of JNK inhibitors demonstrating an increased JNK1 bias. SAR optimization on a series of 2,4-dialkylamino-pyrimidine-5-carboxamides resulted in the identification of compounds possessing low nanomolar JNK inhibitory potency, overall kinome selectivity, and the ability to inhibit cellular phosphorylation of the direct JNK substrate c-Jun. Optimization of physicochemical properties in this series resulted in compounds that demonstrated excellent systemic exposure following oral dosing, enabling in vivo efficacy studies and the selection of a candidate for clinical development, CC-90001, which is currently in clinical trials (Phase II) in patients with idiopathic pulmonary fibrosis (NCT03142191).

Inhibition of c-Jun N-terminal kinase signaling increased apoptosis and prevented the emergence of ALK-TKI-tolerant cells in ALK-rearranged non-small cell lung cancer.

Anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs) have improved clinical outcomes in non-small cell lung cancer (NSCLC) harboring ALK- rearrangements. However, a small population of tumor cells survives due to adaptive resistance under drug pressure and ultimately acquires drug resistance. Thus, it is necessary to elucidate the mechanisms underlying the prevention of drug resistance to improve the prognosis of patients with ALK-rearranged NSCLC. We identified novel adaptive resistance, generated through c-Jun N-terminal kinase (JNK)/c-Jun signaling, to initial ALK-TKIs-alectinib and brigatinib-in ALK-rearranged NSCLC. Inhibition of JNK/c-Jun axis showed suppression of growth and promotion of apoptosis induced by ALK-TKIs in drug-tolerant cells. JNK inhibition, in combination with the use of ALK-TKIs, increased cell apoptosis through repression of the Bcl-xL proteins, compared with ALK-TKI monotherapy. Importantly, combination therapy targeting JNK and ALK significantly delayed the regrowth following cessation of these treatments. Together, our results demonstrated that JNK pathway activation plays a pivotal role in the intrinsic resistance to ALK-TKIs and the emergence of ALK-TKI-tolerant cells in ALK-rearranged NSCLC, thus indicating that optimal inhibition of tolerant signals combined with ALK-TKIs may potentially improve the outcome of ALK-rearranged NSCLC.

Novel c-Jun N-Terminal Kinase (JNK) Inhibitors with an 11H-Indeno[1,2-b]quinoxalin-11-one Scaffold.

c-Jun N-terminal kinase (JNK) plays a central role in stress signaling pathways implicated in important pathological processes, including rheumatoid arthritis and ischemia-reperfusion injury. Therefore, inhibition of JNK is of interest for molecular targeted therapy to treat various diseases. We synthesized 13 derivatives of our reported JNK inhibitor 11H-indeno[1,2-b]quinoxalin-11-one oxime and evaluated their binding to the three JNK isoforms and their biological effects. Eight compounds exhibited submicromolar binding affinity for at least one JNK isoform. Most of these compounds also inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) activation and interleukin-6 (IL-6) production in human monocytic THP1-Blue cells and human MonoMac-6 cells, respectively. Selected compounds (4f and 4m) also inhibited LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. We conclude that indenoquinoxaline-based oximes can serve as specific small-molecule modulators for mechanistic studies of JNKs, as well as potential leads for the development of anti-inflammatory drugs.

Kaempferol Blocks the Skin Fibroblastic Interleukin 1ß Expression and Cytotoxicity Induced by 12-O-tetradecanoylphorbol-13-acetate by Suppressing c-Jun N-terminal Kinase.

Kaempferol, a bioflavonoid present in fruits and vegetables, has a variety of antioxidant and anti-inflammatory capacities, but the functional role of kaempferol in oxidative skin dermal damage has yet to be well studied. In this study, we examine the role of kaempferol during the inflammation and cell death caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) in normal human dermal fibroblasts (NHDF). TPA (5 µM) significantly induced cytotoxicity of NHDF, where a robust increase in the interleukin (IL)-1ß mRNA among the various pro-inflammatory cytokines. The skin fibroblastic cytotoxicity and IL-1ß expression induced by TPA were significantly ameliorated by a treatment with 100 nM of kaempferol. Kaempferol blocked the production of the intracellular reactive oxygen species (ROS) responsible for the phosphorylation of c-Jun N-terminal kinase (JNK) induced by TPA. Interestingly, we found that kaempferol inhibited the phosphorylation of nuclear factor-kappa B (NF-κB) and the inhibitor NF-κB (IκBα), which are necessary for the expression of cleaved caspase-3 and the IL-1ß secretion in TPA-treated NHDF. These results suggest that kaempferol is a functional agent that blocks the signaling cascade of the skin fibroblastic inflammatory response and cytotoxicity triggered by TPA.

TLR-4 Agonist Induces IFN-γ Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway.

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.

Amplification by tramadol of PGD2-induced osteoprotegerin synthesis in osteoblasts: Involvement of µ-opioid receptor and 5-HT transporter.

Tramadol, a weak µ-opioid receptor (MOR) agonist with inhibitory effects on the reuptake of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine, is an effective analgesic to chronic pains. Osteoprotegerin produced by osteoblasts is essential for bone remodeling to suppress osteoclastic bone resorption. We previously reported that prostaglandin D2 (PGD2) induces osteoprotegerin synthesis whereby p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) are involved in osteoblast-like MC3T3-E1 cells. Herein, we investigated the mechanism underlying the effect of tramadol on the PGD2-induced osteoprotegerin synthesis in these cells. Tramadol enhanced the PGD2-induced release and mRNA expression of osteoprotegerin. Naloxone, a MOR antagonist, reduced the amplification by tramadol of the PGD2-stimulated osteoprotegerin release. Not the selective norepinephrine reuptake inhibitor reboxetine but the selective serotonin reuptake inhibitors fluvoxamine and sertraline upregulated the PGD2-induced osteoprotegerin release, which was further amplified by morphine. Tramadol enhanced PGD2-stimulated phosphorylation of p38 MAP kinase and SAPK/JNK, but not p44/p42 MAP kinase. Both SB203580 and SP600125 suppressed the tramadol effect to enhance the PGD2-stimulated osteoprotegerin release. Tramadol enhanced the PGE2-induced osteoprotegerin release as well as PGD2. These results suggest that tramadol amplifies the PGD2-induced osteoprotegerin synthesis at the upstream of p38 MAP kinase and SAPK/JNK in the involvement of both MOR and 5-HT transporter in osteoblasts.

Naja atra Cardiotoxin 1 Induces the FasL/Fas Death Pathway in Human Leukemia Cells.

This study aimed to investigate the mechanistic pathway of Naja atra (Taiwan cobra) cardiotoxin 1 (CTX1)-induced death of leukemia cell lines U937 and HL-60. CTX1 increased cytoplasmic Ca2+ and reactive oxygen species (ROS) production, leading to the death of U937 cells. It was found that Ca2+-induced NOX4 upregulation promoted ROS-mediated p38 MAPK phosphorylation, which consequently induced c-Jun and ATF-2 phosphorylation. Using siRNA knockdown, activated c-Jun and ATF-2 were demonstrated to regulate the expression of Fas and FasL, respectively. Suppression of Ca2+-mediated NOX4 expression or ROS-mediated p38 MAPK activation increased the survival of U937 cells exposed to CTX1. FADD depletion abolished CTX1-induced cell death, caspase-8 activation, and t-Bid production, supporting the correlation between the Fas death pathway and CTX1-mediated cytotoxicity. Among the tested N. atra CTX isotoxins, only CTX1 induced Fas and FasL expression. Chemical modification studies revealed that intact Met residues were essential for the activity of CTX1 to upregulate Fas and FasL expression. Taken together, the data in this study indicate that CTX1 induces c-Jun-mediated Fas and ATF-2-mediated FasL transcription by the Ca2+/NOX4/ROS/p38 MAPK axis, thereby activating the Fas death pathway in U937 cells. Furthermore, CTX1 activates Fas/FasL death signaling in the leukemia cell line HL-60.

Astrocytic c-Jun N-terminal kinase-histone deacetylase-2 cascade contributes to glutamate transporter-1 decrease and mechanical allodynia following peripheral nerve injury in rats.

Decrease of glutamate transporter-1 (GLT-1) in the spinal dorsal horn after nerve injury induces enhanced excitatory transmission and causes persistent pain. Histone deacetylases (HDACs)-catalyzed deacetylation might contribute to the decrease of GLT-1, while the detailed mechanisms have yet to be fully elaborated. Spinal nerve ligation (SNL) induced significant increases of HDAC2 and decreases of GLT-1 in spinal astrocytes. Intrathecal infusion of the HDAC2 inhibitors attenuated the decrease of GLT-1 and enhanced phosphorylation of glutamate receptors. GLT-1 and phosphorylated c-Jun N-terminal kinase (JNK) were highly colocalized in the spinal cord, and a large number of pJNK positive cells were HDAC2 positive. Intrathecally infusion of the JNK inhibitor SP600125 significantly inhibited SNL-induced upregulation of HDAC2. SNL-induced HDAC2 up-regulation could be inhibited by the neutralizing anti-tumor necrosis factor-α (TNF-α) binding protein etanercept or the microglial inhibitor minocycline. In cultured astrocytes, TNF-α induced enhanced phosphorylation of JNK and a significant increase of HDAC2, as well as a remarkable decrease of GLT-1, which could be prevented by SP600125 or the HDAC2 specific inhibitor CAY10683. Our data suggest that astrocytic JNK-HDAC2 cascade contributes to GLT-1 decrease and mechanical allodynia following peripheral nerve injury. Neuroimmune activation after peripheral nerve injury could induce epigenetic modification changes in astrocytes and contribute to chronic pain maintenance.

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.

Understanding Abnormal c-JNK/p38MAPK Signaling Overactivation Involved in the Progression of Multiple Sclerosis: Possible Therapeutic Targets and Impact on Neurodegenerative Diseases.

Demyelination, immune dysregulation, and neuroinflammation are the most common triggers of motor neuron disorders such as multiple sclerosis (MS). MS is a chronic demyelinating neurodegenerative disease of the central nervous system caused by abnormal immune activation, which causes myelin sheath damage. Cell signal transduction pathways are required for a variety of physiological and pathological processes in the brain. When these signaling systems become overactive, they can lead to disease progression. In various physiological conditions, abnormal mitogen-activated protein kinase (MAPK) activation is associated with several physiological dysfunctions that cause neurodegeneration. Previous research indicates that c-JNK and p38MAPK signaling play critical roles in neuronal growth and differentiation. c-JNK/p38MAPK is a member of the MAPK family, which regulates metabolic pathways, cell proliferation, differentiation, and apoptosis that control certain neurological activities. During brain injuries, c-JNK/p38MAPK also affects neuronal elastic properties, nerve growth, and cognitive processing. This review systematically linked abnormal c-JNK/p38MAPK signaling activation to multiple neuropathological pathways in MS and related neurological dysfunctions. MS progression is linked to genetic defects, oligodendrocyte destruction, glial overactivation, and immune dysregulation. We concluded that inhibiting both the c-JNK/p38MAPK signaling pathways can promote neuroprotection and neurotrophic effects against the clinical-pathological presentation of MS and influence other neurological disorders. As a result, the potential benefits of c-JNK/p38MAPK downregulation for the development of disease-modifying treatment interventions in the future could include MS prevention and related neurocomplications.

Irreversible JNK blockade overcomes PD-L1-mediated resistance to chemotherapy in colorectal cancer.

Colorectal cancer (CRC) cells have low or absent tumor cell PD-L1 expression that we previously demonstrated can confer chemotherapy resistance. Here, we demonstrate that PD-L1 depletion enhances JNK activity resulting in increased BimThr116 phosphorylation and its sequestration by MCL-1 and BCL-2. Activated JNK signaling in PD-L1-depeted cells was due to reduced mRNA stability of the CYLD deubiquitinase. PD-L1 was found to compete with the ribonuclease EXOSC10 for binding to CYLD mRNA. Thus, loss of PD-L1 promoted binding and degradation of CYLD mRNA by EXOSC10 which enhanced JNK activity. An irreversible JNK inhibitor (JNK-IN-8) reduced BimThr116 phosphorylation and unsequestered Bim from MCL-1 and BCL-2 to promote apoptosis. In cells lacking PD-L1, treatment with JNK-IN-8, an MCL-1 antagonist (AZD5991), or their combination promoted apoptosis and reduced long-term clonogenic survival by anticancer drugs. Similar effects of the JNK inhibitor on cell viability were observed in CRC organoids with suppression of PD-L1. These data indicate that JNK inhibition may represent a promising strategy to overcome drug resistance in CRC cells with low or absent PD-L1 expression.

Total saponins from Rhizoma Panacis Majoris inhibit proliferation, induce cell cycle arrest and apoptosis and influence MAPK signalling pathways on the colorectal cancer cell.

Colorectal cancer (CRC) ranks third in incidence and second in mortality among all types of cancer, and due to its insidious onset and lack of early symptoms, it is usually diagnosed at a later stage. Saponins, a class of compounds abundant in plants, have been reported to possess prominent anti­tumour properties. The use of ginsenoside Rg3 in the clinical setting was authorized by the National Medicinal Products Administration of China. In the present study, total saponins from Rhizoma Panacis Majoris (RPMTG) were prepared, and the pharmacological mechanisms underlying the anti­CRC effects of RPMTG were investigated. The effect of RPMTG on the proliferation, cell cycle progression and apoptosis of HCT116 and SW620 cells were detected by MTT, flow cytometry and western blotting assays, and it was demonstrated that RPMTG could inhibit the proliferation of HCT116 and SW620 cells with IC50 values of 315.8 and 355.1 µg/ml, respectively, induce cell cycle arrest in the S and G0/G1 phase, and trigger apoptosis by downregulating the expression of the anti­apoptotic proteins Bcl­2, Bcl­xL and induced myeloid leukaemia cell differentiation protein Mcl­1, and increasing the expression of the pro­apoptotic proteins Bax and Bad, cleaved caspased­3 and poly(ADP)­ribose polymerase. These findings suggested that RPMTG induced apoptosis through mitochondrial­related pathways. In addition, RPMTG also decreased the expression of phosphorylated (p)­extracellular signal­regulated kinase and increased p­c­Jun N­terminal kinase (p­JNK) and p­p38. Moreover, the effects of RPMTG on cell proliferation and apoptosis were partially reversed when the JNK and p38 mitogen­activated protein kinase (MAPK) pathways were inhibited, indicating that RPMTG triggered apoptosis mainly via regulating JNK and p38 MAPK signalling. Therefore, RPMTG may have potential as an anti­CRC agent, and further evaluations are needed.

Sestrin Proteins Protect Against Lipotoxicity-Induced Oxidative Stress in the Liver via Suppression of C-Jun N-Terminal Kinases.

BACKGROUND & AIMS: Sestrin 1/2/3 (Sesn1/2/3) belong to a small family of proteins that have been implicated in the regulation of metabolic homeostasis and oxidative stress. However, the underlying mechanisms remain incompletely understood. The aim of this work was to illustrate the collective function of Sesn1/2/3 in the protection against hepatic lipotoxicity. METHODS: We used Sesn1/2/3 triple knockout (TKO) mouse and cell models to characterize oxidative stress and signal transduction under lipotoxic conditions. Biochemical, histologic, and physiological approaches were applied to illustrate the related processes. RESULTS: After feeding with a Western diet for 8 weeks, TKO mice developed remarkable metabolic associated fatty liver disease that was manifested by exacerbated hepatic steatosis, inflammation, and fibrosis compared with wild-type counterparts. Moreover, TKO mice exhibited higher levels of hepatic lipotoxicity and oxidative stress. Our biochemical data revealed a critical signaling node from sestrins to c-Jun N-terminal kinases (JNKs) in that sestrins interact with JNKs and mitogen-activated protein kinase kinase 7 and suppress the JNK phosphorylation and activity. In doing so, sestrins markedly reduced palmitate-induced lipotoxicity and oxidative stress in both mouse and human hepatocytes. CONCLUSIONS: The data from this study suggest that Sesn1/2/3 play an important role in the protection against lipotoxicity-associated oxidative stress and related pathology in the liver.