JNK signaling and its impact on neural cell maturation and differentiation.

C-Jun-N-terminal-kinases (JNKs), members of the mitogen-activated-protein-kinase family, are significantly linked with neurological and neurodegenerative pathologies and cancer progression. However, JNKs serve key roles under physiological conditions, particularly within the central-nervous-system (CNS), where they are critical in governing neural proliferation and differentiation during both embryogenesis and adult stages. These processes control the development of CNS, avoiding neurodevelopment disorders. JNK are key to maintain the proper activity of neural-stem-cells (NSC) and neural-progenitors (NPC) that exist in adults, which keep the convenient brain plasticity and homeostasis. This review underscores how the interaction of JNK with upstream and downstream molecules acts as a regulatory mechanism to manage the self-renewal capacity and differentiation of NSC/NPC during CNS development and in adult neurogenic niches. Evidence suggests that JNK is reliant on non-canonical Wnt components, Fbw7-ubiquitin-ligase, and WDR62-scaffold-protein, regulating substrates such as transcription factors and cytoskeletal proteins. Therefore, understanding which pathways and molecules interact with JNK will bring knowledge on how JNK activation orchestrates neuronal processes that occur in CNS development and brain disorders.

[Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway].

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 µmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 µmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

Oridonin promotes RSL3-induced ferroptosis in breast cancer cells by regulating the oxidative stress signaling pathway JNK/Nrf2/HO-1.

The incidence and mortality of breast cancer, the most common malignant tumor among women in the world, are increasing year by year, which greatly threatens women's health. Ferroptosis is an iron and lipid reactive oxygen species (ROS)-dependent process, a novel form of cell death that is distinct from apoptosis and is closely related to the progression of breast cancer. Inducing the occurrence of ferroptosis in tumor cells can effectively block its malignant progress in vivo. Oridonin (ORI), the primary active ingredient extracted from the Chinese herbal medicine Rabdosia rubescens, has been shown to cause glutathione depletion and directly inhibit glutathione peroxidase 4 induced cell death by ferroptosis, but its mechanism of action in breast cancer remains inadequately elucidated. Therefore, we further investigated whether ORI could promote RSL3-induced ferroptosis in breast cancer cells by regulating the oxidative stress pathway JNK/Nrf2/HO-1. In our study, we assessed cell survival of RSL3 and ORI treatment by MTT assay, and found that co-treatment with RSL3 and ORI inhibited cell proliferation, as evidenced by the cloning assay. To investigate the ability of ORI to promote RSL3-induced ferroptosis in breast cancer cells, we measured levels of ROS, malondialdehyde, glutathione, superoxide dismutase, and Fe2+ content. Lipid peroxidation, ROS, and mitochondrial membrane potential levels induced by co-treatment of ORI with RSL3 were reversed by ferrostatin-1, further confirming that the cell death induced by RSL3 and ORI was ferroptosis rather than other programmed cell death modes. Moreover, RSL3 and ORI co-treatment regulated the JNK/Nrf2/HO-1 axis, as demonstrated by western blotting and target activator validation. Our results showed that ORI could enhance the inhibitory effect of RSL3 on breast cancer cells viability via the induction of ferroptosis. Mechanistically, it potentiated RSL3-induced ferroptosis in breast cancer cells by activating the JNK/Nrf2/HO-1 axis. This study provides a theoretical basis for the application of ORI based on the mechanism of ferroptosis, and provides potential natural drug candidates for cancer prevention and treatment.

The lincRNA JUNI regulates the stress-dependent induction of c-Jun, cellular migration and survival through the modulation of the DUSP14-JNK axis.

Cancer cells employ adaptive mechanisms to survive various stressors, including genotoxic drugs. Understanding the factors promoting survival is crucial for developing effective treatments. In this study, we unveil a previously unexplored long non-coding RNA, JUNI (JUN-DT, LINC01135), which is upregulated by genotoxic drugs through the activation of stress-activated MAPKs, JNK, and p38 and consequently exerts positive control over the expression of its adjacent gene product c-Jun, a well-known oncoprotein, which transduces signals to multiple transcriptional outputs. JUNI regulates cellular migration and has a crucial role in conferring cellular resistance to chemotherapeutic drugs or UV radiation. Depletion of JUNI markedly increases the sensitivity of cultured cells and spheroids to chemotherapeutic agents. We identified 57 proteins interacting with JUNI. The activity of one of them the MAPK phosphatase and inhibitor, DUSP14, is counteracted by JUNI, thereby, facilitating efficient JNK phosphorylation and c-Jun induction when cells are exposed to UV radiation. The antagonistic interplay with DUSP14 contributes not only to c-Jun induction but also augments the survival of UV-exposed cells. In summary, we introduce JUNI as a novel stress-inducible regulator of c-Jun, positioning it as a potential target for enhancing the sensitivity of cancer cells to chemotherapy.

Stress-Activated Protein Kinases in Intervertebral Disc Degeneration: Unraveling the Impact of JNK and p38 MAPK.

Intervertebral disc degeneration (IDD) is a major cause of lower back pain. The pathophysiological development of IDD is closely related to the stimulation of various stressors, including proinflammatory cytokines, abnormal mechanical stress, oxidative stress, metabolic abnormalities, and DNA damage, among others. These factors prevent normal intervertebral disc (IVD) development, reduce the number of IVD cells, and induce senescence and apoptosis. Stress-activated protein kinases (SAPKs), particularly, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), control cell signaling in response to cellular stress. Previous studies have shown that these proteins are highly expressed in degenerated IVD tissues and are involved in complex biological signal-regulated processes. Therefore, we summarize the research reports on IDD related to JNK and p38 MAPK. Their structure, function, and signal regulation mechanisms are comprehensively and systematically described and potential therapeutic targets are proposed. This work could provide a reference for future research and help improve molecular therapeutic strategies for IDD.

Deoxynivalenol leads to endoplasmic reticulum stress-mediated apoptosis via the IRE1/JNK/CHOP pathways in porcine embryos.

The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 µM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling.

Anti-cancer effects of plant-derived Micromonospora sp. M2 against A549 and MCF-7 cell lines.

The huge diversity of secondary bioactive metabolites, such as antibiotic and anticancer compounds produced by Micromonospora sp., makes it an attractive target for study. Here, we explored the anti-proliferative activities of Micromonospora sp. M2 extract (MBE) in relation to its pro-oxidative activities in A549 and MCF7 cell lines. Anti-proliferative effects were assessed by treating cells with MBE. We found that treatment with MBE decreased cell proliferation and increased intracellular reactive oxygen species, and that these observations were facilitated by the suppression of the PI3K-AKT pathway, alterations to the Bcl/Bad ratio, and increased caspase activity. These observations also demonstrated that MBE induced apoptotic cell death in cell lines. In addition, the phosphorylation of P38 and c-Jun N-terminal kinase (JNK) were upregulated following MBE treatment in both cell lines. Collectively, these results indicate that MBE acts as an anticancer agent via oxidative stress and JNK/mitogen-activated protein kinase pathway activation, enhancing apoptotic cell death in cell lines.

Tanshinone IIA induces ER stress and JNK activation to inhibit tumor growth and enhance anti-PD-1 immunotherapy in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) remains at the forefront of new cancer cases, and there is an urgent need to find new treatments or improve the efficacy of existing therapies. In addition to the application in the field of cerebrovascular diseases, recent studies have revealed that tanshinone IIA (Tan IIA) has anticancer activity in a variety of cancers. PURPOSE: To investigate the potential anticancer mechanism of Tan IIA and its impact on immunotherapy in NSCLC. METHODS: Cytotoxicity and colony formation assays were used to detect the Tan IIA inhibitory effect on NSCLC cells. This research clarified the mechanisms of Tan IIA in anti-tumor and programmed death-ligand 1 (PD-L1) regulation by using flow cytometry, transient transfection, western blotting and immunohistochemistry (IHC) methods. Besides, IHC was also used to analyze the nuclear factor of activated T cells 1 (NFAT2) expression in NSCLC clinical samples. Two animal models including xenograft mouse model and Lewis lung cancer model were used for evaluating tumor suppressive efficacy of Tan IIA. We also tested the efficacy of Tan IIA combined with programmed cell death protein 1 (PD-1) inhibitors in Lewis lung cancer model. RESULTS: Tan IIA exhibited good NSCLC inhibitory effect which was accompanied by endoplasmic reticulum (ER) stress response and increasing Ca2+ levels. Moreover, Tan IIA could suppress the NFAT2/ Myc proto oncogene protein (c-Myc) signaling, and it also was able to control the Jun Proto-Oncogene(c-Jun)/PD-L1 axis in NSCLC cells through the c-Jun N-terminal kinase (JNK) pathway. High NFAT2 levels were potential factors for poor prognosis in NSCLC patients. Finally, animal experiments data showed a stronger immune activation phenotype, when we performed treatment of Tan IIA combined with PD-1 monoclonal antibody. CONCLUSION: The findings of our research suggested a novel mechanism for Tan IIA to inhibit NSCLC, which could exert anti-cancer effects through the JNK/NFAT2/c-Myc pathway. Furthermore, Tan IIA could regulate tumor PD-L1 levels and has the potential to improve the efficacy of PD-1 inhibitors.

miR-889-3p targeting BMPR2 promotes the development of retinoblastoma via JNK/MAPK/ERK signaling.

MicroRNAs (miRNAs) are vital regulators of tumor pathogenesis, including that of retinoblastoma (Rb). This study investigated the functions and mechanisms of action of miR-889-3p in Rb. BMPR2 and miR-889-3p levels were assessed by quantitative reverse transcription PCR (qRT-PCR) or western blotting. Through several cell function tests, the effects of miR-889-3p and BMPR2 on cell proliferation, migration, and JNK/MAPK/ERK signaling were evaluated. The interaction between miR-889-3p and BMPR2 was investigated using a luciferase reporter assay. In vivo tumor development was investigated using a xenograft test. The association between miR-889-3p and BMPR2 expression was identified using Pearson's correlation analysis. miR-889-3p was increased in Rb cells, and miR-889-3p knockdown inhibited Rb cell proliferation, migration, and phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and ERK1/2 in vitro, as well as tumor growth in vivo. Further, they were inversely associated in Rb tissues and miR-889-3p may directly attached to the 3'-UTR of BMPR2 mRNA. Finally, the inhibition of BMPR2 inverted the negative effects of the miR-889-3p inhibitor on migration, proliferation, and activation of JNK, p38 MAPK, and ERK1/2 in Rb cells. Our results indicate that miR-889-3p, which targets BMPR2 and promotes Rb growth by controlling the JNK/MAPK/ERK pathway, is an oncogene in Rb. These results suggested that the miR-889-3p/BMPR2 axis may be a new therapeutic target for Rb.

Lycopene Alleviates Endoplasmic Reticulum Stress in Steatohepatitis through Inhibition of the ASK1-JNK Signaling Pathway.

Lycopene has been proven to alleviate nonalcoholic steatohepatitis (NASH), but the precise mechanisms are inadequately elucidated. In this study, we found a previously unknown regulatory effect of lycopene on the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway in both in vivo and in vitro models. Lycopene supplementation (3 and 6 mg/kg/day) exhibited a significant reduction in lipid accumulation, inflammation, and fibrosis of the liver in mice fed with a high-fat/high-cholesterol diet or a methionine-choline-deficient diet. RNA sequencing uncovered that the mitogen-activated protein kinases signaling pathway, which is closely associated with inflammation and endoplasmic reticulum (ER) stress, was significantly downregulated by lycopene. Furthermore, we found lycopene ameliorated ER swelling and decreased the expression levels of ER stress markers (i.e., immunoglobulin heavy chain binding protein, C/EBP homologous protein, and X-box binding protein 1s). Especially, the inositol-requiring enzyme 1α involved in the ASK1 phosphorylation was inhibited by lycopene, resulting in the decline of the subsequent c-Jun N-terminal kinase (JNK) signaling cascade. ASK1 inhibitor DQOP-1 eliminated the lycopene-induced inhibition of the ASK1-JNK pathway in oleic acid and palmitic acid-induced HepG2 cells. Molecular docking further indicated hydrophobic interactions between lycopene and ASK1. Collectively, our research indicates that lycopene can alleviate ER stress and attenuate inflammation cascades and lipid accumulation by inhibiting the ASK1-JNK pathway.

Curcumin Analog L48H37 Induces Apoptosis in Human Oral Cancer Cells by Activating Caspase Cascades and Downregulating the Inhibitor of Apoptosis Proteins through JNK/p38 Signaling.

L48H37 is a synthetic curcumin analog that has anticancer potentials. Here, we further explored the anticancer effect of L48H37 on oral cancer cells and its mechanistic acts. Cell cycle distribution was assessed using flow cytometric analysis. Apoptosis was elucidated by staining with PI/Annexin V and activation of the caspase cascade. Cellular signaling was explored using apoptotic protein profiling, Western blotting, and specific inhibitors. Our findings showed that L48H37 significantly reduced the cell viability of SCC-9 and HSC-3 cells, resulting in sub-G1 phase accumulation and increased apoptotic cells. Apoptotic protein profiling revealed that L48H37 increased cleaved caspase-3, and downregulated cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) in SCC-9 cells, and the downregulated cIAP1 and XIAP in both oral cancer cells were also demonstrated by Western blotting. Meanwhile, L48H37 triggered the activation of caspases and mitogen-activated protein kinases (MAPKs). The involvement of c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) in the L48H37-triggered apoptotic cascade in oral cancer cells was also elucidated by specific inhibitors. Collectively, these findings indicate that L48H37 has potent anticancer activity against oral cancer cells, which may be attributed to JNK/p38-mediated caspase activation and the resulting apoptosis. This suggests a potential benefit for L48H37 for the treatment of oral cancer.

Melanoma differentiation-associated protein 5 prevents cardiac hypertrophy via apoptosis signal-regulating kinase 1-c-Jun N-terminal kinase/p38 signaling.

Pathological cardiac hypertrophy (CH) is driven by maladaptive changes in myocardial cells in response to pressure overload or other stimuli. CH has been identified as a significant risk factor for the development of various cardiovascular diseases, ultimately resulting in heart failure. Melanoma differentiation-associated protein 5 (MDA5), encoded by interferon-induced with helicase C domain 1 (IFIH1), is a cytoplasmic sensor that primarily functions as a detector of double-stranded ribonucleic acid (dsRNA) viruses in innate immune responses; however, its role in CH pathogenesis remains unclear. Thus, the aim of this study was to examine the relationship between MDA5 and CH using cellular and animal models generated by stimulating neonatal rat cardiomyocytes with phenylephrine and by performing transverse aortic constriction on mice, respectively. MDA5 expression was upregulated in all models. MDA5 deficiency exacerbated myocardial pachynsis, fibrosis, and inflammation in vivo, whereas its overexpression hindered CH development in vitro. In terms of the underlying molecular mechanism, MDA5 inhibited CH development by promoting apoptosis signal-regulating kinase 1 (ASK1) phosphorylation, thereby suppressing c-Jun N-terminal kinase/p38 signaling pathway activation. Rescue experiments using an ASK1 activation inhibitor confirmed that ASK1 phosphorylation was essential for MDA5-mediated cell death. Thus, MDA5 protects against CH and is a potential therapeutic target.

Hyperoside inhibits EHV-8 infection via alleviating oxidative stress and IFN production through activating JNK/Keap1/Nrf2/HO-1 signaling pathways.

Equine herpesvirus type 8 (EHV-8) causes abortion and respiratory disease in horses and donkeys, leading to serious economic losses in the global equine industry. Currently, there is no effective vaccine or drug against EHV-8 infection, underscoring the need for a novel antiviral drug to prevent EHV-8-induced latent infection and decrease the pathogenicity of this virus. The present study demonstrated that hyperoside can exert antiviral effects against EHV-8 infection in RK-13 (rabbit kidney cells), MDBK (Madin-Darby bovine kidney), and NBL-6 cells (E. Derm cells). Mechanistic investigations revealed that hyperoside induces heme oxygenase-1 expression by activating the c-Jun N-terminal kinase/nuclear factor erythroid-2-related factor 2/Kelch-like ECH-associated protein 1 axis, alleviating oxidative stress and triggering a downstream antiviral interferon response. Accordingly, hyperoside inhibits EHV-8 infection. Meanwhile, hyperoside can also mitigate EHV-8-induced injury in the lungs of infected mice. These results indicate that hyperoside may serve as a novel antiviral agent against EHV-8 infection.IMPORTANCEHyperoside has been reported to suppress viral infections, including herpesvirus, hepatitis B virus, infectious bronchitis virus, and severe acute respiratory syndrome coronavirus 2 infection. However, its mechanism of action against equine herpesvirus type 8 (EHV-8) is currently unknown. Here, we demonstrated that hyperoside significantly inhibits EHV-8 adsorption and internalization in susceptible cells. This process induces HO-1 expression via c-Jun N-terminal kinase/nuclear factor erythroid-2-related factor 2/Kelch-like ECH-associated protein 1 axis activation, alleviating oxidative stress and triggering an antiviral interferon response. These findings indicate that hyperoside could be very effective as a drug against EHV-8.

Esketamine inhibits the c-Jun N-terminal kinase pathway in the spinal dorsal horn to relieve bone cancer pain in rats.

Cancer-induced bone pain (CIBP) is one of the most common and feared symptoms in patients with advanced tumors. The X-C motif chemokine ligand 12 (CXCL12) and the CXCR4 receptor have been associated with glial cell activation in bone cancer pain. Moreover, mitogen-activated protein kinases (MAPKs), as downstream CXCL12/CXCR4 signals, and c-Jun, as activator protein AP-1 components, contribute to the development of various types of pain. However, the specific CIBP mechanisms remain unknown. Esketamine is a non-selective N-methyl-d-aspartic acid receptor (NMDA) inhibitor commonly used as an analgesic in the clinic, but its analgesic mechanism in bone cancer pain remains unclear. We used a tumor cell implantation (TCI) model and explored that CXCL12/CXCR4, p-MAPKs, and p-c-Jun were stably up-regulated in the spinal cord. Immunofluorescence images showed activated microglia in the spinal cord on day 14 after TCI and co-expression of CXCL12/CXCR4, p-MAPKs (p-JNK, p-ERK, p-p38 MAPK), and p-c-Jun in microglia. Intrathecal injection of the CXCR4 inhibitor AMD3100 reduced JNK and c-Jun phosphorylations, and intrathecal injection of the JNK inhibitor SP600125 and esketamine also alleviated TCI-induced pain and reduced the expression of p-JNK and p-c-Jun in microglia. Overall, our data suggest that the CXCL12/CXCR4-JNK-c-Jun signaling pathway of microglia in the spinal cord mediates neuronal sensitization and pain hypersensitivity in cancer-induced bone pain and that esketamine exerts its analgesic effect by inhibiting the JNK-c-Jun pathway.

The Role of the Dysregulated JNK Signaling Pathway in the Pathogenesis of Human Diseases and Its Potential Therapeutic Strategies: A Comprehensive Review.

JNK is named after c-Jun N-terminal kinase, as it is responsible for phosphorylating c-Jun. As a member of the mitogen-activated protein kinase (MAPK) family, JNK is also known as stress-activated kinase (SAPK) because it can be activated by extracellular stresses including growth factor, UV irradiation, and virus infection. Functionally, JNK regulates various cell behaviors such as cell differentiation, proliferation, survival, and metabolic reprogramming. Dysregulated JNK signaling contributes to several types of human diseases. Although the role of the JNK pathway in a single disease has been summarized in several previous publications, a comprehensive review of its role in multiple kinds of human diseases is missing. In this review, we begin by introducing the landmark discoveries, structures, tissue expression, and activation mechanisms of the JNK pathway. Next, we come to the focus of this work: a comprehensive summary of the role of the deregulated JNK pathway in multiple kinds of diseases. Beyond that, we also discuss the current strategies for targeting the JNK pathway for therapeutic intervention and summarize the application of JNK inhibitors as well as several challenges now faced. We expect that this review can provide a more comprehensive insight into the critical role of the JNK pathway in the pathogenesis of human diseases and hope that it also provides important clues for ameliorating disease conditions.

Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

HIF-1α is a "brake" in JNK-mediated activation of amyloid protein precursor and hyperphosphorylation of tau induced by T-2 toxin in BV2 cells.

Mycotoxins have been shown to activate multiple mechanisms that may potentially lead to the progression of Alzheimer's disease (AD). Overexpression/aberrant cleavage of amyloid precursor protein (APP) and hyperphosphorylation of tau (P-tau) is hallmark pathologies of AD. Recent advances suggest that the neurotoxic effects of mycotoxins involve c-Jun N-terminal kinase (JNK) and hypoxia-inducible factor-1α (HIF-1α) signaling, which are closely linked to the pathogenesis of AD. Due to the high toxicity and broad contamination of T-2 toxin, we assessed how T-2 toxin exposure alters APP and P-tau formation in BV2 cells and determined the underlying roles of HIF-1α and JNK signaling. The findings revealed that T-2 toxin stimulated the expression of HIF-1α and hypoxic stress factors in addition to increasing the expression of APP and P-tau. Additionally, HIF-1α acted as a "brake" on the induction of APP and P-tau expression by negatively regulating these proteins. Notably, T-2 toxin activated JNK signaling, which broke this "brake" to promote the formation of APP and P-tau. Furthermore, the cytoskeleton was an essential target for T-2 toxin to exert cytotoxicity, and JNK/HIF-1α participated in this damage. Collectively, when the T-2 toxin induces the production of APP and P-tau, JNK might interfere with HIF-1α's protective function. This study will provide clues for further research on the neurotoxicity of mycotoxins.

Blockade of isoprenoids biosynthesis by simvastatin induces autophagy-mediated cell death via downstream c-Jun N-terminal kinase activation and cell cycle dysregulation in canine T-cell lymphoma cells.

Statins are inhibitors of the mevalonic acid pathway that mediates cellular metabolism by producing cholesterol and isoprenoids and are widely used in treating hypercholesterolaemia in humans. Lipophilic statins, including simvastatin, induce death in various tumour cells. However, the cytotoxic mechanisms of statins in tumour cells remain largely unexplored. This study aimed to elucidate the cytotoxic mechanisms of simvastatin in canine lymphoma cells. Simvastatin induced cell death via c-Jun N-terminal kinase (JNK) activation and autophagy in canine T-cell lymphoma cell lines Ema and UL-1, but not in B-cell lines. Cell death was mediated by induction of caspase-dependent apoptosis in UL-1 cells, but not in Ema cells. Blockade of autophagy by lysosomal inhibitors attenuated simvastatin-induced JNK activation and cell death. Isoprenoids, including farnesyl pyrophosphate and geranylgeranyl pyrophosphate, attenuated simvastatin-induced autophagy, JNK activation, and cell death. In UL-1 cells, simvastatin treatment resulted in the cell cycle arrest at the G2/M phase, which was altered to G0/1 phase cell cycle arrest by treatment with lysosomal inhibitors. These findings demonstrate that depletion of isoprenoids by simvastatin induces autophagy-mediated cell death via downstream JNK activation and cell cycle dysregulation in canine T-cell lymphoma cells.

Mesenchymal Stem/Stromal Cells Induce Myeloid-Derived Suppressor Cells in the Bone Marrow via the Activation of the c-Jun N-Terminal Kinase Signaling Pathway.

Our previous study demonstrated that mesenchymal stem/stromal cells (MSCs) induce the differentiation of myeloid-derived suppressor cells (MDSCs) in the bone marrow (BM) under inflammatory conditions. In this study, we aimed to investigate the signaling pathway involved. RNA-seq revealed that the mitogen-activated protein kinase (MAPK) pathway exhibited the highest number of upregulated genes in MSC-induced MDSCs. Western blot analysis confirmed the strong phosphorylation of c-Jun N-terminal kinase (JNK) in BM cells cocultured with MSCs under granulocyte-macrophage colony-stimulating factor stimulation, whereas p38 kinase activation remained unchanged in MSC-cocultured BM cells. JNK inhibition by SP600125 abolished the expression of Arg1 and Nos2, hallmark genes of MDSCs, as well as Hif1a, a molecule mediating monocyte functional reprogramming toward a suppressive phenotype, in MSC-cocultured BM cells. JNK inhibition also abrogated the effects of MSCs on the production of TGF-ß1, TGF-ß2 and IL-10 in BM cells. Furthermore, JNK inhibition increased Tnfa expression, while suppressing IL-10 production, in MSC-cocultured BM cells in response to lipopolysaccharides. Collectively, our results suggest that MSCs induce MDSC differentiation and promote immunoregulatory cytokine production in BM cells during inflammation, at least in part, through the activation of the JNK-MAPK signaling pathway.

Sanguinarine Triggers Apoptosis in Cutaneous Squamous Cell Carcinoma Cells through Reactive Oxygen Species-Dependent c-Jun N-Terminal Kinase Signaling Pathway.

BACKGROUND: The benzophenanthridine Sanguinarine (Sng) is one of the most abundant root alkaloids with a long history of investigation and pharmaceutical applications. The cytotoxicity of Sng against various tumor cells is well-established; however, its antiproliferative and apoptotic potential against the cutaneous squamous cell carcinoma (cSCC) cells remains unknown. In the present study, we investigated the anti-cancer potential of Sng against cSCC cells and elucidated the underlying mechanisms relevant to the drug action. METHODS: The inhibitory effect of Sng on cSCC cells was evaluated by analyzing cell viability, colony-forming ability and multi-caspase activity. Apoptosis was quantified through Annexin-V/Propidium iodide flow cytometric assay and antagonized by pan-caspase inhibitor z-VAD-FMK. Mitochondrial membrane potential (ΔΨm) dysfunction was analyzed by JC-1 staining, whereas reactive oxygen species (ROS) generation was confirmed by pretreatment with N-acetylcysteine (NAC) and fluorogenic probe-based flow cytometric detection. The expression of cell cycle regulatory proteins, apoptotic proteins and MAPK signaling molecules was determined by Western blotting. Involvement of JNK, p38-MAPK and MEK/ERK in ROS-mediated apoptosis was investigated by pretreatment with SP600125 (JNK inhibitor), SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor), respectively. The stemness-targeting potential of Sng was assessed in tumor cell-derived spheroids. RESULTS: Treatment with Sng decreased cell viability and colony formation in primary (A431) and metastatic (A388) cSCC cells in a time- and dose-dependent manner. Sng significantly inhibited cell proliferation by inducing sub-G0/G1 cell-cycle arrest and apoptosis in cSCC cells. Sng evoked ROS generation, intracellular glutathione (GSH) depletion, ΔΨm depolarization and the activation of JNK pathway as well as that of caspase-3, -8, -9, and PARP. Antioxidant NAC inhibited ROS production, replenished GSH levels, and abolished apoptosis induced by Sng by downregulating JNK. Pretreatment with z-VAD-FMK inhibited Sng-mediated apoptosis. The pharmacological inhibition of JNK by SP600125 mitigated Sng-induced apoptosis in metastatic cSCC cells. Finally, Sng ablated the stemness of metastatic cSCC cell-derived spheroids. CONCLUSION: Our results indicate that Sng exerts a potent cytotoxic effect against cSCC cells that is underscored by a mechanism involving multiple levels of cooperation, including cell-cycle sub-G0/G1 arrest and apoptosis induction through ROS-dependent activation of the JNK signaling pathway. This study provides insight into the potential therapeutic application of Sng targeting cSCC.