Mitogen-activated Protein Kinase Inhibitor Research Articles

Abstract 5063: Exploring the mutational landscape of KRAS and NRAS in tumors with non-V600 BRAF mutations

Abstract Background: BRAF, a key modulator of the Mitogen-Activated Protein Kinase (MAPK) pathway, is observed in 7% of all cancers. Therapeutic response to MAPK inhibition (MAPKi) often relies on molecular distinctions between members of varying classes: Class 1 (V600), Class 2 and 3 (non-V600) BRAF mutants. Preliminary data indicates that co-occurring RAS mutations in non-V600 BRAF mutant cancers are less responsive to MAPKi treatment. This emphasizes the need to investigate the characteristics of RAS co-mutations in non-V600 BRAF mutant tumors. Methods: Genomic data was obtained from a cohort of 183,292 patients provided by the AACR GENIE database (v14.1). Patient samples were clustered according to their BRAF mutation status and co-occurring K/NRAS mutations: WT BRAF (n=60,845) vs. non-V600 BRAF (n=1009). Samples were grouped based on cancer type: melanoma (n=3841), colorectal (n=15,434), and non-small cell lung cancer (n=14640) and were further categorized according to key biochemical features of RAS GTPase function and overall GTPase activity. Results: This dataset revealed a diverse array of allelic variants of K/NRAS between cancer types and their underlying BRAF mutation status (Table 1). Non-V600 BRAF mutant cancers showed an enrichment for KRAS mutations linked to amplified nucleotide exchange (37.9% vs. 13.4%; p<0.0001) and hydrolysis-impairing NRAS mutations (41.9% vs. 23%; p<0.0001), compared to WT BRAF cancers. Rare allelic variants including KRAS L19F, KRAS A146T, and NRAS G60E were seen in Class 2/3 BRAF mutants. Conclusion: This data suggests that non-V600 BRAF mutant tumors are characterized by a unique distribution of RAS mutations. More research into the difference in downstream effectors of RAS mutants overrepresented in non-V600 BRAF mutant tumors could provide important insights into how these tumors develop and resist targeted therapies. Table 1. Classification of KRAS and NRAS mutations in WT BRAF vs. non-V600 BRAF mutant tumors GTPase Function Cancer Type All cancers NSCLC Colorectal Melanoma BRAF mutation WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value KRASmutation class Impaired hydrolysis n=20731 (79.8%) n=167 (51.9%) <0.0001 n=6068 (86.1%) n=60 (56.6%) <0.0001 n=4799 (68.2%) n=16 (29.1%) <0.0001 n=38 (48.1%) n=13 (50.0%) 0.4047 Nucleotide Exchange n=3475 (13.4%) n=122 (37.9%) n=530 (7.5%) n=36 (34.0%) n=1842 (26.2%) n=36 (65.5%) n=30 (38.0%) n=12 (46.2%) Hybrid n=1765 (6.8%) n=33 (10.2%) n=446 (6.3%) n=10 (9.4%) n=395 (5.6%) n=3 (5.5%) n=11 (13.9%) n=1 (3.8%) NRAS mutation class Impaired hydrolysis n = 974 (23.0%) n=78 (41.9%) <0.0001 n=29 (18.7%) n=9 (39.1%) 0.0196 n=188 (32.6%) n=17 (68.0%) <0.0001 n=86 (5.5%) n=22 (27.8%) <0.0001 Nucleotide Exchange n=459 (10.8%) n=22 (11.8%) n=5 (3.2%) n=2 (8.7%) n=48 (8.3%) n=4 (16.0%) n=93 (5.9%) n=10 (12.7%) Hybrid n=2811 (66.2%) n=86 (46.2%) n=121 (78.1%) n=12 (52.2%) n=341 (59.1%) n=4 (16.0%) n=1385 (88.6%) n=47 (59.5%) GTPase Activity BRAF mutation WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value KRASmutation class Intermediate activity n=14408 (55.3%) n=142 (44.4%) 0.0001 n=5082 (72.3%) n=54 (50.5%) <0.0001 n=3540 (49.9%) n=17 (31.5%) 0.0089 n=46 (56.8%) n=13 (50.0%) 0.6516 High activity n=11630 (44.7%) n=178 (55.6%) n=1947 (27.7%) n=53 (49.5%) n=3552 (50.1%) n=37 (68.5%) n=35 (43.2%) n=13 (50.0%) NRAS mutation class Intermediate activity n=1364 (32.2%) n=101 (55.8%) <0.0001 n=36 (23.4%) n=12 (54.5%) 0.0041 n=239 (41.8%) n=20 (87.0%) <0.0001 n=190 (12.3%) n=34 (43.0%) <0.0001 High activity n=2868 (67.8%) n=80 (44.2%) n=118 (76.6%) n=10 (45.5%) n=333 (58.2%) n=3 (13.0%) n=1357 (87.7%) n=45 (57.0%) Citation Format: Chantel L. Mukonoweshuro, Emmanuelle Rousselle, April A. Rose. Exploring the mutational landscape of KRAS and NRAS in tumors with non-V600 BRAF mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5063.

Abstract 3081: Targeting lipid kinases to enhance autophagy inhibition for the treatment of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized as the most KRAS-addicted cancer type, with KRAS mutations found in >95% of cases. Mutant KRAS relies on dysregulated metabolism to support growth. One dysregulated pathway upregulated in PDAC is autophagy, a lysosome-mediated process that degrades proteins and organelles to supply macromolecules for growth. Our lab and others have shown that inhibition of KRAS or the mitogen activated protein kinase (MAPK) components, MEK or ERK, further increases PDAC dependency on autophagy as a resistance mechanism. Cotreatment with ERK MAPK inhibitors and the autophagy inhibitor chloroquine (CQ) decreased autophagic flux, inhibited proliferation, and induced apoptosis in PDAC cells. CQ is the only FDA-approved autophagy inhibitor, and it demonstrates low potency in vivo, motivating our studies to identify strategies to improve autophagy inhibition in PDAC. We utilized a CRISPR-Cas9 loss-of-function screen to identify sensitizers to CQ in PDAC and identified 25 autophagy-related targets. We hypothesize that targeting the autophagy pathway at multiple nodes will increase the efficacy of CQ treatment in PDAC. To validate hits identified in the CRISPR-Cas9 screen, I designed an siRNA array that contained three distinct oligos for each target. I quantified changes in proliferation in a panel of four PDAC cell lines after siRNA-mediated suppression of the target genes following 3- or 6-day time points. I hypothesized that targets identified to be non-essential on their own would allow for potential synthetic lethality with CQ. To test this, I performed a 3-day knockdown followed by 3-day exposure to a dose response of CQ. I found that suppression of lipid kinases PIP4K2A, PIP4K2C, or PIK3C3 shift the dose response to CQ in a panel of PDAC lines. This indicates that loss of those targets sensitizes PDAC cells to CQ treatment. Furthermore, we found that pharmacological inhibition of VPS34, the lipid kinase encoded by PIK3C3 by the ATP-competitive inhibitor SAR-405 synergizes with CQ. My future studies will be focused on validating other targets identified in the loss-of-function CRISPR-Cas9 screen. I will measure changes in autophagic flux and apoptosis due to the loss of validated targets. The long-term goal of this project is to enhance the translatability of autophagy inhibition for patient care through identifying combinational approaches to improve CQ response in PDAC. Citation Format: Elyse G. Schechter, Jonathan M. DeLiberty, Clint A. Stalnecker, Kirsten L. Bryant. Targeting lipid kinases to enhance autophagy inhibition for the treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3081.

Combined KRAS-MAPK pathway inhibitors and HER2-directed drug conjugate is efficacious in pancreatic cancer

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.

CHF6297: a novel potent and selective p38 MAPK inhibitor with robust anti-inflammatory activity and suitable for inhaled pulmonary administration as dry powder.

Inhibition of p38 mitogen-activated protein kinase (MAPKs) is a potential therapeutic approach for the treatment of acute and chronic pulmonary inflammatory conditions. Here, we report the in vitro and in vivo characterization of the anti-inflammatory effects of CHF6297, a novel potent and selective p38α inhibitor designed for inhalation delivery as a dry powder formulation. CHF6297 has been proven to inhibit p38α enzymatic activity with sub-nanomolar potency (IC50 = 0.14 ± 0.06nM), with >1,000-fold selectivity against p38γ and p38δ. In human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides (LPS), as well as in human bronchial epithelial cells (BEAS2B) stimulated with TNF-α or cigarette smoke extract (CSE), CHF6297 inhibited interleukin (IL)-8 release with low nanomolar potency. CHF6297 administered to rats by using a nose-only inhalation device as a micronized dry powder formulation blended with lactose dose-dependently inhibited the LPS-induced neutrophil influx in the bronchoalveolar lavage fluid (BALF). CHF6297 administered intratracheally to rats dose-dependently counteracted the IL-1β (0.3mg/kg)-induced neutrophil influx (ED50 = 0.22mg/kg) and increase in IL-6 levels (ED50 = 0.82mg/kg) in the BALF. In mice exposed to tobacco smoke (TS), CHF6297, administered intranasally (i.n.) for 4 days at 0.03 or 0.3mg/kg, dose-dependently inhibited the corticosteroid-resistant TS-induced neutrophil influx in the BALF. In a murine house dust mite (HDM) model of asthma exacerbated by influenza virus A (IAV) (H3N3), CHF6297 (0.1mg/kg, i.n.) significantly decreased airway neutrophilia compared to vehicle-treated IAV/HDM-challenged mice. When CHF6297, at a dose ineffective per se (0.03mg/kg), was added to budesonide, it augmented the anti-inflammatory effects of the steroid. Overall, CHF6297 effectively counteracted lung inflammation in experimental models where corticosteroids exhibit limited anti-inflammatory activity, suggesting a potential for the treatment of acute exacerbations associated with chronic obstructive pulmonary disease (COPD) and asthma, acute lung injury (ALI), and viral-induced hyperinflammation.

Cutaneous toxicities of mitogen-activated protein kinase inhibitors in children and young adults with neurofibromatosis-1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder which commonly causes neoplasms leading to disfigurement or dysfunction. Mitogen-activated protein kinase inhibitors (MEKi) are generally well-tolerated treatments which target neural tumor progression in patients with NF1. However, cutaneous adverse events (CAEs) are common and may hinder patients' abilities to remain on treatment, particularly in children. We aim to characterize CAEs secondary to MEKi treatment in pediatric and young adult patients with NF1. We reviewed institutional medical records of patients under 30 years with a diagnosis of "NF1," "NF2," or "other neurofibromatoses" on MEKi therapy between January 1, 2019 and June 1, 2022. We recorded the time-to-onset, type, and distribution of CAEs, non-cutaneous adverse events (AEs), AE management, and tumor response. Our cohort consisted of 40 patients with NF1 (median age, 14 years). Tumor types included low-grade gliomas (51%) and plexiform neurofibromas (38%). MEKi used included selumetinib (69%), trametinib (25%), and mirdametinib (6%). A total of 74 CAEs occurred, with 28 cases of acneiform rash (38%). Other common CAEs were paronychia, seborrheic dermatitis, eczema, xerosis, and oral mucositis. The most common treatments included oral antibiotics and topical corticosteroids. Most patients had clinical (stable or improved) tumor response (71%) while 29% had tumor progression while on a MEKi. There was no significant association between CAE presence and tumor response (p = 0.39). Improvement in characterization of MEKi toxicities and their management is important to develop treatment guidelines for pediatric and young adult patients with NF1 on MEKi therapy.

Design, Synthesis, and Molecular Docking of Novel Miscellaneous Chalcones as p38α Mitogen-Activated Protein Kinase Inhibitors.

New chalcones were synthesized and evaluated to serve as p38-α type of mitogen-activated protein kinase (MAPK) inhibitors. According to the National Cancer Institute, the findings indicated that at a 10 μM dosage, compounds 3a and 6 were the most active among all the compounds examined, with mean growth inhibition% of 94.83 and 58.49, respectively. In 5-dose testing, they showed anticancer activity in the micro-molar range with GI50 in the range of 1.41-46.1 and 2.07-31.3 μM, respectively. Besides, powerful activity, especially against the leukaemia cell lines and good selectivity to cancer cells compared to normal PCS-800-017 with a selectivity index=12.41 and 23.77, respectively. Compounds 3a and 6 inhibited p38α MAPK with IC50 values of 0.1462±0.0063 and 0.4356±0.0189 μM, correspondingly. 3a showed good inhibition for HL-60(TB) cells and induced cell cycle arrest in HL-60(TB) cells at the G2/M phase. Besides, it elevated the total apoptosis by 14.68-fold and increased the caspase-3 level by 3.52-fold compared with doxorubicin, which raised it by 4.30-fold, inducing apoptosis by acting as caspase-dependent inducers. These results suggest that 3a is a promising antiproliferative and p38α MAPK inhibitor, confirmed by molecular docking with high compatibility 3a with the p38α MAPK binding site.

YAP/TEAD involvement in resistance to paclitaxel chemotherapy in lung cancer.

The Yes-associated protein (YAP) oncoprotein has been linked to both metastases and resistance to targeted therapy of lung cancer cells. We aimed to investigate the effect of YAP pharmacological inhibition, using YAP/TEA domain (TEAD) transcription factor interaction inhibitors in chemo-resistant lung cancer cells. YAP subcellular localization, as a readout for YAP activation, cell migration, and TEAD transcription factor functional transcriptional activity were investigated in cancer cell lines with up-regulated YAP, with and without YAP/TEAD interaction inhibitors. Parental (A549) and paclitaxel-resistant (A549R) cell transcriptomes were analyzed. The half-maximal inhibitory concentration (IC50) of paclitaxel or trametinib, which are Mitogen-Activated protein kinase and Erk Kinase (MEK) inhibitors, combined with a YAP/TEAD inhibitor (IV#6), was determined. A three-dimensional (3D) microfluidic culture device enabled us to study the effect of IV#6/paclitaxel combination on cancer cells isolated from fresh resected lung cancer samples. YAP activity was significantly higher in paclitaxel-resistant cell lines. The YAP/TEAD inhibitor induced a decreased YAP activity in A549, PC9, and H2052 cells, with reduced YAP nuclear staining. Wound healing assays upon YAP inhibition revealed impaired cell motility of lung cancer A549 and mesothelioma H2052 cells. Combining YAP pharmacological inhibition with trametinib in K-Ras mutated A549 cells recapitulated synthetic lethality, thereby sensitizing these cells to MEK inhibition. The YAP/TEAD inhibitor lowered the IC50 of paclitaxel in A549R cells. Differential transcriptomic analysis of parental and A549R cells revealed an increased YAP/TEAD transcriptomic signature in resistant cells, downregulated upon YAP inhibition. The YAP/TEAD inhibitor restored paclitaxel sensitivity of A549R cells cultured in a 3D microfluidic system, with lung cancer cells from a fresh tumor efficiently killed by YAP/TEAD inhibitor/paclitaxel doublet. Evidence of the YAP/TEAD transcriptional program's role in chemotherapy resistance paves the way for YAP therapeutic targeting.

Punicalagin promotes mincle-mediated phagocytosis of macrophages via the NF-κB and MAPK signaling pathways

Punicalagin (PUN) is a polyphenol derived from the pomegranate peel. It has been reported to have many beneficial effects, including anti-inflammatory, anti-oxidant, and anti-proliferation. However, the role of PUN in macrophage phagocytosis is currently unknown. In this study, we found that pre-treatment with PUN significantly enhanced phagocytosis by macrophages in a time- and dose-dependent manner in vitro. Moreover, KEGG enrichment analysis by RNA-sequencing showed that differentially expressed genes following PUN treatment were significantly enriched in phagocyte-related receptors, such as the C-type lectin receptor signaling pathway. Among the C-type lectin receptor family, Mincle (Clec4e) significantly increased at the mRNA and protein level after PUN treatment, as shown by qRT-PCR and western blotting. Small interfering RNA (siRNA) mediated knockdown of Mincle in macrophages resulted in down regulation of phagocytosis. Furthermore, western blotting showed that PUN treatment enhanced the phosphorylation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) in macrophages at the early stage. Mincle-mediated phagocytosis by PUN was inhibited by PDTC (a NF-κB inhibitor) and SB203580 (a p38 MAPK inhibitor). In addition, PUN pre-treatment enhanced phagocytosis by peritoneal and alveolar macrophages in vivo. After intraperitoneal injection of Escherichia coli (E.coli), the bacterial load of peritoneal lavage fluid and peripheral blood in PUN pre-treated mice decreased significantly. Similarly, the number of bacteria in the lung tissue significantly reduced after intranasal administration of Pseudomonas aeruginosa (PAO1). Taken together, our results reveal that PUN enhances bacterial clearance in mice by activating the NF-κB and MAPK pathways and upregulating C-type lectin receptor expression to enhance phagocytosis by macrophages.

Inhibitory Effects of Eicosapentaenoic Acid on Vascular Endothelial Growth Factor-Induced Monocyte Chemoattractant Protein-1, Interleukin-6, and Interleukin-8 in Human Vascular Endothelial Cells.

Vascular endothelial growth factor (VEGF) induces monocyte chemoattractant protein-1 (MCP-1) and plays an important role in vascular inflammation and atherosclerosis. We investigated the mechanisms of VEGF-induced MCP-1 expression and the effects of eicosapentaenoic acid (EPA) in human umbilical vein endothelial cells (HUVECs). Real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that VEGF enhanced MCP-1 gene expression and protein secretion in HUVECs. Western immunoblot analysis revealed that VEGF induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor (NF)-κB (IκB). Treatment with pharmacological inhibitors of p38 MAPK (SB203580) or NF-κB (BAY11-7085) significantly suppressed VEGF-induced MCP-1 in HUVECs. EPA inhibited VEGF-induced MCP-1 mRNA, protein secretion, phosphorylation of p38 MAPK, and the translocation of phospho-p65 to the nucleus. Additionally, VEGF also stimulated gene expressions of interleukin (IL)-6 and IL-8, which were suppressed by SB203580, BAY11-7085, and EPA. The present study has demonstrated that VEGF-induced activation of MCP-1, IL-6, and IL-8 involves the p38 MAPK and NF-κB signaling pathways and that EPA inhibits VEGF-induced MCP-1, IL-6, and IL-8 via suppressing these signaling pathways. This study supports EPA as a beneficial anti-inflammatory and anti-atherogenic drug to reduce the VEGF-induced activation of proinflammatory cytokine and chemokines.

Distinct binding modes of a benzothiazole derivative confer structural bases for increasing ERK2 or p38α MAPK selectivity

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors.

Ambroxol-induced leukocytoclastic vasculitis: A case report and literature review

Cutaneous leukocytoclastic vasculitis (LCV) that primarily affects the skin, can be idiopathic or linked to infections, autoimmune diseases, neoplasms, and medications. An immune complex-mediated vasculitis of the dermal capillaries and venules is the histological hallmark of LCV, a small vessel vasculitis. This case report describes a 36-year previously healthy man who was presented to the outpatient department (OPD) with complaints of edema and development of numerous reddish-purple patches (caused by petechiae) after intake of cough syrup containing ambroxol. Ambroxol affects human monocytic cells THP-1, and prevents the generation of platelet-derived growth factor (PDGF)-AB that is produced by lipopolysaccharide (LPS) by inhibiting the expression of PDGF-A messenger Ribonucleic acid (RNA). An extracellular signal-regulated kinase (ERK) on p44/42 is activated by LPS, and ambroxol reduces this activation. In addition, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 98059), a Mitogen-Activated Protein Kinase Inhibitors (MEK), MEK-1-specific inhibitor, prevents p44/42 ERK activation and PDGF synthesis brought on by LPS. Megakaryocytes, erythrocytes, leukocytes, and their progenitors are stimulated to multiply by PDGF to the numerous endogenous growth factors that mesenchymal stem and stromal cells secrete. Ambroxol reduces the number of megakaryocytic progenitors and boosts apoptosis by inhibiting the production of PDGF and related signaling pathways. It is strongly advised that patients must be made more aware of the risks associated with using such over-the-counter medications.

Switching Off Vascular MAPK Signaling: A Novel Strategy to Prevent Delayed Cerebral Ischemia Following Subarachnoid Hemorrhage

AbstractPatients who initially survive the rupture and repair of a brain aneurysm often take a devastating turn for the worse some days later and die or suffer permanent neurologic deficits. This catastrophic sequela is attributed to a delayed phase of global cerebral ischemia (DCI) following aneurysmal subarachnoid hemorrhage (aSAH), but we lack effective treatment. Here we present our view, based on 20 years of research, that the initial drop in blood flow at the time of rupture triggers genomic responses throughout the brain vasculature that manifest days later as increased vasoconstriction and decreased cerebral blood flow. We propose a novel treatment strategy to prevent DCI by early inhibition of the vascular mitogen-activated protein kinase (MAPK) pathway that triggers expression of vasoconstrictor and inflammatory mediators. We summarize evidence from experimental SAH models showing early treatment with MAPK inhibitors “switches off” these detrimental responses, maintains flow, and improves neurological outcome. This promising therapy is currently being evaluated in clinical trials.

Isoflavones Inhibit Hydrogen Peroxide-Induced Angiotensinogen Secretion in Mesangial Cells

The mechanisms underlying increased angiotensinogen secretion in diabetic nephropathy are unknown. This study aimed to examine the mechanism of increased angiotensinogen secretion in mesangial cells. Additionally, we explored the effects of antioxidant compounds, such as isoflavones, on angiotensin secretion. Angiotensinogen expression and secretion were evaluated in mesangial cells treated with hydrogen peroxide. We investigated the effects of pretreatment with catalase, daidzein, and equol and inhibitors of mitogen-activated protein kinase, stress-stimulated kinase p38, or c-Jun NH2-terminal kinase. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay revealed that daidzein and equol have antioxidant properties. Hydrogen peroxide stimulated angiotensinogen expression and secretion in mesangial cells in a concentrationdependent manner. Catalase, daidzein, and equol reduced the enhanced angiotensinogen expression and secretion induced by hydrogen peroxide. We examined the mitogen-activated protein kinase cascade to explore cell signaling mechanisms involved in angiotensinogen induction. We hypothesize that stress-stimulated kinase p38 and c-Jun NH2-terminal kinase were crucial in the mechanisms. We found that hydrogen peroxide enhanced angiotensinogen expression and secretion in mesangial cells. However, daidzein and equol decreased this enhancement. Increased angiotensinogen secretion will enhance stress-stimulated kinase p38 and c-Jun NH2-terminal kinase.

Mitogen-activated protein kinase-guided drug discovery for post-viral and related types of lung disease.

Respiratory viral infections are a major public health problem, with much of their morbidity and mortality due to post-viral lung diseases that progress and persist after the active infection is cleared. This paradigm is implicated in the most common forms of chronic lung disease, such as asthma and COPD, as well as other virus-linked diseases including progressive and long-term coronavirus disease 2019. Despite the impact of these diseases, there is a lack of small-molecule drugs available that can precisely modify this type of disease process. Here we will review current progress in understanding the pathogenesis of post-viral and related lung disease with characteristic remodelling phenotypes. We will also develop how this data leads to mitogen-activated protein kinase (MAPK) in general and MAPK13 in particular as key druggable targets in this pathway. We will also explore recent advances and predict the future breakthroughs in structure-based drug design that will provide new MAPK inhibitors as drug candidates for clinical applications. Each of these developments point to a more effective approach to treating the distinct epithelial and immune cell based mechanisms, which better account for the morbidity and mortality of post-viral and related types of lung disease. This progress is vital given the growing prevalence of respiratory viruses and other inhaled agents that trigger stereotyped progression to acute illness and chronic disease.

Cancer drugs and diabetic retinopathy: a dangerous, underestimated association

The worldwide growing prevalence of diabetes and cancer led to an increase in subjects affected by both these diseases that share several of the involved risk factors and have a complex, multifactorial etiopathogenesis. Cancer therapies could have harmful effects on several organs, particularly in subjects also affected by diabetes and its related comorbidities. Moreover, cancer diagnosis often monopolizes the attention of both patients and caregivers, thus reducing the attention to pre-existent diseases. Retinopathy is one of the most frequent microvascular complications of diabetes, accounting for about 5% of legal blindness worldwide. The retinal neurovascular unit is dysfunctional in diabetes and could represent a frail site when cancer therapies are administered. Nevertheless, the short- and long-term effects of the different anticancer molecules on retinal tissue, especially in diabetic subjects, are poorly known, and no specific recommendations on their prevention and management are available. In this review, we summarised the current data on this topic, focusing on the different cancer class drugs involved in retinal damage: anti-oestrogen, classical cytolytic chemotherapy (alkylating agents, taxanes, topoisomerase inhibitors, and antimetabolites), mitogen-activated protein kinase, tyrosine-kinase, and vascular endothelial growth factor inhibitors are the cancer drugs associated with retinal damage and visual disturbance. However, further studies are necessary to improve knowledge on the molecular and clinical relation between cancer therapies and retinopathy, in order to provide clinicians with evidence-based protocols to optimise the management of these conditions and minimise vision loss occurrence, impaired quality of life, and public health expense.

Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control

p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission.

Antioxidant and Antimelanogenic Activities of Lactobacillus kunkeei NCHBL-003 Isolated from Honeybees.

Excessive reactive oxygen species production can detrimentally impact skin cell physiology, resulting in cell growth arrest, melanogenesis, and aging. Recent clinical studies have found that lactic acid bacteria have a special effect directly or indirectly on skin organs, but the exact mechanism has not been elucidated. In this study, we investigated the mechanisms underlying the antioxidant protective effect and the inhibitory effect on melanin synthesis of Lactobacillus kunkeei culture supernatant (CSK), isolated from Apis mellifera Linnaeus (the Western honeybee). CSK exhibited notable efficacy in promoting cell migration and wound healing under oxidative stress, surpassing the performance of other strains. CSK pretreatment significantly upregulated the expression of Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1), a key player in cellular defenses against oxidative stress, relative to the control H2O2-treated cells. The DCF-DA (dichloro-dihydro-fluorescein diacetate) assay results confirmed that CSK's ability to enhance Nrf2 and HO-1 expression aligns with its robust ability to remove H2O2-induced reactive oxygen species. Furthermore, CSK upregulated MAPK (mitogen-activated protein kinase) phosphorylation, an upstream signal for HO-1 expression, and MAPK inhibitors compromised the wound-healing effect of CSK. Additionally, CSK exhibited inhibitory effects on melanin synthesis, downregulating melanogenesis-related genes in B16F10 cells. Thus, the present study demonstrated that CSK exhibited antioxidant effects by activating the Nrf2/HO-1 pathway through MAPK phosphorylation, thereby restoring cell migration and demonstrating inhibitory effects on melanin production. These findings emphasize the antioxidant and antimelanogenic potential of CSK, suggesting its potential use as a therapeutic agent, promoting wound healing, and as an active ingredient in skin-lightening cosmetics.

Involvement of Sep38β in the Insecticidal Activity of Bacillus thuringiensis against Beet Armyworm, Spodoptera exigua (Lepidoptera).

The p38 mitogen-activated protein kinases (MAPKs) are associated with insect immunity, tissue repair, and the insecticidal activity of Bacillus thuringiensis (Bt). Here, a p38 MAPK family gene (Sep38β) was identified from Spodoptera exigua. Among the developmental stages, the transcription level of Sep38β was the highest in egg, followed by that in prepupa and pupa. Sep38β expression peaked in Malpighian tubules and the hemolymph of fifth instar larvae. Knockdown of Sep38β or injection of SB203580 (a p38 MAPK inhibitor) significantly downregulated the SeDUOX expression and reactive oxygen species (ROS) level in the midgut, accounting for deterioration of the midgut to scavenge pathogens and enhancement of Bt insecticidal activity. In conclusion, all the results demonstrate that Sep38β regulates the immune-related ROS level in the insect midgut, which suppresses the insecticidal activity of Bt against S. exigua by 17-22%. Our study highlights that Sep38β is essential for insect immunity and the insecticidal activity of Bt to S. exigua and is a potential target for pest control.

Baicalin Promotes Apoptosis of Human Medullary Breast Cancer via the ERK/p38 MAPK Pathway

Objectives The present study aims to examine the effects of baicalin on the human medullary breast carcinoma (MBC) cell line Bcap-37 and to determine whether baicalin regulates Bcap-37 cell apoptosis through the ERK/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Materials and Methods 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Transwell assay were utilized to measure the effects of baicalin on the proliferation and migration ability of Bcap-37 cells, respectively. Flow cytometer analysis was implied to detect the effects of baicalin on Bcap-37 cell apoptosis. Real-time quantitative polymerase chain reaction (RT-PCR) was conducted to observe the influence of baicalin on mRNA expressions of apoptosis-related genes. Western blot was executed to further explore the action of baicalin on apoptosis-related proteins. PD98059 (a specific inhibitor of ERK) and SB203580 (a specific inhibitor of p38 MAPK) were used to further clarify the intrinsic mechanism of baicalin regulating apoptosis in Bcap-37 cells. Results Compared with the control group, baicalin significantly inhibits the proliferation activity of Bcap-37 cells in a concentration- and time-dependent manner, with a p-value < 0.05. The transwell assay indicated that the migration viability of cells decreased further, followed by the increased concentration of baicalin, and the p-value had a statistical difference. Besides, flow cytometry was conducted to assess the effects of baicalin on the early and late apoptosis rates of Bcap-37 cells, and results showed that baicalin highly promoted the apoptosis level both at the early and late stages with a statistical difference in a concentration-dependent manner ( p < 0.05). Results of RT-PCR presented that, compared with the control group, baicalin significantly activated the mRNA expression of Bax, p38, and p-ERK1 and abolished the mRNA expression of Bcl-2 in every dosing group in a concentration-dependent way ( p < 0.05). Western blot exhibited that, compared with the control group, baicalin promoted the protein expression of caspase-3, caspase-9, Bax, p38, P-ERK, and p53 while playing an opposite function to Bcl-2 in each dosing group ( p values have statistical differences). At last, PD98059 and SB203580 were applied to explore the potential mechanism of baicalin in apoptosis promotion. And results revealed that, compared with the group treated with baicalin alone, protein expression of Bax, p38, p-ERK, caspase-3, and caspase-9 was downregulated obviously in the group treated with both baicalin and PD98059 or SB203580 ( p < 0.05). The p53 expression inhibition showed a difference only when compared to the group adding PD98059. Compared with the blank group, a statistical difference was only observed in Bcl-2 expression in the group treated with baicalin in combination with PD98059 or SB203580 ( p < 0.05). Interestingly, a p53 expression difference was only displayed between the blank group and the group treated with baicalin and SB203580 ( p < 0.05). Conclusion Our study observed that baicalin inhibits the proliferation and migration ability of Bcap-37 cells. In addition, baicalin induces cell apoptosis via the ERK/p38 MAPK signal pathway.

Roles of MAPK and Nrf2 signaling pathways in quercetin alleviating redox imbalance induced by hydrogen peroxide in mammary epithelial cells

Abstract Oxidative stress is a risk factor for mammary health, resulting in decreased milk yield and milk quality. Application of exogenous bioactive compounds has been a research focus of antioxidation of animals in the mammary gland. Quercetin is a flavonoid extracted from vegetables, fruits and tea and has been shown to have a variety of biological activities, but the effect of quercetin on redox imbalance in mammary epithelial cells is unclear. In this study, cells of HC11, a mouse mammary epithelial cell line, were treated with quercetin, and the effects and molecular mechanisms of quercetin protection on hydrogen peroxide-induced oxidative stress were studied. Results showed that 20 μΜ quercetin attenuated hydrogen peroxide-induced lactate dehydrogenase release and reactive oxygen species (ROS) accumulation and alleviated the reduction of cell viability and antioxidant capacity. Quercetin significantly restored the activation of mitogen-activated protein kinase (MAPK) and nuclear factor E2-related factor 2 (Nrf2) pathways induced by hydrogen peroxide. Importantly, the inhibitors of p38 MAPK and extracellular regulated protein pathways affected the activation of Nrf2 pathway. All inhibitors of MAPK and Nrf2 pathways reduced the protective effects of quercetin on cell proliferation, the activity of catalase and the expression of glutamate-cysteine ligase modifier subunit. Meanwhile, the effects of quercetin on the production of ROS and expression of glutamate/cystine reverse transporter light chain were mainly dependent on Nrf2 pathway. In summary, the protective effect of quercetin in mammary epithelial cells was mediated via MAPK and Nrf2 pathways.