Mitogen-activated Protein Kinase Research Articles

Marine Staurosporine Analogues: Activity and Target Identification in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high mortality and drug resistance and no targeted drug available at present. Compound 4, a staurosporine alkaloid derived from Streptomyces sp. NBU3142 in a marine sponge, exhibits potent anti-TNBC activity. This research investigated its impact on MDA-MB-231 cells and their drug-resistant variants. The findings highlighted that compound 4 inhibits breast cancer cell migration, induces apoptosis, arrests the cell cycle, and promotes cellular senescence in both regular and paclitaxel-resistant MDA-MB-231 cells. Additionally, this study identified mitogen-activated protein kinase kinase kinase 11 (MAP3K11) as a target of compound 4, implicating its role in breast tumorigenesis by affecting cell proliferation, migration, and cell cycle progression.

Identification of growth-related genes based on BSA in Pacific white shrimp Litopenaeus vannamei

The development of SNP genotyping techniques has facilitated the application of genome-wide association studies (GWAS) and genomic selection (GS) in plants and animals. However, the cost of high-throughput SNP genotyping is particularly high in the case of aquaculture animals which can produce a large number of offspring. Consequently, a cost effective method for trait-associated SNP identification and application was urgently needed in aquaculture breeding. In this study, a Bulk Segregation Analysis sequencing (BSA-seq) method was employed to identify growth-related single nucleotide polymorphisms (SNPs) and genes in the Pacific white shrimp Litopenaeus vannamei. Two independent families were used for this analysis to reduce the false positive. The DNA of individuals with the highest and lowest body weights was pooled separately. Two methods including the ED (Euclidean distance) method and the chi-square test were applied to calculate the allele differences between high and low growth rate individuals. A total of 24,325,437 SNPs were identified and consequently 1,299 SNPs and 163 genes were identified to be associated with growth traits. KEGG enrichment analysis showed that these genes were significantly enriched in phosphatidylinositol signaling system and phospholipase D signaling pathway. The diacylglycerol kinase genes, phosphatidylinositol 3-kinase genes, inositol polyphosphate 5-phosphatase genes, mitogen-activated protein kinase genes, myotubularin-related protein genes and epidermal growth factor receptor genes in these pathways were considered as candidate growth genes. This study demonstrated that the BSA-seq based on whole genome resequencing is cost-effective and useful in aquaculture. The results not only provide useful information for understanding the genetic basis of growth traits, but also offer a source of SNPs for marker-assisted selection (MAS) and genomic selection (GS) in shrimp.

9304 Integration of Glucocorticoid and Mineralocorticoid Receptor Signaling in Triple-negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

7873 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

9304 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

Targeting a key protein-protein interaction surface on mitogen-activated protein kinases by a precision-guided warhead scaffold

For mitogen-activated protein kinases (MAPKs) a shallow surface—distinct from the substrate binding pocket—called the D(ocking)-groove governs partner protein binding. Screening of broad range of Michael acceptor compounds identified a double-activated, sterically crowded cyclohexenone moiety as a promising scaffold. We show that compounds bearing this structurally complex chiral warhead are able to target the conserved MAPK D-groove cysteine via reversible covalent modification and interfere with the protein-protein interactions of MAPKs. The electronic and steric properties of the Michael acceptor can be tailored via different substitution patterns. The inversion of the chiral center of the warhead can reroute chemical bond formation with the targeted cysteine towards the neighboring, but less nucleophilic histidine. Compounds bind to the shallow MAPK D-groove with low micromolar affinity in vitro and perturb MAPK signaling networks in the cell. This class of chiral, cyclic and enhanced 3D shaped Michael acceptor scaffolds offers an alternative to conventional ATP-competitive drugs modulating MAPK signaling pathways.

Molecular Mechanism of 5,6-Dihydroxyflavone in Suppressing LPS-Induced Inflammation and Oxidative Stress.

5,6-dihydroxyflavone (5,6-DHF), a flavonoid that possesses potential anti-inflammatory and antioxidant activities owing to its special catechol motif on the A ring. However, its function and mechanism of action against inflammation and cellular oxidative stress have not been elucidated. In the current study, 5,6-DHF was observed inhibiting lipopolysaccharide (LPS)-induced nitric oxide (NO) and cytoplasmic reactive oxygen species (ROS) production with the IC50 of 11.55 ± 0.64 μM and 0.8310 ± 0.633 μM in murine macrophages, respectively. Meanwhile, 5,6-DHF suppressed the overexpression of pro-inflammatory mediators such as proteins and cytokines and eradicated the accumulation of mitochondrial ROS (mtROS). The blockage of the activation of cell surface toll-like receptor 4 (TLR4), impediment of the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 from the mitogen-activated protein kinases (MAPK) pathway, Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) from the JAK-STAT pathway, and p65 from nuclear factor-κB (NF-κB) pathways were involved in the process of 5,6-DHF suppressing inflammation. Furthermore, 5,6-DHF acted as a cellular ROS scavenger and heme-oxygenase 1 (HO-1) inducer in relieving cellular oxidative stress. Importantly, 5,6-DHF exerted more potent anti-inflammatory activity than its close structural relatives, such as baicalein and chrysin. Overall, our findings pave the road for further research on 5,6-DHF in animal models.

The molecular mechanism of berberine affecting psoriasis skin inflammation by regulating keratinocyte pyroptosis via the p38 MAPK/NF-κB pathway.

Berberine (BBR), a Rhizoma Coptis-sourced isoquinoline alkaloid, is an effective drug for psoriasis treatment with its therapeutic mechanism remaining unclear. We delved into the mechanism of BBR affecting psoriatic skin inflammation by regulating keratinocyte pyroptosis. A psoriasis-like skin inflammation mouse model was induced by imiquimod (IMQ) and treated with BBR and a p38 activator anisomycin. Human epidermal keratinocytes (HEKs) were stimulated with five chemokines (M5) [interleukin (IL)-17A, IL-22A, oncostatin M, tumor necrosis factor-α, IL-1α] to simulate psoriasis immune microenvironment, then treated with BBR and anisomycin. Psoriasis skin lesions, skin tissue damage, cell viability and death, and gasdermin D-N (GSDMD-N) and NOD-like receptor protein 3 (NLRP3) positive cell numbers were assessed. The p38 mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) pathway and levels of the NLRP3/GSDMD pathway-related proteins and inflammatory factors were determined. BBR alleviated M5-induced HEK pyroptosis by inactivating NLRP3 inflammasomes. BBR inhibited the p38 MAPK/NF-κB pathway, and its effects on HEKs were partly averted by activating the p38 MAPK/NF-κB pathway. BBR repressed NLRP3 inflammasome activation and pyroptosis by inhibiting the p38 MAPK/NF-κB pathway. Collectively, BBR suppressed keratinocyte NLRP3/GSDMD pathway pyroptosis by suppressing the p38 MAPK/NF-κB pathway, thereby affecting psoriasis skin inflammation.

Punicalagin as a novel selective aryl hydrocarbon receptor (AhR) modulator upregulates AhR expression through the PDK1/p90RSK/AP-1 pathway to promote the anti-inflammatory response and bactericidal activity of macrophages

Aryl hydrocarbon receptor (AhR) plays an important role in inflammation and immunity as a new therapeutic target for infectious disease and sepsis. Punicalagin (PUN) is a Chinese herbal monomer extract of pomegranate peel that has beneficial anti-inflammatory, antioxidant and anti-infective effects. However, whether PUN is a ligand of AhR, its effect on AhR expression, and its signaling pathway remain poorly understood. In this study, we found that PUN was a unique polyphenolic compound that upregulated AhR expression at the transcriptional level, and regulated the AhR nongenomic pathway. AhR expression in lipopolysaccharide-induced macrophages was upregulated by PUN in vitro and in vivo in a time- and dose-dependent manner. Using specific inhibitors and siRNA, induction of AhR by PUN depended on sequential phosphorylation of 90-kDa ribosomal S6 kinase (p90RSK), which was activated by the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-dependent protein kinase (PDK)1 pathways. PUN promoted p90RSK-mediated activator protein-1 (AP-1) activation. AhR knockout or inhibitors reversed suppression of interleukin (IL)-6 and IL-1β expression by PUN. PUN decreased Listeria load and increased macrophage survival via AhR upregulation. In conclusion, we identified PUN as a novel selective AhR modulator involved in AhR expression via the MEK/ERK and PDK1 pathways targeting p90RSK/AP-1 in inflammatory macrophages, which inhibited macrophage inflammation and promoted bactericidal activity.

Calcium-Dependent Protein Kinase 5 and 13 enhance salt tolerance in rice by directly activating OsMPK3/6 kinases.

Mitogen-activated protein kinases (MAPKs/MPKs) are pivotal regulators in many stress-signaling pathways in plants. The dual phosphorylation of the TXY motif by MAP Kinase Kinases (MKKs) is essential for activating MAPKs. Here, we reveal a mechanism for MAPK activation that bypasses the need for MKKs. We identified rice (Oryza sativa) calcium-dependent protein kinase 5 (OsCPK5) and OsCPK13as positive regulators in salt stress tolerance. These kinases are essential for the full activation of OsMPK3 and OsMPK6 in response to elevated sodium levels, with both OsMPK3 and OsMPK6 also acting as positive regulators in rice salt tolerance. Biochemical analysis demonstrated that OsCPK5/13 directly interact with and activate OsMPK3/6 by phosphorylating the TXY motif in vitro and in vivo. Additionally, we have discovered that OsCPK5/13 relocate from the cell membrane to the nucleus in response to salt stress. This process relies on their N-terminal myristoylation and a calcium-dependent phosphorylation event within the N-terminus. Our results elucidate a MAPK activation pathway in rice that is independent of traditional MKK-mediated phosphorylation, highlighting the crucial roles of OsCPK5 and OsCPK13 in directly phosphorylating and activating OsMPK3/6, which are important for rice tolerance to salt stress.

Immunostimulatory Activity of a Mixture of Platycodon grandiflorum, Pyrus serotine, Chaenomeles sinensis, and Raphanus sativus in RAW264.7 Macrophages.

In this study, a mixture of Platycodon grandiflorum, Pyrus serotina, Chaenomeles sinensis, and Raphanus sativus (PPCRE) was investigated for their immuno-enhancing effects, as well as the molecular mechanism of PPCRE in RAW264.7 cells. PPCRE dramatically increased nitric oxide (NO) and prostaglandin E2 (PGE2) generation depending on the concentration while exhibiting no cytotoxicity. PPCRE markedly upregulated the mRNA and protein expression of immune-related cytotoxic factors such as cyclooxygenase (COX)-1, COX-2, and inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), as well as the mRNA level of IL-4. PPCRE increased the mitogen-activated protein kinase (MAPK) signaling pathway by upregulating the phosphorylation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Furthermore, PPCRE considerably activated the nuclear factor kappa B (NF-κB) signaling pathway by increasing phosphorylation of NF-κB-p65. PPCRE-stimulated RAW264.7 cells increased macrophage phagocytic capacity. In conclusion, our study found that PPCRE improved immune function by modulating inflammatory mediators and regulating the MAPK and NF-κB pathway of signaling in macrophages.

Trehalose Attenuates In Vitro Neurotoxicity of 6-Hydroxydopamine by Reducing Oxidative Stress and Activation of MAPK/AMPK Signaling Pathways.

The effects of trehalose, an autophagy-inducing disaccharide with neuroprotective properties, on the neurotoxicity of parkinsonian mimetics 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpiridinium (MPP+) are poorly understood. In our study, trehalose suppressed 6-OHDA-induced caspase-3/PARP1 cleavage (detected by immunoblotting), apoptotic DNA fragmentation/phosphatidylserine externalization, oxidative stress, mitochondrial depolarization (flow cytometry), and mitochondrial damage (electron microscopy) in SH-SY5Y neuroblastoma cells. The protection was not mediated by autophagy, autophagic receptor p62, or antioxidant enzymes superoxide dismutase and catalase. Trehalose suppressed 6-OHDA-induced activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and AMP-activated protein kinase (AMPK), as revealed by immunoblotting. Pharmacological/genetic inhibition of JNK, p38 MAPK, or AMPK mimicked the trehalose-mediated cytoprotection. Trehalose did not affect the extracellular signal-regulated kinase (ERK) and mechanistic target of rapamycin complex 1 (mTORC1)/4EBP1 pathways, while it reduced the prosurvival mTORC2/AKT signaling. Finally, trehalose enhanced oxidative stress, mitochondrial damage, and apoptosis without decreasing JNK, p38 MAPK, AMPK, or AKT activation in SH-SY5Y cells exposed to MPP+. In conclusion, trehalose protects SH-SY5Y cells from 6-OHDA-induced oxidative stress, mitochondrial damage, and apoptosis through autophagy/p62-independent inhibition of JNK, p38 MAPK, and AMPK. The opposite effects of trehalose on the neurotoxicity of 6-OHDA and MPP+ suggest caution in its potential development as a neuroprotective agent.

Network pharmacology combined with molecular docking and molecular dynamics to verify the therapeutic potential of mung beans (Vigna radiata) against prostate cancer

BackgroundProstate cancer is the most common oncological disease in men and one of leading causes of death worldwide. Growing evidence has demonstrated the effectiveness of mung bean bioactive compounds in suppressing various cancer cells. However, their effects and underlying mechanisms on prostate cancer have not been verified. The present study aimed to investigate the therapeutical effects and underlying mechanisms of mung bean compounds against prostate cancer.ResultsThe results revealed that 56 proteins related to prostate cancer could be modulated by mung bean, including several vital proteins of SRC (Sarcoma), Mitogen-Activated Protein Kinase 8 (MAPK8), Heat shock protein 90 kDa alpha member A1 (HSP90AA1), and Harvey Rat sarcoma virus (HRAS). It was also found that the potential pathways associated with prostate cancer pathogenesis comprising pyrimidine metabolism, nitrogen metabolism, and prolactin signaling pathways. Of 19 mung bean compounds docked to four key proteins reveal three promising compound (dulcinoside, peonidin-3-glucoside, and chlorogenic acid) with lower binding affinity score of − 7.7, − 12.2, − 9.0, and − 6.5 kcal/mol against SRC, MAPK8, HSP90AA1, and HRAS, respectively in their site of action. Dynamic simulation results also showed values of − 36.52 ± 2.93, − 35.93 ± 1.67, and − 35.77 ± 1.17 kJ/mol for Dulcinoside-SRC, Dulcinoside-MAPK8, and P3G-HSP90AA1 complexes, respectively. The binding of the compound occur in stable and flexible with the proteins. Moreover, all mung bean compounds predicted to have good ADMET properties.ConclusionsThe study concluded that dulcinoside, peonidin-3-glucoside, and chlorogenic acid potentially exhibited anticancer activity against prostate cancer in silico. Nevertheless, further studies such as in vitro and in vivo are needed to optimize and prove the efficacy of the mung brand and its compounds against prostate cancer.Graphical abstract

Two leucine-rich repeat receptor-like kinases initiate herbivory defense responses in tea plants

Abstract Leucine-rich repeat receptor-like kinases (LRR-RLKs) have emerged as key regulators of herbivory perception and subsequent defense initiation. While their functions in grass plants have been gradually elucidated, the roles of herbivory-related LRR-RLKs in woody plants remain largely unknown. In this study, we mined the genomic and transcriptomic data of tea plants (Camellia sinensis) and identified a total of 307 CsLRR-RLK members. Phylogenetic analysis grouped these CsLRR-RLKs into 14 subgroups along with their Arabidopsis homologs. Gene structure and conserved domain analyses revealed notable similarities among subgroup members. Among the identified CsLRR-RLKs, we focused on two plasma membrane-localized LRR-RLKs, CsLRR-RLK44 and CsLRR-RLK239, which do not form homodimers or heterodimers with each other. Both respond strongly to herbivory, and their expression patterns significantly correlate with herbivore resistance phenotypes across different tea accessions. CsLRR-RLK44 and CsLRR-RLK239 act upstream of mitogen-activated protein kinase (MPK) cascades and modulate the expression of defense-related MPKs and WRKY transcription factors. Additionally, silencing CsLRR-RLK44 or CsLRR-RLK239 reduced the levels of herbivory-induced jasmonates, thereby weakening the plant resistance to tea geometrid larvae (Ectropis obliqua). Our work is the first to demonstrate that in woody plants, LRR-RLKs are essential for enhancing herbivore resistance through the activation of the canonical signaling, including MPKs, WRKYs and jasmonates. Furthermore, our study extends mechanistic insights into how LRR-RLKs initiate plant defenses from grasses to economically important tree species.

MAP kinase kinase 1 (MEK1) within extracellular vesicles inhibits tumour growth by promoting anti-tumour immunity.

Extracellular vesicles (EVs) mediate intercellular communication in many physiologic processes and can modulate immune responses in individuals with cancer. Most studies of EVs in cancer have focused on their tumour promoting properties. Whether and how EVs might mediate tumour regression besides carrying antigens has not been well characterized. Using a mouse model of highly immunogenic regressor versus poorly immunogenic progressor tumour cells, we have characterized the role of EVs in activating macrophages and promoting tumour rejection. We found that the signalling molecule MAP2K1 (MEK1) is enriched in EVs secreted by regressor relative to progressor cells. Progressor EVs engineered to have levels of MEK1 similar to regressor EVs could inhibit tumour growth by indirectly promoting adaptive immunity in both syngeneic and 3rd party tumours. This effect required MEK1 activity and could occur by activating macrophages to promote adaptive immune responses against the tumour via the cytokine interferon-gamma. Our results suggest that MEK inhibition may be deleterious to cancer treatment, since MEK1 plays an important cell-extrinsic, tumour-suppressive role within EVs. Moreover, the delivery of MEK1 to tumour-associated macrophages, either by EVs, nanoparticles, or some other means, could be a useful strategy to treat cancer via the activation of anti-tumour immunity.

Bladder cancer associated with elevated heavy metals: Investigation of probable carcinogenic pathways through mitochondrial dysfunction, oxidative stress and mitogen-activated protein kinase

ObjectiveCarcinogenic mechanisms of heavy metals/ trace elements (HMTE) in bladder cancer (BC) are exactly unknown. Mitochondrial dysfunction (MD), oxidative stress (OS), and mitogen-activated protein kinases (MAPK) are probable carcinogenic mechanisms. The purpose is to investigate probable carcinogenic pathways of HMTE in BC using six MD genes, seven OS markers, and p38-MAPK. MethodsStudy included 125 BC/radical cystectomy (RC) patients between October 2020 and October 2022, and 72 controls. Exclusion criteria included previous neoplasm, chemo- or radiotherapy. Two samples (cancer/noncancer) were taken from RC specimens. Tissues/plasma/urine cadmium (Cd), lead (Pb), cobalt (Co), nickel (Ni), strontium (Sr), aluminium (Al), zinc (Zn), boron (B) were measured by ICP-OES. Tissue MD genes (mt-CO3, mt-CYB, mt-ATP 6, mt-ATP8, mt-CO1, mt-ND1), and serum OS markers (8-OHdG, MDA, 3-NT, AGEs, AOPP, ROS, SOD2), p38-MAPK were assessed by RT-PCR, and ELISA, respectively. ResultsBC and adjacent tissue showed higher (Al, Co, Pb, Ni, Zn, Cd,Sr), lower B concentrations, compared to controls. High tissue concentrations (Cd, Co, Pb, Ni, Sr) were associated with higher MD genes, OS, MAPK and lower SOD2 levels. The same differences were greater in 41 patients with concomitant elevation of two or more HMTE. Noninclusion of BC-related oncogenes (e.g. RAS) is a limitation. ConclusionsEvidence suggests that high BC tissue (Cd, Co, Pb, Ni, Si) concentrations are associated with over-expressed MD genes, OS, p38-MAPK and low SOD2. These findings provide important understanding keys of probable carcinogenic pathways in BC associated with HMTE. So, efforts should be performed to minimize and counteract exposure to toxic HMTE.

The Impact of Hydrogen Sulfide in the Paraventricular Nucleus on the MAPK Pathway in High Salt-Induced Hypertension.

The hypothalamic paraventricular nucleus (PVN) plays a central role in regulating cardiovascular activity and blood pressure. We administered hydroxylamine hydrochloride (HA), a cystathionine-β-synthase inhibitor, into the PVN to suppress endogenous hydrogen sulfide and investigate its effects on the mitogen-activated protein kinase (MAPK) pathway in high salt (HS)-induced hypertension. We randomly divided 40 male Dahl salt-sensitive rats into 4 groups: the normal salt (NS) + PVN vehicle group, the NS + PVN HA group, the HS + PVN vehicle group, and the HS + PVN HA group, with 10 rats in each group. The rats in the NS groups were fed a NS diet containing 0.3% NaCl, while the HS groups were fed a HS diet containing 8% NaCl. The mean arterial pressure was calculated after noninvasive measurement using an automatic sphygmomanometer to occlude the tail cuff once a week. HA or vehicle was infused into the bilateral PVN using Alzet osmotic mini pumps for 6 weeks after the hypertension model was successfully established. We measured the levels of H 2 S in the PVN and plasma norepinephrine using enzyme linked immunosorbent assay. In addition, we assessed the parameters of the MAPK pathway, inflammation, and oxidative stress through western blotting, immunohistochemical analysis, or real-time polymerase chain reaction. In this study, we discovered that decreased levels of endogenous hydrogen sulfide in the PVN contributed to the onset of HS-induced hypertension. This was linked to the activation of the MAPK signaling pathway, proinflammatory cytokines, and oxidative stress in the PVN, as well as the activation of the sympathetic nervous system.

Herbal medicine Ninjinyoeito inhibits RANKL-induced osteoclast differentiation and bone resorption activity by regulating NF-κB and MAPK pathway

ObjectivesOsteoporosis is a systemic bone metabolism disorder characterized by decreased bone mass and strength. Osteoclasts (OCs) are giant multinucleated cells that regulate bone homeostasis by degrading bone matrix. Excessive OC differentiation and activity can lead to serious bone metabolic disorders including osteoporosis. Current treatments, including antiresorptive drugs, exert considerable adverse effects, including jaw osteonecrosis. Herbal medicines, such as Ninjinyoeito (NYT), may also offer efficacy, but with fewer adverse effects. In this study, we investigated NYT's effects on osteoclastogenesis. MethodsTartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays were performed to examine NYT's effects on OC differentiation and function. OC-related gene expression at mRNA and protein levels was investigated to confirm NYT's inhibitory action against osteoclastogenesis. We also demonstrated involvement of signaling pathways mediated by IκBα and mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38] and showed nuclear translocation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and nuclear factor kappa B (NF-κB) p65 during osteoclastogenesis. ResultsTRAP staining and bone resorption assays confirmed that NYT significantly inhibited OC differentiation and function. Western blot and RT-PCR results showed that NYT ameliorated osteoclastogenesis by suppressing mRNA and protein level expression of OC-related genes. Moreover, blots and immunocytochemistry (ICC) data clarified that NYT abrogates signaling pathways mediated by IκBα and MAPK (ERK, JNK, p38), and demonstrated nuclear translocation of NFATc1 and NF-κB p65 during OC differentiation. ConclusionsThese findings suggest NYT is an alternative therapeutic candidate for treating osteoporosis.

Insight in the (Phospho)proteome of Vascular Smooth Muscle Cells Derived From Patients With Abdominal Aortic Aneurysm Reveals Novel Disease Mechanisms.

Abdominal aortic aneurysm (AAA) is characterized by weakening and dilatation of the aortic wall in the abdomen. The aim of this study was to gain insight into cell-specific mechanisms involved in AAA pathophysiology by analyzing the (phospho)proteome of vascular smooth muscle cells derived from patients with AAA compared with those of healthy donors. A (phospho)proteomics analysis based on tandem mass spectrometry was performed on vascular smooth muscle cells derived from patients with AAA (n=24) and healthy, control individuals (C-SMC, n=8). Following protein identification and quantification using MaxQuant, integrative inferred kinase activity analysis was used to calculate kinase activity scores. Expression differences between vascular smooth muscle cells derived from patients with AAA and healthy, control individuals were predominantly found in proteins involved in ECM (extracellular matrix) remodeling (THSD4 [thrombospondin type-1 domain-containing protein 4] and ADAMTS1 [A disintegrin and metalloproteinase with thrombospondin motifs 1]), energy metabolism (GYS1 [glycogen synthase 1] and PCK2 [phosphoenolpyruvate carboxykinase 2, mitochondrial]), and contractility (CACNA2D1 [calcium voltage-dependent channel subunit α-2/δ-1] and TPM1 [tropomyosin α-1 chain]). Phosphorylation patterns on proteins related to actin cytoskeleton organization dominated the phosphoproteome of vascular smooth muscle cells derived from patients with AAA . Besides, phosphorylation changes on proteins related to energy metabolism (GYS1), contractility (PARVA [α-parvin], PPP1R12A [protein phosphatase 1 regulatory subunit 12A], and CALD1 [caldesmon 1]), and intracellular communication (GJA1 [gap junction α-1 protein]) were seen. Kinase activity of NUAK1 (NUAK family SNF1-like kinase 1), FYN (tyrosine-protein kinase Fyn), MAPK7 (mitogen-activated protein kinase 7), and STK10 (serine/threonine kinase 10) was different in vascular smooth muscle cells derived from patients with AAA compared with those from healthy, control individuals. This study revealed changes in expression and phosphorylation levels of proteins involved in various processes responsible for AAA progression and development (eg, energy metabolism, ECM remodeling, actin cytoskeleton organization, contractility, intracellular communication, and cell adhesion). These newly identified proteins, phosphosites, and related kinases provide further insight into the underlying mechanism of vascular smooth muscle cell dysfunction within the aneurysmal wall. Our omics data thereby offer the opportunity to study the relevance, either as drug target or biomarker, of these proteins in AAA development.

Palmitoleate protects against Lipopolysaccharide-induced Inflammation and Inflammasome Activity

Inflammation is part of natural immune defense mechanism against any form of infection or injury. However, prolonged inflammation could perturb cell homeostasis and contribute to the development of metabolic and inflammatory diseases including maternal obesity, diabetes, cardiovascular diseases, and metabolic dysfunction-associated steatotic liver diseases. Polyunsaturated fatty acids have been shown to mitigate inflammatory response by generating specialized pro-resolving lipid mediators which take part in resolution of inflammation. Here, we show that palmitoleate, an omega-7 monounsaturated fatty acid exerts anti-inflammatory properties in response to lipopolysaccharide (LPS)-mediated inflammation. Exposure of bone-marrow derived macrophages (BMDMs) to LPS or TNFα induces robust increase in the expression of pro-inflammatory cytokines and supplementation of palmitoleate inhibited LPS-mediated upregulation of pro-inflammatory cytokines. We also observed that palmitoleate was able to block LPS+ATP-induced inflammasome activation mediated cleavage of pro-caspase 1 and pro-interleukin (IL)-1β. Further, treatment of palmitoleate protects against LPS-induced inflammation in human THP-1 derived macrophages and trophoblasts. Co-exposure of LPS and palmitate (saturated free fatty acid) induces inflammasome and cell death in BMDMs, however, treatment of palmitoleate blocked LPS and palmitate-induced cell death in BMDMs. Further, LPS and palmitate together results in the activation of mitogen activated protein kinases (MAPK) and pretreatment of palmitoleate inhibited the activation of MAPKs and nuclear translocation of nuclear factor kappa B (NF-kB) in BMDMs. In conclusion, palmitoleate shows anti-inflammatory properties against LPS-induced inflammation and LPS+palmitate/ATP-induced inflammasome activity and cell death.