Phosphorylation Of ERK Proteins Research Articles

Plac8-ERK pathway modulation of monocyte function in sepsis

Sepsis, a life-threatening condition caused by infection, is characterized by the dysregulation of immune responses and activation of monocytes. Plac8, a protein, has been implicated in various inflammatory conditions. This study aimed to investigate the effect of Plac8 upregulation on monocyte proliferation and activation in sepsis patients. Peripheral blood samples were collected from healthy individuals and sepsis patients. Monocytes were stimulated with lipopolysaccharide (LPS) to create an in vitro sepsis model, while a murine sepsis model was established using cecal ligation and puncture (CLP). The levels of monocyte markers, proliferation index (PI), and pro-inflammatory cytokines were assessed using flow cytometry and qPCR, respectively. Plac8 and phosphorylated ERK protein levels were determined by western blot, and TNF-α, IL-6, and IL-10 levels were quantified using ELISA. The CCK-8 assay was used to evaluate PBMC proliferation and activation. The results showed that Plac8 was highly expressed in sepsis models, promoting the survival, proliferation, and activation of monocytes. Plac8 upregulation activated the ERK pathway, leading to increased phosphorylation of ERK protein and elevated levels of CD14, CD16, TNF-α, IL-6, Plac8, and IL-10. In sepsis mice, Plac8 overexpression similarly activated the ERK pathway and promoted the survival, proliferation, and activation of monocytes. In conclusion, the upregulation of Plac8 enhances the activation of the ERK pathway and promotes monocyte proliferation and activation in sepsis patients.

The investigation of potential mechanism of Fuzhengkangfu Decoction against Diabetic myocardial injury based on a combined strategy of network pharmacology, transcriptomics, and experimental verification

Background and ObjectivesDiabetic cardiomyopathy (DCM) is a cardiac condition resulting from myocardial damage caused by diabetes mellitus (DM), currently lacking specific therapeutic interventions. Fuzhengkangfu decoction (FZK) plays an important role in the prevention and treatment of various cardiovascular diseases. However, the efficacy and potential mechanisms of FZK are not fully understood. This study aims to investigate the protective effect and mechanisms of FZK against DCM. MethodologiesRats were given a high-calorie diet along with a low dosage of streptozotocin (STZ) to establish a rat model of DCM. The diabetic rats received FZK or normal saline subcutaneously for 12 weeks. Echocardiography was conducted to evaluate their heart function characteristics. Rat heart morphologies were assessed using Sirius Red staining and H&E staining. Transcriptome sequencing analysis and network pharmacology were used to reveal possible targets and mechanisms. Molecular docking was conducted to validate the association between the primary components of FZK and the essential target molecules. Finally, both in vitro and in vivo studies were conducted on the cardioprotective properties and mechanism of FZK. ResultsAccording to the results of network pharmacology, FZK may prevent DCM by reducing oxidative stress and preventing apoptosis. Transcriptomics confirmed that FZK protected against DCM-induced myocardial fibrosis and remodelling, as predicted by network pharmacology, and suggested that FZK regulated the expression of oxidative stress and apoptosis-related proteins. Integrating network pharmacology and transcriptome analysis results revealed that the AGE-RAGE signalling pathway-associated MMP2, SLC2A1, NOX4, CCND1, and CYP1A1 might be key targets. Molecular docking showed that Poricoic acid A and 5-O-Methylvisammioside had the highest docking activities with these targets. We further conducted in vivo experiments, and the results showed that FZK significantly attenuated left ventricular remodelling, reduced myocardial fibrosis, and improved cardiac contractile function. And, our study demonstrated that FZK effectively reduced oxidative stress and apoptosis of cardiomyocytes. The data showed that Erk, NF-κB, and Caspase 3 phosphorylation was significantly inhibited, and Bcl-2/Bax was significantly increased after FZK treatment. In vitro, FZK significantly reduced AGEs-induced ROS increase and apoptosis in cardiomyocytes. Furthermore, FZK significantly inhibited the phosphorylation of Erk and NF-κB proteins and decreased the expression of MMP2. All the results confirmed that FZK inhibited the activation of the Erk/NF-κB pathway in AGE-RAGE signalling and alleviated oxidative stress and apoptosis of cardiomyocytes. In summary, we verified that FZK protects against DCM by inhibiting myocardial apoptotic remodelling through the suppression of the AGE-RAGE signalling pathway. ConclusionIn conclusion, our research indicates that FZK demonstrates anti-cardiac dysfunction properties by reducing oxidative stress and cardiomyocyte apoptosis through the AGE-RAGE pathway in DCM, showing potential for therapeutic use.

Endothelin-1 stimulated human preadipocyte growth through the AMPK, ERK, cJUN, PKC, and STAT3 pathways

High level of plasma endothelin (ET)-1 was noticed in obesity, and it was found to regulate adipogenesis and the endocrine activity of fat cells. However, relatively little is known about the ET-1 signaling pathway in preadipocyte growth. Our previous reports have shown that ET-1 and ET-3 stimulated murine 3T3-L1 preadipocyte growth through different kinase signaling cascades with ET type A receptor (ETAR) but not ETBR dependency. In this study, the hypothesis arose that ET-1 may regulate human visceral white preadipocyte growth through the AMPK, ERK, cJUN, PKC, and STAT3 pathways with ET receptor-specific dependency. As indicated by an increased number of cells and greater conversion of 3-4,5-dimethylthiazol-2-yl-2,5 diphenyl tetrazolium bromide (MTT) to formazan crystals by living cells, the stimulation of human preadipocyte growth by ET-1 depended on concentration. Neither ET-2 nor ET-3 stimulated human preadipocyte growth. When signaling pathways were examined, ET-1 signaling stimulated phosphorylation of AMPK, ERK, cJUN, PKC, and STAT3, proteins but not AKT, JNK, or p38 MAPK. Treatment with an ETAR antagonist, such as BQ610, or an ETBR antagonist, such as BQ788, blocked the ET-1-induced increase in cell growth and phosphorylated levels of AMPK, PKC, and STAT3 proteins. However, treatment with BQ788 but not BQ610 blocked the ET-1-increased levels of ERK and c-JUN protein phosphorylation. In addition, pretreatment with specific inhibitors of AMPK (compound C), ERK1/2 (U0126), JNK (SP600125), JAK2/STAT3 (AG490), or PKC (Ro318220) prevented the ET-1-induced increase in cell growth and reduced the ET-1-stimulated phosphorylation of AMPK, ERK1/2, c-JUN, STAT3, and PKC. However, neither the p38 MAPK antagonist nor the ceramide synthase inhibitor altered the effect of ET-1. The results indicate that the target molecules of ETAR and ETBR signaling pathways transducing ET-1 growth signals in human visceral preadipocytes can be similar (e.g., AMPK, JAK2/STAT3, and PKC) or different (e.g., ERK1/2 and JNK/c-JUN). This work was supported in part by the National Science and Technology Council, Taiwan, Grant NSTC 112-2320-B-008-001 to YHK and the Taoyuan Armed Forces General Hospital, Taiwan, Grant AFTGH to PJH, CCK, LJS, and YHK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bu-Shen-Ning-Xin decoction inhibits macrophage activation to ameliorate premature ovarian insufficiency-related osteoimmune disorder via FSH/FSHR pathway.

Limited studies are associated with premature ovarian insufficiency (POI)-related osteoimmune disorder currently. Bu-Shen-Ning-Xin decoction (BSNXD) displayed a favorable role in treating postmenopausal osteoporosis. However, its impact on the POI-related osteoimmune disorder remains unclear. The study primarily utilized animal experiments and network pharmacology to investigate the effects and underlying mechanisms of BSNXD on the POI-related osteoimmune disorder. First, a 4-vinylcyclohexene dioxide (VCD)-induced POI murine model was conducted to explore the therapeutical action of BSNXD. Second, we analyzed the active compounds of BSNXD and predicted their potential mechanisms for POI-related osteoimmune disorder via network pharmacology, further confirmed by molecular biology experiments. The results demonstrated that VCD exposure led to elevated follicle-stimulating hormone (FSH) levels, a 50% reduction in the primordial follicles, bone microstructure changes, and macrophage activation, indicating an osteoimmune disorder. BSNXD inhibited macrophage activation and osteoclast differentiation but did not affect serum FSH and estradiol levels in the VCD-induced POI model. Network pharmacology predicted the potential mechanisms of BSNXD against the POI-related osteoimmune disorder involving tumor necrosis factor α and MAPK signaling pathways, highlighting BSNXD regulated inflammation, hormone, and osteoclast differentiation. Further experiments identified BSNXD treatment suppressed macrophage activation via downregulating FSH receptor (FSHR) expression and inhibiting the phosphorylation of ERK and CCAAT enhancer binding proteins β. In conclusion, BSNXD regulated POI-related osteoimmune disorder by suppressing the FSH/FSHR pathway to reduce macrophage activation and further inhibiting osteoclastogenesis.

In ovo injection of AZD6244 suppresses feather follicle development by the inhibition of ERK and Wnt/β-catenin pathways in goose embryos (Anser cygnoides)

ABSTRACT 1. Feathers are an important product from poultry, and the state of feather growth and development plays an important role in their economic value. 2. In total, 120 eggs were selected for immunoblotting and immunolocalisation experiments of ERK and β-catenin proteins in different developmental stages of goose embryos. The ERK protein was highly expressed in the early stage of goose embryo development, while β-catenin protein was highly expressed in the middle stage of embryo development. 3. The 120 eggs were divided into four treatment groups, including an uninjected group (BLANK), a group injected with 100 µl of cosolvent (CK), a group injected with 100 µl of AZD6244 containing cosolvent in a dose of 5 mg/kg AZD6244 containing cosolvent (AZD5) and a group injected with 100 µl of AZD6244 containing cosolvent in a dose of 15 mg/kg AZD6244 containing cosolvent (AZD15). The eggs were injected on the ninth day of embryonic development (E9). Samples were collected at E21.5 to observe feather width, feather follicle diameter, ERK and Wnt/β-catenin pathway protein expression. 4. The AZD5 and AZD15 doses were within the embryonic safety range compared to the BLANK and CK groups and had no significant effect on the survival rate and weight at the inflection point, but significantly reduced the feather width and feather follicle diameter (p < 0.05). The AZD6244 treatment inhibited ERK protein phosphorylation levels and blocked the Wnt/β-catenin pathway, which in turn significantly down-regulated the expression levels of FZD4, β-catenin, TCF4 and LEF1 (p < 0.05), with an inhibitory effect in the AZD15 group being more significant. The immunohistochemical results of β-catenin and p-ERK were consistent with Western blot results. 5. The small molecule inhibitor AZD6244 regulated the growth and development of feather follicles in goose embryos by the ERK and Wnt/β-catenin pathways.

Effect of the Pulsatilla decoction n-butanol extract on vulvovaginal candidiasis caused by Candida glabrata and on its virulence factors

Vulvovaginal candidiasis (VVC) caused by Candida glabrata (C. glabrata) is more persistent and resistant to treatment than when caused by Candida albicans (C. albicans) and has been on the rise in recent years. The n-butanol extract of Pulsatilla Decoction (BEPD) has been shown to be effective in treating VVC caused by C. glabrata, but the underlying mechanism of action remains unclear. In this study, the experimenter conducted in vitro and in vivo experiments to explore the effects of BEPD on the virulence factors of C. glabrata, as well as its efficacy, with a focus on possible immunological mechanism in VVC caused by C. glabrata. The contents of Anemoside B4, Epiberberine, Berberine, Aesculin, Aesculetin, Phellodendrine and Jatrorrhizine in BEPD, detected by high-performance liquid chromatography, were 31,736.64, 13,529.66, 105,143.72, 19,406.20, 4952.67, 10,317.03, 2489.93 μg/g, respectively. In vitro experiments indicated that BEPD moderately inhibited the growth of C. glabrata, its adhesion, and biofilm formation, and affected the expression of efflux transporters in the biofilm state. In vivo experiments demonstrated that BEPD significantly reduced vaginal inflammatory manifestation and the release of proinflammatory cytokines and LDH in mice with VVC caused by C. glabrata. Moreover, it inhibited the Phosphorylation of EGFR, ERK, P38, P65, and C-Fos proteins. The results suggested that although BEPD moderately inhibits the growth and virulence factors of C. glabrata in vitro, it can significantly reduce vaginal inflammation by down-regulating the EGFR/MAPK signaling pathway in mice with VVC infected by C. glabrata.

LdCyPA attenuates MAPK pathway to assist Leishmania donovani immune escape in host cells

BackgroundVisceral leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Macrophages serve as the primary host cells for L. donovani, the immune response capability of these host cells is crucial for parasites’ intracellular survival. L. donovani peptidyl-prolyl cis/trans isomerase Cyclophilin A (LdCypA) is a key protein for L. donovani intracellular proliferation, while the molecular mechanism conducive to intracellular survival of parasites remains elusive. MethodsIn this study, we generated a macrophage cell line overexpressing LdCyPA to investigate its role in controlling host immunity and promoting intracellular immune escape of L. donovani. ResultsIt was discovered that the overexpression of the LdCyPA cell line regulated the host immune response following infection by downregulating the proportion of M1-type macrophages, promoting the secretion of the anti-inflammatory factor IL-4, and inhibiting the secretion of pro-inflammatory factors like IL-12, IFN-γ, TNF-α, and INOS. Transcriptome sequencing and mechanistic validation, meanwhile, demonstrated that cells overexpressing LdCyPA controlled the immune responses that followed infection by blocking the phosphorylation of P38 and JNK1/2 proteins in the MAPK signaling pathway and simultaneously increasing the phosphorylation of ERK proteins, which helped the L. donovani escape immune recognition. ConclusionOur findings thus pave the way for the development of host-directed antiparasitic drugs by illuminating the pro-Leishmania survival mechanism of L. donovani cyclophilin A and exposing a novel immune escape strategy for L. donovani that targets host cellular immune regulation.

PF-04449913 Inhibits Proliferation and Metastasis of Colorectal Cancer Cells by Down-regulating MMP9 Expression through the ERK/p65 Pathway.

Colorectal cancer remains a life-threatening malignancy with increasing morbidity and mortality worldwide. Therefore, new and effective anti-colorectal cancer therapeutics are urgently needed. In this study, we have studied the anti-tumor properties and potential mechanisms of PF-04449913. Colorectal cancer cell viability was reduced by PF-04449913 in a dose-dependent manner. The migration and invasion ability of malignant colon cells were attenuated by the drug, as demonstrated by the Transwell test. Moreover, PF-04449913 repressed the phosphorylation levels of ERK and other proteins, and the expression levels of MMP9. The anti-tumor effects of the drug in vivo were demonstrated in BALB/c-nude mice models, and PF-04449913 inhibited the malignant phenotype of colorectal cancer cells, including reduction of tumor size and promotion of apoptosis. At the molecular level, PF-04449913 induced a significant decrease in ERK and p65 protein phosphorylation levels and inhibited MMP9 protein expression. Both in vivo and in vitro results showed PF-04449913 to demonstrate antitumor effects, which have been proposed to be mediated through blockade of the ERK/p65 signaling pathway, and subsequent repression of MMP9 expression. Our study provides a new perspective on the potential clinical application of PF-04449913 in the treatment of colorectal cancer.

Regulatory roles of ACSL5 in the anti-tumor function of palmitic acid (C16:0) via the ERK signaling pathway.

Previous studies have highlighted the susceptibility of cancer to perturbations in lipid metabolism. In particular, C16:0 has emerged as a promising novel treatment for hepatocellular carcinoma. In our study, we investigated the levels of C16:0 in the serum of non-small lung cancer patients were significant downregulation compared to healthy individuals (n=10; p<0.05). Moreover, our in vitro experiments using A549 cells demonstrated that C16:0 effectively inhibited proliferation, apoptosis, migration, and invasion. Despite these promising results, its pathogenesis remains poorly understood. CCK-8 assay, annexin V-FITC/PI double staining assay, wound healing assay and transwell assay were performed to evaluate the effects of C16:0, on proliferation, apoptosis, migration and invasion of A549 cells. RNA sequencing was used to identify essential factors involved in C16:0-growth inhibition in lung cancer. Further, the expression levels of related gene and proteins were detected by quantitative RT-PCR and Western blotting. Mouse NSCLC subcutaneous xenograft tumor model was established, and gastric lavage was given with C16:0. Tumor volume assay and hematoxylin-eosin staining were used to detect tumor growth in vivo. Our analysis revealed a significant upregulation of ACSL5 and its associated proteins in C16:0-treated A549 cells compared to the control group both in vivo and in vitro. Moreover, the knockdown of ACSL5 reversed the anti-tumor effect, resulting in an increased rate of the malignant phenotype mentioned above. Additionally, the expression of phosphorylated ERK protein was significantly inhibited with increasing concentrations of C16:0 in A549 cells. These results reveal for the first time that C16:0, as a novel target, regulates ACLS5 through the ERK signaling pathway, to inhibit the proliferation and apoptosis and inhibits cell migration and invasion of NSCLC. These findings may lead to the development of a novel therapeutic approach for non-small lung cancer.

Sustained CREB phosphorylation by lipid-peptide liquid crystalline nanoassemblies

Cyclic-AMP-response element-binding protein (CREB) is a leucine zipper class transcription factor that is activated through phosphorylation. Ample CREB phosphorylation is required for neurotrophin expression, which is of key importance for preventing and regenerating neurological disorders, including the sequelae of long COVID syndrome. Here we created lipid-peptide nanoassemblies with different liquid crystalline structural organizations (cubosomes, hexosomes, and vesicles) as innovative nanomedicine delivery systems of bioactive PUFA-plasmalogens (vinyl ether phospholipids with polyunsaturated fatty acid chains) and a neurotrophic pituitary adenylate cyclase-activating polypeptide (PACAP). Considering that plasmalogen deficiency is a potentially causative factor for neurodegeneration, we examined the impact of nanoassemblies type and incubation time in an in vitro Parkinson’s disease (PD) model as critical parameters for the induction of CREB phosphorylation. The determined kinetic changes in CREB, AKT, and ERK-protein phosphorylation reveal that non-lamellar PUFA-plasmalogen-loaded liquid crystalline lipid nanoparticles significantly prolong CREB activation in the neurodegeneration model, an effect unattainable with free drugs, and this effect can be further enhanced by the cell-penetrating peptide PACAP. Understanding the sustained CREB activation response to neurotrophic nanoassemblies might lead to more efficient use of nanomedicines in neuroregeneration.

Gastroenteropancreatic neuroendocrine carcinoma tumor spheroid drug screen reveals vulnerability to tyrosyl-DNA phosphodiesterase 1 inhibitors

BackgroundGastroenteropancreatic neuroendocrine carcinomas are rare neoplasms with no effective treatments and poor prognosis. Few reliable preclinical models exist for the study of gastroenteropancreatic neuroendocrine carcinomas, limiting investigation of novel treatments. We used tumor spheroids from our recently established gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models to systematically screen for compounds with diverse structures to identify potential new categories of therapeutic agents that can target gastroenteropancreatic neuroendocrine carcinomas. MethodsTumor spheroids were derived from our NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models. Gastroenteropancreatic neuroendocrine carcinoma spheroids were screened against a library of 885 compounds from the National Cancer Institute Diversity Set VII collection. Cell viability was measured via AlamarBlue assay. After identification of potential therapeutic compounds, synergy screening of a selected group with temozolomide and doxorubicin was performed, and these combinations were further analyzed for γH2AX and phosphorylated-ERK proteins. ResultsWe identified 16 compounds that inhibit over 75% of gastroenteropancreatic neuroendocrine carcinoma spheroid survival. Seven are inhibitors of tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme working closely with the topoisomerase I complex. When combined with temozolomide or doxorubicin, the tyrosyl-DNA phosphodiesterase 1 inhibitor cytarabine increased the cytotoxic effects of these drugs on NEC1452 cells which was further evidenced by increasing γH2AX and decreasing phosphorylated-ERK in combination treatment compared to temozolomide alone. ConclusionBoth NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma spheroid lines are useful preclinical models for drug testing. Our library screen revealed these gastroenteropancreatic neuroendocrine carcinoma spheroids are highly sensitive to a novel class of anti-cancer drugs that target nuclear genome stability.

ERK/NF-kB/COX-2 Signaling Pathway Plays a Key Role in Curcumin Protection against Acetaminophen-Induced Liver Injury.

Recent experimental studies have highlighted the beneficial effects of curcumin on liver injury induced by acetaminophen (APAP). However, the specific molecular mechanisms underlying curcumin's hepatoprotective effects against APAP-induced liver injury remain to be fully elucidated. This study aimed to investigate the therapeutic effect of curcumin on APAP-induced liver injury using a mouse model. In the experiment, mice were subjected to an intraperitoneal hepatotoxic dose of APAP (300 mg/kg) to induce hepatotoxicity. After 30 min of APAP administration, the mice were treated with different concentrations of curcumin (0, 10, 25, or 50 mg/kg). After 16 h, mice with hepatotoxicity showed elevated levels of serum alanine transaminase (ALT), aspartate transaminase (AST), hepatic myeloperoxidase (MPO), TNF-α, and IL-6, and decreased levels of glutathione (GSH). Moreover, there was an increased infiltration of neutrophils and macrophages following intraperitoneal injection of APAP. However, curcumin-treated mice displayed a pronounced reduction in serum ALT, AST, hepatic MPO, TNF-α, and IL-6 levels, coupled with a notable elevation in GSH levels compared to the APAP-treated hepatotoxic mice. Moreover, curcumin treatment led to reduced infiltration of neutrophils and macrophages. Additionally, curcumin inhibited the phosphorylation of ERK and NF-kB proteins while reducing the expression of cyclooxygenase-2 (COX-2). These findings highlight the hepatoprotective potential of curcumin against APAP-induced liver injury through the suppression of the ERK, NF-kB, and COX-2 signaling pathways.

Pro-inflammatory regulatory mechanism of C5a/C5aR pathway in GCRV-infected grass carp (Ctenopharyngodon idella)

Death of grass carp (Ctenopharyngodon idella) caused by grass carp reovirus (GCRV) infection severely restricts the development of the grass carp industry. The interaction of C5a and C5a receptor (C5aR) in the complement system promotes inflammation and aggravates bleeding in grass carps. To reveal the regulatory mechanism of the C5a/C5aR pro-inflammatory response after GCRV infection, the expression of C5 in the liver and muscle tissues of grass carp after GCRV infection was analyzed using immunofluorescence. The effects of over expression of C5a and the C5aR antagonist PMX205 in grass carp kidney cell line (CIK) on the phosphorylation of p38 and ERK and the gene and protein expression of IL-6 and TNF-α were also analyzed using RT-qPCR and western blot. The results showed that the expression of the C5a protein in the liver and muscle of grass carp infected with GCRV first increased and then decreased, and the C5a protein level peaked on the 5th day after infection. In CIK cells, over expression of C5a significantly increased C5aR expression, phosphorylation of p38 and ERK, and overexpression of IL-6 and TNF-α. Treatment of CIK cells with PMX205 significantly decreased the expression of C5a and C5aR, phosphorylation of p38 and ERK proteins, and expression of IL-6 and TNF-α after GCRV infection. These results implied that the interaction of C5a and C5aR in grass carp resulted in the phosphorylation of p38 and ERK proteins, a significant increase in the expression of IL-6 and TNF-α, and ultimately induction of an inflammatory response of grass carp infected with GCRV. These results provide a theoretical basis for relieving grass carp hemorrhagic diseases by regulating the interaction of C5a and C5aR.

Dihydroartemisinin inhibits angiogenesis in breast cancer via regulating VEGF and MMP-2/-9.

Dihydroartemisinin (DHA) is an artemisinin derivative known for its antimalarial properties. It has also shown potential as an anti-tumor and anti-angiogenic agent. However, its specific role in inhibiting angiogenesis in breast cancer is not well understood. We aimed to investigate the anti-angiogenesis effect of DHA on breast cancer and explore its potential as a therapeutic drug. Our objectives were to assess the impact of DHA on neovascularization induced by MDA-MB-231 cells, evaluate its effects on vessel sprout and tube-formation in vascular endothelial cells, and analyze the expression of key angiogenesis-related proteins. Using a chicken chorioallantoic membrane (CAM) model, we cultured MDA-MB-231 cells and treated them with DHA. We assessed neovascularization and cultured vascular endothelial cells with DHA-treated cell media to evaluate vessel sprout and tube-formation. Protein expression levels of VEGF, MMP-2, and MMP-9 were analyzed using Western blotting. DHA significantly attenuated neovascularization induced by MDA-MB-231 cells. It also suppressed vessel sprout and tube-formation of HUVEC cells when exposed to DHA-treated cell media. Furthermore, DHA downregulated the expression of VEGF, MMP-2, and MMP-9 proteins. Mechanistically, DHA inhibited the phosphorylation of PI3K, AKT, ERK, and NF-κB proteins in tumor cells. Our study provides evidence of the inhibitory effect of DHA on breast cancer angiogenesis. These findings support the potential of DHA as an anti-breast cancer drug and warrant further investigation for its therapeutic applications.

FGF12 regulates cell cycle gene expression and promotes follicular granulosa cell proliferation through ERK phosphorylation in geese

The granulosa cells play an important role in the fate of follicular development or atresia in poultry. Fibroblast growth factor 12 (FGF12) is downregulated in atretic follicles and may be involved in regulating granulosa cell survival in previous studies, but its molecular mechanism remains unclear. In this study, FGF12 overexpression and knockdown models of goose granulosa cells were constructed to investigate its function. The downstream expression of the cell cycle pathway was analyzed by qPCR. Granulosa cell proliferative activity and apoptosis were detected by CCK8 and TUNEL. Protein phosphorylation levels of ERK and AKT were measured using Western blotting to analyze the key pathway of FGF12 regulation of granulosa cell proliferation. ERK protein phosphorylation inhibitor was added for further verification. After overexpression of FGF12, cell proliferation activity was increased, the expressions of cell cycle pathway genes CCND1, CCNA2, MAD2, and CHK1 were upregulated, the apoptosis of granulosa cell was decreased, and Caspase 3 gene and protein expression were downregulated. After the knockdown of FGF12, cell proliferation activity decreased, the expression of downstream genes in the cell cycle pathway was downregulated, the apoptosis of granulosa cells was increased, and the Bcl-2 gene and protein were downregulated. Overexpression of FGF12 promoted the synthesis of P4 and upregulates the expression of the STAR gene. Overexpression of FGF12 promoted ERK protein phosphorylation but did not affect AKT phosphorylation. The addition of ERK phosphorylation inhibitors resulted in the elimination of the increase in cell proliferative activity caused by FGF12 overexpression. In conclusion, FGF12 could promote proliferation and inhibit apoptosis of goose granulosa cells by increasing ERK phosphorylation.

P-643 The effect of hyperinsulinemia on FSH-mediated signalling pathways in granulosa cells - implications for follicle growth in women with PCOS

Abstract Study question Does in vitro exposure of granulosa cells (GC) to an equivalent hyperinsulinemic (HI) dose of insulin alter key signalling pathways, potentially slowing follicle growth? Summary answer Exposure of GC to HI dose of insulin down-regulated FSH-mediated phosphorylation of AKT and ERK proteins, which was reversed by metformin but not myoinositol treatment What is known already Antral follicles utilise glycolytic methods of energy production releasing the metabolites pyruvate and lactate, which are then secreted into the follicular fluid or passed directly to the oocyte via gap junctions. Insulin increases GC glucose uptake and cross-talks with FSH-mediated intracellular pathways that determine follicle growth and glucose utilisation. We and others have shown that there is significant perturbation in glucose metabolism (as measured by reduced lactate production) in cultured luteinised GC (GLC) from women with anovulatory PCOS, compared to those with normal ovaries or polycystic ovarian morphology (PCOM), that represents both attenuated glucose uptake and reduced glycolytic activity. Study design, size, duration KGN-GC were exposed to 1000ng/ml insulin for 48h (mimicking prolonged HI in vivo) with/without metformin [10-7M] or myo-inositol [25mM]; followed by acute stimulation with FSH (10ng/ml at 15 and 30 mins) (n = 4-5). GLCs obtained from women with normal ovaries (n = 5), PCOM (n = 7) and PCOS (n = 2) were serum-starved and exposed acutely to insulin (10ng/ml for 30mins), glucose starved (20mins) and treated with a radiolabelled 2-deoxy-glucose mix. Fasting serum glucose and insulin were measured in PCOS women. Participants/materials, setting, methods Western blotting on proteins from KGN-GC was performed using antibodies against total and phosphorylated forms of AKT and ERK. Densitometry measurements of phospho:total forms were taken from individual treatments, normalised to loading controls (either β-actin/Vinculin) and then untreated controls. GLCs were lysed and uptake of 14C-2DG (non-metabolised) measured and expressed as percentage uptake of 14C-2DG in the insulin-treated normalised to untreated/control cells (100%). Insulin resistance (IR) was calculated from fasting glucose and insulin using HOMA2. Main results and the role of chance Chronic exposure of KGNs to high dose insulin significantly down-regulated acute FSH-mediated phosphorylation of AKT and ERK (ANOVA *p=0.03, multiple post-hoc t-tests p &amp;lt; 0.05, two-tailed), without altering insulin receptor levels. Addition of metformin to FSH treatment, significantly reversed the HI-induced reduction in pAKT levels and enhanced it further (*p=0.02) but had no effect on pERK levels. Myoinositol had no effect on either pAKT or pERK levels. These findings demonstrated that HI directly inhibited FSH signalling events downstream of the insulin receptor in GC, but that metformin could counteract some of these detrimental effects. The 2-deoxy form of glucose is taken up into the cell but not metabolised further. Measurement of 14C-2DG showed that acute stimulation with post-prandial levels of insulin of GLCs taken from normal and PCOM women significantly increased glucose uptake (*p&amp;lt;0.05, ratio of paired t-test, 2-tailed); whereas GLCs from women with PCOS and insulin resistance were unable to respond to insulin and take up glucose. Therefore, GLCs from insulin resistant women with PCOS retain their phenotype in vitro and this is not an artefact, since GLCs from normal and PCOM also subjected to the same stimulation regime were able to take up glucose in response to insulin stimulation. Limitations, reasons for caution A clear limitation is the small number of insulin resistant women with PCOS. In addition, human GLCs were collected after hormonal stimulation. Wider implications of the findings Women with IR, HI and PCOS undergoing ART may require extra gonadotrophin stimulation to grow and mature sufficient eggs. Additionally, the oocyte quality maybe compromised, as indicated by reduced glucose uptake. Treatment of women with metformin but not myoinositol may improve the dysregulated FSH-signalling pathways brought about by HI. Trial registration number not applicable

Acetaldehyde induces similar cytotoxic and genotoxic risks in BEAS-2B cells and HHSteCs: involvement of differential regulation of MAPK/ERK and PI3K/AKT pathways.

Long-term use of alcohol and cigarettes is associated with millions of deaths each year, directly or indirectly. The carcinogen acetaldehyde is both a metabolite of alcohol and the most abundant carbonyl compound in cigarette smoke, and co-exposure of them is usual and primarily leads to liver and lung injury, respectively. However, few studies have explored the synchronic risk of acetaldehyde on the liver and lung. Here, we investigated the toxic effects and related mechanisms of acetaldehyde based on normal hepatocytes and lung cells. The results showed that acetaldehyde caused significant dose-dependent increases of cytotoxicity, ROS level, DNA adduct level, DNA single/double-strand breakage, and chromosomal damage in BEAS-2B cells and HHSteCs, with similar effects at the same doses. The gene and protein expression and phosphorylation of p38MAPK, ERK, PI3K, and AKT, key proteins of MAPK/ERK and PI3K/AKT pathways regulating cell survival and tumorigenesis, were significantly upregulated on BEAS-2B cells, while only protein expression and phosphorylation of ERK were upregulated significantly, the other three decreased in HHSteCs. When either the inhibitor of the four key proteins was co-treated with acetaldehyde, cell viabilities were almost unchanged in BEAS-2B cells and HHSteCs. Thus, acetaldehyde could synchronically induce similar toxic effects in BEAS-2B cells and HHSteCs, and MAPK/ERK and PI3K/AKT pathways seem to be involved in different regulatory mechanisms.

Butein inhibits corticosterone-induced apoptosis of Neuro2A cells by maintaining MEK-ERK signaling.

Stress-induced overactivation of glucocorticoid signaling may contribute to mental illness by inducing neuronal death and dysfunction. We previously reported that pretreatment with the plant flavonoid butein inhibits corticosterone (CORT)-induced apoptosis of Neuro2A (N2A) cells. In the current study, we examined whether MEK-ERK and PI3K-AKT signaling pathways are involved in neuroprotection by butein. N2A cells were pre-incubated with serum-free DMEM containing 0.5μM butein for 30min, and then incubated with serum-free DMEM containing 0.5µM butein, 50µM CORT, 50µM LY294002, or 50µM PD98059 as indicated for 24h. We subsequently performed the MTT assay and the western blot analysis. As expected, CORT considerably reduced N2A cell viability and increased relative expression of the apoptosis effector cleaved caspase-3, whereas pretreatment with butein blocked these cytotoxic effects. Treatment with CORT alone also decreased both AKT and ERK protein phosphorylation. Butein pretreatment had no effect on AKT phosphorylation, and only partially reversed the reduction in phosphorylated ERK. However, cotreatment with butein and the PI3K inhibitor LY294002 during CORT exposure enhanced ERK phosphorylation, whereas cotreatment with butein and the ERK phosphorylation/activation inhibitor PD98059 enhanced AKT phosphorylation, suggesting that MEK-ERK negatively regulates AKT phosphorylation. Moreover, the protective efficacy of butein was blocked by PD98059 cotreatment but not LY294002 cotreatment. These findings suggest that butein protects neurons against glucocorticoid-induced apoptosis by sustaining ERK phosphorylation and downstream signaling.

Amphiregulin blockade decreases the levodopa-induced dyskinesia in a 6-hydroxydopamine Parkinson's disease mouse model.

Levodopa (L-DOPA) is considered the most reliable drug for treating Parkinson's disease (PD) clinical symptoms. Regrettably, long-term L-DOPA therapy results in the emergence of drug-induced abnormal involuntary movements (AIMs) in most PD patients. The mechanisms underlying motor fluctuations and dyskinesia induced by L-DOPA (LID) are still perplexing. Here, we first performed the analysis on the microarray data set (GSE55096) from the gene expression omnibus (GEO) repository and identified the differentially expressed genes (DEGs) using linear models for microarray analysis (Limma) R packages from the Bioconductor project. 12 genes (Nr4a2, Areg, Tinf2, Ptgs2, Pdlim1, Tes, Irf6, Tgfb1, Serpinb2, Lipg, Creb3l1, Lypd1) were found to be upregulated. Six genes were validated on quantitative polymerase chain reaction and subsequently, Amphiregulin (Areg) was selected (based on log2 fold change) for further experiments to unravel its involvement in LID. Areg LV_shRNA was used to knock down Areg to explore its therapeutic role in the LID model. Western blotting and immunofluorescence results show that AREG is significantly expressed in the LID group relative to the control. Dyskinetic movements in LID mice were alleviated by Areg knockdown, and the protein expression of delta FOSB, the commonly attributable protein in LID, was decreased. Moreover, Areg knockdown reduced the protein expression of P-ERK. In order to ascertain whether the inhibition of the ERK pathway (a common pathway known to mediate levodopa-induced dyskinesia) could also impede Areg, the animals were injected with an ERK inhibitor (PD98059). Afterward, the AIMs, AREG, and ERK protein expression were measured relative to the control group. A group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group. Taken together, our results indicate unequivocal involvement of Areg in levodopa-induced dyskinesia, thus a target for therapy development.

Abstract 411: PHI-501, a novel pan-RAF/DDRs dual kinase inhibitor, overcomes BRAF or MEK inhibitor resistance in melanoma

Abstract Background: PHI-501 is a newly developed, highly effective, and orally accessible dual inhibitor for pan-RAF and discoidin domain receptor (DDR) that is a collagen-activated receptor tyrosine kinase. Mutations of the BRAF and NRAS are the most common genetic alterations in melanoma and consequently lead to activation of mitogen activated protein kinase (MAPK) signaling. The major drawback of melanoma treatment is the innate and acquired drug resistance to MAPK inhibitors. In this study, we investigated the inhibitory effects of a novel compound, PHI-501 on resistance to known BRAF and MEK inhibitors in melanoma. Methods: After compounds treatment, the growth-inhibiting activity was assessed using the CCK test. Inhibition of RAF/DDR1 pathway signaling was evaluated by western blotting. Using annexin-V/propidium iodide-stained cells and flow cytometry, apoptosis induction was assessed. To quantify cell mobility, monolayer cells were subjected to a wound-healing assay. Long-term therapy with dabrafenib (BRAF inhibitor), cobimetinib or trametinib (MEK inhibitor) led to the establishment of the drug-resistant SK-MEL-3 (BRAF V600E) and SK-MEL-30 (NRAS Q61K) melanoma cell line. Results: PHI-501 demonstrated potent growth inhibition (GI50 &amp;lt;1 µM) in seven melanoma cell lines harboring BRAF V600E or NRAS mutations. Western blot revealed that PHI-501 inhibited the phosphorylation of MEK, ERK, and AKT proteins in the downstream signaling of RAF/DDRs and decreased the levels of cyclin D1 and survivin. In SK-MEL-3, PHI-501 was 47-fold more effective than the class I BRAF inhibitor, dabrafenib (GI50: 0.08 µM vs 3.73 µM). Established dabrafenib- or trametinib-resistant SK-MEL-3 cells were extremely sensitive to PHI-501 (GI50: 0.20 µM and 0.13 µM), whereas their sensitivity to dabrafenib or trametinib was diminished (GI50: NA and 39.61 µM). PHI-501 triggered apoptosis more effectively than these inhibitors, as revealed by a larger proportion of annexin staining and extensive PARP1 cleavage. Comparing the potency of PHI-501 to cobimetinib in acquired resistant SK-MEL-30 cells showed that PHI-501 had 74-fold more inhibitory effect on cell proliferation (GI50: 0.55 µM to 40 µM). Moreover, treatment with PHI-501 strongly suppressed cell migration of cobimetinib-resistant SK-MEL-30 cells. PHI-501 inhibited the growth of drug-resistant melanoma cells as effectively as the original drug-naïve cells, and western blot analysis revealed considerable suppression of DDR1/2, ERK, and AKT phosphorylation in MAPK inhibitor-resistant cells. Conclusion: Melanoma cells resistant to RAF or MEK-targeted treatments or harboring NRAS mutation exhibited strong antiproliferative activity when treated with PHI-501, a highly potent pan-RAF/DDR dual inhibitor. The results of this study suggest that PHI-501, as a single agent, has the potential to overcome the restricted response in the treatment of melanoma. Citation Format: Sue Min Kim, Jung Hee Park, Ky-Youb Nam, June H-J Han, Kyu-Tae Kim, Jeong Hyeok Yoon, Sandip Sengupta, Taebo Sim, Sang Joon Shin. PHI-501, a novel pan-RAF/DDRs dual kinase inhibitor, overcomes BRAF or MEK inhibitor resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 411.