Single-agent TAS102 (trifluridine/tipiracil) and regorafenib are FDA-approved treatments for metastatic colorectal cancer (mCRC). We previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in clinical case reports of multidrug-resistant mCRC patients. We hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers and may in the future provide a treatment option for patients with advanced GI cancer. We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo. TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, as well inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. The TAS102 plus regorafenib drug combination may be further tested in gastric and other GI cancers.

Noise-induced hearing loss (NIHL) is a major cause of hearing impairment and is linked to dementia and mental health conditions, yet no FDA-approved drugs exist to prevent it. Downregulating the mitogen-activated protein kinase (MAPK) cellular pathway has emerged as a promising approach to attenuate NIHL, but the molecular targets and the mechanism of protection are not fully understood. Here, we tested specifically the role of the kinases ERK1/2 in noise otoprotection using a newly developed, highly specific ERK1/2 inhibitor, tizaterkib, in preclinical animal models. Tizaterkib is currently being tested in phase 1 clinical trials for cancer treatment and has high oral bioavailability and low predicted systemic toxicity in mice and humans. In this study, we performed dose-response measurements of tizaterkib\'s efficacy against permanent NIHL in adult FVB/NJ mice, and its minimum effective dose (0.5 mg/kg/bw), therapeutic index (>50), and window of opportunity (<48 h) were determined. The drug, administered orally twice daily for 3 days, 24 h after 2 h of 100 dB or 106 dB SPL noise exposure, at a dose equivalent to what is prescribed currently for humans in clinical trials, conferred an average protection of 20-25 dB SPL in both female and male mice. The drug shielded mice from the noise-induced synaptic damage which occurs following loud noise exposure. Equally interesting, tizaterkib was shown to decrease the number of CD45- and CD68-positive immune cells in the mouse cochlea following noise exposure. This study suggests that repurposing tizaterkib and the ERK1/2 kinases\' inhibition could be a promising strategy for the treatment of NIHL.

As a prevalent neurodegenerative disorder, Parkinson\'s disease is associated with oxidative stress. Our recent investigations revealed that reactive oxygen species (ROS) and PD-toxins like 6-hydroxydopamine (6-OHDA) can induce neuronal apoptosis through over-activation of Akt signaling. Chlorogenic acid (CGA), a natural acid phenol abundant in the human diet, is well-documented for its ability to mitigate intracellular ROS. In this study, we utilized CGA to treat experimental models of PD both in vitro and in vivo. Our study results demonstrated that SH-SY5Y and primary neurons exhibited cell apoptosis in response to 6-OHDA. Pretreatment with CGA significantly attenuated PD toxins-induced large amount of ROS, inhibiting Erk1/2 activation, preventing Akt inhibition, and hindering neuronal cell death. Combining the Erk1/2 inhibitor U0126 with CGA could reverse 6-OHDA-induced Akt inhibition, ROS, and apoptosis in the cells. Crucially, the Akt activator SC79 and ROS scavenger NAC both could eliminate excessive ROS via Akt and Erk1/2 signaling pathways, and CGA further potentiated these effects in PD models. Behavioral experiments revealed that CGA could alleviate gait abnormalities in PD model mice. The neuroprotective effects have been demonstrated in several endocrine regions and in the substantia nigra tissue, which shows the positive tyrosine hydroxylase (TH). Overall, our results suggest that CGA prevents the activation of Erk1/2 and inactivation of Akt by removing excess ROS in PD models. These findings propose a potential strategy for mitigating neuronal degeneration in Parkinson\'s disease by modulating the Akt/Erk1/2 signaling pathway through the administration of CGA and/or the use of antioxidants to alleviate oxidative stress.

UNASSIGNED: Non-alcoholic fatty liver disease (NAFLD) is a chronic steatohepatitis disorder. If left untreated, it can progress to hepatocellular carcinoma. Several studies have shown that saroglitazar, a PPARα/γ dual agonist, and curcumin (the principal constituent of turmeric) may be effective in the treatment of NAFLD. This research aimed to study the pharmacological mechanism of these compounds in rats with NAFLD.
UNASSIGNED: NAFLD was induced in male Wistar rats (aged 6-8 weeks) by feeding them a high-fat diet (HFD) for 6 weeks. Subsequently, the rats were divided into four groups, with Group 1 continuing on HFD, while groups 2, 3, and 4 received HFD supplemented with saroglitazar, curcumin, and both saroglitazar and curcumin, respectively. We evaluated the expression of Nrf2, ERK1/2, NOX1,2,4, antioxidant enzymes, PPARα, γ, and genes regulating lipid metabolism in the liver. Histopathology of liver tissue was also examined. Furthermore, we analyzed serum levels of lipid profiles and hepatic enzymes.
UNASSIGNED: Rats with NAFLD that received treatment involving saroglitazar and curcumin showed a significant decrease in the expression of ERK1/2, SREBP1, PPARγ, pro-inflammatory cytokines, NOXs, and ROS levels. Additionally, the levels of Nrf2, PPARα, and antioxidant enzymes showed a significant increase. The serum levels of lipid profiles and hepatic enzymes also decreased significantly after drug treatment.
UNASSIGNED: Our results confirm that both saroglitazar and curcumin ameliorate NAFLD by regulating the Nrf2 and ERK1/2 signaling pathways. These findings suggest that curcumin could serve as a suitable substitute for saroglitazar, although they appear to have a synergistic effect.

The proliferation and differentiation of skeletal muscle satellite cells is a complex physiological process involving various transcription factors and small RNA molecules. This study aimed to understand the regulatory mechanisms underlying these processes, focusing on interferon-related development factor 2 (IFRD2) as a target gene of miRNA-2400 in bovine skeletal MuSCs (MuSCs). IFRD2 was identified as a target gene of miRNA-2400 involved in regulating the proliferation and differentiation of bovine skeletal MuSCs. Our results indicate that miR-2400 can target binding the 3\'UTR of IFRD2 and inhibit its translation. mRNA and protein expression levels of IFRD2 increased significantly with increasing days of differentiation. Moreover, overexpression of the IFRD2 gene inhibited proliferation and promoted differentiation of bovine MuSCs. Conversely, the knockdown of the gene had the opposite effect. Overexpression of IFRD2 resulted in the inhibition of ERK1/2 phosphorylation levels in bovine MuSCs, which in turn promoted differentiation. In summary, IFRD2, as a target gene of miR-2400, crucially affects bovine skeletal muscle proliferation and differentiation by precisely regulating ERK1/2 phosphorylation.

The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a \"gatekeeper\" in controlling the selection of downstream effectors within the EGFR signaling network.

Protease-activated receptor 2 (PAR2) has garnered attention as a potential therapeutic target in breast cancer. PAR2 is implicated in the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) via G protein and beta-arrestin pathways, contributing to the proliferation and metastasis of breast cancer cells. Despite the recognized role of PAR2 in breast cancer progression, clinically effective PAR2 antagonists remain elusive. To address this unmet clinical need, we synthesized and evaluated a series of novel compounds that target the orthosteric site of PAR2. Using in silico docking simulations, we identified compound 9a, an optimized derivative of compound 1a ((S)-N-(1-(benzylamino)-1-oxo-3-phenylpropan-2-yl)benzamide), which exhibited enhanced PAR2 antagonistic activity. Subsequent molecular dynamics simulations comparing 9a with the partial agonist 9d revealed that variations in ligand-induced conformational changes and interactions dictated whether the compound acted as an antagonist or agonist of PAR2. The results of this study suggest that further development of 9a could contribute to the advancement of PAR2 antagonists as potential therapeutic agents for breast cancer.

Berberrubine (BRB), a main metabolite of berberine, has stronger hypoglycemic and lipid-lowering activity than its parent form. We previously found that BRB could cause obvious nephrotoxicity, but the molecular mechanism involved remains unknown. In this study, we systematically integrated metabolomics and quantitative proteomics to reveal the potential mechanism of nephrotoxicity caused by BRB. Metabolomic analysis revealed that 103 significant- differentially metabolites were changed. Among the mentioned compounds, significantly upregulated metabolites were observed for phosphorylcholine, sn-glycerol-3-phosphoethanolamine, and phosphatidylcholine. The top three enriched KEGG pathways were the mTOR signaling pathway, central carbon metabolism in cancer, and choline metabolism in cancer. ERK1/2 plays key roles in all three metabolic pathways. To further confirm the main signaling pathways involved, a proteomic analysis was conducted to screen for key proteins (such as Mapk1, Mapk14, and Caspase), indicating the potential involvement of cellular growth and apoptosis. Moreover, combined metabolomics and proteomics analyses revealed the participation of ERK1/2 in multiple metabolic pathways. These findings indicated that ERK1/2 regulated the significant- differentially abundant metabolites determined via metabolomics analysis. Notably, through a cellular thermal shift assay (CETSA) and molecular docking, ERK1/2 were revealed to be the direct binding target involved in BRB-induced nephrotoxicity. To summarize, this study sheds light on the understanding of severe nephrotoxicity caused by BRB and provides scientific basis for its safe use and rational development.

RNA virus induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as \'dysregulated immunity,\' is caused by viral single-stranded RNA (ssRNA) and double-stranded-RNA (dsRNA) mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We show that murine bone marrow-derived macrophages (BMDMs) stimulated with TLR3 and TLR7 agonists induce differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified a significantly higher cell-surface and endosomal TLR7 expression compared to TLR3, which further correlated with early and robust MAPK (pERK1/2 and p-P38) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2, p38, and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA-mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG responses, and blocking ERK1/2 activity would mitigate viral-RNA/TLR-induced dysregulated immunity.

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. Progesterone resistance, loss of progesterone receptor -B (PR-B) in the stromal cells of the endometrium, is one of the hallmarks of endometriosis and a major contributing factor for infertility in endometriosis patients. Loss of PR-B in the stromal cells of the endometriotic lesions poses resistance to the success of progesterone-based therapy. The working hypothesis is that PR-B is hypermethylated and epigenetically silenced, and inhibition of AKT and ERK1/2 pathways will decrease the hypermethylation, reverse the epigenetic silencing, and restore the expression of PR-B via DNA methylation and histone modification mechanisms in the endometriotic lesions. The objectives are to (i) determine the effects of dual inhibition of AKT and ERK1/2 pathways on the expression of PR-B and DNA methylation and histone modification protein machinery in the endometriotic lesions and (ii) identify the underlying epigenetic mechanisms of PR-B restoration in the endometriotic lesions. The results indicate that dual inhibition of AKT and ERK1/2 pathways decreases the hypermethylation, reverses the epigenetic silencing, and restores the expression of PR-B via DNA methylation and H3K9 and H3K27 methylation mechanisms in the endometriotic lesions or endometriotic stromal cells of human origin. These results support the novel concept that restored expression of PR-B in the endometriotic lesions and endometrium may improve the clinical outcome of progesterone therapy in endometriosis patients.