Search Results (219)

Resveratrol (RSV), a polyphenol found in a variety of berries and wines, is known for its anti-inflammatory, anticancer, and antioxidant properties. It has been suggested that RSV may play a role in the regulation of metabolic disorders, including diabetes and insulin resistance. However, in recent years, it has been reported to completely inhibit Akt kinase function in liver cells. Akt is a central protein involved in the metabolic function of insulin and is regulated by the phosphatidylinositol-3-kinase (PI3K) pathway. In this study, we examined the effect of RSV on insulin-induced insulin receptor (IR) phosphorylation and proteins involved in the PI3K/Akt pathway in a hepatic cell model, clone 9 (C9), and in hepatoma cells, Hepa 1-6 (H1-6). In both cell lines, RSV inhibited tyrosine phosphorylation of IR and insulin-induced activation of Akt. We also evaluated the effect of RSV on the activation of protein tyrosine phosphatase 1B (PTP1B), which is associated with IR dephosphorylation, and found that RSV increased PTP1B-Tyr¹⁵² phosphorylation in a time- and concentration-dependent manner. Furthermore, we found that the protein kinase C (PKC) inhibitors BIM and Gö6976 prevented the inhibition of Akt phosphorylation by RSV and increased the phosphorylation of Ser/Thr residues in IR, suggesting that PKC is involved in the inhibition of the insulin pathway by RSV. Thus, classical PKC isoforms impair the PI3K/Akt pathway at the IR and GSK3 and GS downstream levels; however, IRS-Tyr⁶³² phosphorylation remains unaffected. These results suggest that RSV can lead to insulin resistance by activating PTP1B and PKC, consequently affecting glucose homeostasis in hepatic cells. Full article

Breast cancer is the second most common cancer in the United States and consists of 30% of all new female cancer each year. PKC iota (PKC-$\iota$) is a bonafide human oncogene and is overexpressed in many types of cancer, including breast cancer. This study explores the role of PKC-$\iota$ in regulating the transcription factor Jun proto-oncogene (c-Jun), pro-inflammatory cytokine Tumor Necrosis Factor-alpha (TNF-$\alpha$), and the Mitogen-Activated Protein Kinase/Jun N-terminal kinase (MAPK/JNK) pathway, which also exhibits an oncogenic role in breast cancer. ICA-1S, a PKC-$\iota$ specific inhibitor, was used to inhibit PKC-$\iota$ to observe the subsequent effect on the levels of c-Jun, TNF-$\alpha$, and the MAPK/JNK signaling pathway. To obtain the results, cell proliferation assay, Western blotting, co-immunoprecipitation, small interfering RNA (siRNA), immunofluorescence, flow cytometry, cycloheximide (CHX) chase assay, and reverse transcription quantitative PCR (RT-qPCR) techniques were implemented. ICA-1S significantly inhibited cell proliferation and induced apoptosis in both breast cancer cell lines. Treatment with ICA-1S and siRNA also reduced the expression levels of the MAPK/JNK pathway protein, c-Jun, and TNF-$\alpha$ in both cell lines. PKC-$\iota$ was also found to be strongly associated with c-Jun, via which it regulated the MAPK/JNK pathway. Additionally, ICA-1S was found to promote the degradation of c-Jun and decrease the mRNA levels of c-Jun. We concluded that PKC-$\iota$ plays a crucial role in regulating breast cancer, and the inhibition of PKC-$\iota$ by ICA-1S reduces breast cancer cell proliferation and induces apoptosis. Therefore, targeting PKC-$\iota$ as a potential therapeutic target in breast cancer could be a significant approach in breast cancer research. Full article

Calcium (Ca²⁺) signaling is a fundamental regulatory mechanism controlling essential processes in the endothelium, vascular smooth muscle cells (VSMCs), and the extracellular matrix (ECM), including maintaining the endothelial barrier, modulation of vascular tone, and vascular remodeling. Cytosolic free Ca²⁺ concentration is tightly regulated by a balance between Ca²⁺ mobilization mechanisms, including Ca²⁺ release from the intracellular stores in the sarcoplasmic/endoplasmic reticulum and Ca²⁺ entry via voltage-dependent, transient-receptor potential, and store-operated Ca²⁺ channels, and Ca²⁺ elimination pathways including Ca²⁺ extrusion by the plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger and Ca²⁺ re-uptake by the sarco(endo)plasmic reticulum Ca²⁺-ATPase and the mitochondria. Some cell membranes/organelles are multifunctional and have both Ca²⁺ mobilization and Ca²⁺ removal pathways. Also, the individual Ca²⁺ handling pathways could be integrated to function in a regenerative, capacitative, cooperative, bidirectional, or reciprocal feed-forward or feed-back manner. Disruption of these pathways causes dysregulation of the Ca²⁺ signaling dynamics and leads to pathological cardiovascular conditions such as hypertension, coronary artery disease, atherosclerosis, and vascular calcification. In the endothelium, dysregulated Ca²⁺ signaling impairs nitric oxide production, reduces vasodilatory capacity, and increases vascular permeability. In VSMCs, Ca²⁺-dependent phosphorylation of the myosin light chain and Ca²⁺ sensitization by protein kinase-C (PKC) and Rho-kinase (ROCK) increase vascular tone and could lead to increased blood pressure and hypertension. Ca²⁺ activation of matrix metalloproteinases causes collagen/elastin imbalance and promotes vascular remodeling. Ca²⁺-dependent immune cell activation, leukocyte infiltration, and cholesterol accumulation by macrophages promote foam cell formation and atherosclerotic plaque progression. Chronic increases in VSMCs Ca²⁺ promote phenotypic switching to mesenchymal cells and osteogenic transformation and thereby accelerate vascular calcification and plaque instability. Emerging therapeutic strategies targeting these Ca²⁺-dependent mechanisms, including Ca²⁺ channel blockers and PKC and ROCK inhibitors, hold promise for restoring Ca²⁺ homeostasis and mitigating vascular disease progression. Full article

Reciprocal signaling between acute myeloid leukemia (AML) cells and the surrounding bone-marrow microenvironment (BMME) promotes AML progression through several mechanisms. One of the most important mechanisms is the induction of Galectin-3 (Gal-3) expression by AML cells and bone marrow mesenchymal stromal cells (BM-MSCs). Emerging evidence indicates that Gal-3 upregulation in the BMME promotes AML cell adhesion and survival, leading to the development of chemotherapy resistance, relapse, and poor prognosis. Identifying the biological function and critical signaling pathways of Gal-3 may contribute to overcoming acquired drug resistance and preventing post-treatment relapse. Gal-3 is involved in several molecular signaling pathways, including PI3K/AKT/mTOR, Ras/Raf/MEK/ERK, JAK/STAT, JNK, Wnt/β-catenin, PLC/PKC and NF-κB, which are interconnected to promote AML cell survival and resistance to chemotherapy. This review focuses on the biological effects, molecular mechanisms of action and regulation of Gal-3 in the pathogenesis and progression of AML. The therapeutic potential of potent synthetic small-molecule Gal-3 inhibitors in high-risk patients with AML is also discussed based on preclinical and clinical evidence from several human diseases. Currently, the effect of these Gal-3 inhibitors in AML has not been investigated either in vitro or in vivo. The findings provide a rationale for targeting Gal-3 that may be a very promising therapeutic approach, especially for patients with relapsed/refractory AML, and may enhance the efficacy of conventional chemotherapeutic drugs and/or immune checkpoint inhibitors. Full article

Background: Uveal melanoma (UM) is a relatively rare malignancy, yet it remains the most common primary intraocular cancer in adults. Several risk factors have been identified, including light iris color, fair skin tone, and cutaneous freckles. Methods: The aim of this article was an overview of the treatment methods for uveal melanoma, with a particular focus on emerging therapies such as tebentafusp and da-rovasertib. The research method was a review of the latest literature. Results: Genetic studies have uncovered key mutations in GNAQ and GNA11, which significantly contribute to UM pathogenesis. Treatment selection depends on tumor location and disease stage. In localized disease, radiotherapy—especially brachytherapy—is commonly used and generally effective. However, the prognosis worsens significantly once distant metastases, most often to the liver, develop, as no standard systemic therapy has demonstrated high efficacy in this setting. Recent years have seen the emergence of promising therapies, including tebentafusp, which stimulates immune responses against gp100-expressing melanoma cells, and darovasertib, a potent PKC inhibitor that targets MAPK pathway activation driven by GNAQ/GNA11 mutations. Both agents have shown encouraging tolerability; tebentafusp has demonstrated clinical benefit in Phase II and III trials, while darovasertib is still under investigation. Additionally, melphalan-based liver-directed therapy, particularly via hepatic arterial infusion (approved by the FDA), has shown potential in controlling liver-dominant disease in metastatic UM. This localized approach may provide significant benefit for patients with limited extrahepatic spread. Conclusions: Future research should focus on optimizing these novel strategies—tebentafusp, darovasertib, melphalan, and combination therapies—and on expanding our understanding of UM’s molecular drivers to enable the development of more effective, personalized treatments. Full article

Although chloroquine appears to inhibit the alpha-1 adrenoceptor, whether the chloroquine-mediated inhibition of phenylephrine-induced contraction is associated with the blockade of alpha-1 adrenoceptors remains unknown. This study examined the effect of chloroquine on contractions elicited by the alpha-1 adrenoceptor agonist phenylephrine in isolated rat aortas and determined the underlying mechanism. The effects of chloroquine and the alpha-1 adrenoceptor inhibitor prazosin on phenylephrine-elicited contractions were examined. The effects of the irreversible alpha-adrenoceptor inhibitor phenoxybenzamine followed by washout with fresh Krebs solution, as well as combined treatment with chloroquine and phenoxybenzamine followed by washout with fresh Krebs solution, on phenylephrine-induced contraction were investigated. Chloroquine and prazosin inhibited phenylephrine-induced contractions. However, pretreatment with prazosin eliminated the chloroquine-induced inhibition of contractions elicited by phenylephrine. Additionally, pretreatment with chloroquine and phenoxybenzamine followed by washout produced a higher contraction elicited by phenylephrine than pretreatment with phenoxybenzamine alone followed by washout. Although chloroquine did not affect the contraction induced by KCl in the endothelium-denuded aorta, it inhibited phenylephrine-induced protein kinase C (PKC) and myosin light-chain (MLC₂₀) phosphorylation and Rho-kinase membrane translocation. These results suggest that chloroquine inhibits vasoconstriction elicited by phenylephrine via alpha-1 adrenoceptor inhibition, which is mediated by decreased MLC₂₀ phosphorylation, the attenuation of PKC phosphorylation, and Rho-kinase membrane translocation. Full article

CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy. Full article

Understanding the mechanisms underlying glucocorticoid (GC) resistance in B-cell acute lymphoblastic leukemia (B-ALL) is essential to improve survival rates in relapsed children. We previously showed that GCs paradoxically induced their own resistance in B-ALL through CXCR4/PLC signaling, and that the inhibition of this pathway significantly reverses GC resistance in B-ALL cells and improves survival of GC-treated NSG mice in vivo. Here, we sought to determine whether the enhancement of GC sensitivity via inhibition of the CXCR4/PLC axis is associated with disruption of the mitochondrial pathway. Analysis of our previous transcriptomic data revealed that in B-ALL, the PLC inhibitor U73122 compromised multiple metabolic pathways related to metabolic reprogramming, mitochondrial function, and oxidative stress. Inhibition of PLC with U73122, protein kinase C with GF109203X, or CXCR4 with AMD3100 significantly potentiated dexamethasone (Dex)-induced mitochondrial membrane potential depolarization, reactive oxygen species production, cytochrome c release, caspase-3 activation, and decreased O₂ consumption in B-ALL cells. These observations were also confirmed after Dex treatment in a B-ALL Nalm-6 cell line transfected with CXCR4 small interfering RNA. Moreover, co-treatment with Dex and CXCR4, PKC, or PLC inhibitors increased the levels of the pro-apoptotic protein BIM (BCL-2 interacting mediator of cell death) and, consequently, promoted the cell death process. Together, these findings suggest that the CXCR4/PLC axis reduces Dex efficacy by limiting mitochondrial apoptotic activity. Full article

Ovarian cancer is the most lethal gynecological cancer, with a 5-year survival rate of approximately 50%. Mutation in the p53 gene and overexpression of the atypical protein kinase C iota (PKC-ι) are two phenomena widely manifested in ovarian cancer. This study investigated the role of PKC-ι-specific inhibitor ICA-1S and proteasome inhibitor MG-132 in ovarian cancer cell lines. To discern the result, cell proliferation assays, cytotoxicity assays, Western blotting, immunofluorescence, flow cytometry, small interfering RNA, and co-immunoprecipitation techniques were applied. ICA-1S and MG-132 were found to inhibit the proliferation of ovarian cancer cell lines significantly. ICA-1S reduced the level of oncogenic PKC-ι as expected. In addition, ICA-1S and MG-132 both were able to decrease the level of mutated p53 in the ES-2 cell line through separate pathways. On the contrary, MG-132 increased the level of wild-type p53 in the HEY-T30 cell line by inhibiting proteasomal degradation. MG-132 also induced apoptosis and autophagy in the ovarian cancer cell lines. We concluded that ICA-1S alone or in combination with MG-132 could be a potential treatment for mutated p53-containing and PKC-ι-overexpressing ovarian cancers. Full article

Berberine has been widely studied for its biological functions in various diseases, including cancer, diabetes, and cardiovascular diseases. Nevertheless, structural modifications of berberine have been demonstrated to augment its pharmacological efficacy in specific biological processes, particularly osteogenesis. In this study, we aimed to explore new berberine derivatives with pro-osteogenic activity and molecular mechanisms. Our results demonstrated that compound 13 is the most effective among the tested compounds. Compound 13 significantly enhanced BMP4-induced alkaline phosphatase (ALP) staining and increased the transcriptional activity of osteogenic markers such as ALP, Runt-related gene 2 (Runx2), and Osterix at both the mRNA and protein levels. Furthermore, we found that the Akt and PKC signaling pathways play crucial roles in compound 13-induced osteogenesis via treatment with specific inhibitors. The molecular docking results supported the potential interaction between compound 13 and these kinases. These findings highlighted the regulatory role of compound 13 in osteoblast differentiation via the Akt and PKC signaling pathways. Overall, our study provides compelling evidence that compound 13 is a promising therapeutic candidate for the treatment of osteoporosis, with the potential for further development and optimization to improve bone health and strength. Full article

Beyond the regulation of cholesterol metabolism, a number of extrahepatic functions of proprotein convertase subtilisin/kexin type 9 (PCSK9) have been increasingly identified. The main purpose of this study was to verify whether PCSK9 expression in vascular smooth muscle cells (VSMC) is influenced by insulin resistance and high glucose (HG). In cultured rat aortic VSMC from lean insulin-sensitive Zucker rats (LZRs) and obese insulin-resistant Zucker rats (OZRs), a classical animal model of insulin resistance, we evaluated PCSK9 expression with or without the monoclonal antibodies against PCSK9 Alirocumab and Evolocumab or the synthetic PCSK9-binding peptide PEP 2-8. Effects and molecular mechanisms underlying altered PCSK9 expression were evaluated by proliferation and migration assay, reactive oxygen species (ROS) production, and involvement of PKC, NADPH-oxidase, MAPK/ERK-1/2 pathway activation. As a result, we found that, in comparison with LZR, VSMC from OZR showed basal PCSK9 overexpression mitigated by Alirocumab, Evolocumab, PEP 2-8, and the inhibitors of PKC, NADPH-oxidase, and MAPK. The finding of PCSK9 upregulation in VSMC from OZR paralleled with increased ROS production, proliferation, and migration. HG increased PCSK9 expression in VSMC from LZR, but not in OZR, via oxidative stress and with effects reduced by PCSK9 inhibitors. These findings suggest that a dysregulation of PCSK9 in VSMC could be involved in vascular damage in metabolic disorders, such as obesity and diabetes. Full article

The receptor transporter protein 4 (RTP4) is a receptor chaperone protein that targets class A G-protein coupled receptor (GPCR)s. Recently, it has been found to play a role in peripheral inflammatory regulation, as one of the interferon-stimulated genes (ISGs). However, the detailed role of RTP4 in response to inflammatory stress in the central nervous system has not yet been fully understood. While we have previously examined the role of RTP4 in the brain, particularly in neuronal cells, this study focuses on its role in microglial cells, immunoreactive cells in the brain that are involved in inflammation. For this, we examined the changes in the RTP4 levels in the microglial cells after exposure to inflammatory stress. We found that lipopolysaccharide (LPS) treatment (0.1~1 µg/mL, 24 h) significantly upregulated the RTP4 mRNA levels in the microglial cell line, SIM-A9. Furthermore, the interferon (IFN)-β mRNA levels and extracellular levels of IFN-β were also increased by LPS treatment. This upregulation was reversed by treatment with neutralizing antibodies targeting either the interferon receptor (IFNR) or toll-like receptor 4 (TLR4), and with a TLR4 selective inhibitor, or a Janus kinase (JAK) inhibitor. On the other hand, the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, significantly enhanced the increase in RTP4 mRNA following LPS treatment, whereas the PKC inhibitor, calphostin C, had no effect. These findings suggest that in microglial cells, LPS-induced inflammatory stress activates TLR4, leading to the production of type I IFN, the activation of IFN receptor and JAK, and finally, the induction of RTP4 gene expression. Based on these results, we speculate that RTP4 functions as an inflammation-responsive molecule in the brain. However, further research is needed to fully understand its role. Full article

SET and MYND Domain-Containing 2 (Smyd-2), a specific protein lysine methyltransferase (PKMT), influences both histones and non-histones. Its role in cerebral ischemia/reperfusion (CIR), particularly in ferroptosis—a regulated form of cell death driven by lipid peroxidation—remains poorly understood. This study identifies the expression of Smyd-2 in the brain and investigates its relationship with neuronal programmed cell death (PCD). We specifically investigated how Smyd-2 regulates ferroptosis in CIR through its interaction with the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway. Smyd-2 knockout protects HT-22 cells from Erastin-induced ferroptosis but not TNF-α + Smac-mimetic-induced apoptosis/necroptosis. This neuroprotective effect of Smyd-2 knockout in HT-22 cells after Oxygen–Glucose Deprivation/Reperfusion (OGD/R) was reversed by Erastin. Smyd-2 knockout in HT-22 cells shows neuroprotection primarily via the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway, despite the concurrent upregulation of Smyd-2 and Nrf-2 observed in both the middle cerebral artery occlusion (MCAO) and OGD/R models. Interestingly, vivo experiments demonstrated that Smyd-2 knockout significantly reduced ferroptosis and lipid peroxidation in hippocampal neurons following CIR. Moreover, the Nrf-2 inhibitor ML-385 abolished the neuroprotective effects of Smyd-2 knockout, confirming the pivotal role of Nrf-2 in ferroptosis regulation. Cycloheximide (CHX) fails to reduce Nrf-2 expression in Smyd-2 knockout HT-22 cells. Smyd-2 knockout suppresses Nrf-2 lysine methylation, thereby promoting the Nrf-2/Keap-1 pathway without affecting the PKC-δ/Nrf-2 pathway. Conversely, Smyd-2 overexpression disrupts Nrf-2 nuclear translocation, exacerbating ferroptosis and oxidative stress, highlighting its dual regulatory role. This study underscores Smyd-2’s potential for ischemic stroke treatment by disrupting the Smyd-2/Nrf-2-driven antioxidant capacity, leading to hippocampal neuronal ferroptosis. By clarifying the intricate interplay between ferroptosis and oxidative stress via the Nrf-2/Keap-1 pathway, our findings provide new insights into the molecular mechanisms of CIR and identify Smyd-2 as a promising therapeutic target. Full article

Anticancer drugs cause anemia in patients through eryptosis and hemolysis. We thus studied the in vitro toxicity of galangin (GAL) in red blood cells (RBCs). RBCs were exposed to 50–500 μM of GAL and analyzed for markers of eryptosis and hemolysis. Ca²⁺ nucleation, phosphatidylserine (PS) externalization, oxidative stress, and cell size were detected via fluorescence-activated cell sorting using Fluo4/AM, annexin-V-FITC, 2′,7′-dichlorodihydrofluorescein diacetate, and forward scatter (FSC), respectively. Acetylcholinesterase (AChE) activity was measured via Ellman’s assay and ultrastructural morphology was examined via scanning electron microscopy. Membrane rupture and extracellular hemoglobin, aspartate transaminase (AST), and lactate dehydrogenase (LDH) were assessed via colorimetric methods. Distinct experiments were carried out to identify protective agents and signaling pathways using small-molecule inhibitors. GAL triggered sucrose-sensitive hemolysis with AST and LDH leakage, increased annexin-V-FITC and Fluo4 fluorescence, and decreased FSC and AChE activity which was associated with the formation of granulated echinocytes. Ca²⁺ omission and energy replenishment with glucose, adenine, and guanosine blunted PS externalization and preserved cellular volume. Moreover, caffeine, Trolox, heparin, and uric acid had similar ameliorative effects. Hemolysis was abrogated via caffeine, Trolox, heparin, mannitol, lactate, melatonin, and PEG 8000. Notably, co-treatment of cells with GAL and staurosporin, D4476, or acetylsalicylic acid prevented PS externalization whereas only the presence of SB203580 and NSC23766 rescued the cells from GAL-induced hemolysis. Ca²⁺ nucleation and metabolic collapse mediated by PKC/CK1α/COX/p38/Rac1 drive GAL-induced eryptosis and hemolysis. These novel findings carry ramifications for the clinical prospects of GAL in anticancer therapy. Full article

Septic patients, coupling severe disseminated intravascular coagulation (DIC) and thrombocytopenia, have poor prognoses and higher mortality. The platelet glycoprotein Ibα (GPIbα) is involved in thrombosis, hemostasis, and inflammation response. However, it remains unclear whether the GPIbα cytoplasmic tail regulates sepsis-mediated platelet activation and inflammation, especially in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections. Using a mouse model of S. aureus-induced bacteremia, we found that both 10 amino acids of GPIbα C-terminal sequence deficiency and pharmacologic inhibition of protein kinase C (PKC) alleviated pathogenesis by diminishing platelet activation and aggregate formation. Furthermore, the GPIbα cytoplasmic tail promoted the phagocytosis of platelets by Kupffer cells in vivo. The genetically deficient GPIbα cytoplasmic tail also downregulated inflammatory cytokines and reduced liver damage, ultimately improving the survival rate of the septic mice. Our results illustrate that the platelet GPIbα cytoplasmic domain exacerbates excessive platelet activation and inflammation associated with sepsis through a PKC-dependent pathway. Thus, our findings provide insights for the development of effective therapeutic strategies using PKC inhibitor treatment against bacterial infection. Full article