Valproic Acid Treatment after Traumatic Brain Injury in Mice Alleviates Neuronal Death and Inflammation in Association with Increased Plasma Lysophosphatidylcholines.

The histone deacetylase inhibitor (HDACi) valproic acid (VPA) has neuroprotective and anti-inflammatory effects in experimental traumatic brain injury (TBI), which have been partially attributed to the epigenetic disinhibition of the transcription repressor RE1-Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). Additionally, VPA changes post-traumatic brain injury (TBI) brain metabolism to create a neuroprotective environment. To address the interconnection of neuroprotection, metabolism, inflammation and REST/NRSF after TBI, we subjected C57BL/6N mice to experimental TBI and intraperitoneal VPA administration or vehicle solution at 15 min, 1, 2, and 3 days post-injury (dpi). At 7 dpi, TBI-induced an up-regulation of REST/NRSF gene expression and HDACi function of VPA on histone H3 acetylation were confirmed. Neurological deficits, brain lesion size, blood-brain barrier permeability, or astrogliosis were not affected, and REST/NRSF target genes were only marginally influenced by VPA. However, VPA attenuated structural damage in the hippocampus, microgliosis and expression of the pro-inflammatory marker genes. Analyses of plasma lipidomic and polar metabolomic patterns revealed that VPA treatment increased lysophosphatidylcholines (LPCs), which were inversely associated with interleukin 1 beta (Il1b) and tumor necrosis factor (Tnf) gene expression in the brain. The results show that VPA has mild neuroprotective and anti-inflammatory effects likely originating from favorable systemic metabolic changes resulting in increased plasma LPCs that are known to be actively taken up by the brain and function as carriers for neuroprotective polyunsaturated fatty acids.

Long-Term Epigenetic Regulation of Foxo3 Expression in Neonatal Valproate-Exposed Rat Hippocampus with Sex-Related Differences.

Perinatal exposure to valproic acid is commonly used for autism spectrum disorder (ASD) animal model development. The inhibition of histone deacetylases by VPA has been proposed to induce epigenetic changes during neurodevelopment, but the specific alterations in genetic expression underlying ASD-like behavioral changes remain unclear. We used qPCR-based gene expression and epigenetics tools and Western blotting in the hippocampi of neonatal valproic acid-exposed animals at 4 weeks of age and conducted the social interaction test to detect behavioral changes. Significant alterations in gene expression were observed in males, particularly concerning mRNA expression of Foxo3, which was significantly associated with behavioral changes. Moreover, notable differences were observed in H3K27ac chromatin immunoprecipitation, quantitative PCR (ChIP-qPCR), and methylation-sensitive restriction enzyme-based qPCR targeting the Foxo3 gene promoter region. These findings provide evidence that epigenetically regulated hippocampal Foxo3 expression may influence social interaction-related behavioral changes. Furthermore, identifying sex-specific gene expression and epigenetic changes in this model may elucidate the sex disparity observed in autism spectrum disorder prevalence.

Reversing valproic acid-induced autism-like behaviors through a combination of low-frequency repeated transcranial magnetic stimulation and superparamagnetic iron oxide nanoparticles.

Transcranial magnetic stimulation (TMS) is a neurostimulation device used to modulate brain cortex activity. Our objective was to enhance the therapeutic effectiveness of low-frequency repeated TMS (LF-rTMS) in a rat model of autism spectrum disorder (ASD) induced by prenatal valproic acid (VPA) exposure through the injection of superparamagnetic iron oxide nanoparticles (SPIONs). For the induction of ASD, we administered prenatal VPA (600 mg/kg, I.P.) on the 12.5th day of pregnancy. At postnatal day 30, SPIONs were injected directly into the lateral ventricle of the brain. Subsequently, LF-rTMS treatment was applied for 14 consecutive days. Following the treatment period, behavioral analyses were conducted. At postnatal day 60, brain tissue was extracted, and both biochemical and histological analyses were performed. Our data revealed that prenatal VPA exposure led to behavioral alterations, including changes in social interactions, increased anxiety, and repetitive behavior, along with dysfunction in stress coping strategies. Additionally, we observed reduced levels of SYN, MAP2, and BDNF. These changes were accompanied by a decrease in dendritic spine density in the hippocampal CA1 area. However, LF-rTMS treatment combined with SPIONs successfully reversed these dysfunctions at the behavioral, biochemical, and histological levels, introducing a successful approach for the treatment of ASD.

Valproate modulates the activity of multidrug resistance efflux pumps, as a chemoresistance factor in gastric cancer cells.

BACKGROUND: Drug resistance is one of the most critical problems in gastric cancer therapy. This study was performed to investigate the valproic acid effects on the proliferation of sensitive and resistant cell lines of human gastric cancer, and to explore the mechanism of the agent on multi drug resistance and apoptosis genes. METHODS: The cytotoxicity effect of valproic acid on the EPG85.257 and EPG85.257RDB cells was assessed by the MTT assay, and the IC50 concentration was evaluated. Apoptosis, genotoxicity, and drug resistance pump activity were evaluated using comet assay, Real-time PCR, and flow cytometry, respectively. Cell proliferation was assayed using a scratch test. RESULTS: Dose-dependent toxicity was recorded after treatment of cells with valproic acid. Valproic acid represented a significant growth inhibition on EPG85.257 cells with IC50 values of 5.84 µM and 4.78 µM after 48 h and 72 h treatment, respectively. In contrast, the drug-resistant counterpart represented 8.7 µM and 7.02 µM IC50 values after the same treatment time. Valproic acid induced PTEN, Bcl2, P53, Bax, P21, and caspase3 expression in EPG85.257 cells, whereas p21, p53, PTEN, and ABCB1 were overexpressed in EPG5.257RDB. Valproic acid hindered cell migration in both cell lines (P < 0.01). Valproate genotoxicity was significantly higher in the parent cells than in their resistant EPG85.257RDB counterparts. Valproate led to a 62% reduction in the daunorubicin efflux of the MDR1 pump activity. CONCLUSIONS: Valproate can affect drug resistance in gastric cancer via a unique mechanism independent of MDR1 expression.

Investigating the synergy of Shikonin and Valproic acid in inducing apoptosis of osteosarcoma cells via ROS-mediated EGR1 expression.

BACKGROUND: Osteosarcoma is the most prevalent malignant bone tumour with a poor prognosis. Shikonin (SHK) is derived from the traditional Chinese medicine Lithospermum that has been extensively studied for its notable anti-tumour effects, including for osteosarcoma. However, its application has certain limitations. Valproic acid (VPA) is a histone deacetylase inhibitor (HDACI) that has recently been employed as an adjunctive therapeutic agent that allows chromatin to assume a more relaxed state, thereby enhancing anti-tumour efficacy. PURPOSE: This study was aimed to investigate the synergistic anti-tumour efficacy of SHK in combination with VPA and elucidate its underlying mechanism. METHODS/STUDY DESIGN: CCK-8 assays were utilized to calculate the combination index. Additional assays, including colony formation, acridine orange/ethidium bromide double fluorescent staining, and flow cytometry, were employed to evaluate the effects on osteosarcoma cells. Wound healing and transwell assays were utilized to assess cell mobility. RNA sequencing, PCR, and Western blot analyses were conducted to uncover the underlying mechanism. Rescue experiments were performed to validate the mechanism of apoptotic induction. The impact of SHK and VPA combination treatment on primary osteosarcoma cells was also assessed. Finally, in vivo experiments were conducted to validate its anti-tumour effects and mechanism. RESULTS: The combination of SHK and VPA synergistically inhibited the proliferation and migration of osteosarcoma cells in vitro and induced apoptosis in these cells. Through a comprehensive analysis involving RNA sequencing, PCR, Western blot, and rescue experiments, we have substantiated our hypothesis that the combination of SHK and VPA induced apoptosis via the ROS-EGR1-Bax axis. Importantly, our in vivo experiments corroborated these findings, demonstrating the potential of the SHK and VPA combination as a promising therapeutic approach for osteosarcoma. CONCLUSION: The combination of SHK and VPA exerted an anti-tumour effect by inducing apoptosis through the ROS-EGR1-Bax pathway. Repurposing the old drug VPA demonstrated its effectiveness as an adjunctive therapeutic agent for SHK, enhancing its anti-tumour efficacy and revealing its potential value. Furthermore, our study expanded the application of natural compounds in the anti-tumour field and overcame some of their limitations through combination therapy. Finally, we enhanced the understanding of the mechanistic pathways linking reactive oxygen species (ROS) accumulation and apoptosis in osteosarcoma cells. Additionally, we elucidated the role of EGR1 in osteosarcoma cells, offering novel strategies and concepts for the treatment of osteosarcoma.

Benefits of bone marrow mesenchymal stem cells compared to their conditioned medium in valproic acid-induced autism in rats.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behaviors, a limited range of activities, and deficiencies in social communications. Bone marrow mesenchymal stem cells (BM-MSCs), which secrete factors that stimulate surrounding microenvironment, and BM-MSCs conditioned medium (BM-MSCs-CM), which contains cell-secreted products, have been speculated to hold potential as a therapy for ASD. This study aimed to compare the therapeutic effects of BM-MSCs and BM-MSCs-CM on behavioral and microglial changes in an animal model of autism induced by valproic acid (VPA). METHODS AND RESULTS: Pregnant Wistar rats were administered by VPA at a dose of 600 mg/kg at 12.5 days post-conception. After birth, male pups were included in the study. At 6 weeks of age, one group of rats received intranasal administration of BM-MSCs, while another group received BM-MSCs-CM. The rats were allowed to recover for 2 weeks. Behavioral tests, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry were performed. Both BM-MSCs and BM-MSCs-CM administration significantly improved some behavioral deficits. Furthermore, these treatments notably reduced Iba-1 marker associated with microgliosis. Additionally, there was a significant reduction in the expression of pro-inflammatory cytokines IL-1ß and IL-6, and an increase in the levels of the anti-inflammatory cytokine IL-10 in rats administered by BM-MSCs and BM-MSCs-CM. CONCLUSIONS: Post-developmental administration of BM-MSCs and BM-MSCs-CM can ameliorate prenatal neurodevelopmental deficits, restore cognitive and social behaviors, and modulate microglial and inflammatory markers. Results indicated that the improvement rate was higher in the BM-MSCs group than BM-MSCs-CM group.

Mechanistic Insights about Sorafenib-, Valproic Acid- and Metformin-Induced Cell Death in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is among the main causes of death by cancer worldwide, representing about 80-90% of all liver cancers. Treatments available for advanced HCC include atezolizumab, bevacizumab, sorafenib, among others. Atezolizumab and bevacizumab are immunological options recently incorporated into first-line treatments, along with sorafenib, for which great treatment achievements have been reached. However, sorafenib resistance is developed in most patients, and therapeutical combinations targeting cancer hallmark mechanisms and intracellular signaling have been proposed. In this review, we compiled evidence of the mechanisms of cell death caused by sorafenib administered alone or in combination with valproic acid and metformin and discussed them from a molecular perspective.

TREM2 improves microglia function and synaptic development in autism spectrum disorders by regulating P38 MAPK signaling pathway.

BACKGROUND: Autism spectrum disorder (ASD) encompasses a diverse range of neurodevelopmental disorders, but the precise underlying pathogenesis remains elusive. This study aim to explore the potential mechanism of TREM2 in regulating microglia function in ASD. MATERIALS AND METHODS: The offspring rat model of ASD was established through prenatal exposure to valproic acid (VPA), and the behavioral symptoms of the ASD model were observed. On postnatal day (PND) 7 and PND 28, the effects of prenatally exposure to VPA on synaptic development and microglia phenotype of offspring rats were observed. Primary microglia were cultured in vitro. Lentivirus and adenovirus were utilized to interfere with TREM2 and overexpress TREM2. RESULTS: Prenatally VPA exposure induced offspring rats to show typical ASD core symptoms, which led to abnormal expression of synapse-related proteins in the prefrontal cortex of offspring rats, changed the phenotype of microglia in offspring rats, promoted the polarization of microglia to pro-inflammatory type, and increased inflammatory response. The experimental results in vitro showed that overexpression of TREM2 could increase the expression of Gephyrin, decrease the content of CD86 protein and increase the content of CD206 protein. In addition, after the expression of TREM2 was interfered, the content of p-P38 MAPK protein increased and the content of p-ELK-1 protein decreased. CONCLUSION: The protective influence of TREM2 on the VPA-induced ASD model is attributed to its inhibition of the P38 MAPK pathway, this protective effect may be achieved by promoting the polarization of microglia to anti-inflammatory phenotype and improving the neuronal synaptic development.

Fucoxanthin mitigates valproic acid-induced autistic behavior through modulation of the AKT/GSK-3ß signaling pathway.

This study aimed to investigate the effects of fucoxanthin, a natural compound found in seaweed, on various aspects of autism using a rat model induced by valproic acid (VPA). Pregnant rats were administered VPA (600 mg/kg) on gestational day 12.5, and male pups were orally administered fucoxanthin at 50, 100, or 200 mg/kg beginning on post-natal day (PND) 23-43. Behavioral assessments were conducted on PND 45-53, and on PND 54, the animals were sacrificed for further biochemical analyses (superoxide dismutase (SOD) and glutathione (GSH), nitric oxide (NO)) via UV spectroscopy. Inflammatory markers (IL-17, TNF-α, and IL-1ß) were also analyzed by sandwich ELISA, and the molecular parameters were evaluated through ELISA. The results revealed that, compared with VPA, fucoxanthin improved behavior and neuronal morphology. Specifically, fucoxanthin administration was found to enhance spatial memory, reduce pain sensitivity, and improve social interaction, locomotor activity, balance, and motor coordination. Fucoxanthin also exhibited anti-inflammatory and antioxidant effects, as indicated by the restoration of SOD and GSH levels and reduced inflammatory cytokine levels. Molecular analyses revealed that fucoxanthin restored the levels of GSK-3ß and AKT. Furthermore, fucoxanthin regulates neurotransmitters, which are related to increasing GABA and reducing glutamate levels in the cortex and cerebellum. The therapeutic effects were dose-dependent, with higher doses (200 mg/kg) showing greater efficacy than lower doses (100 mg/kg) in improving behavioral, biochemical, neurotransmitter, and molecular parameters. Fucoxanthin is a potential treatment for autism, but further research, including clinical trials, is necessary to determine its effectiveness in humans.

Valproic acid regulates the miR-155/Jarid2 axis by affecting miR-155 promoter methylation in glioma.

The most frequent primary brain tumor in adults is glioma, yet no effective curative treatments are currently available. Our previous study demonstrated the enhancing effects of JARID2 on glioma sensitivity to TMZ treatment. In this study, miR-155 is predicted to target JARID2. miR-155 is overexpressed in clinical glioma specimens and cell lines. miR-155 overexpression in glioma cells enhances cell viability and represses cell apoptosis. Through targeting, miR-155 inhibits JARID2 expression. miR-155 inhibition inhibits glioma cell viability and enhances cell apoptosis, whereas JARID2 knockdown enhances cell viability and inhibits cell apoptosis; JARID2 knockdown partially reverses miR-155 inhibition effects on glioma phenotypes. miR-155 inhibition reduces but knockdown of JARID2 promotes the tumor formation ability of glioma cells in vivo. Valproic acid (VPA) upregulates JARID2 expression, inhibits glioma cell viability and enhances cell apoptosis. VPA downregulates the expression level of miR-155 by increasing the methylation level of the miR-155 promoter, suggesting that the miR-155/JARID2 axis is implicated in VPA inhibition of glioma cell viability and enhancement of glioma cell apoptosis. This study demonstrates a new mechanism of VPA treatment of gliomas by affecting the miR-155/JARID2 axis, which could be regarded as a new strategy for the prevention and treatment of glioma.

Effect of Valproic Acid on Promoting the Differentiation of Human Embryonic Stem Cells Into Cholangiocyte-Like Cells.

Cholangiocytes form a complex 3D network of bile ducts in the liver and contribute to liver function. The damage or destruction of cholangiocytes can lead to biliary diseases, and the shortage of cholangiocytes remains an obstacle for drug development targeting biliary diseases. Valproic acid (VPA) is a potent activator of Notch signaling pathway that is essential for cholangiocyte differentiation. Here, we report a VPA-based approach for cholangiocyte differentiation of human pluripotent stem cells. VPA activated Notch2 expression and upregulated HES-1, HEY-1, and Sox9 gene expression in hESC-derived hepatoblast. After 7 days treatment, VPA promoted successful differentiation of hepatoblast into cholangiocytes expressing cholangiocyte marker genes (AE2, AQP1, CFTR) and proteins (CK19 and CK7). In addition, the differentiated cholangiocytes formed bile duct-like structures after implantation into the spleen of NOD/SCID mice. Our results suggested that VPA can promote hESC differentiation to cholangiocyte-like cells. The induced cholangiocytes may serve as a potential cell source for both in vitro modeling and regenerative therapy of cholangiopathies. The findings can also support further development of small-molecule based differentiation protocols for cholangiocyte production.

Valproic acid increased the efficacy of EGFR TKIs on EGFR/TP53 co-mutated lung cancers and downregulated mutant-p53 levels.

The TP53 tumor suppressor is the most frequently mutated gene in human cancers. For p53-targeted therapy, one of the strategies was targeting mutant p53 for degradation. In EGFR-mutated lung cancer patients, concurrent TP53 mutation was associated with faster resistance to EGFR-TKIs. In this study, we discovered that valproic acid (VPA), a widely prescribed antiseizure medication, had a synergic effect on sensitive as well as acquired resistant lung cancers with EGFR/TP53 co-mutation in combination with EGFR-TKIs. In both in vitro and in vivo models, VPA greatly improved the efficacy of EGFR-TKIs, including forestalling the occurrence of acquired resistance and increasing the sensitivity to EGFR-TKIs. Mechanistically, VPA dramatically promoted degradation of mutant p53 in both sensitive and acquired resistant cells while inhibited mutant TP53 mRNA transcription only in sensitive cells. Together, this study suggested that VPA combination treatment could have beneficial effects on EGFR-mutant lung cancers with concurrent p53 mutation in both early and late stages, expanding the potential clinical applications for VPA.

Breast cancer resistance protein polymorphism ABCG2 c.421C>A (rs2231142) moderates the effect of valproate on lamotrigine trough concentrations in adults with epilepsy.

BACKGROUND: Valproate inhibits clearance of lamotrigine and greatly increases its concentrations. We assessed whether this effect was moderated by a polymorphism (ABCG2 c.421C>A) of the breast cancer resistance protein. METHODS: In two consecutive independent studies in adults with epilepsy on lamotrigine monotherapy or cotreated with valproate: (i) Exposure to valproate was considered treatment, (ii) dose-adjusted lamotrigine troughs at steady state were the outcome, and (iii) ABCG2 c.421C>A genotype (wild-type [wt] homozygosity or variant carriage) was the tested moderator. We used entropy balancing (primary analysis) and exact/optimal full matching (secondary analysis) to control for confounding, including polymorphisms (and linked polymorphisms) suggested to affect exposure to lamotrigine (UGT1A4*3 c.142T>G, rs2011425; UGT2B7-161C>T, rs7668258; ABCB1 1236C>T, rs1128503) to generate frequentist and Bayesian estimates of valproate effects (geometric means ratios [GMR]). RESULTS: The two studies yielded consistent results (replicated); hence, we analyzed combined data (total N = 471, 140 treated, 331 controls, 378 ABCG2 c.421C>A wt subjects, 93 variant carriers). Primary analysis: in variant carriers, valproate effect (GMR) on lamotrigine (treated, n = 21 vs. controls, n = 72) was around 60% higher than in wt subjects (treated, n = 119 vs. controls, n = 259)-ratio of GMRs 1.61 (95%CI 1.23-2.11) (frequentist) and 1.63 (95%CrI 1.26-2.10) (Bayes). Similar differences in valproate effects between variant carriers and wt subjects were found in the secondary analysis (valproate troughs up to 364 µmol/L vs. no valproate; or valproate ≥364 µmol/L vs. no valproate). Susceptibility of the estimates to unmeasured confounding was low. CONCLUSION: Data suggest that polymorphism rs2231142 moderates the effect of valproate on exposure to lamotrigine.

Valproic acid ameliorates cauda equina injury by suppressing HDAC2-mediated ferroptosis.

INTRODUCTION: Persistent neuroinflammatory response after cauda equina injury (CEI) lowers nociceptor firing thresholds, accompanied by pathological pain and decreasing extremity dysfunction. Histone deacetylation has been considered a key regulator of immunity, inflammation, and neurological dysfunction. Our previous study suggested that valproic acid (VPA), a histone deacetylase inhibitor, exhibited neuroprotective effects in rat models of CEI, although the underlying mechanism remains elusive. METHODS: The cauda equina compression surgery was performed to establish the CEI model. The Basso, Beattie, Bresnahan score, and the von Frey filament test were carried out to measure the animal behavior. Immunofluorescence staining of myelin basic protein and GPX4 was carried out. In addition, transmission electron microscope analysis was used to assess the effect of VPA on the morphological changes of mitochondria. RNA-sequencing was conducted to clarify the underlying mechanism of VPA on CEI protection. RESULTS: In this current study, we revealed that the expression level of HDAC1 and HDAC2 was elevated after cauda equina compression model but was reversed by VPA treatment. Meanwhile, HDAC2 knockdown resulted in the improvement of motor functions and pathologic pain, similar to treatment with VPA. Histology analysis also showed that knockdown of histone deacetylase (HDAC)-2, but not HDAC1, remarkably alleviated cauda equina injury and demyelinating lesions. The potential mechanism may be related to lowering oxidative stress and inflammatory response in the injured region. Notably, the transcriptome sequencing indicated that the therapeutic effect of VPA may depend on HDAC2-mediated ferroptosis. Ferroptosis-related genes were analyzed in vivo and DRG cells further validated the reliability of RNA-sequencing results, suggesting HDAC2-H4K12ac axis participated in epigenetic modulation of ferroptosis-related genes. CONCLUSION: HDAC2 is critically involved in the ferroptosis and neuroinflammation in cauda equina injury, and VPA ameliorated cauda equina injury by suppressing HDAC2-mediated ferroptosis.

Cholesterol deficiency as a mechanism for autism: A valproic acid model.

Dysregulated cholesterol metabolism represents an increasingly recognized feature of autism spectrum disorder (ASD). Children with fetal valproate syndrome caused by prenatal exposure to valproic acid (VPA), an anti-epileptic and mood-stabilizing drug, have a higher incidence of developing ASD. However, the role of VPA in cholesterol homeostasis in neurons and microglial cells remains unclear. Therefore, we examined the effect of VPA exposure on regulation of cholesterol homeostasis in the human microglial clone 3 (HMC3) cell line and the human neuroblastoma cell line SH-SY5Y. HMC3 and SH-SY5Y cells were each incubated in increasing concentrations of VPA, followed by quantification of mRNA and protein expression of cholesterol transporters and cholesterol metabolizing enzymes. Cholesterol efflux was evaluated using colorimetric assays. We found that VPA treatment in HMC3 cells significantly reduced ABCA1 mRNA, but increased ABCG1 and CD36 mRNA levels in a dose-dependent manner. However, ABCA1 and ABCG1 protein levels were reduced by VPA in HMC3. Furthermore, similar experiments in SH-SY5Y cells showed increased mRNA levels for ABCA1, ABCG1, CD36, and 27-hydroxylase with VPA treatment. VPA exposure significantly reduced protein levels of ABCA1 in a dose-dependent manner, but increased the ABCG1 protein level at the highest dose in SH-SY5Y cells. In addition, VPA treatment significantly increased cholesterol efflux in SH-SY5Y, but had no impact on efflux in HMC3. VPA differentially controls the expression of ABCA1 and ABCG1, but regulation at the transcriptional and translational levels are not consistent and changes in the expression of these genes do not correlate with cholesterol efflux in vitro.

Untargeted LC-MS/MS Metabolomics Study of HO-AAVPA and VPA on Breast Cancer Cell Lines.

Breast cancer (BC) is one of the biggest health problems worldwide, characterized by intricate metabolic and biochemical complexities stemming from pronounced variations across dysregulated molecular pathways. If BC is not diagnosed early, complications may lead to death. Thus, the pursuit of novel therapeutic avenues persists, notably focusing on epigenetic pathways such as histone deacetylases (HDACs). The compound N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA), a derivative of valproic acid (VPA), has emerged as a promising candidate warranting pre-clinical investigation. HO-AAVPA is an HDAC inhibitor with antiproliferative effects on BC, but its molecular mechanism has yet to be deciphered. Furthermore, in the present study, we determined the metabolomic effects of HO-AAVPA and VPA on cells of luminal breast cancer (MCF-7) and triple-negative breast cancer (MDA-MB-231) subtypes. The LC-MS untargeted metabolomic study allowed for the simultaneous measurement of multiple metabolites and pathways, identifying that both compounds affect glycerophospholipid and sphingolipid metabolism in the MCF-7 and MDA-MB-231 cell lines, suggesting that other biological targets were different from HDACs. In addition, there are different dysregulate metabolites, possibly due to the physicochemical differences between HO-AAVPA and VPA.

Modulation of PI3K/Akt/GSK3ß signaling cascade through G protein-coupled receptor 55 (GPR55) activation: Prenatal lysophosphatidylinositol attenuates valproic acid-induced synaptic abnormalities and mitochondrial dysfunction.

AIMS: Dysregulation of PI3K/Akt/GSK3ß signaling has been implicated in various neurological disorders, including autism spectrum disorder (ASD). G protein-coupled receptor 55 (GPR55) has recently emerged as a potential regulator of this signaling cascade. This study explores the intricate modulation of the PI3K/Akt/GSK3ß signaling cascade via GPR55 activation and its potential therapeutic implications in the context of autism-associated neuronal impairments. MAIN METHODS: Valproic acid (VPA) was administered on embryonic day 12 (E12) to induce ASD, and lysophosphatidylinositol (LPI), a GPR55 agonist, was used prenatally to modulate the receptor activity. Golgi-cox staining was performed to observe neuronal morphology, and Hematoxylin and eosin (H and E) staining was carried out to quantify damaged neurons. Enzyme-linked immunosorbent assay (ELISA) was implemented to identify molecular mediators involved in neuroprotection. KEY FINDINGS: Prenatal VPA exposure resulted in significant abnormalities in synaptic development, which were further evidenced by impairments in social interaction and cognitive function. When LPI was administered, most of the synaptic abnormalities were alleviated, as reflected by higher neuron and dendritic spine count. LPI treatment also reduced cytoplasmic cytochrome c concentration and related neuronal cell death. Mechanistically, GPR55 activation by LPI increases the expression of phospho-Akt and phospho-GSK3ß, leading to the activation of this signaling in the process of rescuing synaptic abnormalities and mitochondria-mediated neuronal apoptosis. SIGNIFICANCE: The observed therapeutic effects of GPR55 activation shed light on its significance as a prospective target for ameliorating mitochondrial dysfunction and dendritic spine loss, offering novel prospects for developing targeted interventions to alleviate the neuropathological causes of ASD.

Revisiting Concurrent Radiation Therapy, Temozolomide, and the Histone Deacetylase Inhibitor Valproic Acid for Patients with Glioblastoma-Proteomic Alteration and Comparison Analysis with the Standard-of-Care Chemoirradiation.

BACKGROUND: Glioblastoma (GBM) is the most common brain tumor with an overall survival (OS) of less than 30% at two years. Valproic acid (VPA) demonstrated survival benefits documented in retrospective and prospective trials, when used in combination with chemo-radiotherapy (CRT). PURPOSE: The primary goal of this study was to examine if the differential alteration in proteomic expression pre vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA as compared to standard-of-care CRT. The second goal was to explore the associations between the proteomic alterations in response to VPA/RT/TMZ correlated to patient outcomes. The third goal was to use the proteomic profile to determine the mechanism of action of VPA in this setting. MATERIALS AND METHODS: Serum obtained pre- and post-CRT was analyzed using an aptamer-based SOMAScan® proteomic assay. Twenty-nine patients received CRT plus VPA, and 53 patients received CRT alone. Clinical data were obtained via a database and chart review. Tests for differences in protein expression changes between radiation therapy (RT) with or without VPA were conducted for individual proteins using two-sided t-tests, considering p-values of <0.05 as significant. Adjustment for age, sex, and other clinical covariates and hierarchical clustering of significant differentially expressed proteins was carried out, and Gene Set Enrichment analyses were performed using the Hallmark gene sets. Univariate Cox proportional hazards models were used to test the individual protein expression changes for an association with survival. The lasso Cox regression method and 10-fold cross-validation were employed to test the combinations of expression changes of proteins that could predict survival. Predictiveness curves were plotted for significant proteins for VPA response (p-value < 0.005) to show the survival probability vs. the protein expression percentiles. RESULTS: A total of 124 proteins were identified pre- vs. post-CRT that were differentially expressed between the cohorts who received CRT plus VPA and those who received CRT alone. Clinical factors did not confound the results, and distinct proteomic clustering in the VPA-treated population was identified. Time-dependent ROC curves for OS and PFS for landmark times of 20 months and 6 months, respectively, revealed AUC of 0.531, 0.756, 0.774 for OS and 0.535, 0.723, 0.806 for PFS for protein expression, clinical factors, and the combination of protein expression and clinical factors, respectively, indicating that the proteome can provide additional survival risk discrimination to that already provided by the standard clinical factors with a greater impact on PFS. Several proteins of interest were identified. Alterations in GALNT14 (increased) and CCL17 (decreased) (p = 0.003 and 0.003, respectively, FDR 0.198 for both) were associated with an improvement in both OS and PFS. The pre-CRT protein expression revealed 480 proteins predictive for OS and 212 for PFS (p < 0.05), of which 112 overlapped between OS and PFS. However, FDR-adjusted p values were high, with OS (the smallest p value of 0.586) and PFS (the smallest p value of 0.998). The protein PLCD3 had the lowest p-value (p = 0.002 and 0.0004 for OS and PFS, respectively), and its elevation prior to CRT predicted superior OS and PFS with VPA administration. Cancer hallmark genesets associated with proteomic alteration observed with the administration of VPA aligned with known signal transduction pathways of this agent in malignancy and non-malignancy settings, and GBM signaling, and included epithelial-mesenchymal transition, hedgehog signaling, Il6/JAK/STAT3, coagulation, NOTCH, apical junction, xenobiotic metabolism, and complement signaling. CONCLUSIONS: Differential alteration in proteomic expression pre- vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA. Using pre- vs. post-data, prognostic proteins emerged in the analysis. Using pre-CRT data, potentially predictive proteins were identified. The protein signals and hallmark gene sets associated with the alteration in the proteome identified between patients who received VPA and those who did not, align with known biological mechanisms of action of VPA and may allow for the identification of novel biomarkers associated with outcomes that can help advance the study of VPA in future prospective trials.

Duality of Valproic Acid Effects on Inflammation, Oxidative Stress and Autophagy in Human Eosinophilic Cells.

Eosinophils function in rapid innate immune responses and allergic reactions. Recent research has raised the possibility that the histone deacetylase inhibitor valproic acid (VPA) may be a promising therapeutic agent for treatment of allergic responses and certain cancers. However, its effects on eosinophils remain unclear. Utilizing the EoL-1 human eosinophil cell line as a model, we investigated the effects of VPA on oxidative stress- and autophagy-mediated immune responses. We found that VPA induced reactive oxidative species (ROS) generation and eosinophil activation without affecting cell viability. Moreover, VPA treatment suppressed the negative regulator of antioxidant transcription factor Nrf2, which is known to activate antioxidant defense. Interestingly, VPA was able to increase autophagic markers, as well as NLRP3 and NLRC4 mRNA activation, in Eol-1 cells in a dose-dependent manner. Collectively, our results indicate that VPA could increase the severity of allergic responses, and if so, it clearly would not be a suitable drug for the treatment of allergic reactions. However, VPA does have the potential to induce autophagy and to regulate the inflammatory responses via inflammasome-driven caspase-1 deactivation in a dose-dependent manner.