Inhibition of palmitoyltransferase ZDHHC12 sensitizes ovarian cancer cells to cisplatin through ROS-mediated mechanisms.

Platinum-based therapies have revolutionized the treatment of high-grade serous ovarian cancer (HGSOC). However, high rates of disease recurrence and progression remain a major clinical concern. Impaired mitochondrial function and dysregulated reactive oxygen species (ROS), hallmarks of cancer, hold potential as therapeutic targets for selectively sensitizing cisplatin treatment. Here, we uncover an oncogenic role of the palmitoyltransferase ZDHHC12 in regulating mitochondrial function and ROS homeostasis in HGSOC cells. Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer data revealed significantly elevated ZDHHC12 expression, demonstrating the strongest positive association with ROS pathways among all ZDHHC enzymes. Transcriptomic analysis of independent ovarian cancer datasets and the SNU119 cell model corroborated this association, highlighting a strong link between ZDHHC12 expression and signature pathways involving mitochondrial oxidative metabolism and ROS regulation. Knockdown of ZDHHC12 disrupted this association, leading to increased cellular complexity, ATP levels, mitochondrial activity, and both mitochondrial and cellular ROS. This dysregulation, achieved by the siRNA knockdown of ZDHHC12 or treatment with the general palmitoylation inhibitor 2BP or the fatty acid synthase inhibitor C75, significantly enhanced cisplatin cytotoxicity in 2D and 3D spheroid models of HGSOC through ROS-mediated mechanisms. Markedly, ZDHHC12 inhibition significantly augmented the anti-tumor activity of cisplatin in an ovarian cancer xenograft tumor model, as well as in an ascites-derived organoid line of platinum-resistant ovarian cancer. Our data suggest the potential of ZDHHC12 as a promising target to improve the outcome of HGSOCs in response to platinum-based chemotherapy.

Author Correction: Macrophages confer resistance to PI3K inhibitor GDC-0941 in breast cancer through the activation of NF-κB signaling.

Since publication of this article, the authors have noticed the following errors: (1) Fig. 3c, the image is correct but the authors mistakenly provided incorrect figure legend. The correct figure legend is included below along with the original figure. (2) Supplementary Fig. S2, the authors mistakenly provided the data from ELISA analysis of TNFα and IL-6 in media from co-cultured 4T1 and RAW264.7 cells. As stated in the main text, data from ELISA analysis of TNFα and IL-6 in 4T1 tumors from Balb/c mice treated with GDC-0941 should be provided. The correct figure and figure legend are included below. (3) The authors noticed an error in the manuscript in which "RAW276.7" should be "RAW264.7". The corrections do not alter the conclusions of the paper. The authors apologize for any inconvenience caused. This has been corrected in both the PDF and HTML versions of the Article.

Cryptotanshinone Attenuates Oxygen-Glucose Deprivation/ Recovery-Induced Injury in an in vitro Model of Neurovascular Unit.

Cryptotanshinone (CTs), an active component isolated from the root of Salvia miltiorrhiza (SM), has been shown to exert potent neuroprotective property. We here established an oxygen-glucose deprivation/recovery (OGD/R)-injured Neurovascular Unit (NVU) model in vitro to observe the neuroprotective effects of CTs on cerebral ischemia/reperfusion injury (CIRI), and explore the underlying mechanisms. CTs was observed to significantly inhibit the OGD/R-induced neuronal apoptosis, and decease the activation of Caspase-3 and the degradation of poly-ADP-ribose polymerase (PARP), as well as the increase of Bax/Bcl-2 ratio in neurons under OGD/R condition. The inhibitory effects of CTs on neuron apoptosis were associated with the blocking of mitogen-activated protein kinase (MAPK) signaling pathway. CTs also remarkably ameliorated OGD/R-induced reduction of transepithelial electrical resistance (TEER) values and the increase of transendothelial permeability coefficient (Pe) of sodium fluorescein (SF) by upregulating the expression of ZO-1, Claudin-5, and Occludin in brain microvascular endothelial cells (BMECs), which might be related to the down-regulation of matrix metalloproteinase (MMP)-9 expression. Based on these findings, CTs may play a neuroprotective role in OGD/R injure in NVU models in vitro by inhibiting cell apoptosis and alleviating the damage of blood-brain barrier (BBB).

The effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes in a new in vitro neuron-astrocyte-endothelium co-culture system.

Glutamate-induced excitotoxicity is a contributer to many neurological diseases. Astrocytes may represent a new target for treating glutamate-induced excitotoxicity. However, the in vitro culture system that mimics the in vivo microenvironment is lacking. This study aimed to establish a new in vitro co-culture system including neurons, astrocytes, and endothelial cells (NAE), and to investigate the effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes. A NAE co-culture method was created using a Transwell chamber, in which neurons were seeded on the bottom of the lower chamber, endothelial cells were plated on the top membrane, and astrocytes were plated on the bottom membrane of the insert. Glutamate-induced toxicity was induced using glutamate and glycine, and examined using immunofluorescence and lactate dehydrogenase release assay. Global methylation in astrocytes was analyzed, and the expression of DNMT1 and DNMT3a was examined using Western blot analysis. Glutamate treatment induced less neuronal damage in the NAE system compared with the control group in which neurons and astrocytes were cultured alone. Global DNA methylation was increased and the expression of DNMT1 and DNMT3a in astrocytes was increased after glutamate treatment, which was blocked by application of the NMDAR inhibitor MK-801 and the DNMT inhibitor 5-azaC from the endothelial cells. The in vitro ANE culture system is effective for studying glutamate-induced excitotoxicity, and may be used for testing the passage of drugs across the blood-brain barrier. Inhibition of DNA methylation in astrocytes may be a new therapeutic strategy for treating glutamate-induced excitotoxicity.

Macrophages confer resistance to PI3K inhibitor GDC-0941 in breast cancer through the activation of NF-κB signaling.

The PI3K pathway is one of the most dysregulated signaling pathways in epithelial cancers and has become an attractive therapeutic target under active preclinical and clinical development. However, recent clinical trial studies revealed that blockade of PI3K activity in advanced cancer often leads to the development of resistance and relapse of the diseases. Intense efforts have been made to elucidate resistance mechanisms and identify rational drug combinations with PI3K inhibitors in solid tumors. In the current study, we found that PI3K inhibition by GDC-0941 increased macrophage infiltration and induced the expression of macrophage-associated cytokines and chemokines in the mouse 4T1 breast tumor model. Using the in vitro co-culture system, we showed that the presence of macrophages led to the activation of NF-κB signaling in 4T1 tumor cells, rendering tumor cells resistant to PI3K inhibition by GDC-0941. Furthermore, we found that Aspirin could block the activation of NF-κB signaling induced by PI3K inhibition, and combined use of GDC-0941 and Aspirin resulted in attenuated cell growth and enhanced apoptosis of 4T1 cells in the in vitro co-culture system with the presence of macrophages. Consistently, the combination treatment also effectively reduced tumor burden, macrophage infiltration and pulmonary metastasis in in vivo 4T1 breast tumor model. Together, our results suggested macrophages in microenvironment may contribute to the resistance of breast cancer cells to PI3K inhibition and reveal a new combination paradigm to improve the efficacy of PI3K-targeted therapy.

Neuroprotective effects of Ginkgo biloba extract and Ginkgolide B against oxygen-glucose deprivation/reoxygenation and glucose injury in a new in vitro multicellular network model.

Acute ischemic stroke (AIS), as the third leading cause of death worldwide, is characterized by its high incidence, mortality rate, high incurred disability rate, and frequent reoccurrence. The neuroprotective effects of Ginkgo biloba extract (GBE) against several cerebral diseases have been reported in previous studies, but the underlying mechanisms of action are still unclear. Using a novel in vitro rat cortical capillary endothelial cell-astrocyte-neuron network model, we investigated the neuroprotective effects of GBE and one of its important constituents, Ginkgolide B (GB), against oxygen-glucose deprivation/reoxygenation and glucose (OGD/R) injury. In this model, rat cortical capillary endothelial cells, astrocytes, and neurons were cocultured so that they could be synchronously observed in the same system. Pretreatment with GBE or GB increased the neuron cell viability, ameliorated cell injury, and inhibited the cell apoptotic rate through Bax and Bcl-2 expression regulation after OGD/R injury. Furthermore, GBE or GB pretreatment enhanced the transendothelial electrical resistance of capillary endothelial monolayers, reduced the endothelial permeability coefficients for sodium fluorescein (Na-F), and increased the expression levels of tight junction proteins, namely, ZO-1 and occludin, in endothelial cells. Results demonstrated the preventive effects of GBE on neuronal cell death and enhancement of the function of brain capillary endothelial monolayers after OGD/R injury in vitro; thus, GBE could be used as an effective neuroprotective agent for AIS/reperfusion, with GB as one of its significant constituents.

Integrated network analysis reveals potentially novel molecular mechanisms and therapeutic targets of refractory epilepsies.

Epilepsy is a complex neurological disorder and a significant health problem. The pathogenesis of epilepsy remains obscure in a significant number of patients and the current treatment options are not adequate in about a third of individuals which were known as refractory epilepsies (RE). Network medicine provides an effective approach for studying the molecular mechanisms underlying complex diseases. Here we integrated 1876 disease-gene associations of RE and located those genes to human protein-protein interaction (PPI) network to obtain 42 significant RE-associated disease modules. The functional analysis of these disease modules showed novel molecular pathological mechanisms of RE, such as the novel enriched pathways (e.g., "presynaptic nicotinic acetylcholine receptors", "signaling by insulin receptor"). Further analysis on the relationships between current drug targets and the RE-related disease genes showed the rational mechanisms of most antiepileptic drugs. In addition, we detected ten potential novel drug targets (e.g., KCNA1, KCNA4-6, KCNC3, KCND2, KCNMA1, CAMK2G, CACNB4 and GRM1) located in three RE related disease modules, which might provide novel insights into the new drug discovery for RE therapy.

Network-Based Approach to Identify Potential Targets and Drugs that Promote Neuroprotection and Neurorepair in Acute Ischemic Stroke.

Acute ischemic stroke (AIS) accounts for more than 80% of the approximately 610,000 new stroke cases worldwide every year. Both ischemia and reperfusion can cause death, damage, and functional changes of affected nerve cells, and these alterations can result in high rates of disability and mortality. Therefore, therapies aimed at increasing neuroprotection and neurorepair would make significant contributions to AIS management. However, with regard to AIS therapies, there is currently a large gap between experimental achievements and practical clinical solutions (EC-GAP-AIS). Here, by integrating curated disease-gene associations and interactome network known to be related to AIS, we investigated the molecular network mechanisms of multi-module structures underlying AIS, which might be relevant to the time frame subtypes of AIS. In addition, the EC-GAP-AIS phenomenon was confirmed and elucidated by the shortest path lengths and the inconsistencies in the molecular functionalities and overlapping pathways between AIS-related genes and drug targets. Furthermore, we identified 23 potential targets (e.g. ADORA3, which is involved in the regulation of cellular reprogramming and the extracellular matrix) and 46 candidate drugs (e.g. felbamate, methylphenobarbital and memantine) that may have value for the treatment of AIS.

NPAS3 Regulates Transcription and Expression of VGF: Implications for Neurogenesis and Psychiatric Disorders.

Neuronal PAS domain protein 3 (NPAS3) and VGF (VGF Nerve Growth Factor (NGF) Inducible) are important for neurogenesis and psychiatric disorders. Previously, we have demonstrated that NPAS3 regulates VGF at the transcriptional level. In this study, VGF (non-acronymic) was found regulated by NPAS3 in neuronal stem cells. However, the underlying mechanism of this regulation remains unclear. The aim of this study was to explore the correlation of NPAS3 and VGF, and their roles in neural cell proliferation, in the context of psychiatric illnesses. First, we focused on the structure of NPAS3, to identify the functional domain of NPAS3. Truncated NPAS3 lacking transactivation domain was also found to activate VGF, which suggested that not only transactivation domain but other structural motifs were also involved in the regulation. Second, Mutated enhancer box (E-box) of VGF promoter showed a significant response to this basic helix-loop-helix (bHLH) transcription factor, which suggested an indirect regulatory mechanism for controlling VGF expression by NPAS3. κB site within VGF promoter was identified for VGF activation induced by NPAS3, apart from direct binding to E-box. Furthermore, ectopically expressed NPAS3 in PC12 cells produced parallel responses for nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB (P65)] expression, which specifies that NPAS3 regulates VGF through the NF-κB signaling pathway. Over-expression of NPAS3 also enhances the cell proliferation, which can be blocked by knockdown of VGF. Finally, NPAS3 was found to influence proliferation of neural cells through VGF. Therefore, downstream signaling pathways that are responsible for NPAS3-VGF induced proliferation via glutamate receptors were explored. Combining this work and published literature, a potential network composed by NPAS3, NF-κB, Brain-Derived Neurotrophic Factor (BDNF), NGF and VGF, was proposed. This network collectively detailed how NPAS3 connects with VGF and intersected neural cell proliferation, synaptic activity and psychiatric disorders.

Changes of ubiquitin C-terminal hydrolase-L1 levels in serum and urine of patients with white matter lesions.

OBJECTIVES: Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) has been established as a potential biomarker of neuronal damage. There is not much information about the effects of white matter lesions (WMLs) on serum and urine UCH-L1 levels in white matter disease patients. This study was aimed to assess whether serum or urine UCH-L1 levels are a reliable marker of brain damage in patients with WMLs. DESIGN AND METHODS: Serum and urine levels of UCH-L1 were assessed in 125 patients with dizziness, hypertension, type 2 diabetes mellitus, or dyslipidemia. Of these 125 patient cases, 41 showed periventricular WMLs (P-WMLs), 46 showed subcortical WMLs (S-WMLs), and 38 displayed no well-defined WMLs (controls). RESULTS: Serum UCH-L1 levels were significantly different between the WML group and controls (p<0.05). Further subgroup analysis proved that serum UCH-L1 levels in participants with S-WMLs were significantly increased when compared with controls (p<0.001), but there was no significant differences between controls and patients with P-WMLs (p>0.05). However, urine levels of UCH-L1 were similar between these three groups (p>0.05). In addition, multivariate analysis showed that increased serum UCH-L1 levels were independently associated with the severity of WMLs using Fazekas scale (ß=0.432, p<0.001). CONCLUSIONS: These findings suggest that serum UCH-L1 levels may serve as a novel biomarker for neuronal damage from WMLs, especially S-WMLs.

The use of laminin modified linear ordered collagen scaffolds loaded with laminin-binding ciliary neurotrophic factor for sciatic nerve regeneration in rats.

Nerve conduit provides a promising strategy for nerve injury repair in the peripheral nervous system (PNS). However, simply bridging the transected nerve with an empty conduit is hard to satisfy functional recovery. The regenerated axons may disperse during regeneration in the empty lumen, limiting the functional recovery. Our previous work had reported that linear ordered collagen scaffold (LOCS) could be used as a nerve guidance material. Here we cross-linked LOCS fibers with laminin which was a major component of the extracellular matrix in nervous system. Ciliary neurotrophic factor (CNTF) plays a critical role in peripheral nerve regeneration. But the lack of efficient CNTF delivery approach limits its clinical applications. To retain CNTF on the scaffold, a laminin binding domain (LBD) was fused to the N-terminal of CNTF. Compared with NAT-CNTF, LBD-CNTF exhibited specific laminin-binding ability and comparable neurotrophic bioactivity. We combined LBD-CNTF with the laminin modified LOCS fibers to construct a double-functional bio-scaffold. The functional scaffold was filled in silicon conduit and tested in the rat sciatic nerve transection model. Results showed that this functional biomaterial could guide the axon growth, retain more CNTF on the scaffolds and enhance the nerve regeneration as well as functional recovery.