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Simple Summary: Here, we show that the plant-derived product, sanguinarine, inhibited aerobic glycolysis and oxidative phosphorylation; which resulted in energy alternations and necroptosis of tumor cells. Moreover, we identify the PKM2/β-catenin axis as the main target in sanguinarine treatment against HCC development. Backgrounds: Abnormal metabolism is the hallmark of hepatocellular carcinoma. Targeting energy metabolism has become the major focus of cancer therapy. The natural product, sanguinarine, displays remarkable anti-tumor properties by disturbing energy homeostasis; however, the underlying mechanism has not yet been elucidated. Methods: The anticancer activity of sanguinarine was determined using CCK-8 and colony formation assay. Morphological changes of induced cell death were observed under electron microscopy. Necroptosis and apoptosis related markers were detected using western blotting. PKM2 was identified as the target by transcriptome sequencing. Molecular docking assay was used to evaluate the binding affinity of sanguinarine to the PKM2 molecule. Furthermore, Alb-CreERT2; PKM2loxp/loxp; Rosa26RFP mice was used to construct the model of HCC—through the intervention of sanguinarine in vitro and in vivo—to accurately explore the regulation effect of sanguinarine on cancer energy metabolism. Results: Sanguinarine inhibited tumor proliferation, metastasis and induced two modes of cell death. Molecular docking of sanguinarine with PKM2 showed appreciable binding affinity. PKM2 kinase activity and aerobic glycolysis rate declined, and mitochondrial oxidative phosphorylation was inhibited by sanguinarine application; these changes result in energy deficits and lead to necroptosis. Additionally, sanguinarine treatment prevents the translocation of PKM2 into the nucleus and suppresses the interaction of PKM2 with β-catenin; the transcriptional activity of PKM2/β-catenin signaling and its downstream genes were decreased. Conclusions: Sanguinarine showed remarkable anti-HCC activity via regulating energy metabolism by PKM2/β-catenin signaling. On the basis of these investigations, we propose that sanguinarine might be considered as a promising compound for discovery of anti-HCC drugs. [ABSTRACT FROM AUTHOR]

Simple Summary: The aim of our study is to understand the signaling mechanisms behind the tumor suppressive function of RIPK4 (receptor-interacting serine/threonine protein kinase 4) in squamous cell carcinomas (SCCs) of skin, head, and neck regions. SCCs are some of the deadliest cancers, with poor survival rate, and understanding molecular mechanisms behind the tumor formation will help us to develop effective therapies. Through gene profiling and targeted CRISPR screening approaches in mouse models of cancer, we identified ELOVL4, (elongation of very-long-chain fatty acid-4) as a downstream target of RIPK4. Previous studies have shown that RIPK4 is a NOTCH target gene and RIPK4 phosphorylates IRF6 (interferon regulatory factor 6). Our data from in vitro and in vivo mouse experiments show that the NOTCH-RIPK4-IRF6-ELOVL4 signaling axis functions as a potent tumor suppressor in SCCs. Receptor-interacting serine/threonine protein kinase 4 (RIPK4) and its kinase substrate the transcription factor interferon regulatory factor 6 (IRF6) play critical roles in the development and maintenance of the epidermis. In addition, ourselves and others have previously shown that RIPK4 is a NOTCH target gene that suppresses the development of cutaneous and head and neck squamous cell carcinomas (HNSCCs). In this study, we used autochthonous mouse models, where the expression of Pik3caH1047R oncogene predisposes the skin and oral cavity to tumor development, and show that not only loss of Ripk4, but also loss of its kinase substrate Irf6, triggers rapid SCC development. In vivo rescue experiments using Ripk4 or a kinase-dead Ripk4 mutant showed that the tumor suppressive function of Ripk4 is dependent on its kinase activity. To elucidate critical mediators of this tumor suppressive pathway, we performed transcriptional profiling of Ripk4-deficient epidermal cells followed by multiplexed in vivo CRISPR screening to identify genes with tumor suppressive capabilities. We show that Elovl4 is a critical Notch-Ripk4-Irf6 downstream target gene, and that Elovl4 loss itself triggers SCC development. Importantly, overexpression of Elovl4 suppressed tumor growth of Ripk4-deficient keratinocytes. Altogether, our work identifies a potent Notch1-Ripk4-Irf6-Elovl4 tumor suppressor axis. [ABSTRACT FROM AUTHOR]

The extracts from Psoralea corylifolia Linn. (P. corylifolia) seeds have been shown to display antitumor activity. To date, the prospects of this plant and its active compounds in the treatment of non-small-cell lung cancer (NSCLC) have not been thoroughly studied. In this study, we identified a novel psorachromene compound that displays selective cytotoxic effects on all NSCLC cells tested, including NSCLC cells harboring epidermal growth factor receptor (EGFR) activation mutants (H1975L858R/T790M and H1975-MS35L858R/T790M/C797S ). Psorachromene induces G1 arrest in NSCLC cells harboring wild-type EGFR but induces apoptosis in NSCLC cells harboring activating EGFR mutations. Psorachromene inhibits activated EGFR signaling and kinase activity and suppresses tumor growth of implanted H1975-MS35L858R/T790M/C797S cells in nude mice. Molecular docking analysis revealed that psorachromene could form stronger bonds with mutant EGFR than wild-type EGFR, which might account for the greater cytotoxic effects observed in NSCLC cells harboring activating EGFR mutations (H1975 and H1975-MS35) than wild-type EGFR (A549). In conclusion, it is suggested that psorachromene is an attractive agent to be further explored for its use in the treatment of NSCLC patients harboring EGFR L858R/T790M/C797S. [ABSTRACT FROM AUTHOR]

Despite remarkable initial responses of anaplastic lymphoma kinase (ALK) inhibitors in ALK-positive non-small cell lung cancer (NSCLC) patients, cancers eventually develop resistance within one to two years. This study aimed to compare the properties of iruplinalkib (WX‑0593) with other ALK inhibitors and report the comprehensive characterization of iruplinalkib against the crizotinib resistance. The inhibitory effect of iruplinalkib on kinase activity was detected. A kinase screen was performed to evaluate the selectivity of iruplinalkib. The effect of iruplinalkib on related signal transduction pathways of ALK and c-ros oncogene 1 (ROS1) kinases was examined. The cellular and in vivo activities of ALK inhibitors were compared in engineered cancer-derived cell lines and in mice xenograft models, respectively. Human hepatocytes derived from three donors were used for evaluating hepatic enzyme inducing activity. HEK293 cell lines expressing transportors were used to invesigated the drug interaction potential mediated by several transporters. The results showed iruplinalkib potently inhibited the tyrosine autophosphorylation of wild-type ALK, ALKL1196M, ALKC1156Y and epidermal growth factor receptor (EGFR)L858R/T790M. The inhibitory effects of iruplinalkib in patient-derived xenograft and cell line-derived xenograft models were observed. Moreover, iruplinalkib showed robust antitumor effects in BALB/c nude mice xenograft models with ALK-/ROS1-positive tumors implanted subcutaneously, and the tumor suppressive effects in crizotinib-resistant model was significantly better than that of brigatinib. Iruplinalkib did not induce CYP1A2, CYP2B6 and CYP3A4 at therapeutic concentration, and was also a strong inhibitor of MATE1 and MATE2K transporters, as well as P-gp and BCRP. In conclusion, iruplinalkib, a highly active and selective ALK/ROS1 inhibitor, exhibited strong antitumor effects in vitro and in crizotinib-resistant models. [ABSTRACT FROM AUTHOR]

Background: Inhalational anesthetics are known to disrupt PDZ2 domain-mediated protein-protein interactions of the postsynaptic density (PSD)-95 protein. The aim of this study is to investigate the underlying mechanisms in response to early isoflurane exposure on synaptic PSD-95 PDZ2 domain disruption that altered spine densities and cognitive function. The authors hypothesized that activation of protein kinase-G by the components of nitric oxide (NO) signaling pathway constitutes a mechanism that prevents loss of early dendritic spines and synapse in neurons and cognitive impairment in mice in response to disruption of PDZ2 domain of the PSD-95 protein.Methods: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 wild-type PDZ2 peptide or soluble guanylyl cyclase activator YC-1 along with their respective controls. Primary neurons at 7 days in vitro were exposed to isoflurane or PSD-95 wild-type PDZ2 peptide for 4 h. Coimmunoprecipitation, spine density, synapses, cyclic guanosine monophosphate-dependent protein kinase activity, and novel object recognition memory were assessed.Results: Exposure of isoflurane or PSD-95 wild-type PDZ2 peptide relative to controls causes the following. First, there is a decrease in PSD-95 coimmunoprecipitate relative to N-methyl-d-aspartate receptor subunits NR2A and NR2B precipitate (mean ± SD [in percentage of control]: isoflurane, 54.73 ± 16.52, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 51.32 ± 12.93, P = 0.001). Second, there is a loss in spine density (mean ± SD [spine density per 10 µm]: control, 5.28 ± 0.56 vs. isoflurane, 2.23 ± 0.67, P < 0.0001; and PSD-95 mutant PDZ2 peptide, 4.74 ± 0.94 vs. PSD-95 wild-type PDZ2 peptide, 1.47 ± 0.87, P < 0.001) and a decrease in synaptic puncta (mean ± SD [in percentage of control]: isoflurane, 41.1 ± 14.38, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 50.49 ± 14.31, P < 0.001). NO donor or cyclic guanosine monophosphate analog prevents the spines and synapse loss and decline in the cyclic guanosine monophosphate-dependent protein kinase activity, but this prevention was blocked by soluble guanylyl cyclase or protein kinase-G inhibitors in primary neurons. Third, there were deficits in object recognition at 5 weeks (mean ± SD [recognition index]: male, control, 64.08 ± 10.57 vs. isoflurane, 48.49 ± 13.41, P = 0.001, n = 60; and female, control, 67.13 ± 11.17 vs. isoflurane, 53.76 ± 6.64, P = 0.003, n = 58). Isoflurane-induced impairment in recognition memory was preventable by the introduction of YC-1.Conclusions: Activation of soluble guanylyl cyclase or protein kinase-G prevents isoflurane or PSD-95 wild-type PDZ2 peptide-induced loss of dendritic spines and synapse. Prevention of recognition memory with YC-1, a NO-independent activator of guanylyl cyclase, supports a role for the soluble guanylyl cyclase mediated protein kinase-G signaling in countering the effects of isoflurane-induced cognitive impairment.Editor’s Perspective: [ABSTRACT FROM AUTHOR]

Background: Diabetes is a serious chronic metabolic disorder. Trichosanthes kirilowii Maxim. (TK) is traditionally used for the treatment of diabetes in traditional Chinese medicine (TCM). However, the clinical application of TK on diabetic patients and the hypoglycemic efficacies of TK are still unclear. Methods: A retrospective cohort study was conducted to analyze the usage of Chinese herbs in patients with type 2 diabetes in Taiwan. Glucose tolerance test was performed to analyze the hypoglycemic effect of TK. Proteomic approach was performed to identify the protein constituents of TK. Insulin receptor (IR) kinase activity assay and glucose tolerance tests in diabetic mice were further used to elucidate the hypoglycemic mechanisms and efficacies of TK. Results: By a retrospective cohort study, we found that TK was the most frequently used Chinese medicinal herb in type 2 diabetic patients in Taiwan. Oral administration of aqueous extract of TK displayed hypoglycemic effects in a dose-dependent manner in mice. An abundant novel TK protein (TKP) was further identified by proteomic approach. TKP interacted with IR by docking analysis and activated the kinase activity of IR. In addition, TKP enhanced the clearance of glucose in diabetic mice in a dose-dependent manner. Conclusions: In conclusion, this study applied a bed-to-bench approach to elucidate the hypoglycemic efficacies and mechanisms of TK on clinical usage. In addition, we newly identified a hypoglycemic protein TKP from TK. Our findings might provide a reasonable explanation of TK on the treatment of diabetes in TCM. [ABSTRACT FROM AUTHOR]

Background: Immune system dysfunction, including higher levels of peripheral monocytes and inflammatory cytokines, is an important feature of Parkinson's disease (PD) pathogenesis, although the mechanism underlying the process remains to be investigated. In the central nervous system, it is well-known that α-synuclein (α-syn), a key protein involved in PD, activates microglia potently, and it is also reported that α-syn exists in the peripheral system, especially in erythrocytes or red blood cells (RBC) at exceedingly high concentration. The current study focused on the possibility that RBC-derived α-syn mediates the sensitization of peripheral monocytes in PD patients.Methods: The hyperactivation of monocytes was assessed quantitatively by measuring mRNA levels of typical inflammatory cytokines (including IL-1β, IL-6 and TNF-α) and protein levels of secreted inflammatory cytokines (including pro-inflammatory cytokines: IL-1β, IL-6, TNF-α, IL-8, IFN-γ, IL-2, and IL-12p70 and anti-inflammatory cytokines: IL-4, IL-10, and IL-13). Western blot, nanoparticle tracking analysis and electron microscopy were used to characterize RBC-derived extracellular vesicles (RBC-EVs). Inhibitors of endocytosis and leucine-rich repeat kinase 2 (LRRK2), another key protein involved in PD, were used to investigate how these two factors mediated the process of monocyte sensitization by RBC-EVs.Results: Increased inflammatory sensitization of monocytes was observed in PD patients and PD model mice. We found that α-syn-containing RBC-EVs isolated from PD model mice or free form oligomeric α-syn induced the inflammatory sensitization of THP-1 cells, and demonstrated that endocytosis was a requirement for this pathophysiological pathway. Furthermore, the hyperactivation of THP-1 cells induced by RBC-EVs was associated with increased LRRK2 production and kinase activity. The phenomenon of inflammatory sensitization of human monocytes and increased LRRK2 were also observed by the treatment of RBC-EVs isolated from PD patients.Conclusions: Our data provided new insight into how hyperactivation of monocytes occurs in PD patients, and identified the central role played by α-syn-containing RBC-EVs in this process. [ABSTRACT FROM AUTHOR]

Background: Autosomal recessive mutations in the glucocerebrosidase gene, Beta-glucocerebrosidase 1 (GBA1), cause the lysosomal storage disorder Gaucher's disease. Heterozygous carriers of most GBA1 mutations have dramatically increased Parkinson's disease (PD) risk, but the mechanisms and cells affected remain unknown. Glucocerebrosidase expression is relatively enriched in astrocytes, yet the impact of its mutation in these cells has not yet been addressed.Objectives: Emerging data supporting non-cell-autonomous mechanisms driving PD pathogenesis inspired the first characterization of GBA1-mutant astrocytes. In addition, we asked whether LRRK2, likewise linked to PD and enriched in astrocytes, intersected with GBA1 phenotypes.Methods: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels.Results: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes.Conclusions: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention. © 2020 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3 weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30 mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25 mg/0.1 mL infusion; 10 days of 6 h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50μM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction. [ABSTRACT FROM AUTHOR]

Background: Over-activated microglia and chronic neuroinflammation contribute to dopaminergic neuron degeneration and progression of Parkinson's disease (PD). Leucine-rich repeat kinase 2 (LRRK2), a kinase mutated in autosomal dominantly inherited and sporadic PD cases, is highly expressed in immune cells, in which it regulates inflammation through a yet unclear mechanism.Methods: Here, using pharmacological inhibition and cultured Lrrk2 (-/-) primary microglia cells, we validated LRRK2 as a positive modulator of inflammation and we investigated its specific function in microglia cells.Results: Inhibition or genetic deletion of LRRK2 causes reduction of interleukin-1β and cyclooxygenase-2 expression upon lipopolysaccharide-mediated inflammation. LRRK2 also takes part of the signaling trigged by α-synuclein fibrils, which culminates in induction of inflammatory mediators. At the molecular level, loss of LRRK2 or inhibition of its kinase activity results in increased phosphorylation of nuclear factor kappa-B (NF-κB) inhibitory subunit p50 at S337, a protein kinase A (PKA)-specific phosphorylation site, with consequent accumulation of p50 in the nucleus.Conclusions: Taken together, these findings point to a role of LRRK2 in microglia activation and sustainment of neuroinflammation and in controlling of NF-κB p50 inhibitory signaling. Understanding the molecular pathways coordinated by LRRK2 in activated microglia cells after pathological stimuli such us fibrillar α-synuclein holds the potential to provide novel targets for PD therapeutics. [ABSTRACT FROM AUTHOR]

Background: Transient receptor potential vanilloid-1 (TRPV1), activated by heat, acidic pH, endogenous vanilloids and capsaicin, is essential for thermal hyperalgesia. Under inflammatory conditions, phosphorylation of TRPV1 by protein kinase C (PKC) can sensitize the channel and decrease the activation threshold. Src kinase also phosphorylates TRPV1, promoting channel trafficking to the plasma membrane. These post-translational modifications are important for several chronic pain conditions. This study presents a previously undescribed relationship between Src and PKC phosphorylation of TRPV1, influencing the thermal hypersensitivity associated with TRPV1 activation.Methods: We assessed TRPV1 channel activity using intracellular calcium imaging and patch-clamp electrophysiology in mouse dorsal root ganglion cultures. Additionally, we used behavioural experiments to evaluate plantar thermal sensitivity following intraplantar injections of activators of known modulators of TRPV1 with and without an Src antagonist.Results: Using calcium imaging and patch-clamp techniques, we demonstrated that pharmacological inhibition of Src kinase or mutation of the Src phosphorylation site on TRPV1 prevented PKC but not PKA-mediated sensitization of TRPV1 in vitro. We found that intraplantar injection of the PKC activator phorbol 12-myristate 13-acetate (PMA) or bradykinin produces thermal hypersensitivity that can be attenuated by pharmacological inhibition of Src. Additionally, complete Freund's Adjuvant (CFA)-induced inflammatory hypersensitivity could also be attenuated by local Src kinase inhibition.Conclusions: Our data demonstrate that Src phosphorylation is critical for PKC-mediated sensitization of TRPV1. Further, in a model of inflammatory pain, CFA, Src kinase inhibition could reduce thermal hypersensitivity. Targeting of Src kinase may have analgesic benefits in inflammatory pain conditions.Significance: Src kinase-mediated phosphorylation of TRPV1 is a critical regulator of the PKC-induced sensitization induced by multiple inflammatory mediators. This suggest a new regulatory mechanism governing TRPV1 function and a potential therapeutic target for inflammatory type pain, including cancer pain where Src antagonists are currently utilized. [ABSTRACT FROM AUTHOR]

Interference with DNA damage checkpoints has been demonstrated preclinically to be a highly effective means of increasing the cytotoxicity of a number of DNA-damaging cancer therapies. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA may proceed into premature mitosis followed by cell death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 functionality leads to abrogation of DNA damage checkpoints and has been shown preclinically to enhance the activity of many standard of care chemotherapeutic agents. LY2603618 is a potent and selective small molecule inhibitor of Chk1 protein kinase activity in vitro (IC = 7 nM) and the first selective Chk1 inhibitor to enter clinical cancer trials. Treatment of cells with LY2603618 produced a cellular phenotype similar to that reported for depletion of Chk1 by RNAi. Inhibition of intracellular Chk1 by LY2603618 results in impaired DNA synthesis, elevated H2A.X phosphorylation indicative of DNA damage and premature entry into mitosis. When HeLa cells were exposed to doxorubicin to induce a G2/M checkpoint arrest, subsequent treatment with LY2603618 released the checkpoint, resulting in cells entering into metaphase with poorly condensed chromosomes. Consistent with abrogation of the Chk1 and p53-dependent G2/M checkpoint, mutant TP53 HT-29 colon cancer cells were more sensitive to gemcitabine when also treated with LY2603618, while wild-type TP53 HCT116 cells were not sensitized by LY2603618 to gemcitabine. Treatment of Calu-6 human mutant TP53 lung cancer cell xenografts with gemcitabine resulted in a stimulation of Chk1 kinase activity that was inhibited by co-administration of LY2603618. By all criteria, LY2603618 is a highly effective inhibitor of multiple aspects of Chk1 biology. [ABSTRACT FROM AUTHOR]

IKKβ has previously been implicated in breast cancer bone metastasis and bone remodelling. However, the contribution of IKKβ expressed by bone cells of the tumour microenvironment to breast cancer-induced osteolysis has yet to be investigated. Here, we studied the effects of the verified selective IKKβ inhibitors IKKβIII or IKKβV on osteoclast formation and osteoblast differentiation in vitro and in vivo, human and mouse breast cancer cells' support for osteoclast formation and signalling in vitro and osteolysis ex vivo and in immunocompetent mice after supracalvarial injection of human MDA-MB-231 conditioned medium or intra-cardiac injection of syngeneic 4T1 breast cancer cells. Pre-treatment with IKKβIII or IKKβV prior to exposure to tumour-derived factors from human and mouse breast cancer cell lines protected against breast cancer-induced osteolysis in two independent immunocompetent mouse models of osteolysis and the ex vivo calvarial bone organ system. Detailed functional and mechanistic studies showed that direct inhibition of IKKβ kinase activity in osteoblasts and osteoclasts was associated with significant reduction of osteoclast formation, enhanced osteoclast apoptosis and reduced the ability of osteoblasts to support osteoclastogenesis in vitro. When combined with previous findings that suggest NFκB inhibition reduces breast cancer tumorigenesis and metastasis our present findings have an important clinical implication on raising the possibility that IKKβ inhibitors, as bone anabolics, osteoclast inhibitors as well as anti-metastatic agents, may have advantages over anti-osteoclasts agents in the treatment of both skeletal and non-skeletal complications associated with metastatic breast cancer. [ABSTRACT FROM AUTHOR]

Background: N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin widely used to induce PD models, but the effect of MPTP on the cells and genes of PD has not been fully elucidated. Methods: Single-nucleus RNA sequencing was performed in the Substantia Nigra (SN) of MPTP mice. UMAP analysis was used for the dimensionality reduction visualization of the SN in the MPTP mice. Known marker genes highly expressed genes in each cluster were used to annotate most clusters. Specific Differentially Expressed Genes (DEGs) and PD risk genes analysis were used to find MPTP-associated cells. GO, KEGG, PPI network, GSEA and CellChat analysis were used to reveal cell type-specific functional alterations and disruption of cell-cell communication networks. Subset reconstruction and pseudotime analysis were used to reveal the activation status of the cells, and to find the transcription factors with trajectory characterized. Results: Initially, we observed specific DEGs and PD risk genes enrichment in microglia. Next, We obtained the functional phenotype changes in microglia and found that IGF, AGRN and PTN pathways were reduced in MPTP mice. Finally, we analyzed the activation state of microglia and revealed a pro-inflammatory trajectory characterized by transcription factors Nfe2l2 and Runx1. Conclusion: Our work revealed alterations in microglia function, signaling pathways and key genes in the SN of MPTP mice. [ABSTRACT FROM AUTHOR]

Simple Summary: The prognosis for patients with relapsed neuroblastoma is poor, and novel treatment options for these patients are needed. Some relapsed neuroblastoma tumors harbor activating mutations in the RAS/MAPK pathway. In prior studies, single agent MEK or IGF1R inhibitors induced transient responses as single agents in neuroblastoma models. In this study, we tested the efficacy of a combination of the MEK inhibitor trametinib and the IGF1R inhibitor ganitumab in RAS-mutated neuroblastoma models. While the trametinib/ganitumab combination decreased cell viability and tumor growth, the combination did not prevent metastasis of RAS-mutated neuroblastoma. Therefore, further studies on the effect of trametinib and ganitumab on neuroblastoma metastasis are necessary before initiating clinical trials of this combination of targeted agents in patients with relapsed neuroblastoma. Activating mutations in the RAS/MAPK pathway are observed in relapsed neuroblastoma. Preclinical studies indicate that these tumors have an increased sensitivity to inhibitors of the RAS/MAPK pathway, such as MEK inhibitors. MEK inhibitors do not induce durable responses as single agents, indicating a need to identify synergistic combinations of targeted agents to provide therapeutic benefit. We previously showed preclinical therapeutic synergy between a MEK inhibitor, trametinib, and a monoclonal antibody specific for IGF1R, ganitumab in RAS-mutated rhabdomyosarcoma. Neuroblastoma cells, like rhabdomyosarcoma cells, are sensitive to the inhibition of the RAS/MAPK and IGF1R/AKT/mTOR pathways. We hypothesized that the combination of trametinib and ganitumab would be effective in RAS-mutated neuroblastoma. In this study, trametinib and ganitumab synergistically suppressed neuroblastoma cell proliferation and induced apoptosis in cell culture. We also observed a delay in tumor initiation and prolongation of survival in heterotopic and orthotopic xenograft models treated with trametinib and ganitumab. However, the growth of both primary and metastatic tumors was observed in animals receiving the combination of trametinib and ganitumab. Therefore, more preclinical work is necessary before testing this combination in patients with relapsed or refractory RAS-mutated neuroblastoma. [ABSTRACT FROM AUTHOR]

Background: The restoration of cerebrovascular regulation and improvement of cerebral blood flow in ischaemic regions are crucial for improving the clinical prognosis after stroke. An-Gong-Niu-Huang-Wan (AGNHW) is a famous traditional compound Chinese medicine that has been used for over 220 years to treat acute ischaemic stroke; however, its role in the regulation of cerebral blood flow is still unclear. The aim of the present study was to investigate the regulatory effect of AGNHW on cerebral blood flow and microcirculation after ischaemic stroke and to elucidate the underlying mechanisms involved. Methods: Male C57BL/6 mice were subjected to distal middle cerebral artery occlusion (dMCAO) and randomly assigned to the sham, MCAO, or AGNHW groups. AGNHW was administered intragastrically 1 h after dMCAO. The rotarod test was utilized to evaluate behavioural function; TTC was used to determine the infarct volume; and ischaemic injury was assessed by detecting brain levels of SOD, MDA and NO. Then, cortical perfusion and acetazolamide-induced cerebrovascular reactivity were assessed using laser speckle contrast imaging, and the velocity and flux of red blood cells in cortical capillaries were detected using two-photon laser scanning microscopy. In addition, we employed RNA-Seq to identify variations in gene expression profiles and assessed endothelium-dependent changes in microcirculatory dysfunction by measuring vasoactive mediator levels. Results: AGNHW significantly increased cerebral blood flow, reduced the infarct volume, and promoted functional recovery after cerebral ischaemia. AGNHW increased the velocity and flux of red blood cells in capillaries and improved cerebrovascular reactivity in the ischaemic cortex. Furthermore, AGNHW regulated endothelium-dependent microcirculation, as evidenced by decreases in the expression of endothelins (Edn1, Edn3 and Ednrb) and the ratios of brain and serum TXB2/6-keto-PGF1α and ET-1/CGRP. Conclusions: AGNHW improved cerebral hypoperfusion, regulated cerebrovascular reactivity and attenuated microcirculatory dysfunction within the ischaemic cortex after stroke. This outstanding effect was achieved by modulating the expression of genes related to vascular endothelial cell function and regulating endothelium-dependent vasoactive mediators. [ABSTRACT FROM AUTHOR]

Background: Myocarditis refers to an autoimmune inflammatory response of the myocardium with characterization of self-reactive CD4+ T cell activation, which lacks effective treatment and has a poor prognosis. Acacetin is a natural flavonoid product that has been reported to have anti-inflammatory effects. However, acacetin has not been investigated in myocarditis. Methods: Oral acacetin treatment was administered in an experimental autoimmune myocarditis model established with myosin heavy chain-alpha peptide. Echocardiography, pathological staining, and RT-qPCR were used to detect cardiac function, myocardial injury, and inflammation levels. Flow cytometry was utilized to detect the effect of acacetin on CD4+ T cell function. RNA-seq, molecular docking, and microscale thermophoresis (MST) were employed to investigate potential mechanisms. Seahorse analysis, mitoSOX, JC-1, and mitotracker were utilized to detect the effect of acacetin on mitochondrial function. Results: Acacetin attenuated cardiac injury and fibrosis as well as heart dysfunction, and reduced cardiac inflammatory cytokines and ratio of effector CD4+ T and Th17 cells. Acacetin inhibited CD4+ T cell activation, proliferation, and Th17 cell differentiation. Mechanistically, the effects of acacetin were related to reducing mitochondrial complex II activity thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen species in CD4+ T cells. Conclusion: Acacetin may be a valuable therapeutic drug in treating CD4+ T cell-mediated myocarditis. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma is the most common cancer in infants and the most common solid tumor outside the brain in children, and responds poorly to current therapies. The sphingolipid-modifying drug opaganib, which has anticancer and anti-inflammatory activity in many preclinical models, was tested for inhibition of neuroblastoma cell proliferation in cell culture and in tumors growing in mice. Opaganib inhibited cell proliferation regardless of the MYCN gene status of the neuroblastoma cells. Treatment of tumor-bearing mice with opaganib in combination with the established drugs irinotecan and temozolomide reduced tumor growth and increased survival compared with irinotecan plus temozolomide alone. Because opaganib has already demonstrated safety in patients with cancer, this new drug may provide improved therapy for neuroblastoma patients. Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody. [ABSTRACT FROM AUTHOR]

Simple Summary: Patients with treatable cancers all too often return to the clinic with untreatable tumors, requiring highly toxic secondary treatments or palliative care. In this study, we aim to determine whether activation of the Anaphase Promoting Complex (APC) in multiple-drug-resistant (MDR) breast cancer cells will re-sensitize them to chemotherapeutic agents. The APC is required for the targeted degradation of proteins that inhibit passage through mitosis. We found that APC activity is indeed impaired in MDR cells and that chemical activation of the APC increased the sensitivity of these cells to doxorubicin. Mitotic progression was slowed in MDR cells, compared to matched parental drug sensitive cells, with an accumulation of APC substrate proteins. APC activation in nocodazole-arrested cells resulted in increased passage through mitosis with lower APC substrate levels. Mice growing a patient-derived xenografted (PDX) tumor were treated with increasing doses of a chemical APC activator, resulting in a dose-dependent reduction in tumor size. Taken together, our data show that APC activity is reduced in MDR cells, with APC activation resulting in a species- and cancer-type-independent reversal of the MDR phenotype. The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy. [ABSTRACT FROM AUTHOR]

Background: The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1. Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by 35S-methionine/cysteine incorporation into cellular protein. Results: EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts . Conclusions: EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis. [ABSTRACT FROM AUTHOR]

The fruit body of artificially cultivated Cordyceps bassiana has been reported to exhibit anti-inflammatory and anticancer activities. Although it has been suggested that the fruit body has neutraceutic and pharmaceutic biomaterial potential, the exact anti-inflammatory molecular mechanism has not been fully elucidated. In this study, we demonstrated the immunopharmacologic activity of Cordyceps bassiana under in vitro conditions and investigated its anti-inflammatory mechanism. Water extract (Cm-WE) of the fruit body of artificially cultivated Cordyceps bassiana without polysaccharide fractions reduced the expression of the proinflammatory genes cyclooxygenase (COX)-2, interleukin (IL)-12, and inducible nitric oxide synthase (iNOS) and promoted the expression of the anti-inflammatory gene IL-10 in lipopolysaccharide (LPS)-treated RAW264.7 cells. In addition, this fraction suppressed proliferation and interferon (IFN)- production in splenic T lymphocytes. Cm-WE blocked the activation of nuclear factor (NF)-B and activator protein (AP)-1 and their upstream inflammatory signaling cascades, including Syk, MEK, and JNK. Using kinase assays, Syk was identified as the target enzyme most strongly inhibited by Cm-WE. These results strongly suggest that Cm-WE suppresses inflammatory responses by inhibiting Syk kinase activity, with potential implications for novel neutraceutic and pharmaceutic biomaterials. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNF-α) is an inflammatory cytokine that plays a central role in obesity-induced insulin resistance. It also controls cellular lipid metabolism, but the underlining mechanism is poorly understood. We report in this study that phosphoinositide 3-kinase enhancer A (PIKE-A) is a novel effector of TNF-α to facilitate its metabolic modulation in the skeletal muscle. Depletion of PIKE-A in C2C12 myotubes diminished the inhibitory activities of TNF-α on mitochondrial respiration and lipid oxidation, whereas PIKE-A overexpression exacerbated these cellular responses. We also found that TNF-α promoted the interaction between PIKE-A and AMP-activated protein kinase (AMPK) to suppress its kinase activity in vitro and in vivo. As a result, animals with PIKE ablation in the skeletal muscle per se display an upregulation of AMPK phosphorylation and a higher preference to use lipid as the energy production substrate under high-fat diet feeding, which mitigates the development of diet-induced hyperlipidemia, ectopic lipid accumulation, and muscle insulin resistance. Hence, our data reveal PIKE-A as a new signaling factor that is important for TNF-α-initiated metabolic changes in skeletal muscle. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance. [ABSTRACT FROM AUTHOR]

Introduction: The goal of this study is to explore the pharmacological potential of the amyloid-reducing vasodilator fasudil, a selective Ras homolog (Rho)- associated kinases (ROCK) inhibitor, in the P301S tau transgenic mouse model (Line PS19) of neurodegenerative tauopathy and Alzheimer's disease (AD). Methods: We used LC-MS/MS, ELISA and bioinformatic approaches to investigate the effect of treatment with fasudil on the brain proteomic profile in PS19 tau transgenic mice. We also explored the efficacy of fasudil in reducing tau phosphorylation, and the potential beneficial and/or toxic effects of its administration in mice. Results: Proteomic profiling of mice brains exposed to fasudil revealed the activation of the mitochondrial tricarboxylic acid (TCA) cycle and blood-brain barrier (BBB) gap junction metabolic pathways. We also observed a significant negative correlation between the brain levels of phosphorylated tau (pTau) at residue 396 and both fasudil and its metabolite hydroxyfasudil. Conclusions: Our results provide evidence on the activation of proteins and pathways related to mitochondria and BBB functions by fasudil treatment and support its further development and therapeutic potential for AD. [ABSTRACT FROM AUTHOR]

Simple Summary: Novel therapeutics are urgently needed for high-risk and refractory solid tumors. Clinical studies have demonstrated the safety and efficacy of PV-10 (10% rose bengal sodium) by intralesional injection in skin cancer. However, this agent has not yet been evaluated for the treatment of various adult solid tumors. The aim of our study was to test PV-10 in breast, colorectal, head and neck, and testicular cancers. Using a combination of in vitro and in vivo experiments, we found that PV-10 exhibits anti-cancer activity against a panel of human cell lines derived from these tumors. Our results support further clinical development of PV-10 for the treatment of solid tumors in adults. PV-10 is a 10% formulation of rose bengal sodium that has potent immunotherapeutic and anti-cancer activity against various tumors, including metastatic melanoma and refractory neuroblastoma. Currently, PV-10 is undergoing clinical testing for refractory metastatic neuroendocrine cancer and melanomas. However, preclinical investigation of PV-10 activity and its mechanisms against phenotypically and molecularly diverse adult solid tumors had not been conducted. In a panel of human cell lines derived from breast, colorectal, head and neck, and testicular cancers, we demonstrated that PV-10 induces cytotoxicity by apoptotic and autophagic pathways involving caspase-mediated PARP cleavage, downregulation of SQSTM1/p62, and upregulation of beclin-1. Treatment with PV-10 also consistently reduced phosphorylation of WNK1, which has been implicated in cancer cell migration and autophagy inhibition. By wound healing assay, PV-10 treatment inhibited the migration of cancer cells. Finally, significant inhibition of tumor growth was also noted in tumor-bearing mice treated with PV-10 by intralesional or systemic administration. In addition to known PV-10-mediated tumor-specific cytotoxic effects, we identified the mechanisms of PV-10 and provide new insights into its effect on autophagy and metastasis. Our data provide essential mechanism-based evidence and biomarkers of activity to formulate clinical studies of PV-10 in the future. [ABSTRACT FROM AUTHOR]

Simple Summary: Incidence and mortality rates for prostate cancer remain high due to advanced disease characterized by the heterogeneous activation of numerous molecular pathways. In the current study, utilizing a genetic mouse model of advanced prostate cancer with hyperactivated metastasis-associated protein 1/mammalian target of rapamycin (MTA1/mTOR) tumor-promoting pathway, we show for the first time that gnetin C, a natural compound from the melinjo plant, blocks the progression of prostate cancer by reducing cell proliferation and angiogenesis and promoting cell death through the efficient targeting of the MTA1/mTOR pathway. These data may provide a foundation from which to explore gnetin C as a monotherapy and/or combination therapy with approved drugs against advanced prostate cancer in patients with a loss of phosphatase and tensin homolog (PTEN) expression and activated MTA1/mTOR signaling. The metastasis-associated protein 1/protein kinase B (MTA1/AKT) signaling pathway has been shown to cooperate in promoting prostate tumor growth. Targeted interception strategies by plant-based polyphenols, specifically stilbenes, have shown great promise against MTA1-mediated prostate cancer progression. In this study, we employed a prostate-specific transgenic mouse model with MTA1 overexpression on the background of phosphatase and tensin homolog (Pten) null (R26MTA1; Ptenf/f) and PC3M prostate cancer cells which recapitulate altered molecular pathways in advanced prostate cancer. Mechanistically, the MTA1 knockdown or pharmacological inhibition of MTA1 by gnetin C (dimer resveratrol) in cultured PC3M cells resulted in the marked inactivation of mammalian target of rapamycin (mTOR) signaling. In vivo, mice tolerated a daily intraperitoneal treatment of gnetin C (7 mg/kg bw) for 12 weeks without any sign of toxicity. Treatment with gnetin C markedly reduced cell proliferation and angiogenesis and promoted apoptosis in mice with advanced prostate cancer. Further, in addition to decreasing MTA1 levels in prostate epithelial cells, gnetin C significantly reduced mTOR signaling activity in prostate tissues, including the activity of mTOR-target proteins: p70 ribosomal protein S6 kinase (S6K) and eukaryotic translational initiation factor 4E (elF4E)-binding protein 1 (4EBP1). Collectively, these findings established gnetin C as a new natural compound with anticancer properties against MTA1/AKT/mTOR-activated prostate cancer, with potential as monotherapy and as a possible adjunct to clinically approved mTOR pathway inhibitors in the future. [ABSTRACT FROM AUTHOR]

Background: Cutaneous melanoma is a kind of skin malignancy with low morbidity but high mortality. Cryptotanshinone (CPT), an important component of salvia miltiorrhiza has potent anti-tumor activity and also indicates therapeutic effect on dermatosis. So we thought that CPT maybe a potential agent for therapy of cutaneous melanoma. Methods: B16F10 and A375 melanoma cells were used for in vitro assay. Tumor graft models were made in C57BL/6N and BALB/c nude mice for in vivo assay. Seahorse XF Glycolysis Stress Test Kit was used to detect extracellular acidification rate and oxygen consumption rate. Si-RNAs were used for knocking down adenosine monophosphate-activated protein kinase (AMPK) expression in melanoma cells. Results: CPT could inhibit the proliferation of melanoma cells. Meanwhile, CPT changed the glucose metabolism and inhibited phosphofructokinase (PFK)-mediated glycolysis in melanoma cells to a certain extent. Importantly, CPT activated AMPK and inhibited the expression of hypoxia inducible factor 1α (HIF-1α). Both AMPK inhibitor and silencing AMPK could partially reverse CPT's effect on cell proliferation, cell apoptosis and glycolysis. Finally, in vivo experimental data demonstrated that CPT blocked the growth of melanoma, in which was dependent on the glycolysis-mediated cell proliferation. Conclusions: CPT activated AMPK and then inhibited PFK-mediated aerobic glycolysis leading to inhibition of growth of cutaneous melanoma. CPT should be a promising anti-melanoma agent for clinical melanoma therapy. [ABSTRACT FROM AUTHOR]

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Objective. This study was to construct a ceRNA (lncRNA-miRNA-mRNA) regulatory network for prostate cancer (PCa) and to explore the prognostic correlation, key biological functions, and pathways of core RNAs. Methods. Three subgene expression matrices (miRNA, lncRNA, and mRNA expression matrix) were taken from the TCGA database and used in this investigation. Differential expression analysis and differential expression miRNAs were carried out. Next, the ceRNA (lncRNA-miRNA-mRNA) regulatory complex was used to visualize our data and show how they interacted. Ultimately, the primary molecular roles and biological pathways of PCa were identified using enrichment analysis by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). An effect of AC016773.1 on prostate cancer cell proliferation was investigated by knocking out AC016773.1 in an animal model. The interrelationship between AC016773.1 and hsa-mir-25 was validated using RNA immunoprecipitation technology. Results. The lncRNA, miRNA, and mRNA expression matrices obtained from the TCGA database contain 16901, 1881, and 19962 transcripts, respectively. Through differential expression analysis, we obtained 2010 de lncRNA comparative information, 75 lncRNA and miRNA interaction pairs, and 31 targeted de mRNAs. Through the differential expression analysis of RNA nodes in the ceRNA regulatory network, we found that compared with the NP group, in the PCa group, there were 14 lncRNAs upregulated and 25 lncRNAs downregulated, 1 miRNA upregulated and 3 miRNA downregulated, and 10 mRNA upregulated and 21 mRNA downregulated. In KEGG enrichment analysis, the pathways identified by targeted DE-mRNAs were mainly related to calcium signaling pathway, EGFR tyrosine kinase inhibitor resistance, melanoma, PI3K−Akt signaling pathway, and proteoglycans in cancer. In animal models, it was found that knocking down AC016773.1 significantly reduced tumor volume and weight, indicating that AC016773.1 may promote the proliferation of PCa cells. The use of RNA immunoprecipitation technology indicates a direct binding between AC016773.1 and hsa-mir-25. Conclusion. We elucidated the network regulatory relationship of lncRNA-miRNA-mRNA in PCa and further explored the key molecular functions, biological pathways, and prognostic value of targeted DE-mRNAs. [ABSTRACT FROM AUTHOR]

Simple Summary: Malignant meningiomas have high aggressiveness and recurrence rates and a poor prognosis, with no clear pharmacological treatment available. The study aims to investigate the progression mechanism of malignant meningiomas and the targets of intervention. RACK1 and CSNK2B have been shown to promote tumor progression but their roles in meningiomas are not clear. In this study, we will investigate the roles of RACK1-CSNK2B in meningiomas and search for targets to inhibit the progression of meningiomas. Higher-grade meningiomas (WHO grade II and III) are characterized by aggressive invasiveness and high postoperative recurrence rates. The prognosis remains inadequate even with adjuvant radiotherapy and currently there is no definitive pharmacological treatment strategy and target for malignant meningiomas. This study aims to unveil the mechanisms driving the malignant progression of meningiomas and to identify potential inhibitory targets, with significant clinical implications. Implementing techniques such as protein immunoprecipitation, mass spectrometry, RNA interference, and transcriptome sequencing, we investigated the malignancy mechanisms in meningioma cell lines IOMM-LEE and CH157-MN. Additionally, in vivo experiments were carried out on nude mice. We discovered a positive correlation between meningioma malignancy and the levels of the receptor for activated C kinase 1 (RACK1), which interacts with CSNK2B, the β subunit of casein kinase 2 (CK2), inhibiting its ubiquitination and subsequent degradation. This inhibition allows CK2 to activate the NF-κb pathway, which increases the transcription of CDK4 and cyclin D3, resulting in the transition of the cell cycle into the G2/M phase. The RACK1 inhibitor, harringtonolide (HA), significantly suppressed the malignant tendencies of meningioma cells. Our study suggests that RACK1 may play a role in the malignant progression of meningiomas, and therefore, targeting RACK1 could emerge as an effective strategy for reducing the malignancy of these tumors. [ABSTRACT FROM AUTHOR]

Background: Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma, but the pathways underpinning this effect have not been examined.Methods and Results: To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload-induced hypertrophy but also reverses preestablished cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the antihypertrophic and proapoptotic GSK-3β molecule. Pharmacological inhibition of glycogen synthase kinase-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of Akt(S473), a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2.Conclusions: These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3β activity. [ABSTRACT FROM AUTHOR]

Background We previously reported that acute functional tolerance ( AFT) to the hypnotic effects of alcohol was significantly correlated with drinking in the dark ( DID) in the LXS recombinant inbred panel, but only in mice that had been pretreated with alcohol. Here, we have conducted quantitative trait locus ( QTL) mapping for AFT. DNA sequencing of the progenitor ILS and ISS strains and microarray analyses were also conducted to identify candidate genes and functional correlates. Methods LXS mice were given either saline or alcohol (5 g/kg) on day 1 and then tested for loss of righting reflex AFT on day 2. QTLs were mapped using standard procedures. Two microarray analyses from brain were conducted: (i) naïve LXS mice and (ii) an alcohol treatment time course in the ILS and ISS. The full genomes of the ILS and ISS were sequenced to a depth of approximately 30×. Results A significant QTL for AFT in the alcohol pretreatment group was mapped to distal chromosome 4; numerous suggestive QTLs were also mapped. Preference drinking and DID have previously been mapped to the chromosome 4 locus. The credible interval of the significant chromosome 4 QTL spanned 23 Mb and included 716 annotated genes of which 150 had at least 1 nonsynonymous single nucleotide polymorphism or small indel that differed between the ILS and ISS; expression of 48 of the genes was cis-regulated. Enrichment analysis indicated broad functional categories underlying AFT, including proteolysis, transcription regulation, chromatin modification, protein kinase activity, and apoptosis. Conclusions The chromosome 4 QTL is a key region containing possibly pleiotropic genes for AFT and drinking behavior. Given that the region contains many viable candidates and a large number of the genes in the interval fall into 1 or more of the enriched functional categories, we postulate that many genes of varying effect size contribute to the observed QTL effect. [ABSTRACT FROM AUTHOR]

Salt-inducible kinases (SIKs) are key regulators of cellular metabolism and growth, but their role in cardiomyocyte plasticity and heart failure pathogenesis remains unknown. Here, we showed that loss of SIK1 kinase activity protected against adverse cardiac remodeling and heart failure pathogenesis in rodent models and cardiomyocytes derived from human induced pluripotent stem cells. We found that SIK1 phosphorylated and stabilized histone deacetylase 7 (HDAC7) protein during cardiac stress, an event that is required for pathologic cardiomyocyte remodeling. Gain- and loss-of-function studies of HDAC7 in cultured cardiomyocytes implicated HDAC7 as a prohypertrophic signaling effector that can induce c-Myc expression, indicating a functional departure from the canonical MEF2 corepressor function of class IIa HDACs. Taken together, our findings reveal what we believe to be a previously unrecognized role for a SIK1/HDAC7 axis in regulating cardiac stress responses and implicate this pathway as a potential target in human heart failure. [ABSTRACT FROM AUTHOR]

Background: The calibrator in immunoassay plays an essential role in diagnosing Alzheimer's disease (AD). Presently, the most well-studied biomarkers for AD diagnosis are three phosphorylated Tau (p-Tau): p-Tau231, p-Tau217, and p-Tau181. Glycogen synthase-3beta (GSK3β)-phosphorated Tau-441 is the most commonly used calibrator for p-Tau immunoassays. However, the batch-tobatch inconsistency issue of the commonly used GSK3β-phosphorylated Tau-441 limits its clinical application. Methods: We have successfully generated and characterized 61 Tau monoclonal antibodies (mAbs) with distinct epitopes by using the hybridoma technique and employed them as capture or detection antibodies for p-Tau immunoassays. Through chemical synthesis, we synthesized calibrators, which are three peptides including capture and detection antibody epitopes, for application in immunoassays that detect p-Tau231, p-Tau217, and p-Tau181. The novel calibrators were applied to Enzyme-linked immunosorbent assay (ELISA) and Single-molecule array (Simoa) platforms to validate their applicability and establish a range of p-Tau immunoassays. Results: By employing the hybridoma technique, 49 mAbs recognizing Tau (1-22), nine mAbs targeting p-Tau231, one mAb targeting p-Tau217, and two mAbs targeting p-Tau181 were developed. Peptides, including recognition epitopes of capture and detection antibodies, were synthesized. These peptides were used as calibrators to develop 60 immunoassays on the ELISA platform, of which six highly sensitive immunoassays were selected and applied to the ultrasensitive Simoa platform. Remarkably, the LODs were 2.5, 2.4, 31.1, 32.9, 46.9, and 52.1 pg/ml, respectively. Conclusion: Three novel p-Tau calibrators were successfully generated and validated, which solved the batch-to-batch inconsistency issue of GSK3β-phosphorylated Tau-441. The novel calibrators exhibit the potential to promote the standardization of clinical AD diagnostic calibrators. Furthermore, we established a series of highly sensitive and specific immunoassays on the Simoa platform based on novel calibrators, which moved a steady step forward in p-Tau immunoassay application for AD diagnosis. [ABSTRACT FROM AUTHOR]

Simple Summary: Transforming growth factor-beta (TGFβ) mediates various biological processes including cell growth, cell death, cellular differentiation and stemness, among others. TGFβ also regulates tumor formation and metastasis in a context-dependent manner. This research aims to investigate and define the role of the TGFβ cell signaling pathway in melanoma, which is a deadly form of skin cancer. Using relevant melanoma cancer cell lines and preclinical models of melanoma, we show that TGFβ acts as a potent tumor suppressor and negative regulator of cancer stemness and metastasis in melanoma. These findings will be instrumental for the future development of targeted therapy in melanoma. The secreted protein transforming growth factor-beta (TGFβ) plays essential roles, ranging from cell growth regulation and cell differentiation in both normal and cancer cells. In melanoma, TGFβ acts as a potent tumor suppressor in melanoma by blocking cell cycle progression and inducing apoptosis. In the present study, we found TGFβ to regulate cancer stemness in melanoma through the Smad signaling pathway. We discovered that TGFβ/Smad signaling inhibits melanosphere formation in multiple melanoma cell lines and reduces expression of the CD133+ cancer stem cell subpopulation in a Smad3-dependent manner. Using preclinical models of melanoma, we further showed that preventing Smad3/4 signaling, by means of CRISPR knockouts, promoted both tumorigenesis and lung metastasis in vivo. Collectively, our results define new functions for the TGFβ/Smad signaling axis in melanoma stem-cell maintenance and open avenues for new therapeutic approaches to this disease. [ABSTRACT FROM AUTHOR]

Background: Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder that is common in women of reproductive age. The clinical features of PCOS include hyperandrogenemia and polycystic ovarian changes. Bailing capsule (BL), a proprietary Chinese medicine that contains fermented Cordyceps sinensis powder, has been applied to treat PCOS. However, the specific active ingredients of BL and its mechanisms of action are yet to be elucidated. Methods: Initially, the effectiveness of BL on PCOS model mice was evaluated. Subsequently, the active ingredients of BL were searched in the TCMSP and TCM Systems Pharmacology databases, and their targets were predicted using Swiss Target Prediction and SEA databases. Furthermore, the GEO gene database was used to screen for differentially expressed genes (DEGs) related to PCOS. Data from Gene Card, OMIM, DDT, and Drugbank databases were then combined to establish a PCOS disease gene library. Cross targets were imported into the STRING database to construct a protein–protein interaction network. In addition, GO and KEGG pathway enrichment analyses were performed using Metascape and DAVID databases and visualized using Cytoscape software and R 4.2.3. The core targets were docked with SYBYL-X software, and their expressions in PCOS mice were further verified using qPCR. Results: The core active ingredients of BL were identified to be linoleyl acetate, cholesteryl palmitate, arachidonic acid, among others. Microarray data sets from four groups containing disease and normal samples were obtained from the GEO database. A total of 491 DEGs and 106 drug–disease cross genes were selected. Estrous cycle and ovarian lesions were found to be improved in PCOS model mice following BL treatment. While the levels of testosterone, progesterone, and prolactin decreased, that of estradiol increased. qPCR findings indicated that the expressions of JAK2, PPARG, PI3K, and AKT1 were upregulated, whereas those of ESR1 and IRS1 were downregulated in PCOS model mice. After the administration of BL, the expressions of associated genes were regulated. This study demonstrated that BL exerted anti-PCOS effects via PIK3CA, ESR1, AKT, PPARG, and IRS1 targets affecting PI3K-Akt signaling pathways. Discussion: This research clarified the multicomponent, multitarget, and multichannel action of BL and provided a theoretical reference for further investigations on its pharmacological basis and molecular mechanisms against PCOS. [ABSTRACT FROM AUTHOR]

Background: Alzheimer’s disease (AD) is a multifactorial neurodegenerative condition. The search for multi-target traditional Chinese medicines or ingredients for treating AD has attracted much attention. Corydalis rhizome (CR) is a traditional Chinese medicine. Its main components are alkaloids, which have therapeutic effects that can potentially be used for treating AD. However, no systematic study has been conducted to explore the anti-AD efficacy of CR, as well as its active compounds and mechanisms of action. Objective: The present study aimed to clarify CR’s active constituents and its pharmacological mechanisms in treating AD. Methods: A D-galactose & scopolamine hydrobromide-induced AD mouse model was used and CR was administered orally. The prototypical alkaloid components were identified in the serum. The core components, key targets, and possible mechanisms of action of these alkaloids were revealed through network pharmacology. Molecular docking of the key target was performed. Finally, the mechanism was validated by lipopolysaccharide (LPS)-induced activation of BV2 microglia. Results: The results showed that CR improved anxiety-like behavior, spatial and non-spatial recognition, and memory capacity in AD mice. It also achieved synergistic AD treatment by modulating neurotransmitter levels, antineuroinflammation, and anti-oxidative stress. The core components that enhance CR’s efficacy in treating AD are protoberberine-type alkaloids. The CR may induce the polarization of LPS-activated BV2 microglia from phenotype M1 to M2. This is partially achieved by modulating the IL-6/JAK2/STAT3 signaling pathway, which could be the mechanism by which CR treats AD through anti-inflammation. Conclusion: The present study provided a theoretical and experimental basis for the clinical application of CR in treating AD. It also provides information that aids the secondary development, and precise clinical use of CR. [ABSTRACT FROM AUTHOR]

A feature of most neurodegenerative diseases is the presence of “mis-folded proteins” that form aggregates, suggesting suboptimal activity of neuronal molecular chaperones. Heat shock protein 90 (Hsp90) is the master regulator of cell responses to “proteotoxic” stresses. Some Hsp90 modulators activate cascades leading to upregulation of additional chaperones. Novobiocin is a modulator at the C-terminal ATP-binding site of Hsp90. Of several novobiocin analogs synthesized and tested for protection against amyloid beta (Aβ)- induced neuronal death, “KU-32” was the most potent in protecting primary neurons, but did not increase expression of other chaperones believed to help clear misfolded proteins. However, KU-32 reversed Aβ-induced superoxide formation, activated Complex I of the electron transfer chain in mitochondria, and blocked the Aβ-induced inhibition of Complex I in neuroblastoma cells. A mechanism for these effects of KU-32 on mitochondrial metabolism appeared to be the inhibition of pyruvate dehydrogenase kinase (PDHK), both in isolated brain mitochondria and in SH-SY5Y cells. PDHK inhibition by the classic enzyme inhibitor, dichloroacetate, led to neuroprotection from Aβ25-35-induced cell injury similarly to KU-32. Inhibition of PDHK in neurons would lead to activation of the PDH complex, increased acetyl-CoA generation, stimulation of the tricarboxylic acid cycle and Complex I in the electron transfer chain, and enhanced oxidative phosphorylation. A focus of future studies may be on the potential value of PDHK as a target in AD therapy. [ABSTRACT FROM AUTHOR]

Random skin flaps have limited clinical application as a broad surgical reconstruction treatment because of distal necrosis. The prolyl hydroxylase domain‐containing protein inhibitor roxadustat (RXD) enhances angiogenesis and reduces oxidative stress and inflammation. This study explored the function of RXD in the survival of random skin flaps. Thirty‐six male Sprague–Dawley rats were randomly divided into low‐dose RXD group (L‐RXD group, 10 mg/kg/2 day), high‐dose RXD group (H‐RXD group, 25 mg/kg/2 day), and control group (1 mL of solvent, 1:9 DMSO:corn oil). The proportion of surviving flaps was determined on day 7 after surgery. Angiogenesis was assessed by lead oxide/gelatin angiography, and microcirculation blood perfusion was evaluated by laser Doppler flow imaging. Specimens in zone II were obtained, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as indicators of oxidative stress. Histopathological status was evaluated with haematoxylin and eosin staining. The levels of hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF), and the inflammatory factors interleukin (IL)‐1β, IL‐6, and tumour necrosis factor‐α (TNF‐α) were detected by immunohistochemistry. RXD promoted flap survival and microcirculatory blood perfusion. Angiogenesis was detected distinctly in the experimental group. SOD activity increased and the MDA level decreased in the experimental group. Immunohistochemistry indicated that the expression levels of HIF‐1α and VEGF were increased while the levels of IL‐6, IL‐1β, and TNF‐α were decreased after RXD injection. RXD promoted random flap survival by reinforcing vascular hyperplasia and decreasing inflammation and ischaemia‐reperfusion injury. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor (FGF) receptor 1 (FGFR1) is a potential therapeutic target for treatment of metastatic renal cell carcinoma (RCC). We investigated the preclinical activity of OM-RCA-01, a novel therapeutic humanized anti-FGFR1 antibody in RCC. OM-RCA-01 has been shown to inhibit in vitro kinase activity of FGFR1 and has high affinity (Kd of 1.59 nM). In human renal carcinoma Caki-1 FGFR1-expressing cells, OM-RCA-01 potently inhibited FGF-mediated signaling and proliferation. In vivo, the tumors in untreated mice or mice treated with non-specific IgG continued their aggressive growth to reach the size of 2,000 cm3, at which point the mice were killed. In contrast, treatment with OM-RCA-01 not only significant arrested further growth of the tumors ( P < 0.01) but also demonstrated differences in tumor volume compared with vehicle already on Day 13. A similar anti-tumor activity of OM-RCA-01 was observed when the antibody was given in low (1 mg/kg) or high (10 mg/kg) doses ( P = 0.917). In Matrigel assay, OM-RCA-01 significantly inhibited FGF-induced endothelial cell migration, capillary-like tubular structure and mature vessels formation. Administration of 10 mg/kg antibody for up to 35 days resulted in minimal body weight loss and no observations of gross toxicity were made. Collectively, the data obtained with OM-RCA-01 are consistent with potent inhibition of FGFR1-signaling, angiogenesis, and tumor growth. OM-RCA-01 is being developed clinically as an intravenous therapy for the treatment of clear cell RCC. [ABSTRACT FROM AUTHOR]

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs. [ABSTRACT FROM AUTHOR]

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Exosomes, as functional paracrine units of therapeutic cells, can partially reproduce the reparative properties of their parental cells. The constitution of exosomes, as well as their biological activity, largely depends on the cells that secrete them. We isolated exosomes from explant-derived cardiac stromal cells from patients with heart failure (FEXO) or from normal donor hearts (NEXO) and compared their regenerative activities in vitro and in vivo. Patients in the FEXO group exhibited an impaired ability to promote endothelial tube formation and cardiomyocyte proliferation in vitro. Intramyocardial injection of NEXO resulted in structural and functional improvements in a murine model of acute myocardial infarction. In contrast, FEXO therapy exacerbated cardiac function and left ventricular remodeling. microRNA array and PCR analysis revealed dysregulation of miR-21-5p in FEXO. Restoring miR-21-5p expression rescued FEXO's reparative function, whereas blunting miR-21-5p expression in NEXO diminished its therapeutic benefits. Further mechanistic studies revealed that miR-21-5p augmented Akt kinase activity through the inhibition of phosphatase and tensin homolog. Taken together, the heart failure pathological condition altered the miR cargos of cardiac-derived exosomes and impaired their regenerative activities. miR-21-5p contributes to exosome-mediated heart repair by enhancing angiogenesis and cardiomyocyte survival through the phosphatase and tensin homolog/Akt pathway. [ABSTRACT FROM AUTHOR]

Background: Fundc1 (FUN14 domain containing 1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study, therefore, were to determine whether the diabetes mellitus-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function.Methods: Levels of FUNDC1 were examined in the hearts from diabetic patients and nondiabetic donors. Levels of Fundc1-induced MAMs and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L d-glucose for 48 hours), and in streptozotocin-treated cardiac-specific Fundc1 knockout mice and cardiac-specific Fundc1 knockout diabetic Akita mice, as well.Results: FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients in comparison with those from nondiabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced endoplasmic reticulum-mitochondrial Ca2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L d-glucose). In comparison with nondiabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from streptozotocin-treated mice and Akita mice. Furthermore, in comparison with control hearts, diabetes mellitus markedly increased coimmunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes mellitus-induced MAM formation, prevented mitochondrial Ca2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase activity. Furthermore, in cardiomyocytes of Prkaa2 knockout mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant AMP-activated protein kinase ablated HG-induced MAM formation and mitochondrial dysfunction.Conclusions: We conclude that diabetes mellitus suppresses AMP-activated protein kinase, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that AMP-activated protein kinase-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Recent study indicated that glutamine prevents alcoholic tissue injury in mouse gut and liver. Here we investigated the potential role of Epidermal Growth Factor Receptor (EGFR) in glutamine-mediated prevention of ethanol-induced colonic barrier dysfunction, endotoxemia and liver damage. Wild-type and EGFR*Tg transgenic (expressing dominant negative EGFR) mice were fed 1-6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin, and junctional integrity assessed by confocal microscopy. Liver injury was evaluated by plasma transaminases, histopathology and triglyceride analyses. Glutamine effect on acetaldehyde-induced tight junction disruption was investigated in Caco-2 cell monolayers. Doxycycline-induced expression of EGFR* blocked glutamine-mediated prevention of ethanol-induced disruption of colonic epithelial tight junction, mucosal permeability and endotoxemia. Ethanol activated cofilin and disrupted actin cytoskeleton, which was blocked by glutamine in an EGFR-dependent mechanism. Ethanol down-regulated antioxidant gene expression and up-regulated cytokine and chemokine gene expression, which were blocked by glutamine in wild-type mice in the presence or absence of doxycycline, but not in EGFR*Tg mice in the presence of doxycycline. Histopathology, plasma transaminases, triglyceride and expression of chemokine and antioxidant genes indicated ethanol-induced liver damage, which were blocked by glutamine in an EGFR-dependent mechanism. Src kinase activity and extracellular ligand binding domain of EGFR are required for glutamine-mediated protection of barrier function in Caco-2 cell monolayers. Glutamine released metalloproteinases into the medium, and metalloproteinase inhibitors blocked glutamine-mediated protection of barrier function. Results demonstrate that EGFR plays an important role in glutamine-mediated prevention of alcoholic gut permeability, endotoxemia and liver damage. [ABSTRACT FROM AUTHOR]

Background: The ataxia telangiectasia mutated and Rad3-related kinase (ATR) has a key role in the signalling of stalled replication forks and DNA damage to cell cycle checkpoints and DNA repair. It has long been recognised as an important target for cancer therapy but inhibitors have proved elusive. As NU6027, originally developed as a CDK2 inhibitor, potentiated cisplatin in a CDK2-independent manner we postulated that it may inhibit ATR.Methods: Cellular ATR kinase activity was determined by CHK1 phosphorylation in human fibroblasts with inducible dominant-negative ATR-kinase dead expression and human breast cancer MCF7 cells. Cell cycle effects and chemo- and radiopotentiation by NU6027 were determined in MCF7 cells and the role of mismatch repair and p53 was determined in isogenically matched ovarian cancer A2780 cells.Results: NU6027 is a potent inhibitor of cellular ATR activity (IC(50)=6.7 μM) and enhanced hydroxyurea and cisplatin cytotoxicity in an ATR-dependent manner. NU6027 attenuated G2/M arrest following DNA damage, inhibited RAD51 focus formation and increased the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy but not the antimitotic, paclitaxel. In A2780 cells sensitisation to cisplatin was greatest in cells with functional p53 and mismatch repair (MMR) and sensitisation to temozolomide was greatest in p53 mutant cells with functional MMR. Importantly, NU6027 was synthetically lethal when DNA single-strand break repair is impaired either through poly(ADP-ribose) polymerase (PARP) inhibition or defects in XRCC1.Conclusion: NU6027 inhibits ATR, impairing G2/M arrest and homologous recombination thus increasing sensitivity to DNA-damaging agents and PARP inhibitors. It provides proof of concept data for clinical development of ATR inhibitors. [ABSTRACT FROM AUTHOR]

1 The role of substance P and its high affinity neurokinin-1 receptor in colitis has not been fully elucidated. We assessed the participation of neurokinin-1 receptor in colitis using the 2,4,6,-trinitrobenzensulphonic add and dextran sulphate-induced animal models of colitis and genetically-engineered, neurokinin-1 receptor-deficient mice. 2 Clinical signs, macroscopic and histologic damage associated with 2,4,6,-trinitrobenzensulphonic acid (12 days) and dextran sulphate (5 days) colitis were more severe in neurokinin-1 deficient than in wild-type mice, while immuno reactivities for epidermal growth factor and its receptor were similar in the colon of both mice strains before and after colitis. 3 Substance P. dose-dependently induced intestinal fibroblast proliferation and enhanced epidermal growth factor-induced proliferation in intestinal fibroblasts isolated from wild-type, but not from neurokinin-1 receptor deficient mice. 4 Substance P-induced intestinal fibroblast proliferation required the presence of epidermal growth factor receptor with kinase activity. Furthermore, substance P induced epidermal growth factor tyrosine phosphorylation and activation in normal intestinal fibroblasts. 5 Our results indicate that in mice lacking the neurokinin-I receptor, substance P plays a protective role in prolonged experimental colitis. [ABSTRACT FROM AUTHOR]

Simple Summary: Skin cancer, such as melanoma, is often treatable but becomes deadly when it expands to other places in the body, a process called metastasis. While many current drugs target melanoma tumor growth, no drugs yet exist to specifically prevent metastasis. Improving treatment outcomes, therefore, will require an understanding of the molecular mechanisms leading to metastasis. The AKT family of proteins are important regulators of cellular growth and signaling, which play differing roles in cancer. This work sought to study each of the AKT isoforms and their contributions to melanoma cell migration and metastasis. We found that AKT2 specifically regulates melanoma cell metastasis through effects on metabolism and melanoma cell properties, while AKT1 is involved in cellular proliferation and growth. This study suggests that specifically targeting AKT2 and AKT1 represents novel therapeutic strategies for different-stage melanoma patients. Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer due to its highly metastatic nature. Melanomas harboring oncogenic BRAFV600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain about whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and we investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT isoform-specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo, whereas AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby the inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that the elucidation of AKT2-specific functions in metastasis might inform therapeutic strategies to improve treatment options for melanoma patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Lung cancer (LC) is the leading cause of cancer deaths. While the current immunotherapies are beneficial to patients, the effects are minimal and heterogeneous, which calls for improvements in patient selection and treatment effectiveness. We discovered taxifolin to inhibit LC. The inhibition was associated with alterations of gene expressions. Among those genes affected, a panel of 12 genes, which we named TxflSig (taxifolin signature), and its subpanel of 7 genes (TxflSig1) effectively predicted responses of lung cancer patients to immunotherapy; TxflSig and TxflSig1 are valuable biomarkers for patient selection. Among both TxflSig and TxflSig1 multigene panels are ITGAL, ITGAX, and TMEM119 genes. These three genes were robustly associated with immunosuppressive activities, and their expressions were inhibited by taxifolin. Collectively, this research contributes to improvement in the management of lung cancer patients via patient selection and suggests that taxifolin could be a promising addition to immunotherapy in treating LC patients. Using an LL2 cell-based syngeneic mouse LC model, taxifolin suppressed allografts along with the appearance of 578 differentially expressed genes (DEGs). These DEGs were associated with enhancement of processes related to the extracellular matrix and lymphocyte chemotaxis as well as the reduction in pathways relevant to cell proliferation. From these DEGs, we formulated 12-gene (TxflSig) and 7-gene (TxflSig1) panels; both predicted response to ICB (immune checkpoint blockade) therapy more effectively in non-small-cell lung cancer (NSCLC) than numerous well-established ICB biomarkers, including PD-L1. In both panels, the mouse counterparts of ITGAL, ITGAX, and TMEM119 genes were downregulated by taxifolin. They were strongly associated with immune suppression in LC, evidenced by their robust correlations with the major immunosuppressive cell types (MDSC, Treg, and macrophage) and multiple immune checkpoints in NSCLC and across multiple human cancer types. ITGAL, ITGAX, and IIT (ITGAL-ITGAX-TMEM119) effectively predicted NSCLC's response to ICB therapy; IIT stratified the mortality risk of NSCLC. The stromal expressions of ITGAL and ITGAX, together with tumor expression of TMEM119 in NSCLC, were demonstrated. Collectively, we report multiple novel ICB biomarkers—TxflSig, TxflSig1, IIT, ITGAL, and ITGAX—and taxifolin-derived attenuation of immunosuppressive activities in NSCLC, suggesting the inclusion of taxifolin in ICB therapies for NSCLC. [ABSTRACT FROM AUTHOR]

Simple Summary: Hepatocellular carcinoma (HCC) is a primary liver malignancy that remains a fatal disease with limited therapeutic options. Aberrant activation of c-Met can modulate tumor growth and progression in HCC. Herein, we have examined the anti-neoplastic effects of decanoic acid (DA) in hepatocellular carcinoma cells and in vivo mouse models. DA suppressed the phosphorylation of c-Met and induced apoptosis in HCC cells by inhibiting the expression of various oncogenic proteins. Moreover, DA inhibited the c-Met cascade in the preclinical cancer model. These results support the idea that DA can be considered a new anti-tumor agent for HCC. DA, one of the medium-chain fatty acids found in coconut oil, is suggested to have diverse biochemical functions. However, its possible role as a chemoprevention agent in HCC has not been deciphered. Aberrant activation of c-Met can modulate tumor growth and progression in HCC. Here, we report that DA exhibited pro-found anti-tumor effects on human HCC through the suppression of HGF/c-Met signaling cascades in vitro and in vivo. It was noted that DA inhibited HGF-induced activation of c-Met and its downstream signals. DA induced apoptotic cell death and inhibited the expression of diverse tumorigenic proteins. In addition, DA attenuated tumor growth and lung metastasis in the HCC mouse model. Similar to in vitro studies, DA also suppressed the expression of c-Met and its downstream signals in mice tissues. These results highlight the substantial potential of DA in the prevention and treatment of HCC. [ABSTRACT FROM AUTHOR]