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Metformin may offer benefits to certain cancer populations experiencing metabolic abnormalities. To extend the anticancer studies of metformin, a tumor model was established through the implantation of murine Lewis Lung Carcinoma (LLC) cells to Normal Diet (ND)-fed and High-Fat Diet (HFD)-fed C57BL/6 mice. The HFD-fed mice displayed metabolic and pro-inflammatory alterations together with accompanying aggressive tumor growth. Metformin mitigated tumor growth in HFD-fed mice, paralleled by reductions in circulating glucose, insulin, soluble P-selectin, TGF-β1 and High Mobility Group Box-1 (HMGB1), as well as tumor expression of cell proliferation, aerobic glycolysis, glutaminolysis, platelets and neutrophils molecules. The suppressive effects of metformin on cell proliferation, migration and oncogenic signaling molecules were confirmed in cell study. Moreover, tumor-bearing HFD-fed mice had higher contents of circulating and tumor immunopositivity of Neutrophil Extracellular Traps (NETs)-associated molecules, with a suppressive effect from metformin. Data taken from neutrophil studies confirmed the inhibitory effect that metformin has on NET formation induced by HMGB1. Furthermore, HMGB1 was identified as a promoting molecule to boost the transition process towards NETs. The current study shows that metabolic, pro-inflammatory and NET alterations appear to play roles in the obesity-driven aggressiveness of cancer, while also representing candidate targets for anticancer potential of metformin.

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.

Spinal microglia are crucial to neuronal hyper-excitability and pain hypersensitivity. The local anesthetic bupivacaine is commonly used for both peripheral and spinal anesthesia. The pain-relief effects resulting from the peripheral and systemic administration of bupivacaine have been previously reported. In this study, the preventive effects of intrathecal bupivacaine administration against neuropathic pain were revealed in a rat model of sciatic nerve chronic constriction injury (CCI). Using a CCI rat model, pain hypersensitivity, characterized by mechanical allodynia and thermal hyperalgesia, correlated well with microglia M1 polarization, activation and pro-inflammatory cytokine expression in both spinal cord dorsal horns and sciatic nerves. Bupivacaine attenuated pain behaviors and inflammatory alternations. We further identified that the Interferon Regulatory Factor 5 (IRF5)/P2X Purinoceptor 4 (P2X4R) and High Mobility Group Box 1 (HMGB1)/Toll-Like Receptor 4 (TLR4)/NF-κB inflammatory axes may each play pivotal roles in the acquisition of microglia M1 polarization and pro-inflammatory cytokine expression under CCI insult. The relief of pain paralleled with the suppression of microglia M1 polarization, elevation of microglia M2 polarization, and inhibition of IRF5/P2X4R and HMGB1/TLR4/NF-κB in both the spinal cord dorsal horns and sciatic nerve. Our findings provide molecular and biochemical evidence for the anti-neuropathic effect of preventive bupivacaine.

Elevation of intracellular cAMP levels has been implicated in glioma cell proliferation inhibition, differentiation, and apoptosis. Inhibition of phosphodiesterase is a way to elevate intracellular cAMP levels. The present study aimed to investigate the anti-glioma potential of dipyridamole, an inhibitor of phosphodiesterase. Upon treatment with dipyridamole, human U87 glioma cells decreased cell viability, clonogenic colonization, migration, and invasion, along with Noxa upregulation, Endoplasmic Reticulum (ER) stress, impaired autophagic flux, Yes-associated Protein 1 (YAP1) phosphorylation, and YAP1 reduction. Pharmacological and genetic studies revealed the ability of dipyridamole to initiate Noxa-guided apoptosis through ER stress. Additionally, the current study further identified the biochemical role of YAP1 in communicating with ER stress and autophagy under situations of dipyridamole treatment. YAP1 promoted autophagy and protected glioma cells from dipyridamole-induced apoptotic cell death. Dipyridamole impaired autophagic flux and rendered glioma cells more vulnerable to apoptotic cell death through ER stress-inhibitable YAP1/autophagy axis. The overall cellular changes caused by dipyridamole appeared to ensure a successful completion of apoptosis. Dipyridamole also duplicated the biochemical changes and apoptosis in glioma T98G cells. Since dipyridamole has additional biochemical and pharmacological properties, further research centered on the anti-glioma mechanisms of dipyridamole is still needed.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC–MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

Adequate stress on the Endoplasmic Reticulum (ER) with the Unfolded Protein Response (UPR) could maintain glioma malignancy. Uncontrolled ER stress, on the other hand, predisposes an apoptosis-dominant UPR program. We studied here the proapoptotic actions of the Epidermal Growth Factor Receptor (EGFR) inhibitor gefitinib, with the focus on ER stress. The study models were human H4 and U87 glioma cell lines. We found that the glioma cell-killing effects of gefitinib involved caspase 3 apoptotic cascades. Three branches of ER stress, namely Activating Transcription Factor-6 (ATF6), Protein Kinase R (PKR)-Like ER Kinase (PERK), and Inositol-Requiring Enzyme 1 (IRE1), were activated by gefitinib, along with the elevation of intracellular free Ca2+, Reactive Oxygen Species (ROS), and NADPH Oxidase2/4 (NOX2/4). Specifically, elevated IRE1 phosphorylation, Tumor Necrosis Factor (TNF) Receptor-Associated Factor-2 (TRAF2) expression, Apoptosis Signal-Regulating Kinase-1 (Ask1) phosphorylation, c-Jun N-Terminal Kinase (JNK) phosphorylation, and Noxa expression appeared in gefitinib-treated glioma cells. Genetic, pharmacological, and biochemical studies further indicated an active ROS/ER stress/Ask1/JNK/Noxa axis causing the glioma apoptosis induced by gefitinib. The findings suggest that ER-stress-based therapeutic targeting could be a promising option in EGFR inhibitor glioma therapy, and may ultimately achieve a better patient response.

Protein-bound uremic toxins, such as p-cresol sulfate (PCS), can be accumulated with declined renal function and aging and is closely linked with central nervous system (CNS) diseases. In the periphery, PCS has effects on oxidative stress and inflammation. Since oxidative stress and inflammation have substantial roles in the pathogenesis of neurological disorders, the CNS effects of PCS were investigated in unilateral nephrectomized C57/BL/6 mice. Unlike intact mice, unilateral nephrectomized mice showed increased circulating levels of PCS after exogenous administration. Upon PCS exposure, the unilateral nephrectomized mice developed depression-like, anxiety-like, and cognitive impairment behaviors with brain PCS accumulation in comparison with the nephrectomy-only group. In the prefrontal cortical tissues, neuronal cell survival and neurogenesis were impaired along with increased apoptosis, oxidative stress, and neuroinflammation. Circulating brain-derived neurotrophic factors (BDNF) and serotonin were decreased in association with increased corticosterone and repressor element-1 silencing transcription factor (REST), regulators involved in neurological disorders. On the contrary, these PCS-induced changes were alleviated by uremic toxin absorbent AST-120. Taken together, PCS administration in mice with nephrectomy contributed to neurological disorders with increased oxidative stress and neuroinflammation, which were alleviated by PCS chelation. It is suggested that PCS may be a therapeutic target for chronic kidney disease-associated CNS diseases.

Obesity is a metabolic disorder that results from complex interactions between genetic predisposition and dietary factors. Interleukin-4 (IL-4), besides its role in immunity, has metabolic effects on insulin efficacy. We studied the effects of IL-4 on metabolic abnormalities in a mice model of obesity involving leptin deficiency and leptin resistance. Leptin-deficient 145E and leptin-resistant high-fat diet (HFD) mice showed lower levels of circulating IL-4. 145E and HFD mice showed a number of abnormalities: Obesity, hyperglycemia, hyperinsulinemia, insulin resistance, dyslipidemia, liver injury, and adiposity with concurrent inflammation, decreases in Akt, signal transducer and activator of transcription 3 (STAT3), and STAT6 phosphorylation in the hypothalamus, liver, and epididymal fat. Independent of leptin-deficient obesity and dietary obesity, a course of 8-week IL-4 supplementation improved obesity and impairment in Akt, STAT3, and STAT6 signaling. Amelioration of cytokine expression, despite variable extents, was closely linked with the actions of IL-4. Additionally, the browning of white adipocytes by IL-4 was found in epididymal white adipose tissues and 3T3-L1 preadipocytes. Chronic exercise, weight management, and probiotics are recommended to overweight patients and IL-4 signaling is associated with clinical improvement. Thus, IL-4 could be a metabolic regulator and antiobesity candidate for the treatment of obesity and its complications.

Clinically, high cyclooxygenase-2 expression in malignant glioma correlates well with poor prognosis and the use of aspirin is associated with a reduced risk of glioma. To extend the current understanding of the apoptotic potential of aspirin in most cell types, this study provides evidence showing that aspirin induced glioma cell apoptosis and inhibited tumor growth, in vitro and in vivo. We found that the human H4 glioma cell-killing effects of aspirin involved mitochondria-mediated apoptosis accompanied by endoplasmic reticulum (ER) stress, Noxa upregulation, Mcl-1 downregulation, Bax mitochondrial distribution and oligomerization, and caspase 3/caspase 8/caspase 9 activation. Genetic silencing of Noxa or Bax attenuated aspirin-induced viability loss and apoptosis, while silencing Mcl-1 augmented the effects of aspirin. Data from genetic and pharmacological studies revealed that the axis of ER stress comprised an apoptotic cascade leading to Noxa upregulation and apoptosis. The apoptotic programs and mediators triggered by aspirin in H4 cells were duplicated in human U87 glioma cell line as well as in tumor-bearing BALB/c nude mice. The involvement of ER stress in indomethacin-induced Mcl-1 downregulation was reported in our previous study on glioma cells. Therefore, the aforementioned phenomena indicate that ER stress may be a valuable target for intervention in glioma apoptosis.

Chronic treatment involving opioids exacerbates both the risk and severity of ischemic stroke. We have provided experimental evidence showing the anti-inflammatory and neuroprotective effects of the μ opioid receptor antagonist β-funaltrexamine for neurodegenerative diseases in rat neuron/glia cultures and a rat model of cerebral Ischemia/Reperfusion (I/R) injury. Independent of in vitro Lipopolysaccharide (LPS)/interferon (IFN-γ)-stimulated neuron/glia cultures and in vivo cerebral I/R injury in Sprague–Dawley rats, β-funaltrexamine downregulated neuroinflammation and ameliorated neuronal degeneration. Alterations in microglia polarization favoring the classical activation state occurred in LPS/IFN-γ-stimulated neuron/glia cultures and cerebral I/R-injured cortical brains. β-funaltrexamine shifted the polarization of microglia towards the anti-inflammatory phenotype, as evidenced by decreased nitric oxide, tumor necrosis factor-α, interleukin-1β, and prostaglandin E2, along with increased CD163 and arginase 1. Mechanistic studies showed that the suppression of microglia pro-inflammatory polarization by β-funaltrexamine was accompanied by the reduction of NF-κB, AP-1, cyclic AMP response element-binding protein, along with signal transducers and activators of transcription transcriptional activities and associated upstream activators. The effects of β-funaltrexamine are closely linked with its action on neuroinflammation by switching microglia polarization from pro-inflammatory towards anti-inflammatory phenotypes. These findings provide new insights into the anti-inflammatory and neuroprotective mechanisms of β-funaltrexamine in combating neurodegenerative diseases, such as stroke.

The dormancy of cellular apoptotic machinery has been highlighted as a crucial factor in therapeutic resistance, recurrence, and poor prognosis in patients with malignancy, such as malignant glioma. Increasing evidence indicates that nonsteroidal anti-inflammatory drugs (NSAIDs) confer chemopreventive effects, and indomethacin has been shown to have a novel chemotherapeutic application targeting glioma cells. To extend these findings, herein, we studied the underlying mechanisms of apoptosis activation caused by indomethacin in human H4 and U87 glioma cells. We found that the glioma cell-killing effects of indomethacin involved both death receptor- and mitochondria-mediated apoptotic cascades. Indomethacin-induced glioma cell apoptosis was accompanied by a series of biochemical changes, including reactive oxygen species generation, endoplasmic reticulum (ER) stress, apoptosis signal-regulating kinase-1 (Ask1) activation, p38 hyperphosphorylation, protein phosphatase 2A (PP2A) activation, Akt dephosphorylation, Mcl-1 and FLICE-inhibiting protein (FLIP) downregulation, Bax mitochondrial distribution, and caspases 3/caspase 8/caspase 9 activation. Data on pharmacological inhibition related to oxidative stress, ER stress, free Ca2+, and p38 revealed that the axis of oxidative stress/ER stress/Ask1/p38/PP2A/Akt comprised an apoptotic cascade leading to Mcl-1/FLIP downregulation and glioma apoptosis. Since indomethacin is an emerging choice in chemotherapy and its antineoplastic effects have been demonstrated in glioma tumor-bearing models, the findings further strengthen the argument for turning on the aforementioned axis in order to activate the apoptotic machinery of glioma cells.

The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin β1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-β1 (TGF-β1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-β1 expression. The additions of exogenous fibronectin and TGF-β1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-β1 signaling.

Atypical antipsychotics, such as olanzapine, are commonly prescribed to patients with schizophrenic symptoms and other psychiatric disorders. However, weight gain and metabolic disturbance cause adverse effects, impair patient compliance and limit clinical utility. Thus, a better understanding of treatment-acquired adverse effects and identification of targets for therapeutic intervention are believed to offer more clinical benefits for patients with schizophrenia. Beyond its nutritional effects, studies have indicated that supplementation of chromium brings about beneficial outcomes against numerous metabolic disorders. In this study, we investigated whether olanzapine-induced weight gain and metabolic disturbance involved chromium dynamic mobilization in a female Sprague-Dawley rat model, and whether a dietary supplement of chromium improved olanzapine-acquired adverse effects. Olanzapine medicated rats experienced weight gain and adiposity, as well as the development of hyperglycemia, hyperinsulinemia, insulin resistance, hyperlipidemia, and inflammation. The olanzapine-induced metabolic disturbance was accompanied by a decrease in hepatic Akt and AMP-activated Protein Kinase (AMPK) actions, as well as an increase in serum interleukin-6 (IL-6), along with tissue chromium depletion. A daily intake of chromium supplements increased tissue chromium levels and thermogenic uncoupling protein-1 (UCP-1) expression in white adipose tissues, as well as improved both post-olanzapine weight gain and metabolic disturbance. Our findings suggest that olanzapine medicated rats showed a disturbance of tissue chromium homeostasis by inducing tissue depletion and urinary excretion. This loss may be an alternative mechanism responsible for olanzapine-induced weight gain and metabolic disturbance.

Microglia polarization of classical activation state is crucial to the induction of neuroinflammation, and has been implicated in the pathogenesis of numerous neurodegenerative diseases. Fungal immunomodulatory proteins are emerging health-promoting natural substances with multiple pharmacological activities, including immunomodulation. Herein, we investigated the anti-inflammatory and neuroprotective potential of fungal immunomodulatory protein extracted from Ganoderma microsporum (GMI) in an in vitro rodent model of primary cultures. Using primary neuron/glia cultures consisting of neurons, astrocytes, and microglia, a GMI showed an alleviating effect on lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced inflammatory mediator production and neuronal cell death. The events of neuroprotection caused by GMI were accompanied by the suppression of Nitric Oxide (NO), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Prostaglandin E2 (PGE2) production, along with the inhibition of microglia activation. Mechanistic studies showed that the suppression of microglia pro-inflammatory polarization by GMI was accompanied by the resolution of oxidative stress, the preservation of protein tyrosine phosphatase and serine/threonine phosphatase activity, and the reduction of NF-κB, AP-1, cyclic AMP response element-binding protein (CREB), along with signal transducers and activators of transcription (Stat1) transcriptional activities and associated upstream activators. These findings suggest that GMI may have considerable potential towards the treatment of neuroinflammation-mediated neurodegenerative diseases.

Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners.

Dual leucine zipper kinase (DLK) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family mainly involved in neuronal degeneration. However, the role of DLK signaling in the neuropathic pain has not yet been fully determined. Chronic constrictive injury (CCI) was conducted by four 3-0 chromic gut ligatures loosely ligated around the sciatic nerve. Escalated DLK expression over the dorsal root ganglion was observed from one to four rings of CCI. Remarkable expression of DLK was observed in primary dorsal root ganglion cells culture subjected to electrical stimulation and attenuated by DLK short hairpin RNA (shRNA) treatment. Intrathecal injection of DLK shRNA attenuates the expression of DLK over the dorsal root ganglion and hippocampus neurons and increased the threshold of mechanical allodynia and decreased thermal hyperalgesia. In CatWalk gait analysis, significant decreases of print area, maximum contact maximum intensity, stand phase, single stance, and regular index by CCI were alleviated by the DLK shRNA administration. In conclusion, the expression of DLK was up-regulated in chronic constrictive injury and attenuated by the administration of DLK shRNA, which paralleled the improvement of neurobehavior of neuropathic pain. The modulation of DLK expression is a potential clinic treatment option for neuropathic pain.

Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59-Asp63 and Asp95-Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners. [ABSTRACT FROM AUTHOR]