Malate-Aspartate Shuttle Plays an Important Role in LPS-Induced Neuroinflammation of Mice Due to its Effect on STAT3 Phosphorylation

Neuroinflammation is a key pathological factor in numerous neurological disorders. Cumulating evidence has indicated critical roles of NAD+/NADH metabolism in multiple major diseases, while the role of malate-aspartate shuttle (MAS) - a major NADH shuttle - in inflammation has remained unclear. In this study we investigated the roles of MAS in LPS-induced neuroinflammation both in vivo and in vitro. Immunofluorescence staining, Western blot assay and Real-time PCR assays were conducted to determine the activation of Iba-1, the protein levels of iNOS and COX2 and the mRNA levels of IL-1β, IL-6, and TNF-α in vivo, showing that both pre-treatment and post-treatment of aminooxyacetic acid (AOAA) - an MAS inhibitor - profoundly decreased the LPS-induced neuroinflammation in mice. BV2 microglia was also used as a cellular model to investigate the mechanisms of this finding, in which such assays as Western blot assay and nitrite assay. Our study further indicated that AOAA produced its effects on LPS-induced microglial activation by its effects on MAS: Pyruvate treatment reversed the effects of AOAA on the cytosolic NAD+/NADH ratio, which also restored the LPS-induced activation of the AOAA-treated microglia. Moreover, the lactate dehydrogenase (LDH) inhibitor GSK2837808A blocked the effects of pyruvate on the AOAA-produced decreases in both the cytosolic NAD+/NADH ratio and LPS-induced microglial activation. Our study has further suggested that AOAA produced inhibition of LPS-induced microglial activation at least partially by decreasing STAT3 phosphorylation. Collectively, our findings have indicated AOAA as a new and effective drug for inhibiting LPS-induced neuroinflammation. Our study has also indicated that MAS is a novel mediator of LPS-induced neuroinflammation due to its capacity to modulate LPS-induced STAT3 phosphorylation, which has further highlighted a critical role of NAD+/NADH metabolism in inflammation.

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Prodigiosin inhibits cholangiocarcinoma cell proliferation and induces apoptosis via suppressing SNAREs-dependent autophagy

Cancer Cell International ◽

10.1186/s12935-021-02355-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Dijie Zheng ◽

Shiyu Chen ◽

Kun Cai ◽

Linhan Lei ◽

Chunchen Wu ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Cancer Biology ◽

Bacterial Species ◽

Inhibitory Effect ◽

Cell Counting ◽

Dependent Manner ◽

Western Blot Assay

Abstract Background Prodigiosin (PG), a natural red pigment produced by numerous bacterial species, has been a eye-catching research point in recent years for its anticancer activity. However, the role of PG in the cancer biology of cholangiocarcinoma (CCA) remains vague. Methods The proliferation of CCA cells was detected by Cell Counting Kit-8(CCK-8), Colony formation assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was evaluated by flow cytometry assay and western blot assay. The effects of PG or SNAREs on cell autophagy were measured by autophagy flux assay and western blot assay. Xenograft mouse models were used to assess the role of PG in CCA cells in vivo. Results PG could inhibit the proliferation and viability of CCA cells in a concentration- and time-dependent manner via suppressing the late stage of autophagy. Mechanistically, PG inhibits the fusion of autophagosomes and lysosomes by blocking STX17 and SNAP29, components of soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs)complex. When STX17 and SNAP29 were overexpressed, the inhibitory effect of PG on CCA cells autophagy was relieved. In addition, PG showed obvious inhibitory effects on cancer cell viability but no toxic effects on organs in xenotransplantation models. Conclusion Taken together, our results demonstrated that PG inhibits CCA cell proliferation via suppressing SNAREs-dependent autophagy, implying that PG could be a potential chemotherapy drug for advanced CCA.

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The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666191022160024 ◽

2020 ◽

Vol 19 (17) ◽

pp. 2108-2119

Author(s):

Yang Jin ◽

Li Lv ◽

Shu-Xiang Ning ◽

Ji-Hong Wang ◽

Rong Xiao

Keyword(s):

Wound Healing ◽

Western Blot ◽

Morphological Changes ◽

In Vivo Studies ◽

Migration And Invasion ◽

Tunel Staining ◽

Dna Ladder ◽

Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

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The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620999200818155814 ◽

2020 ◽

Vol 20 ◽

Author(s):

Hongtao Li ◽

Peng Chen ◽

Lei Chen ◽

Xinning Wang

Keyword(s):

Cell Growth ◽

Western Blot ◽

Wilms Tumor ◽

Critical Role ◽

Time Dependent ◽

Luciferase Assay ◽

Dependent Manner ◽

Tlr4 Expression ◽

Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

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Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

Human & Experimental Toxicology ◽

10.1177/09603271211023789 ◽

2021 ◽

pp. 096032712110237

Author(s):

L Zhou ◽

S Li ◽

J Sun

Keyword(s):

Endometrial Cancer ◽

B Cells ◽

Western Blot ◽

Signal Pathway ◽

Mtor Pathway ◽

Related Protein ◽

Western Blot Assay ◽

Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

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LncRNA PVT1 promotes cervical cancer progression by sponging miR-503 to upregulate ARL2 expression

Open Life Sciences ◽

10.1515/biol-2021-0002 ◽

2021 ◽

Vol 16 (1) ◽

pp. 1-13

Author(s):

Weiwei Liu ◽

Dongmei Yao ◽

Bo Huang

Keyword(s):

Cervical Cancer ◽

Cancer Progression ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Nude Mouse Model ◽

Western Blot Assay ◽

Non Coding Rna ◽

Long Non Coding Rna

Abstract Cervical cancer (CC) is a huge threat to the health of women worldwide. Long non-coding RNA plasmacytoma variant translocation 1 gene (PVT1) was proved to be associated with the development of diverse human cancers, including CC. Nevertheless, the exact mechanism of PVT1 in CC progression remains unclear. Levels of PVT1, microRNA-503 (miR-503), and ADP ribosylation factor-like protein 2 (ARL2) were measured by quantitative reverse transcription-polymerase chain reaction or western blot assay. 3-(4,5)-Dimethylthiazole-2-y1)-2,5-biphenyl tetrazolium bromide (MTT) and flow cytometry were used to examine cell viability and apoptosis, respectively. For migration and invasion detection, transwell assay was performed. The interaction between miR-503 and PVT1 or ARL2 was shown by dual luciferase reporter assay. A nude mouse model was constructed to clarify the role of PVT1 in vivo. PVT1 and ARL2 expressions were increased, whereas miR-503 expression was decreased in CC tissues and cells. PVT1 was a sponge of miR-503, and miR-503 targeted ARL2. PVT1 knockdown suppressed proliferation, migration, and invasion of CC cells, which could be largely reverted by miR-503 inhibitor. In addition, upregulated ARL2 could attenuate si-PVT1-mediated anti-proliferation and anti-metastasis effects on CC cells. Silenced PVT1 also inhibited CC tumor growth in vivo. PVT1 knockdown exerted tumor suppressor role in CC progression via the miR-503/ARL2 axis, at least in part.

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The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Nature Communications ◽

10.1038/s41467-021-24418-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Brianna J. Klein ◽

Anagha Deshpande ◽

Khan L. Cox ◽

Fan Xuan ◽

Mohamad Zandian ◽

...

Keyword(s):

Critical Role ◽

Cellular Transformation ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Nucleosome Core ◽

Stem And Progenitor Cells ◽

Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

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Essential role of IPS-1 in innate immune responses against RNA viruses

Journal of Experimental Medicine ◽

10.1084/jem.20060792 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1795-1803 ◽

Cited By ~ 345

Author(s):

Himanshu Kumar ◽

Taro Kawai ◽

Hiroki Kato ◽

Shintaro Sato ◽

Ken Takahashi ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Critical Role ◽

Nuclear Factor Κb ◽

Type I ◽

Innate Immune Responses ◽

Antiviral Responses ◽

Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

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A Yes-Associated Protein (YAP) and Insulin-Like Growth Factor 1 Receptor (IGF-1R) Signaling Loop Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma

Cancers ◽

10.3390/cancers13153812 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3812

Author(s):

Mai-Huong T. Ngo ◽

Sue-Wei Peng ◽

Yung-Che Kuo ◽

Chun-Yen Lin ◽

Ming-Heng Wu ◽

...

Keyword(s):

Nuclear Translocation ◽

Combination Index ◽

Specific Inhibitor ◽

In Vivo Model ◽

Mrna Levels ◽

Margin Distribution ◽

Related Proteins ◽

Emt Markers

The role of a YAP-IGF-1R signaling loop in HCC resistance to sorafenib remains unknown. Method: Sorafenib-resistant cells were generated by treating naïve cells (HepG2215 and Hep3B) with sorafenib. Different cancer cell lines from databases were analyzed through the ONCOMINE web server. BIOSTORM–LIHC patient tissues (46 nonresponders and 21 responders to sorafenib) were used to compare YAP mRNA levels. The HepG2215_R-derived xenograft in SCID mice was used as an in vivo model. HCC tissues from a patient with sorafenib failure were used to examine differences in YAP and IGF-R signaling. Results: Positive associations exist among the levels of YAP, IGF-1R, and EMT markers in HCC tissues and the levels of these proteins increased with sorafenib failure, with a trend of tumor-margin distribution in vivo. Blocking YAP downregulated IGF-1R signaling-related proteins, while IGF-1/2 treatment enhanced the nuclear translocation of YAP in HCC cells through PI3K-mTOR regulation. The combination of YAP-specific inhibitor verteporfin (VP) and sorafenib effectively decreased cell viability in a synergistic manner, evidenced by the combination index (CI). Conclusion: A YAP-IGF-1R signaling loop may play a role in HCC sorafenib resistance and could provide novel potential targets for combination therapy with sorafenib to overcome drug resistance in HCC.

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Circadian clock protein Rev-erbα regulates neuroinflammation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812405116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 5102-5107 ◽

Cited By ~ 36

Author(s):

Percy Griffin ◽

Julie M. Dimitry ◽

Patrick W. Sheehan ◽

Brian V. Lananna ◽

Chun Guo ◽

...

Keyword(s):

Circadian Clock ◽

Microglial Activation ◽

Time Of Day ◽

Resting State Functional Connectivity ◽

Promoter Regions ◽

Clock Component ◽

Glial Cultures ◽

Inflammatory Phenotype

Circadian dysfunction is a common attribute of many neurodegenerative diseases, most of which are associated with neuroinflammation. Circadian rhythm dysfunction has been associated with inflammation in the periphery, but the role of the core clock in neuroinflammation remains poorly understood. Here we demonstrate that Rev-erbα, a nuclear receptor and circadian clock component, is a mediator of microglial activation and neuroinflammation. We observed time-of-day oscillation in microglial immunoreactivity in the hippocampus, which was disrupted in Rev-erbα−/− mice. Rev-erbα deletion caused spontaneous microglial activation in the hippocampus and increased expression of proinflammatory transcripts, as well as secondary astrogliosis. Transcriptomic analysis of hippocampus from Rev-erbα−/− mice revealed a predominant inflammatory phenotype and suggested dysregulated NF-κB signaling. Primary Rev-erbα−/− microglia exhibited proinflammatory phenotypes and increased basal NF-κB activation. Chromatin immunoprecipitation revealed that Rev-erbα physically interacts with the promoter regions of several NF-κB–related genes in primary microglia. Loss of Rev-erbα in primary astrocytes had no effect on basal activation but did potentiate the inflammatory response to lipopolysaccharide (LPS). In vivo, Rev-erbα−/− mice exhibited enhanced hippocampal neuroinflammatory responses to peripheral LPS injection, while pharmacologic activation of Rev-erbs with the small molecule agonist SR9009 suppressed LPS-induced hippocampal neuroinflammation. Rev-erbα deletion influenced neuronal health, as conditioned media from Rev-erbα–deficient primary glial cultures exacerbated oxidative damage in cultured neurons. Rev-erbα−/− mice also exhibited significantly altered cortical resting-state functional connectivity, similar to that observed in neurodegenerative models. Our results reveal Rev-erbα as a pharmacologically accessible link between the circadian clock and neuroinflammation.

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Brain-Specific SNAP-25 Deletion Leads to Elevated Extracellular Glutamate Level and Schizophrenia-Like Behavior in Mice

Neural Plasticity ◽

10.1155/2017/4526417 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 12

Author(s):

Hua Yang ◽

Mengjie Zhang ◽

Jiahao Shi ◽

Yunhe Zhou ◽

Zhipeng Wan ◽

...

Keyword(s):

Cerebral Cortex ◽

Mouse Model ◽

Glutamate Release ◽

Critical Role ◽

Conditional Knockout ◽

Snare Complex ◽

Extracellular Glutamate ◽

Glutamate Level

Several studies have associated reduced expression of synaptosomal-associated protein of 25 kDa (SNAP-25) with schizophrenia, yet little is known about its role in the illness. In this paper, a forebrain glutamatergic neuron-specific SNAP-25 knockout mouse model was constructed and studied to explore the possible pathogenetic role of SNAP-25 in schizophrenia. We showed that SNAP-25 conditional knockout (cKO) mice exhibited typical schizophrenia-like phenotype. A significantly elevated extracellular glutamate level was detected in the cerebral cortex of the mouse model. Compared with Ctrls, SNAP-25 was dramatically reduced by about 60% both in cytoplasm and in membrane fractions of cerebral cortex of cKOs, while the other two core members of SNARE complex: Syntaxin-1 (increased ~80%) and Vamp2 (increased ~96%) were significantly increased in cell membrane part. Riluzole, a glutamate release inhibitor, significantly attenuated the locomotor hyperactivity deficits in cKO mice. Our findings provide in vivo functional evidence showing a critical role of SNAP-25 dysfunction on synaptic transmission, which contributes to the developmental of schizophrenia. It is suggested that a SNAP-25 cKO mouse, a valuable model for schizophrenia, could address questions regarding presynaptic alterations that contribute to the etiopathophysiology of SZ and help to consummate the pre- and postsynaptic glutamatergic pathogenesis of the illness.

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