Chronoeffects of the Herbal Medicines Puerariae radix and Coptidis rhizoma in Mice: A Potential Role of REV-ERBα

Identifying drugs with dosing time-dependent effects (chronoeffects) and understanding the underlying mechanisms would help to improve drug treatment outcome. Here, we aimed to determine chronoeffects of the herbal medicines Puerariae radix (PR) and Coptidis rhizoma (CR), and investigate a potential role of REV-ERBα as a drug target in generating chronoeffects. The pharmacological effect of PR on hyperhomocysteinemia in mice was evaluated by measuring total homocysteine, triglyceride levels and lipid accumulation. PR dosed at ZT10 generated a stronger effect on hyperhomocysteinemia than drug dosed at ZT2. Furthermore, PR increased the expression levels of REV-ERBα target genes Bhmt, Cbs and Cth (encoding three key enzymes responsible for homocysteine catabolism), thereby alleviating hyperhomocysteinemia in mice. Moreover, CR attenuated chronic colitis in mice in a dosing time-dependent manner based on measurements of disease activity index, colon length, malondialdehyde/myeloperoxidase activities and IL-1β/IL-6 levels. ZT10 dosing generated a stronger anti-colitis effect as compared to ZT2 dosing. This was accompanied by lower production of colonic inflammatory cytokines (i.e., Nlrp3, IL-1β, IL-6, Tnf-α and Ccl2, REV-ERBα target genes) in colitis mice dosed at ZT10. The diurnal patterns of PR and CR effects were respectively consistent with those of puerarin (a main active constituent of PR, a REV-ERBα antagonist) and berberine (a main active constituent of CR, a REV-ERBα agonist). In addition, loss of Rev-erbα in mice abolished the dosing time-dependency in PR and CR effects. In conclusion, the therapeutic effects of PR and CR depend on dosing time in mice, which are probably attributed to diurnal expression of REV-ERBα as the drug target. Our findings have implications for improving therapeutic outcomes of herbal medicines with a chronotherapeutic approach.

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Chronoefficacy of the herbal medicines Puerariae radix and Coptidis rhizoma in mice: A potential role of REV-ERBα

10.21203/rs.3.rs-151541/v1 ◽

2021 ◽

Author(s):

Jinming Liu ◽

Haiman Xu ◽

Li Zhang ◽

Shuai Wang ◽

Danyi Lu ◽

...

Keyword(s):

Drug Target ◽

Potential Role ◽

Target Genes ◽

Herbal Medicines ◽

Chronic Colitis ◽

Active Constituent ◽

Circadian Time ◽

Coptidis Rhizoma ◽

Underlying Mechanisms

Abstract Background Identifying drugs with circadian time-dependent efficacy (chronoefficacy) and understanding the underlying mechanisms would help to improve drug treatment outcome. Here, we aimed to determine chronoefficacy of the herbal medicines Puerariae radix (PR) and Coptidis rhizoma (CR), and investigate a potential role of REV-ERBα as a drug target in generating chronoefficacy. Materials and methods PR and CR efficacy were assessed based on the diseases hyperhomocysteinemia and chronic colitis, respectively. The efficacy of PR against hyperhomocysteinemia in mice was evaluated by measuring total homocysteine, triglyceride levels and lipid accumulation. The efficacy of CR against chronic colitis in mice was evaluated by measuring disease activity index, colon length, malondialdehyde/myeloperoxidase activities and IL-1β/IL-6 levels. The underlying mechanisms related to REV-ERBα target genes were analyzed by qPCR. Puerarin in PR and berberine in CR were analyzed by UPLC-QTOF/MS. Results PR dosed at ZT10 generated a better efficacy against hyperhomocysteinemia than drug dosed at ZT2. Furthermore, PR increased the expressions of REV-ERBα target genes Bhmt, Cbs and Cth (encoding three key enzymes responsible for homocysteine catabolism), thereby alleviating hyperhomocysteinemia in mice. Moreover, CR attenuated chronic colitis in mice in a circadian time-dependent manner. ZT10 dosing generated a better anti-colitis efficacy as compared to ZT2 dosing. This was accompanied by lower production of colonic inflammatory cytokines (i.e, Nlrp3, IL-1β, IL-6, Tnf-α and Ccl2, REV-ERBα target genes) in colitis mice at ZT10 dosing. The circadian patterns of PR and CR effects were respectively consistent with those of puerarin (a main active constituent of PR, a REV-ERBα antagonist) and berberine (a main active constituent of CR, a REV-ERBα agonist). Conclusion The therapeutic effects of PR and CR depend on dosing time in mice, which are probably attributed to circadian expression of REV-ERBα as the drug target. Our findings have implications for improving therapeutic outcomes of herbal medicines with a chronotherapeutics approach.

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Role of CD37SMIP a Novel Engineered Small Modular Immunopharmaceutical in the Treatment of CLL.

Blood ◽

10.1182/blood.v110.11.801.801 ◽

2007 ◽

Vol 110 (11) ◽

pp. 801-801 ◽

Cited By ~ 1

Author(s):

Rosa Lapalombella ◽

Xiaobin B. Zhao ◽

Peter R. Baum ◽

Jeffrey A. Ledbetter ◽

Natarajan Muthusamy ◽

...

Keyword(s):

B Cell ◽

Tyrosine Phosphorylation ◽

Potential Role ◽

Time Dependent ◽

Dependent Manner ◽

B Cell Malignancies ◽

Phosphorylated Proteins ◽

Apoptotic Proteins ◽

Induced Apoptosis

Abstract CD37 is a lineage-specific B-cell antigen highly expressed on both normal and transformed B-cells. The significant B-cell selective CD37 expression makes it a valuable target for therapy against B cell malignancies including chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), and non-Hodgkins lymphoma (B-NHL). A novel CD37-specific small modular immunopharmaceutical (CD37SMIP) was engineered to contain a single chain variable region (scFv) linked to a human IgG1 hinge, CH2 and CH3 domains. CD37SMIP induces potent apoptosis in the presence of a crosslinker and antibody dependent cellular cytotoxicity (ADCC) against primary CLL cells. Significant in vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model that is dependent upon NK cell function. The induced cytotoxicity in primary CLL cells was independent of activation of caspase cascade, and consistent with this, the pan-caspase inhibitor z-VAD-fmk failed to rescue CD37 SMIP drug induced cytotoxicity. In an attempt to define the CD37 mediated early signaling events and their role in cytotoxicity, we investigated protein tyrosine phosphorylation as a potential early activation event responsible for CD37 SMIP drug mediated cytotoxicity. Primary B CLL cells were stimulated with increasing concentration of CD37 SMIP with or without crosslinker. Western blot analysis of cellular lysates with anti-phosphotyrosine antibody revealed several tyrosine phosphorylated proteins including a predominant ∼65KDa protein in response to the cross-linking of CD37 SMIP within 10 minutes. Further, pre-treatment with the tyrosine kinase inhibitor herbimycin prevented tyrosine phosphorylation of the predominant 65KDa protein and inhibited CD37 SMIP induced apoptosis in a dose dependent manner. Attempts to identify the tyrosine phosphorylated proteins, using MALDI-TOF mass spectrometry, and to define the potential role of these target proteins in the apoptotic pathway are ongoing. To further examine the molecular mechanism whereby CD37 SMIP triggers cell death, we investigated the involvement of mitochondrial pathways including changes in mitochondrial potential (Δψm) and generation of reactive oxygen species. Treatment with CD37 SMIP induces a time dependent mitochondrial membrane depolarization and increased production of ROS. Further, primary B CLL cells cultured with CD37SMIP induced cleavage and mitochondrial translocation of Bax and upregulation of Bim in a time dependent manner indicating a potential role for Bim and Bax pro apoptotic proteins in CD37 induced apoptosis. These findings provide strong justification for CD37 as a therapeutic target aimed at modulation of pro apoptotic proteins and introduce small modular immunopharmaceuticals as a novel class of targeted therapies for B-cell malignancies. (SMIP trademark is owned by Trubion Pharmaceuticals).

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EGFR-ERK induced activation of GRHL1 promotes cell cycle progression by up-regulating cell cycle related genes in lung cancer

Cell Death and Disease ◽

10.1038/s41419-021-03721-9 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Yiming He ◽

Mingxi Gan ◽

Yanan Wang ◽

Tong Huang ◽

Jianbin Wang ◽

...

Keyword(s):

Lung Cancer ◽

Cell Cycle ◽

Cell Cycle Progression ◽

Potential Role ◽

Target Genes ◽

Nuclear Translocation ◽

Phosphorylation Site ◽

Promoter Regions ◽

Cycle Progression

AbstractGrainyhead-like 1 (GRHL1) is a transcription factor involved in embryonic development. However, little is known about the biological functions of GRHL1 in cancer. In this study, we found that GRHL1 was upregulated in non-small cell lung cancer (NSCLC) and correlated with poor survival of patients. GRHL1 overexpression promoted the proliferation of NSCLC cells and knocking down GRHL1 inhibited the proliferation. RNA sequencing showed that a series of cell cycle-related genes were altered when knocking down GRHL1. We further demonstrated that GRHL1 could regulate the expression of cell cycle-related genes by binding to the promoter regions and increasing the transcription of the target genes. Besides, we also found that EGF stimulation could activate GRHL1 and promoted its nuclear translocation. We identified the key phosphorylation site at Ser76 on GRHL1 that is regulated by the EGFR-ERK axis. Taken together, these findings elucidate a new function of GRHL1 on regulating the cell cycle progression and point out the potential role of GRHL1 as a drug target in NSCLC.

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Endothelial cell-related autophagic pathways in Sugen/hypoxia-exposed pulmonary arterial hypertensive rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00527.2016 ◽

2017 ◽

Vol 313 (5) ◽

pp. L899-L915 ◽

Cited By ~ 8

Author(s):

Fumiaki Kato ◽

Seiichiro Sakao ◽

Takao Takeuchi ◽

Toshio Suzuki ◽

Rintaro Nishimura ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cell ◽

Vascular Remodeling ◽

Time Dependent ◽

Causative Role ◽

Dependent Manner ◽

Vascular Endothelial ◽

Pulmonary Vascular Remodeling ◽

Pulmonary Arterial

Pulmonary arterial hypertension (PAH) is characterized by progressive obstructive remodeling of pulmonary arteries. However, no reports have described the causative role of the autophagic pathway in pulmonary vascular endothelial cell (EC) alterations associated with PAH. This study investigated the time-dependent role of the autophagic pathway in pulmonary vascular ECs and pulmonary vascular EC kinesis in a severe PAH rat model (Sugen/hypoxia rat) and evaluated whether timely induction of the autophagic pathway by rapamycin improves PAH. Hemodynamic and histological examinations as well as flow cytometry of pulmonary vascular EC-related autophagic pathways and pulmonary vascular EC kinetics in lung cell suspensions were performed. The time-dependent and therapeutic effects of rapamycin on the autophagic pathway were also assessed. Sugen/hypoxia rats treated with the vascular endothelial growth factor receptor blocker SU5416 showed increased right ventricular systolic pressure (RVSP) and numbers of obstructive vessels due to increased pulmonary vascular remodeling. The expression of the autophagic marker LC3 in ECs also changed in a time-dependent manner, in parallel with proliferation and apoptotic markers as assessed by flow cytometry. These results suggest the presence of cross talk between pulmonary vascular remodeling and the autophagic pathway, especially in small vascular lesions. Moreover, treatment of Sugen/hypoxia rats with rapamycin after SU5416 injection activated the autophagic pathway and improved the balance between cell proliferation and apoptosis in pulmonary vascular ECs to reduce RVSP and pulmonary vascular remodeling. These results suggested that the autophagic pathway can suppress PAH progression and that rapamycin-dependent activation of the autophagic pathway could ameliorate PAH.

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CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Blood ◽

10.1182/blood-2019-129873 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1734-1734

Author(s):

Orit Uziel ◽

Zinab Sarsur- Amer ◽

Einat Beery ◽

Pia Raanani ◽

Uri Rozovski

Keyword(s):

B Cells ◽

Time Dependent ◽

Dependent Manner ◽

Environmental Resources ◽

Type B ◽

Wild Type ◽

Cell Competition ◽

Western Immunoblotting ◽

Competition Theory

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

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Transition kinetics of impurity doped quantum dots under time-dependent confinement potential: role of noise

The European Physical Journal B ◽

10.1140/epjb/e2020-10102-x ◽

2020 ◽

Vol 93 (5) ◽

Author(s):

Anuja Ghosh ◽

Sk. Md. Arif ◽

Aindrila Bera ◽

Manas Ghosh

Keyword(s):

Quantum Dots ◽

Potential Role ◽

Time Dependent ◽

Confinement Potential ◽

Doped Quantum Dots ◽

Impurity Doped ◽

Kinetics Of

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Inhibition of Wnt/β-Catenin Signaling in Neuroendocrine Tumors In Vitro: Antitumoral Effects

Cancers ◽

10.3390/cancers12020345 ◽

2020 ◽

Vol 12 (2) ◽

pp. 345

Author(s):

Xi-Feng Jin ◽

Gerald Spöttl ◽

Julian Maurer ◽

Svenja Nölting ◽

Christoph Josef Auernhammer

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Lines ◽

Neuroendocrine Tumors ◽

Density Lipoprotein ◽

Time Dependent ◽

Dependent Manner ◽

Antitumor Properties

Background and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cytometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose- and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose- and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling (including pAKT/mTOR, pEGFR and pIGFR signaling). Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target.

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The action of α-adrenergic agonists on plasma-membrane calcium fluxes in perfused rat liver

Biochemical Journal ◽

10.1042/bj2200043 ◽

1984 ◽

Vol 220 (1) ◽

pp. 43-50 ◽

Cited By ~ 35

Author(s):

P H Reinhart ◽

W M Taylor ◽

F L Bygrave

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Potential Role ◽

Dependent Manner ◽

Perfused Rat Liver ◽

Adrenergic Agonists ◽

Mediated Effects ◽

Alpha Agonist ◽

Alpha Adrenergic Agonist

The effect of alpha-adrenergic agonists on Ca2+ fluxes was examined in the perfused rat liver by using a combination of Ca2+-electrode and 45Ca2+-uptake techniques. We showed that net Ca2+ fluxes can be described by the activities of separate Ca2+-uptake and Ca2+-efflux components, and that alpha-adrenergic agonists modulate the activity of both components in a time-dependent manner. Under resting conditions, Ca2+-uptake and -efflux activities are balanced, resulting in Ca2+ cycling across the plasma membrane. The alpha-adrenergic agonists vasopressin and angiotensin, but not glucagon, stimulate the rate of both Ca2+ efflux and Ca2+ uptake. During the first 2-3 min of alpha-agonist administration the effect on the efflux component is the greater, the net effect being efflux of Ca2+ from the cell. After 3-4 min of phenylephrine treatment, net Ca2+ movements are essentially complete, however, the rate of Ca2+ cycling is significantly increased. After removal of the alpha-agonist a large stimulation of the rate of Ca2+ uptake leads to the net accumulation of Ca2+ by the cell. The potential role of these Ca2+ flux changes in the expression of alpha-adrenergic-agonist-mediated effects is discussed.

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Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2007.2041 ◽

2007 ◽

Vol 362 (1483) ◽

pp. 1149-1163 ◽

Cited By ~ 117

Author(s):

Maria Sanchez-Contreras ◽

Wolfgang D Bauer ◽

Mengsheng Gao ◽

Jayne B Robinson ◽

J Allan Downie

Keyword(s):

Quorum Sensing ◽

Potential Role ◽

Active Role ◽

Dependent Manner ◽

Homoserine Lactones ◽

Signalling Molecules ◽

Sensing Systems ◽

Symbiotic Interactions ◽

Regulatory Systems

Legume-nodulating bacteria (rhizobia) usually produce N -acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regulation in rhizobia and the potential role of legumes in influencing and responding to this signalling system.

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