Microtubule-associated Protein Kinase Research Articles

To explore the mechanism of Yigong San anti-gastric cancer and immune regulation

BACKGROUND Yigong San (YGS) is a representative prescription for the treatment of digestive disorders, which has been used in clinic for more than 1000 years. However, the mechanism of its anti-gastric cancer and regulate immunity are still remains unclear. AIM To explore the mechanism of YGS anti-gastric cancer and immune regulation. METHODS Firstly, collect the active ingredients and targets of YGS, and the differentially expressed genes of gastric cancer. Secondly, constructed a protein-protein interaction network between the targets of drugs and diseases, and screened hub genes. Then the clinical relevance, mutation and repair, tumor microenvironment and drug sensitivity of the hub gene were analyzed. Finally, molecular docking was used to verify the binding ability of YGS active ingredient and hub genes. RESULTS Firstly, obtained 55 common targets of gastric cancer and YGS. The Kyoto Encyclopedia of Genes and Genomes screened the microtubule-associated protein kinase signaling axis as the key pathway and IL6, EGFR, MMP2, MMP9 and TGFB1 as the hub genes. The 5 hub genes were involved in gastric carcinogenesis, staging, typing and prognosis, and their mutations promote gastric cancer progression. Finally, molecular docking results confirmed that the components of YGS can effectively bind to therapeutic targets. CONCLUSION YGS has the effect of anti-gastric cancer and immune regulation.

Abstract LB219: DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays

Abstract Gastrointestinal cancers, including gastric cancer (GC) and colorectal cancer (CRC), continue to be devastating diseases despite advances in screening, early detection, and removal of pre-neoplastic and early cancerous lesions. Even with advancements in targeted chemotherapy and immunotherapies, advanced and metastatic diseases continue to be responsible for the high mortality associated with GC and CRC. Newer, safer, and more effective therapies to prevent, inhibit, or reverse advanced disease progression are urgently needed. Doublecortin-like kinase 1 (DCLK1) is a microtubule associated protein kinase that has been reported to drive tumor initiation, progression, metastasis, and drug resistance in many solid tumor cancers including GC and CRC, where it is upregulated in patient tumors and associated with poor overall survival. An anti-DCLK1 monoclonal antibody has demonstrated potential therapeutic activity against CRC. In this study, we established patient-derived organoids (PDOs) to evaluate the effects of inhibiting DCLK1 kinase activity (DCLK1-IN-1) or targeting DCLK1 with a highly specific CAR-T cells on the treated PDOs. Two GC PDOs were treated with various concentrations of DCLK1 kinase inhibitor DCLK1-IN-1 (0-32 μM) for 72 hrs, viability of the PDOs were determined, histochemistry and immunohistochemistry staining were used to image the live cells and DCLK1 expression. For CRC PDOs, five CRC PDOs were cultured in the presence or absence of PBMC, treated with Mock CAR-T cells or DCLK1 CAR-T cells (three doses) for 4 days. Tumor size, tumor total area, and viability of these PDOs were determined. Here, we demonstrate that treating GC PDOs with DCLK1 kinase inhibitor resulted in a significant dose-dependent inhibition in PDO numbers in both PDOs (two-way ANOVA P<0.0001), a decrease of cell viability, revealing IC50 values of 7.566 and 9.958 μM respectively, and a near total loss of GC PDO viability at 10 μM using CellTiterGlo assay. In CRC PDOs, two of five CRC PDOs showed a significant decrease in total tumor size, tumor areas, and an increase in cell death measured by the DRAQ7 method. Compared to Mock CAR-T cells, treatment with DCLK1 CAR-T cells resulted in a dose dependent decrease of tumor size and total tumor area, with more than a 50% reduction at the 50000 cells dose (p<0.05). Treatment with DCLK1 CAR-T cells also resulted in a dose dependent increase in cell death, with approximately 20% cell death without PBMC, and 50% cell death with PBMC at the 50000 cells dose (p<0.05). These results suggest that targeting DCLK1 represents a potential novel therapeutic strategy for GI cancers, especially in combination with FDA approved drugs: 5-fluorouracil (5-FU) or Gefitinib. Citation Format: Dongfeng Qu, Jinsen Shi, Zhiyun Cao, Eddie BAnnerman-Menson, Nathaniel Weygant, Courtney Houchen. DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB219.

Mangiferin, a Potential Supplement to Improve Metabolic Syndrome: Current Status and Future Opportunities.

Metabolic syndrome (MetS) represents a considerable clinical and public health burden worldwide. Mangiferin (MF), a flavonoid compound present in diverse species such as mango (Mangifera indica L.), papaya (Pseudocydonia sinensis (Thouin) C. K. Schneid.), zhimu (Anemarrhena asphodeloides Bunge), and honeybush tea (Cyclopia genistoides), boasts a broad array of pharmacological effects. It holds promising uses in nutritionally and functionally targeted foods, particularly concerning MetS treatment. It is therefore pivotal to systematically investigate MF's therapeutic mechanism for MetS and its applications in food and pharmaceutical sectors. This review, with the aid of a network pharmacology approach complemented by this experimental studies, unravels possible mechanisms underlying MF's MetS treatment. Network pharmacology results suggest that MF treats MetS effectively through promoting insulin secretion, targeting obesity and inflammation, alleviating insulin resistance (IR), and mainly operating via the phosphatidylinositol 3 kinase (PI3K)/Akt, nuclear factor kappa-B (NF-[Formula: see text]B), microtubule-associated protein kinase (MAPK), and oxidative stress signaling pathways while repairing damaged insulin signaling. These insights provide a comprehensive framework to understand MF's potential mechanisms in treating MetS. These, however, warrant further experimental validation. Moreover, molecular docking techniques confirmed the plausibility of the predicted outcomes. Hereafter, these findings might form the theoretical bedrock for prospective research into MF's therapeutic potential in MetS therapy.

Discovery of a doublecortin-like kinase 1 inhibitor to prevent inflammatory responses in acute lung injury

Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated protein kinase involved in neurogenesis and human cancer. Recent studies have revealed a novel functional role for DCLK1 in inflammatory signaling, thus positioning it as a novel target kinase for respiratory inflammatory disease treatment. In this study, we designed and synthesized a series of NVP-TAE684-based derivatives as novel anti-inflammatory agents targeting DCLK1. Bio-layer interferometry binding screening and kinase assays of the NVP-TAE684 derivatives led to the discovery of an effective DCLK1 inhibitor (a24), with an IC50 of 179.7 nM. Compound a24 effectively inhibited lipopolysaccharide (LPS)-induced inflammation in macrophages with higher potency than the lead compound. Mechanistically, compound a24 inhibited LPS-induced inflammation by inhibiting DCLK1-mediated IKKβ phosphorylation. Furthermore, compound a24 showed in vivo anti-inflammatory activity in an LPS-challenged acute lung injury model. These findings suggest that compound a24 may serve as a novel candidate for the development of DCLK1 inhibitors and a potential therapeutic agent for the treatment of inflammatory diseases.

Identification of several inflammation-related genes based on bioinformatics and experiments.

Osteoarthritis (OA) is a common disease of elderly individuals, with an unclear pathogenesis and limited treatment options to date. Inflammation occurs prominently in osteoarthritis, thereby making anti-inflammatory treatments promising in clinical outcomes. Therefore, it is of diagnostic and therapeutic significance to explore more inflammatory genes. In this study, appropriate datasets were first acquired through gene set enrichment analysis (GSEA), followed by inflammation-related genes through weighted gene coexpression network analysis (WGCNA). Two machine learning algorithms (random forest-RF and support vector machine-recursive feature elimination, SVM-RFE) were used to capture the hub genes. In addition, two genes negatively associated with inflammation and osteoarthritis were identified. Afterwards, these genes were verified through experiments and network pharmacology. Due to the association between inflammation and many diseases, the expression levels of the above genes in various inflammatory diseases were determined through literature and experiments. Two hub genes closely related to osteoarthritis and inflammation were obtained, namely, lysyl oxidase-like 1 (LOXL1) and pituitary tumour-transforming gene (PTTG1), which were shown to be highly expressed in osteoarthritis according to the literature and experiments. However, the expression levels of receptor expression-enhancing protein (REEP5) and cell division cycle protein 14B (CDC14B) remained unchanged in osteoarthritis. This finding was consistent with our verification from the literature and experiments that some genes were highly expressed in numerous inflammation-related diseases, while REEP5 and CDC14B were almost unchanged. Meanwhile, taking PTTG1 as an example, we found that inhibition of PTTG1 expression could suppress the expression of inflammatory factors and protect the extracellular matrix through the microtubule-associated protein kinase (MAPK) signalling pathway. LOXL1 and PTTG1 were highly expressed in some inflammation-related diseases, while that of REEP5 and CDC14B were almost unchanged. PTTG1 may be a potential target for the treatment of osteoarthritis.

Doublecortin-like kinase 1 activates NF-κB to induce inflammatory responses by binding directly to IKKβ.

Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase, is involved in neurogenesis, and its levels are elevated in various human cancers. Recent studies suggest that DCLK1 may relate to inflammatory responses in the mouse model of colitis. However, cellular pathways engaged by DCLK1, and potential substrates of the kinase remain undefined. To understand how DCLK1 regulates inflammatory responses, we utilized the well-established lipopolysaccharide (LPS)-stimulated macrophages and mouse model. Through a range of macrophage-based and cell-free platforms, we discovered that DCLK1 binds directly with the inhibitor of κB kinase β (IKKβ) and induces IKKβ phosphorylation on Ser177/181 to initiate nuclear factor-κB (NF-κB) pathway. Deficiency in DCLK1, achieved by silencing or through pharmacological inhibition, prevented LPS-induced NF-κB activation and cytokine production in macrophages. We further show that mice with myeloid-specific DCLK1 knockout or DCLK1 inhibitor treatment are protected against LPS-induced acute lung injury and septic death. Our studies report a novel functional role of macrophage DCLK1 as a direct IKKβ regulator in inflammatory signaling and suggest targeted therapy against DCLK1 for inflammatory diseases.

Macrophage DCLK1 promotes atherosclerosis via binding to IKKβ and inducing inflammatory responses

Atherosclerosis is a chronic inflammatory disease with high morbidity and mortality rates worldwide. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase, is involved in neurogenesis and human cancers. However, the role of DCLK1 in atherosclerosis remains undefined. In this study, we identified upregulated DCLK1 in macrophages in atherosclerotic lesions of ApoE-/- mice fed an HFD and determined that macrophage-specific DCLK1 deletion attenuates atherosclerosis by reducing inflammation in mice. Mechanistically, RNA sequencing analysis indicated that DCLK1 mediates oxLDL-induced inflammation via NF-κB signaling pathway in primary macrophages. Coimmunoprecipitation followed by LC-MS/MS analysis identified IKKβ as a binding protein of DCLK1. We confirmed that DCLK1 directly interacts with IKKβ and phosphorylates IKKβ at S177/181, thereby facilitating subsequent NF-κB activation and inflammatory gene expression in macrophages. Finally, a pharmacological inhibitor of DCLK1 prevents atherosclerotic progression and inflammation both in vitro and in vivo. Our findings demonstrated that macrophage DCLK1 promotes inflammatory atherosclerosis by binding to IKKβ and activating IKKβ/NF-κB. This study reports DCLK1 as a new IKKβ regulator in inflammation and a potential therapeutic target for inflammatory atherosclerosis.

GINS2 regulates epithelial mesenchymal transformation and cell cycle in endometrial carcinoma by stimulating ERK/MAPK signaling

Endometrial cancer (EC) is one of the three main gynecological cancers. Identifying new therapeutic targets and further elucidating the molecular mechanisms of EC tumorigenesis have important implications for women’s health. The Go-Ichi-Ni-San (GINS) family, which includes four subunits (GINS1–4), has specific functions in DNA replication and cell cycle. The Cancer Genome Atlas (TCGA) data showed that GINS2 transcription level is upregulated in endometrial cancer tissue. However, the possible role of GINS2 in EC progression is still unknown. Herein, we explored the role of GINS2 in EC. We noticed that GINS2 was overexpressed in EC cells. GINS2 knockdown suppressed the proliferation of EC cells and induced cell cycle arrest. We further noticed that GINS2 knockdown restrained the Epithelial mesenchymal transformation (EMT) of EC cells. Mechanically, its downregulation suppressed the extracellular regulated protein kinase (ERK)/Microtubule-Associated Protein Kinase (MAPK) pathway, thereby suppressing EC progression. Thus, GINS2 has the potential to act as a therapeutic target for EC.

Chikusetsusaponin Iva induces apoptosis and inhibits proliferation in endometrial cancer via promoting reactive oxygen species (ROS) production

Endometrial carcinoma (EC) is an epithelial malignant tumor that occurs in the endometrium. It is of great significance to explore new targets for improving the prognosis of endometrial cancer. Chikusetsusaponin Iva (CHI) is a plant compound extracted from Panacis japonica which has multiple biological activities, including anti-inflammation and anti-tumor effects. However, its possible effects on EC are unclear. Herein, we found that CHI inhibited EC cell proliferation and cell cycle, confirmed by colony formation and flow cytometry (FCM) assays. The results further confirmed that CHI stimulated the apoptosis of EC cells. Furthermore, we noticed that CHI treatment induced reactive oxygen species (ROS) in EC cells. In addition, it could suppress the Microtubule-Associated Protein Kinase (MAPK) pathway in EC cells, thereby affecting cell proliferation and apoptosis. Therefore, CHI could serve as a potential drug for EC treatment.

Doublecortin-like kinase 2 promotes breast cancer cell invasion and metastasis.

Doublecortin-like kinase 2 (DCLK2) is a microtubule-associated protein kinase that participates in neural development and maturation; however, whether it is involved in tumour progression remains unclear. DCLK2 overexpression and knockdown clones were established by lentivirus transfection. Western blot, PCR assays and bioinformatics analyses were conducted to observe the expression of DCLK2. CCK8, colony formation, scratch migration and Transwell assays were used to detect cell proliferation, migration and invasion, respectively. Tumour metastasis was evaluated in vivo using a tail vein metastasis model. Bioinformatics analyses were performed to analyse the expression correlation between DCLK2 and TCF4, or EMT markers in breast cancer. Our data indicate that DCLK2 is highly expressed in breast cancer cells and is associated with poor prognosis. Silencing DCLK2 does not affect the proliferation rate of tumour cells, but significantly suppresses migration and invasion as well as lung metastasis processes. Overexpression of DCLK2 can enhance the migratory and invasive abilities of normal breast epithelial cells. Moreover, TCF4/β-catenin inhibitor LF3 downregulates the expression of DCLK2 and inhibits the migration and invasion of breast cancer cells. Furthermore, we found that the downregulation of DCLK2 blocks the epithelial-mesenchymal transition (EMT) process. Our study indicates that DCLK2 plays an important role in EMT, cell invasion and metastasis, suggesting that DCLK2 is a potential target for the treatment of metastatic breast cancer.

Capsaicin affects macrophage anti-inflammatory activity via the MAPK and NF-κB signaling pathways.

Capsaicin, the main constituent in chili, is an extremely spicy vanillin alkaloid and is found in several Capsicum species in China. Traditionally, it has been used to treat inflammatory diseases such as allergic rhinitis, neuralgia after shingles, refractory female urethral syndrome, spontaneous recalcitrant anal pruritus, and solid tumors. Constant stimulation of the body by inflammatory factors can lead to chronic inflammation. Capsaicin possesses anti-inflammatory activity; however, the underlying mechanism is unknown. We investigated the effect of capsaicin on the secretion of macrophage inflammatory factors in a lipopolysaccharide-induced inflammation model using 56 healthy, SPF grade, BALB/c mice. To this end, mice peritoneal macrophages were isolated and stimulated with lipopolysaccharide (1 μg/mL) and capsaicin (25, 50, 75, or 100 μg/mL) for 24 h. At all concentrations tested, capsaicin significantly promoted the phagocytosis of neutral red dye by macrophages. Furthermore, the gene expression and secretion of inflammatory cytokines significantly increased after induction with lipopolysaccharide (P<0.01); the interleukin (IL)-6 level was 204 μg/mL, tumor necrosis factor (TNF)-α level was 860 μg/mL, and nitric oxide (NO) level was 19.8 μg/mL. However, the treatment with capsaicin reduced their levels (P<0.01) and protein expression of lipopolysaccharide-induced extracellular signal-related kinase 1/2 and p65 (P<0.05). Overall, capsaicin reduced the secretion of inflammatory cytokines (P<0.01), interleukins, TNF-α (P<0.01), and NO by inhibiting the nuclear factor-kappa B and microtubule-associated protein kinase signaling pathways, and thereby reduced lipopolysaccharide-induced inflammatory response in macrophages.

Role of exosomal miRNAs in brain metastasis affected by radiotherapy.

In oncogenesis and development of malignant tumor, microRNAs (miRNAs) regulate the complex gene expression associated with the tumor pathogenesis. Currently, only few studies have been conducted to identify miRNAs and the potential pathways involved in the pathogenesis of brain metastasis in patients who underwent radiotherapy, especially miRNAs in the plasma exosomes. Therefore, this study is aimed to use small RNA analysis to identify miRNAs and their potential target genes in plasma exosomes during the initiation and development of brain metastasis in patients who underwent radiotherapy. Using high-throughput sequencing technologies, we identified 35 differentially expressed miRNAs in patients with brain metastasis who had undergone radiotherapy. In annotation of miRNA targets, gene ontology enrichment analysis revealed that the targets of the differentially expressed miRNAs were significantly enriched in the regulation of cellular processes. Kyoto Encyclopedia of Genes and Genomes revealed that most of the miRNA targets were cancer-related, including genes involved in the mitogen-activated protein kinase signaling pathway, cancer-related pathways, phosphatidylinositol 3-kinase-protein kinase B signaling pathway, microtubule-associated protein kinase signaling pathway, Ras signaling pathway, regulation of the actin cytoskeleton, and axon guidance. In conclusion, this study provides a new perspective to understand the possible function of these miRNAs in the pathogenesis of brain metastasis. This was the first time that a pilot study identified plasma exosomal miRNAs in five patients with brain metastasis before and after radiotherapy. This study is the beginning; more specimen and further research are needed to explore the functional role of specific miRNAs and their potential as therapeutic targets for brain metastasis.

DNA Methyltransferase 3A Is Involved in the Sustained Effects of Chronic Stress on Synaptic Functions and Behaviors

Abstract Emerging evidence suggests that epigenetic mechanisms regulate aberrant gene transcription in stress-associated mental disorders. However, it remains to be elucidated about the role of DNA methylation and its catalyzing enzymes, DNA methyltransferases (DNMTs), in this process. Here, we found that male rats exposed to chronic (2-week) unpredictable stress exhibited a substantial reduction of Dnmt3a after stress cessation in the prefrontal cortex (PFC), a key target region of stress. Treatment of unstressed control rats with DNMT inhibitors recapitulated the effect of chronic unpredictable stress on decreased AMPAR expression and function in PFC. In contrast, overexpression of Dnmt3a in PFC of stressed animals prevented the loss of glutamatergic responses. Moreover, the stress-induced behavioral abnormalities, including the impaired recognition memory, heightened aggression, and hyperlocomotion, were partially attenuated by Dnmt3a expression in PFC of stressed animals. Finally, we found that there were genome-wide DNA methylation changes and transcriptome alterations in PFC of stressed rats, both of which were enriched at several neural pathways, including glutamatergic synapse and microtubule-associated protein kinase signaling. These results have therefore recognized the potential role of DNA epigenetic modification in stress-induced disturbance of synaptic functions and cognitive and emotional processes.

Highly elastic, electroconductive, immunomodulatory graphene crosslinked collagen cryogel for spinal cord regeneration

Novel amino-functionalized graphene crosslinked collagen based nerve conduit having appropriate electric (3.8 ± 0.2 mSiemens/cm) and mechanical cues (having young modulus value of 100–347 kPa) for stem cell transplantation and neural tissue regeneration was fabricated using cryogelation. The developed conduit has shown sufficiently high porosity with interconnectivity between the pores. Raman spectroscopy analysis revealed the increase in orderliness and crosslinking of collagen molecules in the developed cryogel due to the incorporation of amino-functionalized graphene. BM-MSCs grown on graphene collagen cryogels have shown enhanced expression of CD90 and CD73 gene upon electric stimulation (100 mV/mm) contributing towards maintaining their stemness. Furthermore, an increased secretion of ATP from BM-MSCs grown on graphene collagen cryogel was also observed upon electric stimulation that may help in regeneration of neurons and immuno-modulation. Neuronal differentiation of BM-MSCs on graphene collagen cryogel in the presence of electric stimulus showed an enhanced expression of MAP-2 kinase and β-tubulin III. Immunohistochemistry studies have also demonstrated the improved neuronal differentiation of BM-MSCs. BM-MSCs grown on electro-conductive collagen cryogels under inflammatory microenvironment in vitro showed high indoleamine 2,3 dioxygenase activity. Moreover, macrophages cells grown on graphene collagen cryogels have shown high CD206 (M2 polarization marker) and CD163 (M2 polarization marker) and low CD86 (M1 polarization marker) gene expression demonstrating M2 polarization of macrophages, which may aid in tissue repair. In an organotypic culture, the developed cryogel conduit has supported cellular growth and migration from adult rat spinal cord. Thus, this novel electro-conductive graphene collagen cryogels have potential for suppressing the neuro-inflammation and promoting the neuronal cellular migration and proliferation, which is a major barrier during the spinal cord regeneration.

MAP/ERK Signaling in Developing Cognitive and Emotional Function and Its Effect on Pathological and Neurodegenerative Processes.

The signaling pathway of the microtubule-associated protein kinase or extracellular regulated kinase (MAPK/ERK) is a common mechanism of extracellular information transduction from extracellular stimuli to the intracellular space. The transduction of information leads to changes in the ongoing metabolic pathways and the modification of gene expression patterns. In the central nervous system, ERK is expressed ubiquitously, both temporally and spatially. As for the temporal ubiquity, this signaling system participates in three key moments: (i) Embryonic development; (ii) the early postnatal period; and iii) adulthood. During embryonic development, the system is partly responsible for the patterning of segmentation in the encephalic vesicle through the FGF8-ERK pathway. In addition, during this period, ERK directs neurogenesis migration and the final fate of neural progenitors. During the early postnatal period, ERK participates in the maturation process of dendritic trees and synaptogenesis. During adulthood, ERK participates in social and emotional behavior and memory processes, including long-term potentiation. Alterations in mechanisms related to ERK are associated with different pathological outcomes. Genetic alterations in any component of the ERK pathway result in pathologies associated with neural crest derivatives and mental dysfunctions associated with autism spectrum disorders. The MAP-ERK pathway is a key element of the neuroinflammatory pathway triggered by glial cells during the development of neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis, as well as prionic diseases. The process triggered by MAPK/ERK activation depends on the stage of development (mature or senescence), the type of cellular element in which the pathway is activated, and the anatomic neural structure. However, extensive gaps exist with regards to the targets of the phosphorylated ERK in many of these processes.

Phenotypic and microRNA characterization of the neglected CD24+ cell population in MCF-7 breast cancer 3-dimensional spheroid culture.

In vitro 3-dimensional (3D) spheroid culture has been widely used as model to enrich CD44CD24 cancer stem cells (CSC) with high aldehyde dehydrogenase 1 (ALDH1) activity. Although CD24 subpopulation was known to be present in 3D spheroids and may influence cancer drug therapies, its characteristics and CSC properties were not well defined. In this study, CD24 population from the Michigan Cancer Foundation-7 (MCF-7) spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion and migration test, as well as microRNA expression profiling. Sorted MCF-7 CD24 cells from primary spheroids were able to reform its 3D spheroid shape after 7 days in nonadherent culture conditions. In contrast to the primary spheroids, the expression of SOX-2, CD44, CD49f, and Nanog was dim in MCF-7 CD24 cells. Remarkably, MCF-7 CD24 cells were found to show high expression of ALDH1 protein which may have resulted in these cells exhibiting higher resistance against doxorubicin and cisplatin when compared with that of the parental cells. Moreover, microRNA profiling has shown that the absence of CSC properties was consistent with the downregulation of major CSCs-related pathways including Hedgehog, wingless-related integration site (Wnt), and microtubule associated protein kinase (MAPK) signaling pathways. However, the upregulated pathways such as adherens junctions, focal adhesion, and tight junction suggest that CD24 cells were probably at an epithelial-like state of cell transition. In conclusion, neglected CD24 cells in MCF-7 spheroid did not exhibit typical breast CSCs properties. The presence of miRNAs and their analyzed pathways suggested that these cells could be a distinct intermediate cell state in breast CSCs.

Tryptophan photo-product FICZ upregulates AHR/MEK/ERK-mediated MMP1 expression: Implications in anti-fibrotic phototherapy

BackgroundScleroderma is caused by aberrant transforming growth factor-ß signaling. The degradation of extracellular matrix proteins is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Ultraviolet (UV) radiation has been a therapy for scleroderma. 6-Formylindolo[3,2-b]carbazole (FICZ), an endogenous aryl hydrocarbon receptor (AHR) ligand, is a tryptophan metabolite generated by UV exposure. Nonetheless, whether FICZ regulates MMPs and TIMPs has not been investigated. ObjectiveTo elucidate the regulatory roles of FICZ in the expression of MMPs and TIMPs in normal human dermal fibroblasts (NHDFs). MethodsQuantitative real-time polymerase chain reaction was performed to determine the expression of MMPs or TIMPs in the NHDFs treated with FICZ or UVB. The MMPs levels were measured by enzyme-linked immunosorbent assay. The actions of FICZ on MMPs were analyzed using AHR-knockdown NHDFs or selective inhibitors of mitogen-activated protein kinases (MAPKs). Microtubule-associated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation was examined by western blotting. ResultsUVB increased the mRNA and protein levels of MMP1 and MMP3 in NHDFs, while FICZ upregulated those of MMP1, but not MMP3. The effects of FICZ on TIMPs were negligible. FICZ increased MMP1 expression in an AHR-dependent manner. The FICZ-induced MMP1 upregulation was ameliorated with MEK/ERK inhibitors, whereas the effects of UVB were canceled with c-Jun N-terminal kinase (JNK) and p38-MAPK as well as MEK/ERK inhibitors. FICZ-induced ERK phosphorylation is dependent on AHR. ConclusionFICZ contributes to the UV-mediated anti-fibrotic effects via the AHR/MEK/ERK signal pathway in NHDFs. FICZ is a potential therapeutic agent for scleroderma.

Molecular advances in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a group of malignancies derived from skin-homing T cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common CTCL variants. In recent years, the genetic landscape of SS/MF has been characterized using genome-wide nextgeneration sequencing approaches. These studies have revealed that genes subjected to oncogenic mutations take part in cell cycle regulation, chromatin modification, Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling, T-cell receptor (TCR)/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and microtubule associated protein kinase (MAPK) signaling, which suggests that deregulation of these cellular processes underlies lymphomagenesis. These studies provide the groundwork for functional and clinical studies that will lead to better risk assessment and more effective therapeutic approach in CTCL patients.

Defect in Synaptic Pruning of Motor Cortex Neurons is Associated with Early Perturbed Dopaminergic System

Background: Early stress exposure during neurodevelopmental stages has been linked to some adult neuropsychiatric disorders. The dopaminergic system which has been implicated in movement and reward system has been linked to movement and mood disorders when perturbed at early development. This study is designed to check the mechanisms involved in movement disorders such as dyskinesia, associated with early perturbed dopaminergic system in the motor cortex Methods: Haloperidol was used to block D2R in neonatal albino Wistar rats in utero by administering 20 mg/kg BW (intraperitoneally) to pregnant adult wistar rats (n=8) in the third week of gestation. Behavioural studies such as the rotarod test were carried out on the neonatal animals (n=5) to test their motor function at postnatal day twentyeight (P28). Electrophysiological recordings were carried out on the motor cortex (M1) to determine the significance of D2R inhibition on calcium neural activity. Immunofluorescence was done to demonstrate synaptic vesicle protein (SV) and microtubule associated protein kinases (MAP K) as a measure of synapses count and microtubule phosphorylation respectively. Results: Behavioural studies showed a decline in motor function of animals exposed to haloperidol in utero compared to the control. This motor deficit was accompanied by a significant increase in the Ca2+ neural activity of the motor cortex as shown by electrophysiological recordings. Immunofluorescence staining showed there was significant increase in the number of MAPK+ and SV+ cells in the motor cortex of haloperidol exposed animals compared to the control. Conclusion: These findings showed that early perturbation in dopaminergic system is associated with an increase in synapses and neuronal density, as well as an increase in phosphorylation of microtubules of neurons in the motor cortex.

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 coimmunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.