Cell Growth Factor Receptor Kit Research Articles

Inhibition of CSF1R and KIT With Pexidartinib Reduces Inflammatory Signaling and Cell Viability in Endometriosis.

Endometriosis is a common and debilitating disease, affecting ∼170 million women worldwide. Affected patients have limited therapeutic options such as hormonal suppression or surgical excision of the lesions, though therapies are often not completely curative. Targeting receptor tyrosine kinases (RTKs) could provide a nonhormonal treatment option for endometriosis. We determined that 2 RTKs, macrophage-colony stimulating factor 1 receptor (CSF1R) and mast/stem cell growth factor receptor KIT (KIT), are overexpressed in endometriotic lesions and could be novel nonhormonal therapeutic targets for endometriosis. The kinase activity of CSF1R and KIT is suppressed by pexidartinib, a small molecule inhibitor that was recently approved by the US Food and Drug Administration. Using immunohistochemistry, we detected CSF1R and KIT in endometriotic tissues obtained from peritoneal lesions, colorectal lesions, and endometriomas. Specifically, we show that KIT is localized to the epithelium of the lesions, while CSF1R is expressed in the stroma and macrophages of the endometriotic lesions. Given the high epithelial expression of CSF1R and KIT, 12Z endometriotic epithelial cells were used to evaluate the efficacy of dual CSF1R and KIT inhibition with pexidartinib. We found that pexidartinib suppressed activation in 12Z cells of JNK, STAT3, and AKT signaling pathways, which control key proinflammatory and survival networks within the cell. Using quantitative real-time polymerase chain reaction, we determined that pexidartinib suppressed interleukin 8 (IL8) and cyclin D1 (CCND1) expression. Lastly, we demonstrated that pexidartinib decreased cell growth and viability. Overall, these results indicate that pexidartinib-mediated CSF1R and KIT inhibition reduces proinflammatory signaling and cell viability in endometriosis.

Factors Influencing Marker Expressions of Cultured Human Cord Blood-Derived Mast Cells.

Mast cells (MCs) are tissue-resident immune cells of a hematopoietic origin that play vital roles in innate and adaptive immunity. Human MCs can be isolated and differentiated from various tissue sources, including cord blood, when supplemented with cytokines such as stem cell factor, interleukin 3, and interleukin 6. Our current research study has shown significant differences in the marker expressions of human cord blood-derived mast cells (hCBMCs) based on donor dependency and the type of medium used for culturing and differentiation. These findings are particularly relevant given the challenges of obtaining specialty media influencing MC phenotypic marker expressions. We found that hCBMCs cultured in StemSpanTM-XF medium had a moderate expression of mast/stem cell growth factor receptor Kit (c-KIT) (mRNA and protein), low expressions of FcεRI (mRNA) and TLR2 (mRNA and protein) but had high levels of MRGPRX2 (mRNA and protein) expressions. In contrast, hCBMCs cultured in Stem Line II medium expressed FcεRI and TLR2 (mRNA and protein) with higher c-KIT but had lower MRGPRX2 expressions compared to the hCBMCs cultured in the StemSpanTM-XF medium. These results suggest that it is crucial to consider both donor dependency and the medium when investigating MC functions and that further research is needed to fully understand the impact of these factors on the hCBMC marker expressions.

Novel somatic Missense Mutations in Exon 11 of the KIT Gene are Detected in Melanoma.

The aim of the present study was to analyze mutations of the mast/stem cell growth factor receptor Kit (KIT) gene in patients with melanoma from Eastern Siberia regions of the Russian Federation. KIT gene mutations in exons 11 and 13 were analyzed by Sanger sequencing in 57 tumor samples obtained from patients with KIT-positive melanomas localized in preferable locations. Mutations were identified in 21% of patients. Among them, multiple mutations were identified in five patients. A total of 18 mutations were observed in the KIT gene, of which three were deletions and fourteen substitution mutations. Age, gender and clinicopathological characteristics of patients with cutaneous KIT-positive melanoma in Eastern Siberia corresponded to the European population. According to computational prediction tools, all mutations were evaluated as potentially harmful. The six novel mutations reported in the present study expand our knowledge on the molecular pathogenesis of melanoma, which can be used to further explore methods to improve disease therapeutic strategies.

Pharmacology and Rationale for Seralutinib in the Treatment of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a complex disorder characterized by vascular remodeling and a consequent increase in pulmonary vascular resistance. The histologic hallmarks of PAH include plexiform and neointimal lesions of the pulmonary arterioles, which are composed of dysregulated, apoptosis-resistant endothelial cells and myofibroblasts. Platelet-derived growth factor receptors (PDGFR) α and β, colony stimulating factor 1 receptor (CSF1R), and mast/stem cell growth factor receptor kit (c-KIT) are closely related kinases that have been implicated in PAH progression. In addition, emerging data indicate significant crosstalk between PDGF signaling and the bone morphogenetic protein receptor type 2 (BMPR2)/transforming growth factor β (TGFβ) receptor axis. This review will discuss the importance of the PDGFR-CSF1R-c-KIT signaling network in PAH pathogenesis, present evidence that the inhibition of all three nodes in this kinase network is a potential therapeutic approach for PAH, and highlight the therapeutic potential of seralutinib, currently in development for PAH, which targets these pathways.

Identification of novel c-Kit inhibitors from natural sources using virtual screening and molecular dynamics simulations

The Mast/Stem cell growth factor receptor Kit (c-Kit), a Proto-oncogene c-Kit, is a tyrosine-protein kinase involved in cell differentiation, proliferation, migration, and survival. Its role in developing certain cancers, particularly gastrointestinal stromal tumors (GISTs) and acute myeloid leukemia (AML), makes it an attractive therapeutic target. Several small molecule inhibitors targeting c-Kit have been developed and approved for clinical use. Recent studies have focused on identifying and optimizing natural compounds as c-Kit inhibitors employing virtual screening. Still, drug resistance, off-target side effects, and variability in patient response remain significant challenges. From this perspective, phytochemicals could be an important resource for discovering novel c-Kit inhibitors with less toxicity, improved efficacy, and high specificity. This study aimed to uncover possible c-Kit inhibitors by utilizing a structure-based virtual screening of active phytoconstituents from Indian medicinal plants. Through the screening stages, two promising candidates, Anilinonaphthalene and Licoflavonol, were chosen based on their drug-like features and ability to bind to c-Kit. These chosen candidates were subjected to all-atom molecular dynamics (MD) simulations to evaluate their stability and interaction with c-Kit. The selected compounds Anilinonaphthalene from Daucus carota and Licoflavonol from Glycyrrhiza glabra showed their potential to act as selective binding partners of c-Kit. Our results suggest that the identified phytoconstituents could serve as a starting point to develop novel c-Kit inhibitors for developing new and effective therapies against multiple cancers, including GISTs and AML. The use of virtual screening and MD simulations provides a rational approach to discovering potential drug candidates from natural sources. Communicated by Ramaswamy H. Sarma

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal–regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Correlation between stem cell molecular phenotype and atherosclerotic plaque neointima formation and analysis of stem cell signal pathways.

Vascular stem cells exist in the three-layer structure of blood vessel walls and play an indispensable role in angiogenesis under physiological conditions and vascular remodeling under pathological conditions. Vascular stem cells are mostly quiescent, but can be activated in response to injury and participate in endothelial repair and neointima formation. Extensive studies have demonstrated the differentiation potential of stem/progenitor cells to repair endothelium and participate in neointima formation during vascular remodeling. The stem cell population has markers on the surface of the cells that can be used to identify this cell population. The main positive markers include Stem cell antigen-1 (Sca1), Sry-box transcription factor 10 (SOX10). Stromal cell antigen 1 (Stro-1) and Stem cell growth factor receptor kit (c-kit) are still controversial. Different parts of the vessel have different stem cell populations and multiple markers. In this review, we trace the role of vascular stem/progenitor cells in the progression of atherosclerosis and neointima formation, focusing on the expression of stem cell molecular markers that occur during neointima formation and vascular repair, as well as the molecular phenotypic changes that occur during differentiation of different stem cell types. To explore the correlation between stem cell molecular markers and atherosclerotic diseases and neointima formation, summarize the differential changes of molecular phenotype during the differentiation of stem cells into smooth muscle cells and endothelial cells, and further analyze the signaling pathways and molecular mechanisms of stem cells expressing different positive markers participating in intima formation and vascular repair. Summarizing the limitations of stem cells in the prevention and treatment of atherosclerotic diseases and the pressing issues that need to be addressed, we provide a feasible scheme for studying the signaling pathways of vascular stem cells involved in vascular diseases.

Inhaled seralutinib exhibits potent efficacy in models of pulmonary arterial hypertension.

Signalling through platelet-derived growth factor receptor (PDGFR), colony-stimulating factor 1 receptor (CSF1R) and mast/stem cell growth factor receptor kit (c-KIT) plays a critical role in pulmonary arterial hypertension (PAH). We examined the preclinical efficacy of inhaled seralutinib, a unique small-molecule PDGFR/CSF1R/c-KIT kinase inhibitor in clinical development for PAH, in comparison to a proof-of-concept kinase inhibitor, imatinib. Seralutinib and imatinib potency and selectivity were compared. Inhaled seralutinib pharmacokinetics/pharmacodynamics were studied in healthy rats. Efficacy was evaluated in two rat models of PAH: SU5416/Hypoxia (SU5416/H) and monocrotaline pneumonectomy (MCTPN). Effects on inflammatory/cytokine signalling were examined. PDGFR, CSF1R and c-KIT immunohistochemistry in rat and human PAH lung samples and microRNA (miRNA) analysis in the SU5416/H model were performed. Seralutinib potently inhibited PDGFRα/β, CSF1R and c-KIT. Inhaled seralutinib demonstrated dose-dependent inhibition of lung PDGFR and c-KIT signalling and increased bone morphogenetic protein receptor type 2 (BMPR2). Seralutinib improved cardiopulmonary haemodynamic parameters and reduced small pulmonary artery muscularisation and right ventricle hypertrophy in both models. In the SU5416/H model, seralutinib improved cardiopulmonary haemodynamic parameters, restored lung BMPR2 protein levels and decreased N-terminal pro-brain natriuretic peptide (NT-proBNP), more than imatinib. Quantitative immunohistochemistry in human lung PAH samples demonstrated increased PDGFR, CSF1R and c-KIT. miRNA analysis revealed candidates that could mediate seralutinib effects on BMPR2. Inhaled seralutinib was an effective treatment of severe PAH in two animal models, with improved cardiopulmonary haemodynamic parameters, a reduction in NT-proBNP, reverse remodelling of pulmonary vascular pathology and improvement in inflammatory biomarkers. Seralutinib showed greater efficacy compared to imatinib in a preclinical study.

Diagnostic and Prognostic Protein Biomarkers of β-Cell Function in Type 2 Diabetes and Their Modulation with Glucose Normalization.

Development of type-2 diabetes(T2D) is preceded by β-cell dysfunction and loss. However, accurate measurement of β-cell function remains elusive. Biomarkers have been reported to predict β-cell functional decline but require validation. Therefore, we determined whether reported protein biomarkers could distinguish patients with T2D (onset < 10-years) from controls. A prospective, parallel study in T2D (n = 23) and controls (n = 23) was undertaken. In T2D subjects, insulin-induced blood glucose normalization from baseline 7.6 ± 0.4 mmol/L (136.8 ± 7.2 mg/dL) to 4.5 ± 0.07 mmol/L (81 ± 1.2 mg/dL) was maintained for 1-h. Controls were maintained at 4.9 ± 0.1 mmol/L (88.2 ± 1.8 mg/dL). Slow Off-rate Modified Aptamer (SOMA) -scan plasma protein measurement determined a 43-protein panel reported as diagnostic and/or prognostic for T2D. At baseline, 9 proteins were altered in T2D. Three of 13 prognostic/diagnostic proteins were lower in T2D: Adiponectin (p < 0.0001), Endocan (p < 0.05) and Mast/stem cell growth factor receptor-Kit (KIT) (p < 0.01). Two of 14 prognostic proteins [Cathepsin-D (p < 0.05) and Cadherin-E (p < 0.005)], and four of 16 diagnostic proteins [Kallikrein-4 (p = 0.001), Aminoacylase-1 (p = 0.001), Insulin-like growth factor-binding protein-4 (IGFBP4) (p < 0.05) and Reticulon-4 receptor (RTN4R) (p < 0.001)] were higher in T2D. Protein levels were unchanged following glucose normalization in T2D. Our results suggest that a focused biomarker panel may be useful for assessing β-cell dysfunction and may complement clinical decision-making on insulin therapy. Unchanged post-glucose normalization levels indicate these are not acute-phase proteins or affected by glucose variability.

Using clinically derived human tissue to 3-dimensionally bioprint personalized testicular tubules for in vitro culturing: first report

To study the feasibility and spermatogenic potential of 3-dimensional (3D) bioprinting personalized human testicular cells derived from a patient with nonobstructive azoospermia (NOA). A human testicular biopsy from a single donor with NOA was dissociated into single cells, expanded invitro, and 3D bioprinted into tubular structures akin to the seminiferous tubule using AGC-10 bioink and an RX1 bioprinter with a CENTRA coaxial microfluidic printhead from Aspect Biosystems. Three-dimensional organoid cultures were used as a nonbioprinted invitro control. Academic medical center. A 31-year-old man with NOA with testis biopsy demonstrating Sertoli cell-only syndrome. Three-dimensional bioprinting and invitro culturing of patient-derived testis cells. Cellular viability after printing was determined, along with the expression of phenotypic and spermatogenic functional genetic markers after 12 days of invitro culture. Testicular cultures were expandable invitro and generated sufficiently large numbers for 3D bioprinting at 35 million cells per mL of bioink. Viability 24 hours after printing was determined to be 93.4% ± 2.4%. Immunofluorescence staining for the phenotype markers SRY-Box transcription factor 9, insulin-like 3, actin alpha 2 smooth muscle, and synaptonemal complex protein 3 after 12 days was positive, confirming the presence of Sertoli, Leydig, peritubular myoid, and meiotic germ cells. Reverse transcription qualitative polymerase chain reaction analysis showed that after 12 days in spermatogenic media, the bioprints substantially up-regulated spermatogenic gene expression on par with nonbioprinted controls and showed a particularly significant improvement in genes involved in spermatogonial stem cell maintenance: inhibitor of deoxyribonucleic acid binding 4 by 365-fold; fibroblast growth factor 3 by 94,152-fold; stem cell growth factor receptor KIT by twofold; stimulated by retinoic acid 8 by 125-fold; deleted in azoospermia-like by 114-fold; synaptonemal complex protein 3 by sevenfold; zona pellucida binding protein by twofold; transition protein 1 by 2,908-fold; and protamine 2 by 11-fold. This study demonstrates for the first time the feasibility of 3D bioprinting adult human testicular cells. We show that the bioprinting process is compatible with high testicular cell viability and without loss of the main somatic phenotypes within the testis tissue. We demonstrate an increase in germ cell markers in the 3D bioprinted tubules after 12 days of invitro culture. This platform may carry future potential for disease modeling and regenerative opportunities in a personalized medicine framework.

Autocrine/Paracrine Loop Between SCF+/c-Kit+ Mast Cells Promotes Cutaneous Melanoma Progression.

c-Kit, or mast/stem cell growth factor receptor Kit, is a tyrosine kinase receptor structurally analogous to the colony-stimulating factor-1 (CSF-1) and platelet-derived growth factor (PDGF) CSF-1/PDGF receptor Tyr-subfamily. It binds the cytokine KITLG/SCF to regulate cell survival and proliferation, hematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration and function, and it plays an essential role in melanogenesis. SCF and c-Kit are biologically active as membrane-bound and soluble forms. They can be expressed by tumor cells and cells of the microenvironment playing a crucial role in tumor development, progression, and relapses. To date, few investigations have concerned the role of SCF+/c-Kit+ mast cells in normal, premalignant, and malignant skin lesions that resemble steps of malignant melanoma progression. In this study, by immunolabeling reactions, we demonstrated that in melanoma lesions, SCF and c-Kit were expressed in mast cells and released by themselves, suggesting an autocrine/paracrine loop might be implicated in regulatory mechanisms of neoangiogenesis and tumor progression in human melanoma.

Understanding human mast cells: lesson from therapies for allergic and non-allergic diseases.

Mast cells have crucial roles in allergic and other inflammatory diseases. Preclinical approaches provide circumstantial evidence for mast cell involvement in many diseases, but these studies have major limitations - for example, there is still a lack of suitable mouse models for some mast cell-driven diseases such as urticaria. Some approaches for studying mast cells are invasive or can induce severe reactions, and very few mediators or receptors are specific for mast cells. Recently, several drugs that target human mast cells have been developed. These include monoclonal antibodies and small molecules that can specifically inhibit mast cell degranulation via key receptors (such as FcεRI), that block specific signal transduction pathways involved in mast cell activation (for example, BTK), that silence mast cells via inhibitory receptors (such as Siglec-8) or that reduce mast cell numbers and prevent their differentiation by acting on the mast/stem cell growth factor receptor KIT. In this Review, we discuss the existing and emerging therapies that target mast cells, and we consider how these treatments can help us to understand mast cell functions in disease.

A shift of dynamic equilibrium between the KIT active and inactive states causes drug resistance.

Tyrosine phosphorylation, a highly regulated post-translational modification, is carried out by the enzyme tyrosine kinase (TK). TKs are important mediators in signaling cascades, facilitating diverse biological processes in response to stimuli. TKs may acquire mutations leading to malignancy and are viable targets for anti-cancer drugs. Mast/stem cell growth factor receptor KIT is a TK involved in cell differentiation, whose dysregulation leads to various types of cancer, including gastrointestinal stromal tumors, leukemia, and melanoma. KIT can be targeted by a range of inhibitors that predominantly bind to the inactive state of the enzyme. A mutation Y823D in the activation loop of KIT is known to be responsible for the loss of sensitivity to some drugs in metastatic tumors. We used all-atom molecular dynamics simulations to study the impact of Y823D on the KIT conformation and dynamics and compared it to the effect of phosphorylation of Y823. We simulated in total 6.4 μs of wild-type, mutant and phosphorylated KIT in the active- and inactive-state conformations. We found that Y823D affects the protein dynamics differently: in the active state, the mutation increases the protein stability, whereas in the inactive state it induces local destabilization, thus shifting the dynamic equilibrium towards the active state, altering the communication between distant regulatory regions. The observed dynamics of the Y823D mutant is similar to the dynamics of KIT phosphorylated at position Y823, thus we hypothesize that this mutation mimics a constitutively active kinase, which is not responsive to inhibitors that bind its inactive conformation.

Clinicopathological significance and prognostic implication of CD44 and its splice variants (v3 and v6) in colorectal cancer.

BackgroundCD44 is a marker for colorectal cancer (CRC) stem cells (CCSCs), but its prognostic value remains controversial. Furthermore, few studies have investigated the expression profile of CD44 variants in CRC. The proto-oncogene mast/stem cell growth factor receptor Kit (c-Kit) was previously found to play an important role in supporting CRC cells; however, the associations between c-Kit and CD44 (and its splice variants) remain largely unknown.MethodsA total of 148 patients with CRC were enrolled in the present study, and the levels of CD44, CD44 splice variant (v)3, CD44v6 and c-Kit were examined by immunohistochemical staining. Associations between these markers and clinicopathological parameters, as well as the extent to which these markers predict progression free-survival (PFS), were analyzed.ResultsA total of 29 (19.6%), 60 (40.5%) and 66 (44.6%) patients were CD44-, CD44v3- and CD44v6-positive, respectively. No clear c-Kit-positivity was detected in any of the patients. Analyses of clinicopathological features indicated that positive expression of CD44v3 was significantly associated with cell differentiation (P=0.03), and N (P=0.04), M (P<0.01) and tumor-node-metastasis (TNM) stages (P<0.01). Univariate analysis demonstrated that T, N, M and TNM stages (all P<0.01), and CD44 (P<0.01), CD44v3 (P=0.03) and CD44v6 (P=0.02) levels were significantly associated with PFS. Furthermore, multivariate analysis indicated that patients without CD44 expression (P<0.01) but with CD44v3 (P=0.04) and CD44v6 (P=0.02) expression exhibited significantly shortened PFS.ConclusionsCD44, CD44v3 and CD44v6 were determined to be heterogeneously expressed in CRC, and may aid in the prognostic prediction of patients with this disease.

Predictive and prognostic value of phosphorylated c-KIT and PDGFRA in advanced non-small cell lung cancer harboring ALK fusion.

Secondary KIT gene amplification leads to tyrosine kinase inhibitor resistance in anaplastic lymphoma kinase (ALK) fusion-positive advanced non-small cell lung cancer (NSCLC). The presence of the 4q12 amplicon causes the activation of downstream mast/stem cell growth factor receptor Kit (c-Kit) or platelet-derived growth factor receptor α (PDGFRA) signaling pathways. Therefore, in the present study, the association between the functional proteins phosphorylated c-Kit (p-c-Kit) and phosphorylated PDGFRA (p-PDGFRA) and the prognosis of ALK fusion NSCLC was investigated. Advanced stage NSCLC samples with ALK fusion were tested for their p-c-Kit and p-PDGFRA content by immunohistochemical staining, and for its association with crizotinib efficacy and the survival of the patients. Of 64 eligible ALK-positive patients with NSCLC, 30 (46.9%) were p-c-Kit-positive and 10 (15.7%) were p-PDGFRA-positive. Brain metastases were more common in ALK-positive cases that were p-PDGFRA-positive compared with those who were p-PDGFRA-negative. ALK-positive patients treated with crizotinib, who exhibited high levels of p-c-Kit had significantly lower progression-free survival times than those with low levels. In addition, the patients with high levels of p-c-Kit exhibited lower overall survival times than those with low levels. Furthermore, multivariate analysis indicated that high levels of p-c-Kit in patients with ALK fusion was the only significant predictive factor for crizotinib efficacy and was a prognostic factor for poor overall survival time. However, no statistically significant difference was observed in the survival of patients with different p-PDGFRA levels. p-PDGFRA was more frequently expressed in the ALK-positive cases with brain metastasis. c-Kit signaling activation may be associated with poor efficacy of crizotinib and poor prognosis in advanced ALK fusion NSCLC.

Combination of comprehensive two-dimensional prostate cancer cell membrane chromatographic system and network pharmacology for characterizing membrane binding active components from Radix et Rhizoma Rhei and their targets

Prostate cancer (PCa) is a common and fatal cancer for men and effective treatment options are still not enough for patients. Radix et Rhizoma Rhei had been applied to treat PCa long-termly and effectively when combined with surgical treatment and chemotherapy. However, its active components and target proteins are still not quite clear. As membrane receptors play a vital role in PCa, in this study, a novel strategy that combines comprehensive 2D 3-aminopropyltriethoxysilane-decorated prostate cancer cell (DU145) membrane chromatographic (CMC) system with network pharmacology approach was developed to characterize membrane binding active components proteins from Radix et Rhizoma Rhei and their targets. Thirteen active components were screened out by CMC system, among which emodin and rhapontigrnin with good membrane binding behaviors were validated to show ideal inhibitory effects on DU145 cells by cell viability and cell apoptosis assays. Five membrane proteins were predicted as the potential targets by the a specific network pharmacology approach, among which mast/stem cell growth factor receptor Kit (KIT) was identified as the most possible target by network data mining. Surface plasmon resonance analysis verified that the dissociation constant (KD) of rhapontigrnin and emodin with KIT was 6.06 × 10−5 M and 8.82 × 10−5 M, respectively. Our results showed that the combination of comprehensive 2D CMC system and network pharmacology based target identification could not only rapidly identify the membrane binding components but also find the potential membrane protein targets with high confidence, which could broaden the range of application scope of CMC, especially for the screening of active compounds from complex chemical samples using primary pathologic cell lines.

The human CSF pain proteome

Chronic pain represents one of the major medical challenges in the 21st century, affecting >1.5 billion of the world population. Overlapping and heterogenous symptoms of various chronic pain conditions complicate their diagnosis, emphasizing the need for more specific biomarkers to improve the diagnosis and understand the disease mechanisms.We have here investigated proteins found in human CSF with respect to known “pain” genes and in a cohort of patients with dysfunctional pain (fibromyalgia, FM), inflammatory pain (rheumatoid arthritis patients, RA) and non-pain controls utilized semi-quantitative proteomics using mass spectrometry (MS) to explore quantitative differences between these cohorts of patients.We found that “pain proteins” detected in CSF using MS are typically related to synaptic transmission, inflammatory responses, neuropeptide signaling- and hormonal activity. In addition, we found ten proteins potentially associated with chronic pain in FM and RA: neural cell adhesion molecule L1, complement C4-A, lysozyme C, receptor-type tyrosine-protein phosphatase zeta, apolipoprotein D, alpha-1-antichymotrypsin, granulins, calcium/calmodulin-dependent protein kinase type II subunit alpha, mast/stem cell growth factor receptor Kit, prolow-density lipoprotein receptor-related protein 1. These proteins might be of importance for understanding the mechanisms of dysfunctional/inflammatory chronic pain and also for use as potential biomarkers. SignificanceChronic pain is a common disease and it poses a large burden on worldwide health. Fibromyalgia (FM) is a heterogeneous disease of unknown etiology characterized by chronic widespread pain (CWP). The diagnosis and treatment of FM is based on the analysis of clinical assessments and no measurable biomarkers are available. Cerebrospinal fluid (CSF) has been historically considered as a rich source of biomarkers for diseases of nervous system including chronic pain. Here, we explore CSF proteome of FM patients utilizing mass spectrometry based quantitative proteomics method combined with multivariate data analysis in order to monitor the dynamics of the CSF proteome. Our findings in this exploratory study support notable presence of pain related proteins in CSF yet with specific domains including inflammatory responses, neuropeptide signaling- and hormonal activity. We have investigated molecular functions of significantly altered proteins and demonstrate presence of 176 known pain related proteins in CSF. In addition, we found ten proteins potentially associated with pain in FM and RA: neural cell adhesion molecule L1, complement C4-A, lysozyme C, receptor-type tyrosine-protein phosphatase zeta, apolipoprotein D, alpha-1-antichymotrypsin, granulins, calcium/calmodulin-dependent protein kinase type II subunit alpha, mast/stem cell growth factor receptor Kit, prolow-density lipoprotein receptor-related protein 1. These proteins are novel in the context of FM but are known to be involved in pain mechanisms including inflammatory response and signal transduction. These results should be of clear significance and interest for researchers and clinicians working in the field of pain utilizing human CSF and MS based proteomics.

Inhibitors to Overcome Secondary Mutations in the Stem Cell Factor Receptor KIT.

In modern cancer therapy, the use of small organic molecules against receptor tyrosine kinases (RTKs) has been shown to be a valuable strategy. The association of cancer cells with dysregulated signaling pathways linked to RTKs represents a key element in targeted cancer therapies. The tyrosine kinase mast/stem cell growth factor receptor KIT is an example of a clinically relevant RTK. KIT is targeted for cancer therapy in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). However, acquired resistance mutations within the catalytic domain decrease the efficacy of this strategy and are the most common cause of failed therapy. Here, we present the structure-based design and synthesis of novel type II kinase inhibitors to overcome these mutations in KIT. Biochemical and cellular studies revealed promising molecules for the inhibition of mutated KIT.

Contrast-enhanced computerized tomography combined with a targeted nanoparticle contrast agent for screening for early-phase non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a major cause of morbidity and mortality, and patients with NSCLC are frequently diagnosed at an advanced stage. This is primarily due to a lack of advanced and sensitive protocols for the detection of early stage NSCLC. Therefore, methods for the accurate diagnosis of early stage NSCLC are urgently required to improve survival rates. The present study investigated the use of contrast-enhanced computerized tomography (CECT) combined with a targeted nanoparticle contrast agent (TNCA) to diagnose early-stage NSCLC in a mice xenograft model. The TNCA used was lenvatinib, a multi-target tyrosine kinase inhibitor that inhibits vascular endothelial growth factor receptor 1-3, fibroblast growth factor receptor 1-4, platelet-derived growth factor receptor β, proto-oncogene tyrosine-protein kinase receptor Ret and mast/stem cell growth factor receptor Kit. Xenograft NSCLC mice were established and used to analyze the efficacy of CECT-TNCA compared with CT scanning alone. The TNCA was inhaled with the use of an atomizer. The results demonstrated that CECT-TNCA improved the sensitivity of the diagnosis of early stage NSCLC. In addition, imaging using the TNCA enabled the visualization of nodules in the lung in mice with early stage NSCLC. In addition, lung nodule signal enhancement was increased in CECT-TNCA compared with CT, suggesting a high accurate accumulation of the TNCA in tumor nodules. Mice diagnosed with early stage NSCLC exhibited a higher eradication rate of NSCLC after treatment with cisplatin compared with mice with advanced stage NSCLC. These data indicate that the sensitivity and accuracy of CT imaging for the diagnosis of early stage NSCLC was improved through combination with the liposome-encapsulated TNCA.

Bmi1 and BRG1 drive myocardial repair by regulating cardiac stem cell function in acute rheumatic heart disease.

Rheumatic heart disease (RHD) occurs due to the accumulation of complications associated with rheumatic fever, and it results in high morbidity and mortality. The majority of cases of RHD are diagnosed in the chronic stages, when treatment options are limited. A small reservoir of cardiac stem cells is responsible for maintaining cardiac homeostasis and repairing tissue damage. Understanding the role of cardiac stem cells and the various proteins responsible for their functions in different pathological stages of RHD is an important area of investigation. Polycomb complex protein BMI-1 (Bmi1) and transcription activator BRG1 (BRG1) are associated with the maintenance of stemness in various types of stem cells. The present study investigated the role served by Bmi1 and BRG1 in cardiac stem cells during various pathological stages of RHD through immunohistochemistry and western blotting. A rat model of RHD was established via immunization with the Group A Streptococcus M5 protein. The rat was demonstrated to develop acute RHD 2 months after the final immunization, characterized by cardiac inflammation and tissue damage. Chronic RHD was identified 4 months after the final immunization, revealed by cardiac tissue compression and shrinkage. Expression of the cardiac stem cell marker mast/stem cell growth factor receptor kit was identified to be elevated during acute RHD, but downregulated in the chronic stages of RHD. A similar pattern of expression was revealed for Bmi1 and BRG1, indicating that they serve a role in regulating cardiac stem cell proliferation during acute RHD. These results suggest that cardiac stem cells serve a supportive role in the acute, but not chronic, stages of RHD via expression of Bmi1 and BRG1.