Small molecule inhibitor agerafenib effectively suppresses neuroblastoma tumor growth in mouse models via inhibiting ERK MAPK signaling.

Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. Despite intensive multimodal therapy, nearly half of children with high-risk disease will relapse with therapy-resistant tumors. Dysregulation of MAPK pathway has been implicated in the pathogenesis of relapsed and refractory NB patients, which underscores the possibility of targeting MAPK signaling cascade as a novel therapeutic strategy. In this study, we found that high expressions of RAF family kinases correlated with advanced tumor stage, high-risk disease, tumor progression, and poor overall survival. Targeted inhibition of RAF family kinases with the novel small molecule inhibitor agerafenib abrogated the activation of ERK MAPK pathway in NB cells. Agerafenib significantly inhibited the cell proliferation and colony formation ability of NB cells in vitro, and its combination with traditional chemotherapy showed a synergistic pro-apoptotic effect. More importantly, agerafenib exhibited a favorable toxicity profile, potently suppressed tumor growth, and prolonged survival in NB mouse models. In conclusion, our preclinical data suggest that agerafenib might be an effective therapeutic agent for NB treatment, both as a single-agent and in combination with chemotherapy.

Effect of NELL1 on lung cancer stem­like cell differentiation.

The cancer stem cell theory recently has received enormous attention in cancer biology. Lung cancer stem­like cells are a subpopulation of undifferentiated lung tumor cells critical for lung cancer tumorigenesis, metastasis and resistance to therapy and disease relapse. The neural EGFL like 1 (NELL1) is a potent growth factor believed to preferentially target cells committed to the osteochondral lineage; yet, its expression and function in lung cancer are largely unknown. In the present study, we used specific medium to accumulate lung cancer stem­like cells of 95­D cells in spheres and obtained these highly expressed CD133 cells through flow cytometric cell sorting of CD133­stained cells which were termed 95­D lung cancer stem­like cells (95­D LCSCs). These 95­D LCSCs highly expressed stemness genes CD133, Oct4 and Sox2 determined by western blot analysis and quantitative real­time polymerase chain reaction (qPCR) analysis. Notably, we found that overexpression of NELL1 significantly reduced colony formation and invasion of 95­D LCSCs tested by soft agar colony formation and cell invasion assay. In addition, as determined by cell proliferation assay, overexpression of NELL1 increased the chemotherapeutic sensitivity of 95­D LCSCs to carboplatin and cisplatin. NELL1 also reduced the expression of phospho­MET (p­MET), Notch3 and HES1, which suggests that NELL1 may induce 95­D LCSC differentiation by inhibiting the expression of c­MET­Notch signaling. Our results suggest that NELL1 induces lung cancer stem­like cell differentiation, which provides a new potential therapeutic target for cancer stem cells.

A combination of valproic acid sodium salt, CHIR99021, E-616452, tranylcypromine, and 3-Deazaneplanocin A causes stem cell-like characteristics in cancer cells.

Many studies are based on the hypothesis that recurrence and drug resistance in lung carcinoma are due to a subpopulation of cancer stem-like cells (CSLCs) in solid tumors. Therefore it is crucial to screen for and recognize lung CSLCs. In this study, we stimulated non-small cell lung cancer (NSCLC) A549 cells to display stem cell-like characteristics using a combination of five small molecule compounds. The putative A549 stem cells activated an important CSLC marker, CD133 protein, as well multiple CSLC-related genes including ATP-binding cassette transporter G2 (ABCG2), C-X-C chemokine receptor type 4 (CXCR4), NESTIN, and BMI1. The A549 stem-like cells displayed resistance to the chemotherapeutic drugs etoposide and cisplatin, epithelial-to-mesenchymal transition properties, and increased protein expression levels of NOTCH1 and Hes Family bHLH Transcription Factor 1 (HES1). When A549 cells were pretreated with a NOTCH signaling pathway inhibitor before compound induction, expression of the NOTCH1 target gene HES1 was reduced. This demonstrated that the NOTCH signaling pathway in the putative A549 stem-like cells had been activated. Together, the results of our study showed that a combination of five small molecule agents could transform A549 cells into putative stem-like cells, and that these compounds could also elevate CD133 and ABCG2 protein expression levels in H460 cells. This study provides a convenient method for obtaining lung CSLCs, which may be an effective strategy for developing lung carcinoma treatments.

Interleukin 37 promotes angiogenesis through TGF-ß signaling.

IL-37 is a novel pro-angiogenic cytokine that potently promotes endothelial cell activation and pathological angiogenesis in our previous study, but the mechanisms behind the pro-angiogenic effect of IL-37 are less well understood. Extending our observations, we found that TGF-ß interacts with IL-37, and potently enhances the binding affinity of IL-37 to the ALK1 receptor complex, thus allowing IL-37 to signal through ALK1 to activate pro-angiogenic responses. We further show that TGF-ß and ALK1 are required in IL-37 induced pro-angiogenic response in ECs and in the mouse model of Matrigel plug and oxygen-induced retinopathy. The result suggests that IL-37 induces pro-angiogenic responses through TGF-ß, which may act as the bridging molecule that mediates IL-37 binding to the TGF-ß receptor complex.

Molecular Chaperone Calnexin Regulates the Function of Drosophila Sodium Channel Paralytic.

Neuronal activity mediated by voltage-gated channels provides the basis for higher-order behavioral tasks that orchestrate life. Chaperone-mediated regulation, one of the major means to control protein quality and function, is an essential route for controlling channel activity. Here we present evidence that Drosophila ER chaperone Calnexin colocalizes and interacts with the α subunit of sodium channel Paralytic. Co-immunoprecipitation analysis indicates that Calnexin interacts with Paralytic protein variants that contain glycosylation sites Asn313, 325, 343, 1463, and 1482. Downregulation of Calnexin expression results in a decrease in Paralytic protein levels, whereas overexpression of the Calnexin C-terminal calcium-binding domain triggers an increase reversely. Genetic analysis using adult climbing, seizure-induced paralysis, and neuromuscular junction indicates that lack of Calnexin expression enhances Paralytic-mediated locomotor deficits, suppresses Paralytic-mediated ghost bouton formation, and regulates minature excitatory junction potentials (mEJP) frequency and latency time. Taken together, our findings demonstrate a need for chaperone-mediated regulation on channel activity during locomotor control, providing the molecular basis for channlopathies such as epilepsy.

The hypoparathyroidism-associated mutation in Drosophila Gcm compromises protein stability and glial cell formation.

Differentiated neurons and glia are acquired from immature precursors via transcriptional controls exerted by factors such as proteins in the family of Glial Cells Missing (Gcm). Mammalian Gcm proteins mediate neural stem cell induction, placenta and parathyroid development, whereas Drosophila Gcm proteins act as a key switch to determine neuronal and glial cell fates and regulate hemocyte development. The present study reports a hypoparathyroidism-associated mutation R59L that alters Drosophila Gcm (Gcm) protein stability, rendering it unstable, and hyperubiquitinated via the ubiquitin-proteasome system (UPS). GcmR59L interacts with the Slimb-based SCF complex and Protein Kinase C (PKC), which possibly plays a role in its phosphorylation, hence altering ubiquitination. Additionally, R59L causes reduced Gcm protein levels in a manner independent of the PEST domain signaling protein turnover. GcmR59L proteins bind DNA, functionally activate transcription, and induce glial cells, yet at a less efficient level. Finally, overexpression of either wild-type human Gcmb (hGcmb) or hGcmb carrying the conserved hypoparathyroidism mutation only slightly affects gliogenesis, indicating differential regulatory mechanisms in human and flies. Taken together, these findings demonstrate the significance of this disease-associated mutation in controlling Gcm protein stability via UPS, hence advance our understanding on how glial formation is regulated.

Drug-induced premature senescence model in human dental follicle stem cells.

Aging is identified by a progressive decline of physiological integrity leading to age-related degenerative diseases, but its causes is unclear. Human dental pulp stem cells (hDPSCs) has a remarkable rejuvenated capacity that relies on its resident stem cells. However, because of the lack of proper senescence models, exploration of the underlying molecular mechanisms has been hindered. Here, we established a cellular model utilizing a hydroxyurea (HU) treatment protocol and effectively induced Human dental pulp stem cells to undergo cellular senescence. Age-related phenotypic changes were identified by augmented senescence-associated-ß-galactosidase (SA-ß-gal) staining, declined proliferation and differentiation capacity, elevated G0/G1 cell cycle arrest, increased apoptosis and reactive oxygen species levels. Furthermore, we tested the expression of key genes in various DNA repair pathways including nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. In addition, our results showed that Dental pulp stem cells from young donors are more resistant to apoptosis and exhibit increased non-homologous end joining activity compared to old donors. Further transcriptome analysis demonstrate that multiple pathways are involved in the HU-induced Dental pulp stem cells ageing, including genes associated with DNA damage and repair, mitochondrial dysfunction and increased reactive oxygen species levels. Taken together, the cellular model have important implications for understanding the molecular exploration of Dental pulp stem cells senescence and aging.

The expression profile and promoter analysis of ß-N-acetylglucosaminidases in the silkworm Bombyx mori.

ß-N-acetylglucosaminidase (GlcNAcase) is a key enzyme in the chitin decomposition process. In this study, we investigated the gene expression profile of GlcNAcases and the regulation mechanism for one of these genes, BmGlcNAcase1, in the silkworm. We performed sequence analysis of GlcNAcase. Using dual-spike-in qPCR method, we examined the expression of Bombyx ß-N-acetylglucosaminidases (BmGlcNAcases) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. Using Bac-to-Bac system and luciferase reporter vectors, we further analyzed the promoter sequence of BmGlcNAcase1. The results showed that these proteins have a highly conserved catalytic domain. The expression levels of the BmGlcNAcase genes varied in different tissues, and were increased 48 h after exposure to ecdysone. BmGlcNAcase1 gene promoter with 5'-end serial deletions showed different levels of activity in various tissues, higher in the blood, skin and fat body. Deletion of the region from -347 to -223 upstream of BmGlcNAcase-1 gene abolished its promoter activity. This region contains the binding sites for key transcription factors including Hb, BR-C Z, the HSF and the typical TATA-box element. These results indicate that BmGlcNAcases are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. BmGlcNAcase1 promoter analysis has paved a foundation for further study of the gene expression patterns.

Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm Bombyx mori.

In general, for real-time quantitative polymerase chain reaction (qPCR), normalization strategies use a reference gene as a control and to avoid the introduction of experimental errors expression of this gene should not vary in response to changing conditions. However, the expression of many reference genes has been reported to vary considerably and, without appropriate normalization, the expression profile of a target gene can be misinterpreted. In this study, the expression levels of seven commonly used reference genes (ACT, GAPDH, 28srRNA, RPL3, α-tubulin, UBC, and TBP) were detected at different development time points and in response to treatment with 20-hydroxyecdysone (20E) and with rutin. The expression stability was analyzed using geNorm and NormFinder software. Significant variations were found among normal tissues and between experimentally treated tissues. The dual spike-in strategy also revealed significant variations of the expression levels of the reference genes among normal tissues and between experimentally treated tissues. Glutathione-S-transferase sigma 1 (GSTs1), which has a high expression level in fat body and is related to the mechanism of resistance, was used as a target gene to validate the feasibility and difference of these two approaches.