DRR1 promotes neuroblastoma cell differentiation by regulating CREB expression.

BACKGROUND: Neuroblastoma is the most common cancer in infants and the most common extracranial solid tumor in childhood. DRR1 was identified to be downregulated in poorly differentiated ganglion cells from neuroblastoma model mice. However, the roles of DRR1 in neuroblastoma remain largely unclear. METHODS: The neuroblastoma cells were induced to differentiate, and the expression of DRR1 was detected. The expression of the neuroblastoma cell differentiation markers was analyzed in DRR1 shRNA- or DRR1-expressing vector-treated neuroblastoma cells. The downstream genes of DRR1 were screened with ChIP-seq assay. Finally, TNB1 cells were infected with DRR1 shRNA and CREB expressing vector containing lentivirus, and the expression of the cell differentiation markers, cell cycle distribution and tumor growth were analyzed. RESULTS: The expression of DRR1 was increased in differentiated neuroblastoma cells, and downregulation of DRR1 expression inhibited the differentiation of neuroblastoma cells. Further experiments indicated that CREB is a candidate downstream gene of DRR1, and it mediates neuroblastoma cell differentiation. Moreover, overexpression of CREB rescued the effect of DRR1 shRNA on cell differentiation, cell cycle distribution and tumor growth in neuroblastoma. CONCLUSIONS: DRR1-CREB axis modulates the differentiation of neuroblastoma cells and is associated with the outcome of neuroblastoma patients. IMPACT: DRR1 is involved in regulation of the differentiation of neuroblastoma. Binding with actin is essential for DRR1 to regulate neuroblastoma cell differentiation. CREB is a candidate downstream gene of DRR1 in regulating of the differentiation of neuroblastoma.

SIRT1 Is Involved in the Neuroprotection of Pterostilbene Against Amyloid ß 25-35-Induced Cognitive Deficits in Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-ß (Aß) deposits and neurofibrillary tangles. Pterostilbene (PTE), a bioactive component mainly in blueberries, is found to have neuroprotective properties. However, the specific underlying mechanisms of PTE in protecting AD remain unclear. Herein, we explored its effects on Aß25-35-induced neuronal damage in vivo and in vitro and further compared the roles with its structural analog resveratrol (RES) in improving learning-memory deficits. We found that intragastric administration of PTE (40 mg/kg) displayed more effective neuroprotection on Aß25-35-induced cognitive dysfunction assessed using the novel object test, Y-maze test, and Morris water maze test. Then, we found that PTE improved neuronal plasticity and alleviated neuronal loss both in vivo and in vitro. Additionally, PTE upregulated the expression of sirtuin-1 (SIRT1) and nuclear factor erythroid 2-related factor 2 (Nrf2) and the level of superoxide dismutase (SOD), and inhibited mitochondria-dependent apoptosis in the Aß25-35-treated group. However, SIRT1 inhibitor EX527 reversed the neuroprotection and induced a drop in mitochondrial membrane potential in PTE-treated primary cortical neurons. Our data suggest that PTE's enhancing learning-memory ability and improving neuroplasticity might be related to inhibiting mitochondria-dependent apoptosis via the antioxidant effect regulated by SIRT1/Nrf2 in AD.

MYCN Directly Targets NeuroD1 to Promote Cellular Proliferation in Neuroblastoma.

NeuroD1 is a neuronal differentiation factor that contains a basic helixloophelix (bHLH) motif. Recently, NeuroD1 was found to be associated with tumorigenesis in neuroblastoma (NB) and is known to promote cell proliferation and migration in these cells. Here we found that MYCN regulates the expression of NeuroD1 in NB cells and that the downregulation of MYCN using short hairpin RNAs (shRNA) results in the inhibition of cellular proliferation in NB cells. Moreover, the phenotype induced by MYCN shRNA was rescued by the exogenous expression of NeuroD1. Chromatin immunoprecipitation (ChIP) assay showed that MYCN directly binds to the E-box element in the NeuroD1 promoter region. In addition, our evaluation of two clinical databases showed that there was a positive correlation between the expression of MYCN and NeuroD1 in NB patients, which supports our in vitro data. In conclusion, this study demonstrates that MYCN-regulated NeuroD1 expression is one of the important mechanisms underlying enhanced cellular proliferation induced by the increase in MYCN expression in NB, and our results provide an important therapeutic target for NB in the future.

Downregulated in renal carcinoma 1 (DRR1) mediates the differentiation of neural stem cells through transcriptional regulation.

Downregulated in renal carcinoma 1 (DRR1), also called family with sequence similarity 107, member A (FAM107A), is highly expressed in the nervous system. DRR1 has been found to be involved in neuronal survival, spine formation, and synaptic function. Recently, several studies have reported that DRR1 is expressed in neural stem cells (NSCs) and neural progenitor cells during the early stages of brain development. However, the mechanisms underlying the role and function of DRR1 in NSCs are poorly understood. To clarify the role of DRR1 in NSCs, we transfected DRR1 shRNA into primary NSCs and found that downregulation of DRR1 suppressed the differentiation of NSCs. To investigate the underlying mechanism in this case, chromatin immunoprecipitation sequencing (ChIP-seq) analysis was performed to identify the genes downstream of DRR1. Several genes, such as AHNAK, VAMP8, NOD1, and ACVR2B were identified to be downstream of DRR1 in NSCs.

Down-regulated in renal cell carcinoma 1 (DRR1) regulates axon outgrowth during hippocampal neuron development.

Down-regulated in renal cell carcinoma 1 (DRR1), a unique stress-induced protein, is highly expressed in the nervous system. This study investigated the roles of DRR1 in the brain by examining its expression pattern at different developmental stages of a rat brain and in cultured primary hippocampal neurons. High expression of DRR1 was observed in all developmental stages of a rat brain and cultured primary hippocampal neurons. We then focused on the role of DRR1 in promoting neurite outgrowth during the early stage of hippocampal neuron development. Results showed that down-regulation of DRR1 suppressed axon outgrowth. Mass spectrometry analysis revealed that tropomodulin-2 (Tmod2) is a novel binding partner of DRR1. Our results showed that both DRR1 and Tmod2 mediate axon formation during the early stage of hippocampal neuron development. Suppression of TMOD2 expression rescued the abnormal axon outgrowth induced by DRR1 knockdown during the early stage of hippocampal neuron development.

Amyloid-ß (25-35) regulates neuronal damage and memory loss via SIRT1/Nrf2 in the cortex of mice.

Alzheimer's disease (AD) is the most common type of dementia. AD is pathologically characterized by synaptic dysfunction and cognitive decline due to the aggregation of a large amount of amyloid-ß (Aß) protein in the brain. However, recent studies have discovered that the Aß is produced as an antimicrobial peptide that acts against bacteria and viruses. This has renewed interest in the effect of Aß on AD. Thus, in this study, we investigated the different concentrations of Aß25-35 on neuroprotection and further explore the related mechanisms. Firstly, we detected the cognitive function using the Y-maze test, novel object recognition memory task and Morris water maze test. Then, we analyzed the ultrastructure of synapses and mitochondria, in addition to evaluating SOD levels. We also examined the effect of Aß25-35 on the viability and structure of the primary neurons. The western blot analysis was used to measure the protein levels. The results showed that mice treated with high concentration of Aß25-35 impaired the learning-memory ability and disordered the structure of neurons and mitochondria. Meanwhile, high concentration of Aß25-35 decreased the SIRT1/Nrf2 related antioxidant capacity and induced apoptosis. In contrast, mice treated with low concentration of Aß25-35 increased SOD levels and SIRT1/Nrf2 expressions, and induced autophagy. Our data suggest that low concentration of Aß25-35 may increase SOD levels through SIRT1/Nrf2 and induce autophagy.

Downregulation of TMOD1 promotes cell motility and cell proliferation in cervical cancer cells.

Tropomodulin-1 (TMOD1) is a key regulator of actin dynamics, which caps the pointed end of actin filaments. TMOD1 has been reported to be involved in several cellular processes, including neurite outgrowth, spine formation and cell migration. Increasing evidence demonstrates that TMOD1 is implicated in several aspects of cancer development. The present study aimed to investigate the role of TMOD1 in cervical cancer. HeLa and CaSki cell lines, derived from human cervical cancer, were used to evaluate the function of TMOD1. Cell motility was measured via a wound-healing assay, with the TMOD1 short hairpin (sh)RNAs transfected cells. Subsequently, cell proliferation was assessed using low serum cell culture condition, while cell cycle distribution was analyzed via flow cytometry. The results demonstrated that downregulated TMOD1 promoted cell motility and proliferation, which is attributed to promotion of G1/S phase transition in HeLa and CaSki cells. Furthermore, it was indicated that co-expression of shRNA resistant TMOD1 rescued these phenomena. The clinical data demonstrated that high TMOD1 expression is associated with good pathological status in patients with cervical cancer. Overall, the results of the present study indicated that TMOD1 may act as a tumor suppressor in cervical cancer, whereby its downregulated expression was demonstrated to have direct effects on cell motility and cell proliferation. These results provide new evidence for the prognostic prediction of cervical cancer, which may serve as a promising therapeutic strategy for patients with cervical cancer.

Molecular mechanisms of apoptosis and autophagy elicited by combined treatment with oridonin and cetuximab in laryngeal squamous cell carcinoma.

Combined oridonin (ORI), a natural and safe kaurene diterpenoid isolated from Rabdosia rubescens, and cetuximab (Cet), an anti-EGFR monoclonal antibody, have been reported to exert synergistic anti-tumor effects against laryngeal squamous cell carcinoma (LSCC) both in vitro and in vivo by our group. In the present study, we further found that ORI/Cet treatment not only resulted in apoptosis but also induced autophagy. AMPK/mTOR signaling pathway was found to be involved in the activation of autophagy in ORI/Cet-treated LSCC cells, which is independent of p53 status. Additionally, chromatin immunoprecipitation (ChIP) assay showed that ORI/Cet significantly increased the binding NF-κB family member p65 with the promotor of BECN 1, and p65-mediated up-regulation of BECN 1 caused by ORI/Cet is coupled to increased autophagy. On the other hand, we demonstrated that either Beclin 1 SiRNA or autophagy inhibitors could increase ORI/Cet induced-apoptosis, indicating that autophagy induced by combination of the two agents plays a cytoprotective role. Interestingly, 48 h after the combined treatment, autophagy began to decrease but apoptosis was significantly elevated. Our findings suggest that autophagy might be strongly associated with the antitumor efficacy of ORI/Cet, which may be beneficial to the clinical application of ORI/Cet in LSCC treatment.

[Chemistry and biology research on bitter-taste Chinese materia medica with function of regulating glycolipid metabolism].

The bitter taste is one of the important properties among five flavors of Chinese materia medica (CMM), characterized by downbearing and discharging, drying dampness, and consolidating Yin. In common CMM, bitter-taste CMM accounts for a large proportion, indicating the importance of it. Through the efficacy of clearing away heat and dampness, reducing fire and removing toxin, bitter-taste CMM has achieved good results in treating diabetes in clinical application, proving their definite therapeutic effect on regulating glucose and lipid metabolism (main features of diabetes). At present, there are many reports about the chemical constituents and pharmacological effects of CMM on diabetes, but there are few reviews on the chemistry and biology of bitter-taste CMM. This study summarized the properties and compatibility characteristics of bitter-taste CMM for treating diabetes, and mainly analyzed the chemistry and biology basis of bitter-taste CMM with function of regulating glycolipid metabolism, laying foundation for further researches on properties theory of CMM.

Downregulation of CREB Promotes Cell Proliferation by Mediating G1/S Phase Transition in Hodgkin Lymphoma.

The cyclic-AMP response element-binding protein (CREB), a well-known nuclear transcription factor, has been shown to play an essential role in many cellular processes, including differentiation, cell survival, and cell proliferation, by regulating the expression of downstream genes. Recently, increased expression of CREB was frequently found in various tumors, indicating that CREB is implicated in the process of tumorigenesis. However, the effects of CREB on Hodgkin lymphoma (HL) remain unknown. To clarify the role of CREB in HL, we performed knockdown experiments in HL. We found that downregulation of CREB by short hairpin RNA (shRNA) resulted in enhancement of cell proliferation and promotion of G1/S phase transition, and these effects can be rescued by expression of shRNA-resistant CREB. Meanwhile, the expression level of cell cycle-related proteins, such as cyclin D1, cyclin E1, cyclin-dependent kinase 2 (CDK2), and CDK4, was elevated in response to depletion of CREB. Furthermore, we performed chromatin immunoprecipitation (ChIP) assay and confirmed that CREB directly bound to the promoter regions of these genes, which consequently contributed to the regulation of cell cycle. Consistent with our results, a clinical database showed that high expression of CREB correlates with favorable prognosis in B-cell lymphoma patients, which is totally different from the function of CREB in other cancers such as colorectal cancer, acute myeloid leukemia, and some endocrine cancers. Taken together, all of these features of CREB in HL strongly support its role as a tumor suppressor gene that can decelerate cell proliferation by inhibiting the expression of several cell cycle-related genes. Our results provide new evidence for prognosis prediction of HL and a promising therapeutic strategy for HL patients.

NeuroD1 promotes neuroblastoma cell growth by inducing the expression of ALK.

Neuroblastoma is derived from the sympathetic neuronal lineage of neural crest cells, and is the most frequently observed of the extracranial pediatric solid tumors. The neuronal differentiation factor, NeuroD1, has previously been shown to promote cell motility in neuroblastoma by suppressing the expression of Slit2. Here we report that NeuroD1 is also involved in the proliferation of neuroblastoma cells, including human cell lines and primary tumorspheres cultured from the tumor tissues of model mice. Interestingly, the growth inhibition of neuroblastoma cells induced by knockdown of NeuroD1 was accompanied by a reduction of ALK expression. ALK is known to be one of the important predisposition genes for neuroblastoma. The phenotype resulting from knockdown of NeuroD1 was suppressed by forced expression of ALK and, therefore, NeuroD1 appears to act mainly through ALK to promote the proliferation of neuroblastoma cells. Furthermore, we showed that NeuroD1 directly bound to the promoter region of ALK gene. In addition, the particular E-box in the promoter was responsible for NeuroD1-mediated ALK expression. These results indicate that ALK should be a direct target gene of NeuroD1. Finally, the expressions of NeuroD1 and ALK in the early tumor lesions of neuroblastoma model mice coincided in vivo. We conclude that the novel mechanism would regulate the expression of ALK in neuroblastoma and that NeuroD1 should be significantly involved in neuroblastoma tumorigenesis.