RESUMO
Rationale: Vitamin D (VD) has been suggested to have antitumor effects, however, research on the role of its transporter vitamin D-binding protein (VDBP, gene name as GC) in tumors is limited. In this study, we demonstrated the mechanism underlying the inhibition of vasculogenic mimicry (VM) by VDBP in hepatocellular carcinoma (HCC) and proposed an anti-tumor strategy of combining anti-PD-1 therapy with VD. Methods: Three-dimensional cell culture models and mice with hepatocyte-specific GC deletion were utilized to study the correlation between VDBP expression and VM. A patient-derived tumor xenograft (PDX) model was further applied to validate the therapeutic efficacy of VD in combination with an anti-PD-1 drug. Results: The study revealed that VDBP expression is negatively correlated with VM in HCC patients and elevated VDBP expression is associated with a favorable prognosis. The mechanism studies suggested VDBP hindered the binding of Twist1 on the promoter of VE-cadherin by interacting with its helix-loop-helix DNA binding domain, ultimately leading to the inhibition of VM. Furthermore, VD facilitated the translocation of the vitamin D receptor (VDR) into the nucleus where VDR interacts with Yin Yang 1 (YY1), leading to the transcriptional activation of VDBP. We further demonstrated that the combination of VD and anti-PD-1 led to an improvement in the anti-tumor efficacy of an anti-PD-1 drug. Conclusion: Collectively, we identified VDBP as an important prognostic biomarker in HCC patients and uncovered it as a therapeutic target for enhancing the efficacy of immune therapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/patologia , Proteína de Ligação a Vitamina D/uso terapêutico , Neoplasias Hepáticas/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Diferenciação Celular , Linhagem Celular TumoralRESUMO
Intense ultraviolet (UV) exposure can cause phototoxic reactions, such as skin inflammation, resulting in injury. UV is a direct cause of DNA damage, but the mechanisms underlying transcriptional regulation within cells after DNA damage are unclear. The bioinformatics analysis of transcriptome sequencing data from UV-irradiated and non-UV-irradiated skin showed that transcription-related proteins, such as HSF4 and COIL, mediate cellular response to UV irradiation. HSF4 and COIL can form a complex under UV irradiation, and the preference for binding target genes changed because of the presence of a large number of R-loops in cells under UV irradiation and the ability of COIL to recognize R-loops. The regulation of target genes was altered by the HSF4-COIL complex, and the expression of inflammation and ageing-related genes, such as Atg7, Tfpi, and Lims1, was enhanced. A drug screen was performed for the recognition sites of COIL and R-loop. N6-(2-hydroxyethyl)-adenosine can competitively bind COIL and inhibit the binding of COIL to the R-loop. Thus, the activation of downstream inflammation-related genes and inflammatory skin injury was inhibited.
Assuntos
Estruturas R-Loop , Pele , Regulação da Expressão Gênica , Fatores de Transcrição de Choque Térmico/metabolismo , Inflamação/genética , Inflamação/metabolismo , Pele/metabolismo , TranscriptomaRESUMO
Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, including Alzheimer's, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that Usp14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). Usp14-deficient axJ mice display a resting tremor, a reduction in muscle mass, and notable hindlimb rigidity without any detectable loss of motor neurons. Instead, loss of Usp14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting, and poor arborization of the motor nerve terminals, whereas postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of Usp14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the axJ mice. These data define a critical role for Usp14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.
Assuntos
Junção Neuromuscular/enzimologia , Junção Neuromuscular/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/metabolismo , Sinapses/enzimologia , Ubiquitina Tiolesterase/fisiologia , Ubiquitina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Junção Neuromuscular/metabolismo , Ubiquitina Tiolesterase/deficiênciaRESUMO
Transient forebrain ischemia induces delayed, selective neuronal death in the CA1 region of the hippocampus. The underlying molecular mechanisms are as yet unclear, but it is known that activation of L-type Ca2+ channels specifically increases the expression of a group of genes required for neuronal survival. Accordingly, we examined temporal changes in L-type calcium-channel activity in CA1 and CA3 pyramidal neurons of rat hippocampus after transient forebrain ischemia by patch-clamp techniques. In vulnerable CA1 neurons, L-type Ca2+-channel activity was persistently downregulated after ischemic insult, whereas in invulnerable CA3 neurons, no change occurred. Downregulation of L-type calcium channels was partially caused by oxidation modulation in postischemic channels. Furthermore, L-type but neither N-type nor P/Q-type Ca2+-channel antagonists alone significantly inhibited the survival of cultured hippocampal neurons. In contrast, specific L-type calcium-channel agonist remarkably reduced neuronal cell death and restored the inhibited channels induced by nitric oxide donor. More importantly, L-type calcium-channel agonist applied after reoxygenation or reperfusion significantly decreased neuronal injury in in vitro oxygen-glucose deprivation ischemic model and in animals subjected to forebrain ischemia-reperfusion. Together, the present results suggest that ischemia-induced inhibition of L-type calcium currents may give rise to delayed death of neurons in the CA1 region, possibly via oxidation mechanisms. Our findings may lead to a new perspective on neuronal death after ischemic insult and suggest that a novel therapeutic approach, activation of L-type calcium channels, could be tested at late stages of reperfusion for stroke treatment.
Assuntos
Isquemia Encefálica/metabolismo , Canais de Cálcio Tipo L/metabolismo , Hipocampo/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Infarto Encefálico/metabolismo , Infarto Encefálico/fisiopatologia , Isquemia Encefálica/fisiopatologia , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Hipocampo/efeitos dos fármacos , Masculino , Degeneração Neural/etiologia , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/fisiopatologia , Fatores de TempoAssuntos
Ácido 3-Hidroxibutírico/metabolismo , Carcinoma Hepatocelular/enzimologia , Histona Desacetilases/metabolismo , Neoplasias Hepáticas/enzimologia , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/enzimologia , Proteínas Repressoras/metabolismo , Ácido 3-Hidroxibutírico/genética , Carcinoma Hepatocelular/genética , Histona Desacetilases/genética , Humanos , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Proteínas Repressoras/genéticaRESUMO
Buyang Huanwu Decoction (BYHWD), a traditional Chinese medicine, has been developed as a drug to be used for treatment of stroke for hundreds of years. However, the underlying mechanisms remain unknown. In the present study, the effects of BYHWD on delayed neuronal death of hippocampus after transient forebrain ischemia were examined in rats. Transient forebrain ischemia in a duration of 15 min was induced with the four-vessel occlusion method. BYHWD (per 6.65 g/kg) was given orally to rats twice each day for 7 days before ischemia. In BYHWD-pretreated rats, the neuronal injury in the hippocampal CA1 region was significantly less than that of controls. Oral administration of BYHWD also markedly attenuated the number of TUNEL-positive neurons and suppressed the expression of caspase-3p20, a product of catalytically active caspase-3, in the CA1 region. Our results suggest that an inhibition of caspase-3 and apoptosis by BYHWD may partially account for its neuroprotection against ischemic injury in the hippocampal CA1 region.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Ataque Isquêmico Transitório/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Prosencéfalo/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Medicamentos de Ervas Chinesas/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Ataque Isquêmico Transitório/patologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Prosencéfalo/irrigação sanguínea , Prosencéfalo/patologia , Células Piramidais/patologia , Ratos , Ratos WistarRESUMO
An improved method is described for fast and reliable isolation of neurons from hippocampus of adult rats by a combination of mechanical and enzymatic means. The procedure allows the isolation of neurons from 500-600-d-old rats (over 300 g), preserving the proximal dendritic structure without impairing the electrical characteristics of the cells. Morphologically distinct neurons can be recognized. Using cell-attached, inside-out and whole-cell configurations of patch clamp technique, it was shown that the enzymatically isolated neurons in hippocampus from rats weighing more than 300 g exhibited voltage-gated calcium, sodium and potassium currents, outwardly rectifying chloride channel and large conductance Ca(2+)-activated potassium channel currents. Approximately, 95% of healthy cells allowed the formation of giga-ohm seals.