Sulforaphane-N-Acetyl-Cysteine Induces Autophagy Through Activation of ERK1/2 in U87MG and U373MG Cells.

BACKGROUND/AIMS: Sulforaphane-N-acetyl-cysteine (SFN-NAC) is a sulforaphane (SFN) metabolite with a longer half-life and better blood-brain barrier permeability than those of SFN. Previous studies have found that SFN-NAC can act via ERK to destroy microtubules and inhibit cell growth in lung cancer cells. However, the underlying mechanisms are unclear, and it is unknown whether SFN-NAC can inhibit the growth of glioma. Here, we have demonstrated for the first time that SFN-NAC activates autophagy-mediated downregulation of α-tubulin expression via the ERK pathway. METHODS: U87MG and U373MG cells, two widely used glioma cell lines, were utilized in this study. Apoptosis assay, western blot analysis, co-immunoprecipitation, immunostaining, and electron microscopy were used to analyze the effect of SFN-NAC on α-tubulin and its interaction with microtube-associated protein 1 light-chain 3 (LC3). RESULTS: SFN-NAC induced cell-cycle arrest in the G2/M phase and dose-dependently induced intracellular ERK activation, autophagy, and α-tubulin downregulation. These SFN-NAC-induced effects were reversed by inhibiting the ERK pathway with its inhibitor PD98059. U87MG and U373MG cells were transfected with LC3 small interfering RNA, and the subsequent inhibition of autophagy reversed the downregulation of α-tubulin by SFN-NAC. Furthermore, co-immunoprecipitation experiments and confocal microscopy confirmed that SFN-NAC promotes the binding of LC3 with α-tubulin in the cytoplasm. Cell viability experiments demonstrate that SFN-NAC inhibits the growth of U87MG and U373MG cell colonies. CONCLUSION: These findings suggest that SFN-NAC is a novel potential anti-glioma agent.

The Differentially Expressed Circular RNAs in the Substantia Nigra and Corpus Striatum of Nrf2-Knockout Mice.

BACKGROUND/AIMS: The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a protective role in both acute neuronal damage and chronic neurodegeneration-related oxidative stress. Circular RNAs (circRNAs) are involved with various diseases in the central nervous system (CNS). This study aimed to identify the key circRNAs involved in Nrf2-neuroprotection against oxidative stress. METHODS: The differentially expressed circRNAs (DEcircRNAs) in the substantia nigra and corpus striatum between Nrf2 (-/-) and Nrf2 (+/+) mice were identified by microarray analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) was then used to validate the expression of selected DEcircRNAs in the substantia nigra and corpus striatum between Nrf2 (-/-) and Nrf2 (+/+) mice. Based on our previous microarray analysis of the differentially expressed mRNAs (DEmRNAs) in the substantia nigra and corpus striatum between Nrf2 (-/-) and Nrf2 (+/+) mice, the DEcircRNA-miRNA-DEmRNA interaction network was constructed. Functional annotation of DEmRNAs that shared the same binding miRNAs with DEcircRNAs was performed using gene ontology (GO) and pathway analyses. RESULTS: A total of 65 and 150 significant DEcircRNAs were obtained in the substantia nigra and corpus striatum of Nrf2 (-/-) mice, respectively, and seventeen shared DEcircRNAs were found in both these two tissues. The qRT-PCR results were generally consistent with the microarray results. The DEcircRNA-miRNA-DEmRNA interaction network and pathway analysis indicated that mmu_circRNA_34132, mmu_circRNA_017077 and mmu-circRNA-015216 might be involved with Nrf2-mediated neuroprotection against oxidative stress. Mmu_circRNA_015216 and mmu_circRNA_017077 might play roles in the Nrf2-related transcriptional misregulation and Nrf2-mediated processes of rheumatoid arthritis, respectively. In addition to these two processes, mmu_circRNA_34132 may be a potential regulator of Nrf2-mediated protection for diabetes mellitus and Nrf2-mediated defence against ROS in hearts. CONCLUSION: In conclusion, our study identified the key DEcircRNAs in the substantia nigra and corpus striatum of Nrf2 (-/-) mice, which might provide new clues for further exploring the mechanism of Nrf2-mediated neuroprotection against oxidative stress and other Nrf2-mediated processes.

Finasteride inhibited brain dopaminergic system and open-field behaviors in adolescent male rats.

AIMS: Finasteride inhibits the conversion of testosterone to dihydrotestosterone. Because androgen regulates dopaminergic system in the brain, it could be hypothesized that finasteride may inhibit dopaminergic system. The present study therefore investigates the effects of finasteride in adolescent and early developmental rats on dopaminergic system, including contents of dopamine and its metabolites (dihydroxy phenyl acetic acid and homovanillic acid) and tyrosine hydroxylase expressions both at gene and protein levels. Meanwhile, open-field behaviors of the rats are examined because of the regulatory effect of dopaminergic system on the behaviors. METHODS: Open-field behaviors were evaluated by exploratory and motor behaviors. Dopamine and its metabolites were assayed by liquid chromatography-mass spectrometry. Tyrosine hydroxylase mRNA and protein expressions were determined by real-time qRT-PCR and western blot, respectively. RESULTS: It was found that in adolescent male rats, administration of finasteride at doses of 25 and 50 mg/kg for 14 days dose dependently inhibited open-field behaviors, reduced contents of dopamine and its metabolites in frontal cortex, hippocampus, caudate putamen, nucleus accumbens, and down-regulated tyrosine hydroxylase mRNA and protein expressions in substantia nigra and ventral tegmental area. However, there was no significant change of these parameters in early developmental rats after finasteride treatment. CONCLUSION: These results suggest that finasteride inhibits dopaminergic system and open-field behaviors in adolescent male rats by inhibiting the conversion of testosterone to dihydrotestosterone, and imply finasteride as a potential therapeutic option for neuropsychiatric disorders associated with hyperactivities of dopaminergic system and androgen.

[Effect of testosterone propionate on the distribution pattern of calcitonin gene-related peptide in different motoneuron pools].

OBJECTIVE: To study the effect of testosterone propionate (TP) on the distribution pattern of calcitonin gene-related peptide (CGRP) in two types of motoneuron (Mn) pools in rats. METHOD: The double labeling of cholera toxin B subunit coupled with colloidal gold (CB-Au) retrograde identification combining with immunocytochemistry was mainly used to reveal the distribution pattern of CGRP-like immunoreactivity (CGRP-LI) and its changes in the motoneuron pools labeled by CB-Au. RESULT: TP injected intramuscularly 28 days later significantly decreased CGRP expression in Mn pool innervating extensor digitorum longus (EDL, fast-twitch), comparing with corresponding control and castration group respectively (P < 0.001), while no significant effect on Mn pools innervating soleus (SOL, slow-twitch, P > 0.05) was observed. CONCLUSION: EDL-Mn pool is more sensitive to testosterone propionate than SOL-Mn pool in regulating CGRP expression.

Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIß pathway.

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIß (TopoIIß), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIß expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80µM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin ßIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIß, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIß gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIß expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIß signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIß in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system.

[Apoptosis of human oral epidermoid carcinoma KB cells and multidrug resistant KBv200 cells induced by matrine].

OBJECTIVE: To investigate the induction of apoptosis on human oral epidermoid carcinoma KB cells and multidrug resistant KBv200 cells by Matrine. METHODS: MTT assay was used to investigate the inhibition ability of Matrine on the cells in vitro. Transmission electron microscope was used to observe the ultrastructure feature of cells. after treated by Matrine. Acridine orange (AO)/Ethidium bromide (EB) fluorescent staining and flow cytometry were used to observe apoptosis induced by Matrine. Flow cytometry was applied to study the effects of the drug on cell cycles of the cells. RESULTS: When 0.50, 1.00, 1.50, 2.00 mg/ml of Matrine was used, the vital rates of KB and KBv200 cells were decreased according to Matrine's concentration. The IC50 concentrations of Matrine on KB and KBv200 cells were 1.35 mg/ml and 1.43 mg/ml individually. The results of AO/EB fluorescent staining and flow cytometry showed that Matrine could induce apoptosis of two kinds of cells. While observed by transmission electron microscope, there were more contraction of cells, condensation of nuclei, bubble of cytoplasm in both kinds of cells after treated by Matrine. Matrine could stop the growth of KB and KBv200 cells at S period and restrain mitosis of cells. CONCLUSION: Matrine can inhibit the growth of KB and KBv200 cells by inducing apoptosis. The apoptosis effect is dose-dependent and it has certain relation to the blocking of S period cells.