Rab11a Controls Cell Shape via C9orf72 Protein: Possible Relationships to Frontotemporal Dementia/Amyotrophic Lateral Sclerosis (FTDALS) Type 1.

Abnormal nucleotide insertions of C9orf72, which forms a complex with Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8) protein and WD repeat-containing protein 41 (WDR41) protein, are associated with an autosomal-dominant neurodegenerative frontotemporal dementia and/or amyotrophic lateral sclerosis type 1 (FTDALS1). The differentially expressed in normal and neoplastic cells (DENN) domain-containing C9orf72 and its complex with SMCR8 and WDR41 function as a guanine-nucleotide exchange factor for Rab GTP/GDP-binding proteins (Rab GEF, also called Rab activator). Among Rab proteins serving as major effectors, there exists Rab11a. However, it remains to be established which Rab protein is related to promoting or sustaining neuronal morphogenesis or homeostasis. In this study, we describe that the knockdown of Rab11a decreases the expression levels of neuronal differentiation marker proteins, as well as the elongation of neurite-like processes, using N1E-115 cells, a well-utilized neuronal differentiation model. Similar results were obtained in primary cortical neurons. In contrast, the knockdown of Rab11b, a Rab11a homolog, did not significantly affect their cell morphological changes. It is of note that treatment with hesperetin, a citrus flavonoid (also known as Vitamin P), recovered the neuronal morphological phenotypes induced by Rab11a knockdown. Also, the knockdown of Rab11a or Rab11b led to a decrease in glial marker expression levels and in morphological changes in FBD-102b cells, which serve as the oligodendroglial differentiation model. Rab11a is specifically involved in the regulation of neuronal morphological differentiation. The knockdown effect mimicking the loss of function of C9orf72 is reversed by treatment with hesperetin. These findings may reveal a clue for identifying one of the potential molecular and cellular phenotypes underlying FTDALS1.

Pathogenic role of NAMPT in the perivascular regions after ischemic stroke in mice with type 2 diabetes mellitus.

Ischemic stroke in patients with abnormal glucose tolerance results in poor outcomes. Nicotinamide phosphoribosyltransferase (NAMPT), an adipocytokine, exerts neuroprotective effects. However, the pathophysiological role of NAMPT after ischemic stroke with diabetes and the relationship of NAMPT with cerebrovascular lesions are unclear. The purpose of this study was to clarify the pathophysiological role of NAMPT in cerebral ischemia with diabetes, using db/db mice as a type 2 diabetes animal model. The number of degenerating neurons increased after middle cerebral artery occlusion and reperfusion (MCAO/R) in db/db mice compared with the degenerating neurons in db/+ mice. Extracellular NAMPT (eNAMPT) levels, especially monomeric eNAMPT, increased significantly in db/db MCAO/R mice but not db/+ mice in isolated brain microvessels. The increased eNAMPT levels were associated with increased expression of inflammatory cytokine mRNA. Immunohistochemical analysis demonstrated that NAMPT colocalized with GFAP-positive cells after MCAO/R. In addition, both dimeric and monomeric eNAMPT levels increased in the conditioned medium of primary cortical astrocytes under high glucose conditions subsequent oxygen/glucose deprivation. Our findings are the first to demonstrate the ability of increased monomeric eNAMPT to induce inflammatory responses in brain microvessels, which may be located near astrocyte foot processes.

Role of senkyunolide I in the promotion of neural stem/progenitor cell proliferation via the Akt/ß-catenin pathway.

Following brain injury, neural stem cells (NSCs) can generate mature neurons and replace damaged cells. However, the capacity of endogenous NSCs to self-repair from injured brain is limited as most NSCs die before becoming mature neurons. Therefore, a boosting endogenous NSCs by pharmacological support offers the potential to repair the damaged brain. Recently, small molecules have hold considerable promise for neuron regeneration and repair as they can penetrate the blood-brain barrier easily. Senkyunolide I (SEI) is a bioactive constituent derived from traditional Chinese medicines Ligusticum chuanxiong Hort. and Angelica sinensis (Oliv.) Diels, and was found to able to prevent ischemic stroke. This study examined the effects of SEI on the proliferation and neuronal lineage differentiation of prepared neural stem/progenitor cells (NS/PCs). The NS/PC proliferation was determined by 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, and neurosphere formation assays. The NS/PC differentiation was also investigated by immunocytochemistry, and western blotting was employed to measure phosphorylated Akt (pAkt) and GSK-3ß (pGSK-3ß), and active-ß-catenin protein levels. We showed that the NS/PC proliferation was enhanced after SEI exposure. Elevated cell numbers were also observed in neurospheres, which were incubated with SEI for 3 days, whereas the NS/PC differentiation was decreased after SEI exposure for 5 days. Furthermore, SEI upregulated pAkt/Akt and active-ß-catenin levels and increased NS/PC proliferation after SEI treatment was reversed by phosphatidylinositol 3-kinase inhibitor LY294002. downregulated differentiated processes. Thus, SEI promoted the NS/PC proliferation and suppressed NS/PC differentiation into neurons and/or astrocytes, therefore SEI could be an interesting and promising candidate for stimulating NSCs.

Cytotoxic Effects of Darinaparsin, a Novel Organic Arsenical, against Human Leukemia Cells.

To explore the molecular mechanisms of action underlying the antileukemia activities of darinaparsin, an organic arsenical approved for the treatment of peripheral T-cell lymphoma in Japan, cytotoxicity of darinaparsin was evaluated in leukemia cell lines NB4, U-937, MOLT-4 and HL-60. Darinaparsin was a more potent cytotoxic than sodium arsenite, and induced apoptosis/necrosis in NB4 and HL-60 cells. In NB4 cells exhibiting the highest susceptibility to darinaparsin, apoptosis induction was accompanied by the activation of caspase-8/-9/-3, a substantial decrease in Bid expression, and was suppressed by Boc-D-FMK, a pancaspase inhibitor, suggesting that darinaparsin triggered a convergence of the extrinsic and intrinsic pathways of apoptosis via Bid truncation. A dramatic increase in the expression level of γH2AX, a DNA damage marker, occurred in parallel with G2/M arrest. Activation of p53 and the inhibition of cdc25C/cyclin B1/cdc2 were concomitantly observed in treated cells. Downregulation of c-Myc, along with inactivation of E2F1 associated with the activation of Rb, was observed, suggesting the critical roles of p53 and c-Myc in darinaparsin-mediated G2/M arrest. Trolox, an antioxidative reagent, suppressed the apoptosis induction but failed to correct G2/M arrest, suggesting that oxidative stress primarily contributed to apoptosis induction. Suppression of Notch1 signaling was also confirmed. Our findings provide novel insights into molecular mechanisms underlying the cytotoxicity of darinaparsin and strong rationale for its new clinical application for patients with different types of cancer.

Possible Involvement of DNA Methylation and Protective Effect of Zebularine on Neuronal Cell Death after Glutamate Excitotoxity.

Neuronal cell death after cerebral ischemia consists various steps including glutamate excitotoxity. Excessive Ca2+ influx through the N-methyl-D-aspartate (NMDA) receptor, which is one of the ionotropic glutamate receptors, plays a central role in neuronal cell death after cerebral ischemia. We previously reported that DNA methylation is transiently increased in neurons during ischemic injury and that this aberrant DNA methylation is accompanied by neuronal cell death. Therefore, we performed the present experiments on glutamate excitotoxicity to gain further insight into DNA methylation involvement in the neuronal cell death. We demonstrated that knockdown of DNA methyltransferase (DNMT)1, DNMT3a, or DNMT3b gene in Neuro2a cells was performed to examine which DNMTs were more important for neuronal cell death after glutamate excitotoxicity. Although we confirmed a decrease in the levels of the target DNMT protein after small interfering RNA (siRNA) transfection, the Neuro2a cells were not protected from injury by transfection with siRNA for each DNMT. We next revealed that the pharmacological inhibitor of DNMTs protected against glutamate excitotoxicity in Neuro2a cells and also in primary cultured cortical neurons. This protective effect was associated with a decrease in the number of 5-methylcytosine (5 mC)-positive cells under glutamate excitotoxicity. In addition, the increased level of cleaved caspase-3 was also reduced by a DNMT inhibitor. Our results suggest the possibility that at least 2 or all DNMTs functionally would cooperate to activate DNA methylation after glutamate excitotoxicity and that inhibition of DNA methylation in neurons after cerebral ischemia might become a strategy to reduce the neuronal injury.

Effect of Progranulin on Proliferation and Differentiation of Neural Stem/Progenitor Cells after Oxygen/Glucose Deprivation.

We previously demonstrated that sivelestat, a selective neutrophil elastase inhibitor, attenuates the cleavage of progranulin (PGRN) and ischemia-induced cell injury in the brain. To obtain further insight into the role of PGRN, in the present study we evaluated the direct effects of sivelestat and recombinant PGRN (rPGRN) on the proliferation and differentiation of neural stem cells in cultures of neural stem/progenitor cells (NS/PC) under the ischemic condition in vitro. We demonstrated that oxygen/glucose deprivation (OGD)-induced cell proliferation of NS/PC was increased by rPGRN treatment. In addition, this increase was accompanied by increased phosphorylation of Akt and GSK-3ß (Ser9) after OGD. But none of these responses occurred by treatment with sivelestat. Therefore, activation of the Akt/GSK-3ß pathway could well be involved in this proliferative effect of rPGRN. Although OGD and reoxygenation-induced changes in the differentiation of NS/PC into neurons or astrocytes was not affected by treatment with rPGRN or sivelestat, it is noteworthy that rPGRN enhanced neurite outgrowth of ß3-tubulin-positive neurons that had differentiated from the NS/PC. These findings suggest that enhancement of proliferation of endogenous NS/PC and neurite outgrowth of differentiated neurons from NS/PC by PGRN could be useful for a new therapeutic approach for cerebral ischemia.

Corrigendum: Cytotoxic Effects of Arsenite in Combination With Gamabufotalin Against Human Glioblastoma Cell Lines.

[This corrects the article DOI: 10.3389/fonc.2021.628914.].

Apolipoprotein E-Containing Lipoproteins and LRP1 Protect From NMDA-Induced Excitotoxicity Associated With Reducing α2-Macroglobulin in Müller Glia.

Purpose: Optic nerve damage leads to impairment of visual functions. We previously demonstrated that apolipoprotein E-containing lipoproteins (E-LPs) protect retinal ganglion cells (RGCs) from degeneration in a glaucoma model of glutamate/aspartate transporter-deficient mice. This study aimed to determine whether E-LPs protect RGCs from N-methyl-D-aspartate (NMDA)-induced excitotoxicity, and to investigate the details of an indirect neuroprotective mechanism of E-LPs by reducing α2-macroglobulin, which interferes with the neuroprotective effect of E-LPs, in Müller glia. Methods: Excitotoxicity was caused by intravitreal injection of NMDA, and then retinae were subjected to immunoblotting or quantitative reverse transcription-PCR. Primary cultures of mouse mixed retinal cells and mouse Müller glia were used for evaluating the effects of E-LPs on the expression of α2-macroglobulin. Results: Intravitreal injection of E-LPs protected the optic nerve from degeneration and attenuated the increase in α2-macroglobulin in aqueous humor and retina of rats. E-LPs directly decreased the expression and secretion of α2-macroglobulin in primary cultures of Müller glia; this decrease in production of α2-macroglobulin was blocked by knockdown of the low-density lipoprotein receptor-related protein 1 (LRP1) with small interfering RNA. E-LPs promoted the phosphorylation of STAT3, whereas Stattic, an inhibitor of STAT3, restored the expression of α2-macroglobulin decreased by E-LPs. Conclusions: In addition to our previous findings of the protection of RGCs by E-LPs, the new observations in Müller glia indicate that a reduction of the intraocular α2-macroglobulin, regulated by the E-LP-LRP1-STAT3 pathway, might be an additional protective mechanism against excitotoxicity in the retina.

Cytotoxic Effects of Hellebrigenin and Arenobufagin Against Human Breast Cancer Cells.

Development of new therapeutic strategies for breast cancer is urgently needed due to the sustained emergence of drug resistance, tumor recurrence and metastasis. To gain a novel insight into therapeutic approaches to fight against breast cancer, the cytocidal effects of hellebrigenin (Helle) and arenobufagin (Areno) were investigated in human estrogen receptor (ER)-positive breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231. Helle exhibited more potent cytotoxicity than Areno in both cancer cells, and MCF-7 cells were more susceptible to both drugs in comparison with MDA-MB-231 cells. Apoptotic-like morphological characteristics, along with the downregulation of the expression level of Bcl-2 and Bcl-xL and the upregulation of the expression level of Bad, were observed in Helle-treated MCF-7 cells. Helle also caused the activation of caspase-8, caspase-9, along with the cleavage of poly(ADP-ribose) polymerase in MCF-7 cells. Helle-mediated necrosis-like phenotype, as evidenced by the increased propidium iodide (PI)-positive cells was further observed. G2/M cell cycle arrest was also induced by Helle in the cells. Upregulation of the expression level of p21 and downregulation of the expression level of cyclin D1, cyclin E1, cdc25C and survivin were observed in MCF-7 cells treated with Helle and occurred in parallel with G2/M arrest. Autophagy was triggered in MCF-7 cells and the addition of wortmannin or 3-MA, two well-known autophagy inhibitors, slightly but significantly rescued the cells. Furthermore, similar alterations of some key molecules associated with the aforementioned biological phenomena were observed in MDA-MB-231 cells. Intriguingly, the numbers of PI-positive cells in Helle-treated MCF-7 cells were significantly reduced by wortmannin and 3-MA, respectively. In addition, Helle-triggered G2/M arrest was significantly corrected by wortmannin, suggesting autophagy induction contributed to Helle-induced cytotoxicity of breast cancer cells by modulating necrosis and cell cycle arrest. Collectively, our results suggested potential usefulness of both Helle and Areno in developing therapeutic strategies to treat patients with different types of breast cancer, especially ER-positive breast cancer.

Ternary Complexes of pDNA, Neuron-Binding Peptide, and PEGylated Polyethyleneimine for Brain Delivery with Nano-Bubbles and Ultrasound.

In brain-targeted delivery, the transport of drugs or genes across the blood-brain barrier (BBB) is a major obstacle. Recent reports found that focused ultrasound (FUS) with microbubbles enables transient BBB opening and improvement of drug or gene delivery. We previously developed nano-sized bubbles (NBs), which were prepared based on polyethylene glycol (PEG)-modified liposomes containing echo-contrast gas, and showed that our NBs with FUS could also induce BBB opening. The aim of this study was to enhance the efficiency of delivery of pDNA into neuronal cells following transportation across the BBB using neuron-binding peptides. This study used the RVG-R9 peptide, which is a chimeric peptide synthesized by peptides derived from rabies virus glycoprotein and nonamer arginine residues. The RVG peptide is known to interact specifically with the nicotinic acetylcholine receptor in neuronal cells. To enhance the stability of the RVG-R9/pDNA complex in vivo, PEGylated polyethyleneimine (PEG-PEI) was also used. The ternary complexes composed of RVG-R9, PEG-PEI, and pDNA could interact with mouse neuroblastoma cells and deliver pDNA into the cells. Furthermore, for the in vivo experiments using NBs and FUS, gene expression was observed in the FUS-exposed brain hemispheres. These results suggest that this systemic gene delivery system could be useful for gene delivery across the BBB.

Oral Limonite Supplement Ameliorates Glucose Intolerance in Diabetic and Obese Mice.

INTRODUCTION: Diabetes mellitus is a serious threat to public health worldwide. It causes a substantial economic burden, mental and physical disabilities, poor quality of life, and high mortality. Limonite is formed when iron-rich materials from the underground emerge and oxidized on the ground surface. It is currently used to purify contaminated water, absorption of irritant gases, and improve livestock breeding. Limonite can change the composition of environmental microbial communities. In the present study, we evaluated whether limonite can ameliorate glucose metabolism abnormalities by remodeling the gut microbiome. METHODS: The investigation was performed using mouse models of streptozotocin-induced diabetes mellitus and high-calorie diet-induced metabolic syndrome. RESULTS: Oral limonite supplement was associated with significant body weight recovery, reduced glycemia with improved insulin secretion, increased number of regulatory T cells, and abundant beneficial gut microbial populations in mice with diabetes mellitus compared to control. Similarly, mice with obesity fed with limonite supplements had significantly reduced body weight, insulin resistance, steatohepatitis, and systemic inflammatory response with significant gut microbiome remodeling. CONCLUSION: This study demonstrates that limonite supplement ameliorates abnormal glucose metabolism in diabetes mellitus and obesity. Gut microbiome remodeling, inhibition of inflammatory cytokines, and the host immune response regulation may explain the limonite's beneficial activity under pathological conditions in vivo.

Neuroprotective effects of Senkyunolide I against glutamate-induced cells death by attenuating JNK/caspase-3 activation and apoptosis.

Glutamate-induced neurotoxicity is one of the most important pathogenic mechanisms in neurological diseases and is widely used as an in vitro model for ischemic stroke. Senkyunolide I (SEI), an active constituent derived from traditional Chinese medicine Ligusticum chuanxiong Hort. and Angelica sinensis (Oliv.) Diels, has been shown to have beneficial effects against focal cerebral ischemia-reperfusion in rats. However, the mechanisms underlying SEI-mediated neuroprotection remain not well understood. Thus, we explored the influence of SEI in glutamate-mediated injury to mouse neuroblastoma (Neuro2a) cells and determined the mechanisms involved. Neuro2a cells were treated with SEI under exposure to glutamate for 24 h. Cell viability was assessed by using WST-1 reagents, and apoptosis was evaluated using Annexin V-FITC and a PI double staining kit. The protein expression levels of p-AKT, AKT, p-GSK3ß, GSK3ß, p-p38, p38, p-ERK, ERK, p-JNK, JNK, Bcl-2, Bax, Bcl-xl, p-Bad, Bad, p53, and cleaved caspase-3 were determined by Western blot analysis. Glutamate significantly decreased cell viability and elevated the level of apoptosis. Treatment with SEI reversed those effects. Furthermore, the expression of p-JNK/JNK and cleaved caspase-3 were also reduced after treatment with SEI. Our findings demonstrate that SEI protected Neuro2a cells against glutamate toxicity by regulating JNK/caspase-3 pathway and apoptosis. Thus, SEI maybe a promising candidate for neuroprotection.

Possible involvement of progranulin in the protective effect of elastase inhibitor on cerebral ischemic injuries of neuronal and glial cells.

In a previous study, we demonstrated that neutrophil elastase is activated in the brain parenchyma after cerebral ischemia, which enzyme cleaves progranulin (PGRN), an anti-inflammatory factor. In that study, we also found that sivelestat, a selective neutrophil elastase inhibitor, attenuates ischemia-induced inflammatory responses. However, it was not clear whether this anti-inflammatory effect was due to the direct effect of sivelestat. In this study, we evaluated the effects of sivelestat or recombinant PGRN (rPGRN) on cell injuries in cultured neurons, astrocytes, and microglia under oxygen/glucose deprivation (OGD) conditions. We demonstrated that OGD-induced neuronal cell injury, astrocyte activation, and increased proinflammatory cytokines caused by microglial activation, were suppressed by rPGRN treatment, whereas sivelestat had no effect on any of these events. These results indicate that the anti-inflammatory responses after in vivo cerebral ischemia were not due to the direct action of sivelestat but due to the suppression of PGRN cleavage by inhibition of elastase activity. It was also suggested that the pleiotropic effect of rPGRN could be attributed to the differentiation of M1 microglia into anti-inflammatory type M2 microglia. Therefore, the inhibition of PGRN cleavage by sivelestat could contribute to the establishment of a new therapeutic approach for cerebral ischemia.

Cytotoxic Effects of Arsenite in Combination With Gamabufotalin Against Human Glioblastoma Cell Lines.

Glioblastoma is a fatal primary malignant brain tumor, and the 5-year survival rate of treated glioblastoma patients still remains <5%. Considering the sustained development of metastasis, tumor recurrence, and drug resistance, there is an urgent need for the novel therapeutic approaches to combat glioblastoma. Trivalent arsenic derivative (arsenite, AsIII) with remarkable clinical efficacy in leukemia has been shown to exert cytocidal effect against glioblastoma cells. Gamabufotalin, an active bufadienolide compound, also shows selective cytocidal effect against glioblastoma cells, and has been suggested to serve as a promising adjuvant therapeutic agent to potentiate therapeutic effect of conventional anticancer drugs. In order to gain novel insight into therapeutic approaches against glioblastoma, the cytotoxicity of AsIII and gamabufotalin was explored in the human glioblastoma cell lines U-87 and U-251. In comparison with U-251 cells, U-87 cells were highly susceptible to the two drugs, alone or in combination. More importantly, clinically achieved concentrations of AsIII combined with gamabufotalin exhibited synergistic cytotoxicity against U-87 cells, whereas showed much less cytotoxicity to human normal peripheral blood mononuclear cells. G2/M cell cycle arrest was induced by each single drug, and further augmented by their combination in U-87 cells. Downregulation of the expression levels of cdc25C, Cyclin B1, cdc2, and survivin was observed in U-87 cells treated with the combined regimen and occurred in parallel with G2/M arrest. Concomitantly, lactate dehydrogenase leakage was also observed. Intriguingly, SB203580, a specific inhibitor of p38 MAPK, intensified the cytotoxicity of the combined regimen in U-87 cells, whereas wortmannin, a potent autophagy inhibitor, significantly rescued the cells. Collectively, G2/M arrest, necrosis and autophagy appeared to cooperatively contribute to the synergistic cytotoxicity of AsIII and gamabufotalin. Given that p38 MAPK serves an essential role in promoting glioblastoma cell survival, developing a possible strategy composed of AsIII, gamabufotalin, and a p38 MAPK inhibitor may provide novel insight into approaches designed to combat glioblastoma.

Effects of laminin-111 peptide coatings on rat neural stem/progenitor cell culture.

Neurons require adhesive scaffolds for their growth and differentiation. Laminins are a major cell adhesive component of basement membranes and have various biological activities in the peripheral and central nervous systems. Here, we evaluated the biological activities of 5 peptides derived from laminin-111 as a scaffold for mouse neuroblastoma Neuro2a cells and rat neural stem/progenitor cells (NPCs). The 5 peptides showed Neuro2a cell attachment activity similar to that of poly-d-lysine. However, when NPCs were cultured on the peptides, 2 syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139-150), demonstrated significantly higher cell attachment and neurite extension activities than other peptides including integrin-binding ones. Long-term cell culture experiments showed that both AG73 and C16 supported the growth of neurons and astrocytes that had differentiated from NPCs. Furthermore, C16 markedly promoted the expression of neuronal markers such as synaptosomal-associated protein-25 and syntaxin 1A. These results indicate that AG73 and C16 are useful for NPC cultures and that C16 can be applied to specialized research on synapses in differentiated neurons. These peptides have the potential for use as valuable biomaterials for NPC research.

Investigating the Relationship Between Neuronal Cell Death and Early DNA Methylation After Ischemic Injury.

Cerebral ischemia induces neuronal cell death and causes various kinds of brain dysfunction. Therefore, prevention of neuronal cell death is most essential for protection of the brain. On the other hand, it has been reported that epigenetics including DNA methylation plays a pivotal role in pathogenesis of some diseases such as cancer. Accumulating evidences indicate that aberrant DNA methylation is related to cell death. However, DNA methylation after cerebral ischemia has not been fully understood yet. The aim of this present study was to investigate the relationships between DNA methylation and neuronal cell death after cerebral ischemia. We examined DNA methylation under the ischemic condition by using transient middle cerebral artery occlusion and reperfusion (MCAO/R) model rats and N-methyl-D-aspartate (NMDA)-treated cortical neurons in primary culture. In this study, we demonstrated that DNA methylation increased in these neurons 24 h after MCAO/R and that DNA methylation, possibly through activation of DNA methyltransferases (DNMT) 3a, increased in such neurons immediately after NMDA treatment. Furthermore, NMDA-treated neurons were protected by treatment with a DNMT inhibitor that were accompanied by inhibition of DNA methylation. Our results showed that DNA methylation would be an initiation factor of neuronal cell death and that inhibition of such methylation could become an effective therapeutic strategy for stroke.

JNK and Autophagy Independently Contributed to Cytotoxicity of Arsenite combined With Tetrandrine via Modulating Cell Cycle Progression in Human Breast Cancer Cells.

Novel therapeutic strategies for breast cancer are urgently needed due to the sustained development of drug resistance and tumor recurrence. Trivalent arsenic derivative (arsenite, AsIII) has been reported to induce cytotoxicity in breast cancer cells. We recently demonstrated that AsIII plus tetrandrine (Tetra), a Chinese plant-derived alkaloid, exerted potent antitumor activity against human breast cancer cells, however, the underlying mechanisms for their action have not been well defined. In order to provide fundamental insights for understanding the action of AsIII plus Tetra, the effects of the combined regimen on two breast cancer cell lines T47D and MDA-MB-231 were evaluated. Compared to T47D cells, MDA-MB-231 cells were much more susceptible to the synergistic cytotoxic effects of AsIII and Tetra. Besides the induction of apoptotic/necrotic cell death, S-phase arrest and autophagic cell death were also observed in MDA-MB-231 cells. Exposure of MDA-MB-231 cells to AsIII and Tetra caused the activation of MAPKs. Cytotoxicity of the combined regimen in MDA-MB-231 cell was significantly abrogated by SP600125, a potent c-Jun N-terminal kinase (JNK) inhibitor. However, similar abrogation was not caused by p38 and ERK inhibitors. The addition of either autophagy inhibitors (3-methyladenine or wortmannin) or SP600125 corrected the combined regimen-triggered S-phase arrest, whereas had little effect on the apoptosis/necrosis induction in the cells. Surprisingly, SP600125NC, a negative control for SP600125, significantly strengthened S-phase arrest and the cytotoxicity induced by the combined regimen. The addition of SP600125 did not alter autophagy induction. In conclusion, the cytotoxicity of AsIII combined with Tetra was attributed to the induction of S-phase arrest, apoptotic/necrotic and autophagic cell death. The enhanced cytotoxicity of the two drugs by SP600125NC might be explained by its capability to strengthen S-phase arrest. Our results suggested that JNK and autophagy independently contributed to the cytotoxicity via modulating cell cycle progression. The study further provides fundamental insights for the development of AsIII in combination with Tetra for patients with different types of breast cancer.

[Corrigendum] Antitumor effects of arsenic disulfide on the viability, migratory ability, apoptosis and autophagy of breast cancer cells.

Following the publication of the above paper, an interested reader drew to our attention the fact that the same ß­actin bands had been included in the western blots featured in Figs. 7 and 10. Upon consulting the authors in relation to this matter, they were able to offer a legitimate explanation for this apparent duplication of the bands; essentially, in the western blotting experiments, different antibodies were used in one membrane to present different target proteins by stripping and reprobing the membrane. That the authors had performed this additional step in their protocol was inadvertently omitted from the 'Western blot analyses' subsection of the Materials and methods section. The authors are grateful to the interested reader for drawing this matter to their attention, and apologize for any inconvenience caused to the readership of the Journal. [the original article was published in Oncology Reports 41: 27-42, 2019; DOI: 10.3892/or.2018.6780].

Antineuropathic pain actions of Wu-tou decoction resulted from the increase of neurotrophic factor and decrease of CCR5 expression in primary rat glial cells.

Wu-tou decoction (WTD), a classic Traditional Chinese medicine formula, has been extensively used in the treatment of neuropathic pain (NP) such as chronic inflammatory pain, trigeminal neuralgia, and cancer-induced pain. Our previous studies have shown that the severity of mechanical allodynia and thermo hypersensitivity in NP rats are reduced by WTD, of which analgesic candidates are paeoniflorin (Pae) and liquiritin (Liq). The aim of this study was to clarify the molecular mechanisms of WTD, Pae and Liq against NP based on the primary rat glial cells in vitro. The gene expression levels of neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and Artemin and C-C chemokine receptor type 5 (CCR5) were augmented by inflammatory cytokines, while chemokines increased only CCR5 gene expression. The constitutive and cytokine-augmented neurotrophic factor gene expression was enhanced by WTD, Pae, and Liq through PI3K- and PKA-dependent pathways in rat glial cells, leading to the increase of NGF and BDNF production. Furthermore, the CCR5 gene expression under basal and chemokine-treated conditions was suppressed by these reagents, in which signal pathway(s) was independent on the activation of PI3K and PKA. Moreover, there was no cytotoxicity in the WTD, Pae, and Liq treatments in glial cells. Thus, these results provide a novel evidence that WTD may exert the anti-NP actions by predominantly increasing the production of neurotrophic factors through PI3K- and PKA-signaling pathways in rat glial cells. Furthermore, Pae and Liq may play as analgesic candidates in WTD-mediated NP management.

Ligusticum chuanxiong exerts neuroprotection by promoting adult neurogenesis and inhibiting inflammation in the hippocampus of ME cerebral ischemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemia, also known as stroke, can stimulate the proliferation and migration of endogenous neural stem cells (NSCS) in subventricular zone of the lateral ventricle and subgranularzone of the dentate gyrus in the adult hippocampus as a defense response to damage. However, the proliferation of endogenous NSCS is insufficient for central nervous system repair. Neurogenesis and anti-neuroinflammation are two important aspects for neuroprotection. Rhizome Ligusticum chuanxiong (LC), the dried rhizomes of Ligusticum striatum DC., has been widely used to treat stroke for over hundreds of years in Traditional Chinese Medicine. PURPOSE: of the study: Previous reports on pharmacological mechanism of LC mainly focus on the cerebral blood flow and thrombolysis. We aim to explore whether LC provides neuroprotective effect by increasing neurogenesis and inhibiting the IL-1ß, TNF-α and expressions of glial fibrillary acidic protein. MATERIALS AND METHODS: LC extract was delivered to microsphere-embolized (ME) cerebral ischemia Wister rats to examine its neuroprotection. Body weight, neurological scores, hematoxylin-eosin staining (HE), TUNEL assay were conducted for neurological damage. Neurogenesis was evaluated by assessing the expression of Doublecortin (DCX) and neurogenic differentiation1 (NeuroD1) through immunofluorescence staining. Western blot performed to measure the protein levels of growth associated protein-43(GAP-43), glial fibrillary acidic protein (GFAP). IL-1ß and TNF-α was detected by Elisa. RESULTS: LC alleviated pathomorphological change and apoptosis of neurons in the hippocampus caused by ME surgery. Furthermore, LC significantly increased the DCX in the DG of adult rat hippocampus at 14 days after surgery. A significant upregulation of GAP-43 compared to the ME after LC was administered. Besides, LC decreased pro-inflammatory cytokine (IL-1ß, TNF-α) and protein level of GFAP. CONCLUSION: The finding suggested that LC had the ability to protect neurons by promoting the endogenous proliferation of neuroblast and production of neural differentiation factor in rats after ischemia injury. Meanwhile, LC can anti-neuroinflammation, which is important for the treatment of neuron injury. Accordingly, LC perhaps a promising medicine for neuron damage therapy after cerebral ischemia.