It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.

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BackgroundThe efficacy and safety of multikinase inhibitor anlotinib have been confirmed in the treatment of advanced non-small cell lung cancer (NSCLC). However, the direct functional mechanisms of tumor lethality mediated by anlotinib were not fully elucidated, and the underlying mechanisms related to resistance remain largely elusive.MethodsCell viability, colony formation, apoptosis and tumor growth assays were performed to examine the effect of anlotinib on lung cancer cells in vitro and in vivo. The punctate patterns of LC3-I/II were detected by confocal microscopy. HUVECs motility was detected using Transwell and scratch wound-healing assay. To visualize the microvessels, tubular formation assay was performed. The expression of LC3-I/II and beclin-1 and the changes of JAK2/STAT3/VEGFA pathway were detected by western blotting. The VEGFA levels in tumor supernatant were measured by ELISA.ResultsAnlotinib treatment decreased cell viability and induced apoptosis in Calu-1 and A549 cells. Moreover, anlotinib induced human lung cancer cell autophagy in a dose- and time-dependent manner. Blocking autophagy enhanced the cytotoxicity and anti-angiogenic ability of anlotinib as evidenced by HUVECs migration, invasion, and tubular formation assay. Co-administration of anlotinib and chloroquine (CQ) further reduced VEGFA level in the tumor supernatant, compared with that of anlotinib or CQ treatment alone. When autophagy was induced by rapamycin, the JAK2/STAT3 pathway was activated and VEGFA was elevated, which was attenuated after deactivating STAT3 by S3I-201. Further in vivo studies showed that anlotinib inhibited tumor growth, induced autophagy and suppressed JAK2/STAT3/VEGFA pathway, and CQ enhanced this effect.ConclusionAnlotinib induced apoptosis and protective autophagy in human lung cancer cell lines. Autophagy inhibition further enhanced the cytotoxic effects of anlotinib, and potentiated the anti-angiogenic property of anlotinib through JAK2/STAT3/VEGFA signaling.

Prequenching and selective activation of photosensitizer (PS) are highly desired in photodynamic therapy (PDT) to avoid off-target effect due to nonspecific activation and poor targeting selectivity of PS. In this study, nanographene materials as a unique π-conjugated planar system for electronic transfer were employed as the robust platform for temporarily quenching of PS. Photosensitizer chlorin e6 (Ce6) was integrated onto planar structure of graphene quantum dot (GQD) or graphene oxide (GO) via a reduction cleavable disulfide linker. The formed hybrid nanosystem displayed considerable fluorescence quenching and slight phototoxicity, even under the condition of light irradiation, while the photoactivity of PS could be selectively recovered in the presence of the reducing agent. Compared with graphene oxide system with larger size (around 200 nm), GQD nanosystem exhibited significantly improved tumor accumulation via enhanced permeation and retention effect (EPR effect). The in vivo study demonstrated extremely effective suppression of tumor growth for the group treated with the GQD nanosystem with cleavable linker, revealing the promising application of the presented novel strategy.

Inflammatory responses play a critical role in ischemic brain injury. induces the expression of inflammatory cytokines, and acetylbritannilactone (ABL) exerts potent antiinflammatory actions by inhibiting expression of inflammation-related genes. However, the functions of miR-155 and the actual relationship between ABL and miR-155 in ischemia-induced cerebral inflammation remain unclear. In this study, cerebral ischemia of wild-type (WT) and miR-155 -/-mice was induced by permanent middle cerebral artery occlusion (MCAO). pAd-miR-155 was injected into the lateral cerebral ventricle 24 h before MCAO to induce miR-155 overexpression. MCAO mice and oxygen-glucose deprivation (OGD)-treated BV2 cells were used to examine the effects of ABL and miR-155 overexpression or deletion on the expression of proinflammatory cytokines. We demonstrated that ABL treatment significantly reduced neurological deficits and cerebral infarct volume by inhibiting tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression in ischemic cerebral tissue and OGD-treated BV2 cells. Mechanistic studies suggested that the observed decrease in TNF-α and IL-1β expression was attributable to the ABL-induced suppression of the expression of nuclear factorkappa B (NF-κB) and Toll-like receptor 4 (TLR4). We further found that miR-155 promoted TNF-α and IL-1β expression by upregulating TLR4 and downregulating the expression of suppressor of cytokine signaling 1 (SOCS1) and myeloid differentiation primary response gene 88 (MyD88), while ABL exerted an inhibitory effect on miR-155-mediated gene expression. In conclusion, miR-155 mediates inflammatory responses in ischemic cerebral tissue by modulating TLR4/MyD88 and SOCS1 expression, and ABL exerts its antiinflammatory action by suppressing miR-155 expression, suggesting a novel miR-155-based therapy for ischemic stroke. scription factors at the transcriptional level but also by microRNAs (miRNAs, miRs) at the posttranscriptional level. Emerging evidence suggest that miRNAs are involved in regulating several aspects of inflammation (10,11) and the response to cerebral ischemia (12). Among various miRNAs, miR-155 is an inflammation-related miRNA. Its expression is induced in inflammatory macrophages (11,13), as well as in the ischemic cerebral cortex by middle cerebral artery occlusion (MCAO) surgery (14) and, in turn, it enhances proinflammatory cytokine expression in macrophages by regulating the NF-κB signaling pathway (11,15). In addition, miR-155 can exert both pro-and antiinflammatory effects by targeting different mediators of inflammatory signaling, such as SHIP1, SOCS1, SMAD2 and TAB2 (16,17). Given the importance of miR-155 in the development of postischemic brain inflammation, understanding the role of miR-155 and the mechanism of miR-155 actions in ischemic cerebral injury will provide novel insights into ischemic stroke therapy. Pharmacological alleviation of inflammatory response is one of the most promising avenues for stroke therapy. Acetylbritannilactone (ABL) is a new antiin...

Known high-risk cutaneous malignant melanoma (CMM) genes account for melanoma risk in <40% of melanoma-prone families, suggesting the existence of additional high-risk genes or perhaps a polygenic mechanism involving multiple genetic modifiers. The goal of this study was to systematically characterize rare germline variants in 42 established melanoma genes among 144 CMM patients in 76 American CMM families without known mutations using data from whole-exome sequencing. We identified 68 rare (<0.1% in public and in-house control datasets) nonsynonymous variants in 25 genes. We technically validated all loss-of-function, inframe insertion/deletion, and missense variants predicted as deleterious, and followed them up in 1, 559 population-based CMM cases and 1, 633 controls. Several of these variants showed disease co-segregation within families. Of particular interest, a stopgain variant in TYR was present in five of six CMM cases/obligate gene carriers in one family and a single population-based CMM case. A start gain variant in the 5'UTR region of PLA2G6 and a missense variant in ATM were each seen in all three affected people in a single family, respectively. Results from rare variant burden tests showed that familial and population-based CMM patients tended to have higher frequencies of rare germline variants in albinism genes such as TYR, TYRP1, and OCA2 (P < 0.05). Our results suggest that rare nonsynonymous variants in low- or intermediate-risk CMM genes may influence familial CMM predisposition, warranting further investigation of both common and rare variants in genes affecting functionally important pathways (such as melanogenesis) in melanoma risk assessment.

BACKGROUND AND PURPOSERecent findings suggest the importance of inflammation in the pathogenesis of cerebral ischaemia and its potential as a therapeutic target. Cinnamaldehyde is a diterpene with a wide range of anti-inflammatory effects thus may be advantageous in the treatment of cerebral ischaemia. The present study examined the potential therapeutic effects of cinnamaldehyde on cerebral ischaemia using a mouse model with permanent middle cerebral artery occlusion. EXPERIMENTAL APPROACHMale CD-1 mice, which had the middle cerebral artery occluded, were treated (i.p.) with cinnamaldehyde. Neuroprotection by cinnamaldehyde was analysed by evaluating neurological deficit scores, brain oedema and infarct volume. Expressionsof signal transduction molecules and inflammatory mediators were measured by Western blotting, qRT-PCR and immunohistochemical staining. Activation of NF-κB was assessed by Western blotting, immunohistochemistry and immunofluorescence. KEY RESULTSCinnamaldehyde reduced the neurological deficit scores, brain oedema and infarct volume. Cinnamaldehyde suppressed the activation of signal transduction molecules including toll-like receptor 4, tumour necrosis receptor-associated factor 6 and NF-κB, attenuated the increased levels of TNF-α, IL-1β, CCL2 and endothelial-leukocyte adhesion molecule-1 and ultimately reduced leukocyte infiltration into the ischaemic brain areas after cerebral ischaemia. CONCLUSIONS AND IMPLICATIONSCinnamaldehyde protects against cerebral ischaemia injury by inhibiting inflammation, partly mediated by reducing the expression of toll-like receptor 4, tumour necrosis receptor-associated factor 6 and the nuclear translocation of NF-κB. Our findings suggest that cinnamaldehyde may serve as a new candidate for further development as a treatment for stroke. AbbreviationsBBB, blood brain barrier; ELAM-1, endothelial leukocyte adhesion molecule-1; IHC, immunohistochemical staining; IRAK, IL-1 receptor-associated kinase; MAPKs, mitogen-activated protein kinases; MPO, myeloperoxidase; pMCAO, permanent middle cerebral artery occlusion; qRT-PCR, quantitative real-time polymerase chain reaction; rCBF, relative regional cerebral blood flow; TRAF6, tumour necrosis factor receptor-associated factor 6; TTC, 2,3,5-triphenyltetrazolium chloride BJP British Journal of Pharmacology

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