TAT-Modified ω-Conotoxin MVIIA for Crossing the Blood-Brain Barrier.

As the first in a new class of non-opioid drugs, ω-Conotoxin MVIIA was approved for the management of severe chronic pains in patients who are unresponsive to opioid therapy. Unfortunately, clinical application of MVIIA is severely limited due to its poor ability to penetrate the blood-brain barrier (BBB), reaching the central nervous system (CNS). In the present study, we have attempted to increase MVIIA's ability to cross the BBB via a fusion protein strategy. Our results showed that when the TAT-transducing domain was fused to the MVIIA C-terminal with a linker of varied numbers of glycine, the MVIIA-TAT fusion peptide exhibited remarkable ability to cross the bio-membranes. Most importantly, both intravenous and intranasal administrations of MVIIA-TAT in vivo showed therapeutic efficacy of analgesia. Compared to the analgesic effects of intracerebral administration of the nascent MVIIA, these systemic administrations of MVIIA-TAT require higher doses, but have much prolonged effects. Taken together, our results showed that TAT conjugation of MVIIA not only enables its peripheral administration, but also maintains its analgesic efficiency with a prolonged effective time window. Intranasal administration also rendered the MVIIA-TAT advantages of easy applications with potentially reduced side effects. Our results may present an alternative strategy to improve the CNS accessibility for neural active peptides.

Cloning, Synthesis and Functional Characterization of a Novel α-Conotoxin Lt1.3.

α-Conotoxins (α-CTxs) are small peptides composed of 11 to 20 amino acid residues with two disulfide bridges. Most of them potently and selectively target nicotinic acetylcholine receptor (nAChR) subtypes, and a few were found to inhibit the GABAB receptor (GABABR)-coupled N-type calcium channels (Cav2.2). However, in all of α-CTxs targeting both receptors, the disulfide connectivity arrangement "C¹-C³, C²-C4" is present. In this work, a novel α4/7-CTx named Lt1.3 (GCCSHPACSGNNPYFC-NH2) was cloned from the venom ducts of Conus litteratus (C. litteratus) in the South China Sea. Lt1.3 was then chemically synthesized and two isomers with disulfide bridges "C¹-C³, C²-C4" and "C¹-C4, C²-C³" were found and functionally characterized. Electrophysiological experiments showed that Lt1.3 containing the common disulfide bridges "C¹-C³, C²-C4" potently and selectively inhibited α3ß2 nAChRs and not GABABR-coupled Cav2.2. Surprisingly, but the isomer with the disulfide bridges "C¹-C4, C²-C³" showed exactly the opposite inhibitory activity, inhibiting only GABABR-coupled Cav2.2 and not α3ß2 nAChRs. These findings expand the knowledge of the targets and selectivity of α-CTxs and provide a new structural motif to inhibit the GABABR-coupled Cav2.2.

Salvianolic acid A suppresses CCL-20 expression in TNF-α-treated macrophages and ApoE-deficient mice.

OBJECTIVES: The CC chemokine ligand-20 (CCL-20)/macrophage inflammatory protein-3α has been seen as one of the most important chemokines and played a key role in atherogenesis, but the mechanism that underlies the regulation of CCL-20 has not been established clearly yet. The aim of this study was to investigate the influence of salvianolic acid A (SAA) on the expression of CCL-20 in macrophages and ApoE-deficient (ApoE) mice. METHODS: The expression of CCL-20 was detected both at protein and messenger RNA levels in RAW264.7 cells. We validated the result in ApoE mice that were intraperitoneally injected with SAA. Phosphorylation of p38 mitogen-activated protein kinase was detected with Western blot, and inhibitor of p38 was used to investigate the mechanism of regulation of CCL-20. Hematoxylin and eosin and Oil-Red-O staining were used to evaluate the atherosclerotic lesions and lipid accumulation in ApoE mice. Immunohistochemical analysis was used to detect the expressions of CCL-20 and CCR6 in the atherosclerotic lesions. Immunofluorescent analysis was used to certify the origination of CCL-20. RESULTS: Recombinant tumor necrosis factor-α (TNF-α) upregulated CCL-20 production in dose- and time-dependent manners in RAW264.7 cells. The activity of TNF-α-induced CCL-20 production seemed to be significantly suppressed by SAA. Using p38 mitogen-activated protein kinase inhibitor, we found that p38 mediated the effects of TNF-α- and SAA-induced CCL-20 expression changes. In addition, immunohistochemical analysis of aortic root of ApoE mice also demonstrated that the expressions of CCL-20 and CCR6 were both downregulated significantly with SAA treatment. Furthermore, treatment of SAA inhibited the progression of the atherosclerotic plaques and lipid accumulation. CONCLUSIONS: These results demonstrate that TNF-α increased but SAA suppressed CCL-20 production significantly via a novel mechanism.

Current status and breakthroughs in treating advanced non-small cell lung cancer with EGFR exon 20 insertion mutations.

Recently, targeted therapy and immunotherapy have emerged as effective treatment options for non-small cell lung cancer (NSCLC). This progress has been facilitated by the rapid development of diagnostic and therapeutic technologies and the continuous research and development of new drugs, leading to a new era in precision medicine for NSCLC. This is a breakthrough for patients with common mutations in the human epidermal growth factor receptor (EGFR) gene in NSCLC. Consequently, the use of targeted drugs has significantly improved survival. Nevertheless, certain rare genetic mutations are referred to as EGFR exon 20 insertion (ex20ins) mutations, which differ in structure from conventional EGFR gene mutations, namely, exon 19 deletion mutations (19-Del) and exon 21 point mutations. Owing to their distinct structural characteristics, patients harboring these EGFR ex20ins mutations are unresponsive to traditional tyrosine kinase inhibitor (TKI) therapy. This particular group of patients did not fall within the scope of their applicability. However, the activating A763_Y764insFQEA mutation elicits a more pronounced response than mutations in the near and far regions of the C-helix immediately following it and should, therefore, be treated differently. Currently, there is a lack of effective treatments for EGFR ex20ins mutations NSCLC. The efficacy of chemotherapy has been relatively favorable, whereas the effectiveness of immunotherapy remains ambiguous owing to inadequate clinical data. In addition, the efficacy of the first- and second-generation targeted drugs remains limited. However, third-generation and novel targeted drugs have proven to be effective. Although novel EGFR-TKIs are expected to treat EGFR ex20ins mutations in patients with NSCLC, they face many challenges. The main focus of this review is on emerging therapies that target NSCLC with EGFR ex20ins and highlight major ongoing clinical trials while also providing an overview of the associated challenges and research advancements in this area.

Long non-coding RNA SLC25A25-AS1 exhibits oncogenic roles in non-small cell lung cancer by regulating the microRNA-195-5p/ITGA2 axis.

Long non-coding RNA SLC25A25 antisense RNA 1 (SLC25A25-AS1) exerts antitumour activity in colorectal cancer. The present study investigated whether SLC25A25-AS1 is implicated in the aggressiveness of non-small cell lung cancer (NSCLC) and the possible underlying mechanism. SLC25A25-AS1 expression in NSCLC was determined by reverse transcription-quantitative PCR. The proliferation, apoptosis, migration and invasion of NSCLC cells were tested in vitro through cell counting kit-8 assay, flow cytometry analysis, Transwell migration and invasion assays, followed by in vivo validation using animal experiments. Additionally, the competitive endogenous RNA theory for SLC25A25-AS1, microRNA-195-5p (miR-195-5p) and integrin α2 (ITGA2) was identified using subcellular fractionation, bioinformatics analysis, reverse transcription-quantitative PCR, western blotting, a luciferase assay and RNA immunoprecipitation. As compared with normal lung tissues, increased expression of SLC25A25-AS1 was demonstrated in NSCLC tissues using The Cancer Genome Atlas database.. In addition, SLC25A25-AS1 was overexpressed in both NSCLC tissues and cell lines. High SLC25A25-AS1 expression was markedly associated with shorter overall survival time of patients with NSCLC. SLC25A25-AS1 silencing impeded NSCLC cell proliferation and triggered apoptosis, while restricting cell migration and invasion. Tumour growth in vivo was also impaired by SLC25A25-AS1 silencing. Mechanistically, SLC25A25-AS1 was demonstrated to be an miR-195-5p sponge in NSCLC cells. miR-195-5p mimics decreased ITGA2 expression in NSCLC cells by directly targeting ITGA2, and SLC25A25-AS1 interference decreased ITGA2 expression by sequestering miR-195-5p. Furthermore, the antitumour effects of SLC25A25-AS1 silencing on malignant behaviours were counteracted when ITGA2 was restored or when miR-195-5p was silenced. In summary, by controlling the miR-195-5p/ITGA2 axis, SLC25A25-AS1 served tumour-promoting roles in NSCLC cells. Therefore, the SLC25A25-AS1/miR-195-5p/ITGA2 signalling pathway might be an attractive target for future therapeutic options in NSCLC.

Silencing of the long noncoding RNA LINC01132 alleviates the oncogenicity of epithelial ovarian cancer by regulating the microRNA­431­5p/SOX9 axis.

To date, the role of lncRNA long intergenic non­protein­coding RNA 1132 (LINC01132) expression in epithelial ovarian cancer (EOC) has not been explored. Thus, LINC01132 expression in EOC was assessed and the regulatory activity of LINC01132 on the malignant behaviours of EOC cells was investigated. Additionally, the molecular events that occurred downstream of LINC01132 in EOC cells were also revealed. In the present study, LINC01132 expression in EOC was verified by employing RT­qPCR. The effects of LINC01132 on the aggressive behaviours of EOC cells were revealed utilizing multiple functional experiments. The targeting interaction among LINC01132, microRNA­431­5p (miR­431­5p) and SRY­box 9 (SOX9) was demonstrated by RNA immunoprecipitation and luciferase reporter assay. Herein, LINC01132 was overexpressed in EOC and was significantly associated with poor patient prognosis. Functionally, cell experiments revealed that LINC01132 depletion produced cancer­suppressive effects in EOC cells and regulated cell proliferation, migration, invasion and apoptosis in vitro. Additionally, the loss of LINC01132 attenuated tumour growth in vivo. Mechanistically, LINC01132 acted as a competing endogenous RNA by sequestering miR­431­5p and consequently overexpressing SOX9 in EOC cells, forming a LINC01132/miR­431­5p/SOX9 axis. In rescue experiments, miR­431­5p inhibition or SOX9 reintroduction eliminated the anti­tumour effects of LINC01132 silencing on the pathological behaviours of EOC cells. Generally, LINC01132 exhibited oncogenic activities in EOC cells by regulating the outcome of the miR­431­5p/SOX9 axis, providing an effective target for EOC diagnosis, therapy and prognosis evaluation.

A novel ω-conotoxin Bu8 inhibiting N-type voltage-gated calcium channels displays potent analgesic activity.

ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple ß-strand. The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists.

Serum Amyloid A Induces a Vascular Smooth Muscle Cell Phenotype Switch through the p38 MAPK Signaling Pathway.

Atherosclerosis is an important pathological condition which is accompanied by a vascular smooth muscle cell (VSMC) phenotype switch toward a synthetic phenotype. As an acute-phase protein, Serum Amyloid A (SAA) is thought to have a close relationship to atherosclerosis development. However, no study has investigated the direct effect of SAA on the VSMC phenotype switch, as well as the underlying mechanisms. The purpose of our study was to explore the effect of SAA on the VSMC phenotype switch and the potential mechanisms involved. In our study, we found that SAA induced the VSMC phenotype switch which reduced expression of the smooth muscle cell (SMC) marker and enhanced expression of the matrix synthesis related marker. The proliferative ability of VSMCs was also increased by SAA treatment. Furthermore, our research found that SAA activated the ERK1/2 and p38 MAPK signaling pathways. Finally, by applying the ERK1/2 and p38 inhibitors, U0126 and SB203580, we demonstrated that the SAA-induced VSMC phenotype switch was p38-dependent. Taken together, these results indicated that SAA may play an important role in promoting the VSMC phenotype switch through the p38 MAPK signaling pathway.