Development of hypoxia-activated PROTAC exerting a more potent effect in tumor hypoxia than in normoxia.

Hypoxia is a hallmark of many solid tumors, and it causes the overexpression of a variety of proteins including the epidermal growth factor receptor (EGFR). Many antitumor prodrugs have been designed to target hypoxia. Here we report the identification of a kind of hypoxia-activated proteolysis targeting chimera (ha-PROTAC) by introducing the hypoxia-activated leaving group (1-methyl-2-nitro-1H-imidazol-5-yl)methyl or 4-nitrobenzyl into the structure of an EGFRDel19-based PROTAC. Among the obtained molecules, ha-PROTAC 13 exhibits a more potent degradation activity for EGFRDel19 in hypoxia than in normoxia in HCC4006 cells. This is the first example of identifying a PROTAC to selectively act on tumors utilizing the characteristic of tumor hypoxia and provides a new approach for PROTAC development.

UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model.

BACKGROUND: Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA. METHODS: An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. RESULTS: In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40-20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2-100 ng/mL for guanosine, and 2-1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment. CONCLUSION: The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.

Identification of integrative molecular and clinical profiles of Fibrinogen-like protein 2 in gliomas using 1323 samples.

BACKGROUND: Fibrinogen-like protein 2 (FGL2), a member of the fibrinogen superfamily, has been described to augment immunosuppression in gliomas. However, the precise clinical molecular features and the prognostic relevance of FGL2 in gliomas remain unclear. Therefore, a comprehensive analysis of the role of FGL2 in gliomas would provide insights into the therapeutic implications for this disease. METHODS: Totally, 1323 glioma samples with RNA-seq and microarray data from TCGA and CGGA databases were used to clarify the clinical significance and molecular profile of FGL2 in glioma. The findings were further validated through immunohistochemistry (IHC). RESULTS: The transcriptional level of FGL2 was positively associated with tumor grade in gliomas, which was confirmed at the protein level through IHC staining. Consistently, FGL2 was significantly enriched in isocitrate dehydrogenase wild-type tumors and the mesenchymal subtype of gliomas. We also demonstrated FGL2 expression correlated with high immune scores and infiltration of immune cell populations, including T cells, macrophages and B cells. Pearson's correlation analysis revealed that FGL2-related genes correlated with inflammatory-immune responses, particularly T cell-mediated immune response. Additionally, FGL2 expression was found tightly associated with immune checkpoints PD-L1 and PD-L2. Clinically, patients with high FGL2 expression exhibited unfavorable overall survival. CONCLUSION: Our results provide the integrative molecular and clinical profiles of FGL2 in gliomas and emphasize the importance of prospective studies on the FGL2-related immune-inflammatory network.

Analysis of lncRNA, miRNA and mRNA-associated ceRNA networks and identification of potential drug targets for drug-resistant non-small cell lung cancer.

Background: Drug resistance to chemotherapeutic drugs or targeted medicines is an obstacle encountered in the treatment of non-small-cell lung cancer (NSCLC). However, the mechanisms of competing endogenous RNA (ceRNA) on the drug resistance in NSCLC are rarely reported. In this paper, the comprehensive expression profiles of lncRNAs and mRNAs in drug-resistant NSCLC cells were obtained by RNA sequencing. Methods: The dysregulated lncRNAs, miRNAs and mRNAs in drug-resistant NSCLC cell lines were identified by RNA-sequencing and bioinformatics methods. Results: A total of 39 dysregulated lncRNAs and 650 dysregulated mRNAs were identified between drug-resistant NSCLC cell lines and their parental cell lines. Additionally, 33 lncRNA-miRNA-mRNA pathways in the ceRNA network in drug-resistant NSCLC were constructed through bioinformatics methods and ceRNA regulatory rules. These comprised 12 dysregulated lncRNAs, five dysregulated miRNAs, and eight dysregulated mRNAs. In addition, lncRNA ATP2B1/miR-222-5p/TAB2 and lncRNA HUWE1/miR-222-5p/TAB2 were identified as potential ceRNA networks involved in drug resistance to NSCLC. Conclusions: The current study provides a promising therapeutic strategy against the lncRNA-miRNA-mRNA ceRNA regulatory network for NSCLC treatment and deepens our comprehension of the ceRNA regulatory mechanisms related to drug resistance to NSCLC.

Polyphyllin VI induces apoptosis and autophagy in human osteosarcoma cells by modulation of ROS/JNK activation.

PURPOSE: Polyphyllin VI, a main active saponin isolated from traditional medicinal plant Paris polyphylla, has exhibited antitumor activities in several cancer cell lines. In the present study, we investigated the antitumor effect of Polyphyllin VI against human osteosarcoma cells (U2OS) and the underlying molecular mechanisms. METHODS: The U2OS cell lines were used to determine the antiproliferative effect of Polyphyllin VI by CCK8 assay. Cell cycle was analyzed by flow cytometry. The Polyphyllin VI-induced apoptosis was determined by Annexin V-APC/7-AAD apoptosis detection kit and JC-1 staining. Meanwhile, the autophagy was determined by acridine orange staining. The apoptosis and autophagy-related proteins were monitored by Western blot assay. Subsequently, intracellular hydrogen peroxide (H2O2) and the activation of ROS/JNK pathway were detected. RESULTS: Polyphyllin VI could potently inhibit cell proliferation by causing G2/M phase arrest. Polyphyllin VI induced mitochondria-mediated apoptosis with the upregulation of proapoptotic proteins Bax and poly ADP-ribose polymerase, and downregulation of antiapoptotic protein Bcl-2 in U2OS cells. Concomitantly, Polyphyllin VI provoked autophagy with the upregulation of critical Atg proteins and accumulation of LC3B-II. Intracellular H2O2 production was triggered upon exposure to Polyphyllin VI, which could be blocked by ROS scavenger. Polyphyllin VI dramatically promoted JNK phosphorylation, whereas it decreased the levels of phospho-p38 and ERK. CONCLUSION: Our results reveal that Polyphyllin VI may effectively induce apoptosis and autophagy to suppress cell growth via ROS/JNK activation in U2OS cells, suggesting that Polyphyllin VI is a potential drug candidate for the treatment of osteosarcomas.

In vitro metabolic profiles of motolimod by using liquid chromatography tandem mass spectrometry: Metabolic stability, metabolite characterization and species comparison.

Motolimod (VTX-2337) is an agonist of toll-like receptor 8 (TLR8) with potential immune-stimulating and antineoplastic activities. The purpose of this study was to investigate the in vitro metabolic profiles of VTX-2337. The average in vitro T1/2 values were 6.93, 8.71, 7.39, 2.85, and 10.58 min in the liver microsomes of mouse, rat, dog, monkey and human respectively, suggesting that VTX-2337 suffered from extensive metabolism. The metabolites were further profiled and identified by using ultra-high performance liquid chromatography coupled with diode array detector and Q-Exactive-Orbitrap tandem mass spectrometer (UHPLC-DAD-Q-Exactive-Orbitrap-MS) operated in positive ion mode. A total of 20 metabolites were detected and their identities were characterized based on their accurate masses, fragment ions and retention times. M13 (depropylation) was the most abundant metabolite in all species. M14 (oxygenation) was also the major metabolite in the liver microsomes of mouse, rat, monkey and human. M1, M5, M10, M15, and M16 were specifically detected in mouse, while M6 and M17 were monkey-specific. All the metabolites present in human could be found in animal species. The metabolic pathways of VTX-2337 referred to oxygenation, hydrolysis, depropylation, and dehydrogenation. Rat had the similar metabolic profiles to humans. The current study provided overall metabolic profiles of VTX-2337, which would be of great help in predicting in vivo pharmacokinetic profiles and in understanding the effectiveness and safety of this drug.

Expression Profiles of microRNAs in Drug-Resistant Non-Small Cell Lung Cancer Cell Lines Using microRNA Sequencing.

BACKGROUND/AIMS: Drug resistance remains a main obstacle to the treatment of non- small cell lung cancer (NSCLC). The aim of this study was to identify the expression profiles of microRNAs (miRNAs) in drug-resistant NSCLC cell lines. METHODS: The expression profiles of miRNAs in drug-resistant NSCLC cell lines were examined using miRNA sequencing, and the common dysregulated miRNAs in these cell lines were identified and analyzed by bioinformatics methods. RESULTS: A total of 29 upregulated miRNAs and 36 downregulated miRNAs were found in the drug-resistant NSCLC cell lines, of which 26 upregulated and 36 downregulated miRNAs were found to be involved in the Ras signaling pathway. The expression levels, survival analysis, and receiver operating characteristic curve of the dysregulated miRNAs based on The Cancer Genome Atlas database for lung adenocarcinoma showed that hsa-mir-192, hsa-mir-1293, hsa-mir-194, hsa-mir-561, hsa-mir-205, hsa-mir-30a, and hsa-mir-30c were related to lung cancer, whereas only hsa-mir-1293 and hsa-mir-561 were not involved in drug resistance. CONCLUSION: The results of this study may provide novel biomarkers for drug resistance in NSCLC and potential therapies for overcoming drug resistance, and may also reveal the potential mechanisms underlying drug resistance in this disease.

Retigabine ameliorates acute stress-induced impairment of spatial memory retrieval through regulating USP2 signaling pathways in hippocampal CA1 area.

Acute stress could trigger maladaptive changes associated with stress-related cognitive and emotional deficits. Dysfunction of ion channel or receptor in the hippocampal area has been linked to the cognitive deficits induced by stress. It is known that Kv7 channel openers, including FDA-approved drug retigabine, show cognitive protective efficacy. However, the underlying molecular mechanisms remain elusive. Here we showed that exposing adult male rats to acute stress significantly impaired the spatial memory, a cognitive process controlled by the hippocampus. Concomitantly, significantly reduced AMPA receptor expression was found in hippocampal CA1 area from acute stressed rats. This effect relied on the down-regulation of deubiquitinating enzyme USP2 and its upstream regulators (PGC-1α and ß-catenin), and the subsequent enhancement of mTOR-related autophagy which is regulated by USP2. These findings suggested that acute stress dampened AMPA receptor expression by controlling USP2-related signaling, which caused the detrimental effect on hippocampus-dependent cognitive processes. We also found that retigabine alleviated acute stress-induced spatial memory retrieval impairment through adjusting the aberrance of USP2, its upstream regulators (PGC-1α, E4BP4 and ß-catenin) and its downstream targets (mTOR, autophagy and GluA1). Our results have identified USP2 as a key molecule that mediates stress-induced spatial memory retrieval impairment, which provides a framework for new druggable targets to conceptually treat stress-associated cognitive deficits.

Angelica sinensis polysaccharide attenuates concanavalin A-induced liver injury in mice.

Angelica sinensis polysaccharide (ASP), extracted from the roots of A. sinensis (Oliv.) Diels, is a ß-D-pyranoid polysaccharide with an average molecular weight of 72,900 Da. In this study, we investigated the protective effects of ASP against concanavalin A-induced liver failure and the underlying mechanisms. Concentrations of ASP ranging from 5 to 125 µg/mL could inhibit concanavalin A (ConA)-induced lymphoproliferative response. The potential hepatoprotective activity of ASP was demonstrated by the significant decrease in serum transaminase (ALT and AST) levels and the attenuation of liver inflammation damage exhibited by H&E stain of the liver. Furthermore, ASP pretreatment significantly decreased proinflammatory cytokines (TNF-α, IFN-γ, IL-2 and IL-6) and alleviated oxidative stress by reducing MDA and ROS levels and by enhancing SOD activity after ConA administration in mice. Results of Western blot analysis indicated that ASP attenuated Caspase-3-dependent apoptosis by Caspase-8 and JNK-mediated pathway and inhibited the activation of IL-6/STAT3 and NF-κB signaling pathways in ConA-induced liver damage in mice. In conclusion, ASP pretreatment could attenuate concanavalin A-induced liver injury through its anti-inflammatory and anti-oxidant actions in mice.