A novel lncRNA-hidden polypeptide regulates malignant phenotypes and pemetrexed sensitivity in A549 pulmonary adenocarcinoma cells.

The advance of high-throughput sequencing enhances the discovery of short ORFs embedded in long non-coding RNAs (lncRNAs). Here, we uncovered the production and biological activity of lncRNA-hidden polypeptides in lung adenocarcinoma (LUAD). In the present study, bioinformatics was used to screen the lncRNA-hidden polypeptides in LUAD. Analysis of protein expression was done by western blot or immunofluorescence assay. The functions of the polypeptide were determined by detecting its effects on cell viability, proliferation, migration, invasion, and pemetrexed (PEM) sensitivity. The protein interactors of the polypeptide were analyzed by mass spectrometry after Co-immunoprecipitation (Co-IP) assay. The results showed that the lncRNA LINC00954 was confirmed to encode a novel polypeptide LINC00954-ORF. The polypeptide had tumor-suppressor features in A549 cells by repressing cell growth, motility and invasion. Moreover, the polypeptide enhanced PEM sensitivity and suppressed growth in A549/PEM cells. The protein interactors of this polypeptide had close correlations with RNA processing, amide metabolic process, translation, RNA binding, RNA transport, and DNA replication. As a conclusion, the LINC00954-ORF polypeptide embedded in lncRNA LINC00954 possesses tumor-suppressor features in A549 and PEM-resistant A549 cells and sensitizes PEM-resistant A549 cells to PEM, providing evidence that the LINC00954-ORF polypeptide is a potential anti-cancer agent in LUAD.

Pemetrexed alleviates piglet diarrhea by blocking the interaction between porcine epidemic diarrhea virus nucleocapsid protein and Ezrin.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes high mortality in piglets, thus posing a serious threat to the world pig industry. Porcine epidemic diarrhea (PED) is related to the imbalance of sodium absorption by small intestinal epithelial cells; however, the etiology of sodium imbalanced diarrhea caused by PEDV remains unclear. Herein, we first proved that PEDV can cause a significant decrease in Na+/H+ exchanger 3 (NHE3) expression on the cell membrane, in a viral dose-dependent manner. Further study showed that the PEDV nucleocapsid (N) protein participates in the regulation of NHE3 activity through interacting with Ezrin. Flame atomic absorption spectroscopy results indicated a serious imbalance in Na+ concentration inside and outside cells following overexpression of PEDV N. Meanwhile, molecular docking technology identified that the small molecule drug Pemetrexed acts on the PEDV N-Ezrin interaction region. It was confirmed that Pemetrexed can alleviate the imbalanced Na+ concentration in IPEC-J2 cells and the diarrhea symptoms of Rongchang pigs caused by PEDV infection. Overall, our data suggest that the interaction between PEDV N and Ezrin reduces the level of phosphorylated Ezrin, resulting in a decrease in the amount of NHE3 protein on the cell membrane. This leads to an imbalance of intracellular and extracellular Na+, which causes diarrhea symptoms in piglets. Pemetrexed is effective in relieving diarrhea caused by PEDV. Our results provide a reference to screen for anti-PEDV targets and to develop drugs to prevent PED.IMPORTANCEPorcine epidemic diarrhea (PED) has caused significant economic losses to the pig industry since its initial outbreak, and the pathogenic mechanism of porcine epidemic diarrhea virus (PEDV) is still under investigation. Herein, we found that the PEDV nucleocapsid protein interacts with Ezrin to regulate Na+/H+ exchanger 3 activity. In addition, we screened out Pemetrexed, a small molecule drug, which can effectively alleviate pig diarrhea caused by PEDV. These results provide support for further exploration of the pathogenesis of PEDV and the development of drugs to prevent PED.

miR-145-5p Targets Sp1 in Non-Small Cell Lung Cancer Cells and Links to BMI1 Induced Pemetrexed Resistance and Epithelial-Mesenchymal Transition.

Pemetrexed is a folic acid inhibitor used as a second-line chemotherapeutic agent for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancers. However, prolonged treatment with pemetrexed may cause cancer cells to develop resistance. In this study, we found increased expressions of BMI1 (B Lymphoma Mo-MLV insertion region 1 homolog) and Sp1 and a decreased expression of miR-145-5p was found in pemetrexed-resistant A400 cells than in A549 cells. Direct Sp1 targeting activity of miR-145-5p was demonstrated by a luciferase based Sp1 3'-UTR reporter. Changed expression of miR-145-5p in A400 or A549 cells by transfection of miR-145-5p mimic or inhibitor affected the sensitivity of the cells to pemetrexed. On the other hand, the overexpression of Sp1 in A549 cells caused the decreased sensitivity to pemetrexed, induced cell migratory capability, and epithelial-mesenchymal transition (EMT) related transcription factors such as Snail Family Transcriptional Repressor 1 and Zinc Finger E-Box Binding Homeobox 1. In addition, the overexpression of BMI1 in the A549 cells resulted in an increase in Sp1 and a decrease in miR-145-5p accompanied by the elevations of cell proliferation and EMT transcription factors, which could be reduced by the overexpression of miR-145-5p or by treatment with the Sp1 inhibitor of mithramycin A. In conclusion, the results of this study suggest that the downregulation of miR-145-5p by BMI1 overexpression could lead to the enhanced expression of Sp1 to induce the EMT process in pemetrexed-resistant NSCLC cells. These results suggest that increasing miR-145-5p expression by delivering RNA drugs may serve as a sensitizing agent for pemetrexed-resistant NSCLC patients.

Montelukast ameliorated pemetrexed-induced cytotoxicity in hepatocytes by mitigating endoplasmic reticulum (ER) stress and nucleotide oligomerization domain-like receptor protein 3 (NLRP3) activation.

Pemetrexed (PEM) is an effective chemotherapeutic drug used for the treatment of clinical non-small-cell lung cancer (NSCLC) and is reported to induce severe hepatotoxicity. Exploring potential drugs which could counteract the side effects of PEM is of great clinical interest. Here, we aim to examine the beneficial effects of Montelukast, a novel anti-asthma drug, against PEM-induced cytotoxicity in hepatocytes, and to explore the underlying mechanism. We found that Montelukast reduces cytotoxicity of PEM in hepatocytes, confirmed by its increasing cell viability and reducing lactate dehydrogenase (LDH) release. In addition, Montelukast attenuated PEM-induced oxidative stress by reducing mitochondrial reactive oxygen species (ROS), increasing reduced glutathione (GSH), and downregulating NADPH oxidase 4 (NOX-4) expression. Importantly, Montelukast suppressed PEM-induced activation of the nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome and mitigated endoplasmic reticulum (ER) stress by reducing NLRP3, growth arrest, and DNA damage-inducible protein 34 (GADD34), CEBP-homologous protein (CHOP), and also blocking the eukaryotic initiation factor 2 (eIF-2α)/activating transcription factor 4 (ATF4) signaling pathway. Lastly, we found that Montelukast inhibited the transcriptional activity of nuclear factor kappa-B (NF-κB). Montelukast exerted a protective action against PEM-induced cytotoxicity in hepatocytes by mitigating ER stress and NLRP3 activation.

Towards establishment of a plant-based model to assess the novel anti-cancerous lead molecule(s): An in silico, in vivo and in vitro assessment of some potential anti-cancerous drugs on Lathyrus sativus L.

The drug development process is one of the important aspects of medical biology. The classical lead identification strategy in the way of drug development based on animal cell is time-consuming, expensive and involving ethical issues. The following study aims to develop a novel plant-based screening of drugs. Study shows the efficacy of certain anti-cancerous drugs (Pemetrexed, 5-Fluorouracil, Methotrexate, Topotecan and Etoposide) on a plant-based (Lathyrus sativus L.) system. Two important characteristics of cancer cells were observed in the colchicine-treated polyploid cell and the callus, where the chromosome numbers were unusual and the division of cells were uncontrolled respectively. With increasing concentration, the drugs significantly reduced the mitotic index, ploidy level and callus growth. Increasing Pemetrexed concentration decreased the plant DHFR activity. A decrease in total RNA content was observed in 5-FU and Methotrexate with increasing concentrations of the drugs. Etoposide and Topotecan inhibited plant topoisomerase II and topoisomerase I activities, which was justified through plasmid nicking and comet assay, respectively. Molecular and biochemical study revealed similar results to the animal system. The in silico study had been done, and the structural similarity of drug binding domains of L. sativus and human beings had also been established. The binding site of the selected drugs to the domains of plant target proteins was also determined. Experimental results are significant in terms of the efficacy of known anti-cancerous drugs on the plant-based system. The proposed assay system is a cost-effective, convenient and less time-consuming process for primary screening of anti-cancerous lead molecules.

Structural basis of antifolate recognition and transport by PCFT.

Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.

Substituted-cysteine accessibility and cross-linking identify an exofacial cleft in the 7th and 8th helices of the proton-coupled folate transporter (SLC46A1).

The proton-coupled folate transporter (PCFT-SLC46A1) is required for folate transport across the apical membrane of the small intestine and across the choroid plexus. This study focuses on the structure/function of the 7th transmembrane domain (TMD), and its relationship to the 8th TMD as assessed by the substituted cysteine accessibility method (SCAM) and dicysteine cross-linking. Nine exofacial residues (I278C; H281C-L288C) of 23 residues in the 7th TMD were accessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin). Pemetrexed, a high-affinity substrate for PCFT, decreased or abolished biotinylation of seven of these residues consistent with their location in or near the folate binding pocket. Homology models of PCFT based on Glut5 fructose transporter structures in both inward- and outward- open conformations were constructed and predicted that two pairs of residues (T289-I304C and Q285-Q311C) from the 7th and 8th TMDs should be in sufficiently close proximity to form a disulfide bond when substituted with cysteines. The single Cys-substituted mutants were accessible to MTSEA-biotin and functional with and without pretreatment with dithiotreitol. However, the double mutants were either not accessible at all, or accessibility was markedly reduced and function markedly impaired. This occurred spontaneously without inclusion of an oxidizing agent. Dithiotreitol restored accessibility and function consistent with disulfide bond disruption. The data establish the proximity of exofacial regions of the 7th and 8th TMDs and their role in defining the aqueous translocation pathway and suggest that these helices may be a component of an exofacial cleft through which substrates enter the protein binding pocket in its outward-open conformation.

Development of a Prodrug of Levofloxacin to Avoid Chelation with Al3+ and of Pemetrexed Dimedoxomil Esters for Oral Administration.

An ethoxycarbonyl 1-ethyl hemiacetal ester of levofloxacin (LVFX-EHE) avoids insoluble chelate formation with metal-containing drugs in the intestinal tract and is rapidly hydrolyzed to the parent drug. Furthermore, the minimum inhibitory concentration confirms that LVFX-EHE is less likely to cause pseudomembranous colitis because of less susceptibility to normal intestinal bacteria flora. Pemetrexed dimedoxomil, the prodrug of pemetrexed, was synthesized via reaction with medoxomil bromide after modification of L-glutamate with the tert-butyloxycarbonyl protecting group (BOC), followed by hydrolysis of the BOC moiety with trifluoroacetic acid (TFA) in CH2Cl2 at a temperature of 0°C for 2 h. A serum pemetrexed concentration of >2 µg/mL was observed after oral administration of pemetrexed dimedoxomil at a dose of 60 mg/kg to rats.

Determination of the Degradation Chemistry of the Antitumor Agent Pemetrexed Disodium.

Stress-testing (forced degradation) studies have been conducted on pemetrexed disodium heptahydrate (1) (LY231514·2Na·7H2O) drug substance in order to identify its likely degradation products and establish its degradation pathways. Solid samples of the drug substance were stressed under various conditions of heat, humidity, and light, and solutions of the drug substance were stressed under various conditions of heat, light, oxidation, and over a wide pH range (1-13). The stressed samples were analyzed using a gradient elution reversed-phase HPLC method. The 7 major degradation products detected in the stress-testing studies were isolated, and the structures were elucidated via spectroscopic characterization. The structures of the degradation products and their proposed mechanisms of formation indicate that 1 degrades via 2 main pathways: oxidation and hydrolysis. Of the 7 identified degradation products, 6 are proposed to result from oxidation and 1 from hydrolysis.