PD-L1 enhanced cisplatin resistance in non-small cell lung cancer through PI3K/AKT/mTOR

Objective: Non-small cell lung cancer (NSCLC) is a disease with high morbidity and mortality. Cisplatin (DDP) is one of the most widely used chemotherapy regimens. However, patients who take cisplatin-based drugs for a long time are prone to develop side effects such as ototoxicity, nephrotoxicity and drug resistance, and these side effects seriously affect patients' quality of life and treatment outcomes. The current findings suggest that chemotherapeutics can suppress tumor immune responses and achieve tumor immune escape by remodeling immune cells and altering PD-L1 expression in tumor cells. Therefore, it is of clinical concern whether cisplatin may limit the anti-tumor effect of the organism by inducing immunosuppression in lung cancer or not. Studies on the effects of cisplatin on PD-L1 expression in NSCLC cell lines are scarce, and the intracellular regulatory mechanisms are unclear. Our study focused on unraveling the effects and on clarifying whether the PI3K/Akt/mTOR signaling pathway (PATSP) also played a regulatory role in this process. We also compared the difference in PD-L1 expression between NSCLC cisplatin-resistant cell lines (NSCLC/DDP) and NSCLC naïve cell lines. This study aimed to reveal that NSCLC/DDP cell lines contribute to tumor immune escape. This phenomenon might be caused by the high expression of PD-L1 regulated by PATSP. Our research also provided new evidence that low concentrations of cisplatin promote tumor immune evasion in NSCLC cell lines. Providing a new direction for better immunotherapy in cisplatin-resistant patients is the clinical significance of this study. Methods: We successfully induced A549/DDP cells that were not available in the laboratory by incrementing the concentration method. Therefore, A549, HCC827, H1975 cell lines, and A549/DDP, HCC827/DDP, H1975/DDP cell lines were selected for this research. Cisplatin, XL-765 (dual PI3K/mTOR inhibitor), and 740Y-P (PI3K agonist) were selected as stimulating drugs. We first detected the IC50s and EC50s of the above six cell lines stimulated by the above drugs with CCK-8 kit and determined the optimal concentration range of the drugs. After that, we detected the invasion capability, migration capability, proliferation rate and apoptosis rate of the cell lines after drug stimulation by Transwell, Wound healing, CellTiter-Glo ® 2.0 and Flow cytometry assays. These experiments were able to show the variations in cellular function after stimulation of NSCLC cell lines with cisplatin, XL-765 and 740Y-P. In the following experiments, we initially detected the expression differences of PD-L1 in the above cells before and after drug stimulation by Immunohistochemistry and then the differences in expression of PD-L1, p-AKT (phosphorylated AKT), AKT, p-S6 (phosphorylated S6) and S6 proteins were then detected by Western blot. Finally, we also compared PD-L1 mRNA and membrane PD-L1 expression differences in the cell lines before and after drug stimulation by qRT-PCR and Flow cytometry. Results: There are five primary outcomes that we can learn. 1. The cell activity, invasion capability, and migration capability of NSCLC/DDP cell lines were higher than those of the corresponding NSCLC naïve cell lines, but the apoptosis rates of NSCLC/DDP cell lines were lower than that of the corresponding NSCLC naïve cell lines. Cisplatin and 740Y-P both had a promoting effect on the cellular function of NSCLC naïve cell lines, while XL-765 had an inhibitory effect. 2. Low concentrations of cisplatin significantly up-regulated the expression of cell membrane PD-L1 and total PD-L1 in NSCLC naïve cell lines, and the expression levels tended to increase with increasing concentrations of cisplatin.3. PD-L1 expression was significantly higher in NSCLC/DDP cells than in NSCLC naïve cells but was significantly reduced after stimulation with XL-765. 4.In H1975 and H1975/DDP cell lines, a low concentration of cisplatin and 740Y-P could increase the expression of p-AKT, AKT, p-S6, S6, and PD-L1, while XL-765 could decrease the expression. 5. The degree of increase and decrease in the expression of p-AKT, AKT, p-S6, S6, and PD-L1 correlated with the drugs' treatment time and treatment concentration. Conclusion: Our results suggested that low concentrations of cisplatin could promote PD-L1 expression in NSCLC cells, leading to cisplatin resistance and immune escape in NSCLC cell lines. The over-activated PATSP was involved in cisplatin-induced up-regulation of PD-L1 expression. Inhibition of this signaling pathway resulted in decreased expression of PD-L1 and, to some extent, reversed immune escape caused by cisplatin resistance in NSCLC cell lines.Zielsetzung: Nichtkleinzelliger Lungenkrebs (NSCLC) ist eine Krankheit mit hoher Morbidität und Mortalität. Cisplatin (DDP) ist eines der am häufigsten eingesetzten Chemotherapieschemata. Patienten, die Cisplatin-haltige Medikamente über einen langen Zeitraum einnehmen, neigen jedoch dazu, Nebenwirkungen wie Ototoxizität, Nephrotoxizität und Arzneimittelresistenz zu entwickeln, und diese Nebenwirkungen beeinträchtigen die Lebensqualität der Patienten und die Behandlungsergebnisse erheblich. Die aktuellen Erkenntnisse deuten darauf hin, dass Chemotherapeutika die Immunreaktion von Tumoren unterdrücken und durch den Umbau von Immunzellen und die Veränderung der PD-L1-Expression in Tumorzellen eine Flucht des Tumorimmunsystems ermöglichen können. Daher ist es von klinischem Interesse, ob Cisplatin die Anti-Tumor-Wirkung des Organismus durch Induktion von Immunsuppression bei Lungenkrebs einschränken kann oder nicht. Es gibt nur wenige Studien über die Auswirkungen von Cisplatin auf die PD-L1-Expression in NSCLC-Zelllinien, und die intrazellulären Regulationsmechanismen sind unklar. Unsere Studie konzentrierte sich darauf, die Auswirkungen zu entschlüsseln und zu klären, ob der PI3K/Akt/mTOR-Signalweg (PATSP) bei diesem Prozess ebenfalls eine regulatorische Rolle spielt. Außerdem verglichen wir den Unterschied in der PD-L1-Expression zwischen cisplatinresistenten NSCLC-Zelllinien (NSCLC/DDP) und naiven NSCLC-Zelllinien. Ziel dieser Studie war es, zu zeigen, dass NSCLC/DDP-Zelllinien zur Immunflucht des Tumors beitragen. Dieses Phänomen könnte auf die hohe Expression von PD-L1 zurückzuführen sein, die durch PATSP reguliert wird. Unsere Forschung lieferte auch neue Beweise dafür, dass niedrige Konzentrationen von Cisplatin die Umgehung des Tumorimmunsystems in NSCLC-Zelllinien fördern. Die klinische Bedeutung dieser Studie liegt darin, dass sie eine neue Richtung für eine bessere Immuntherapie bei cisplatinresistenten Patienten aufzeigt. Methoden: Wir haben erfolgreich A549/DDP-Zellen induziert, die im Labor nicht verfügbar waren, indem wir die Konzentrationsmethode erhöht haben. Daher wurden die Zelllinien A549, HCC827, H1975 und A549/DDP, HCC827/DDP, H1975/DDP für diese Untersuchung ausgewählt. Cisplatin, XL-765 (dualer PI3K/mTOR-Inhibitor) und 740Y-P (PI3K-Agonist) wurden als stimulierende Medikamente ausgewählt. Zunächst wurden die IC50- und EC50-Werte der oben genannten sechs Zelllinien, die durch die oben genannten Medikamente stimuliert wurden, mit dem CCK-8-Kit ermittelt und der optimale Konzentrationsbereich der Medikamente bestimmt. Danach haben wir die Invasionsfähigkeit, die Migrationsfähigkeit, die Proliferationsrate und die Apoptoserate der Zelllinien nach Medikamentenstimulation durch Transwell-, Wundheilungs-, CellTiter-Glo ® 2.0- und Durchflusszytometrie-Assays ermittelt. Diese Experimente konnten die Veränderungen der Zellfunktionen nach Stimulation der NSCLC-Zelllinien mit Cisplatin, XL-765 und 740Y-P aufzeigen. In den folgenden Experimenten wiesen wir zunächst die Unterschiede in der Expression von PD-L1 in den oben genannten Zellen vor und nach der Medikamentenstimulation durch Immunhistochemie nach. Anschließend wurden die Unterschiede in der Expression von PD-L1, p-AKT (phosphoryliertes AKT), AKT, p-S6 (phosphoryliertes S6) und S6-Proteinen durch Western Blotting nachgewiesen. Schließlich verglichen wir auch die Unterschiede in der PD-L1-mRNA- und Membran-PD-L1-Expression in den Zelllinien vor und nach der Medikamentenstimulation mittels qRT-PCR und Durchflusszytometrie. Ergebnisse: Es gibt fünf Hauptergebnisse, die wir lernen können. 1. Die Zellaktivität, Invasionsfähigkeit und Migrationsfähigkeit der NSCLC/DDP-Zelllinien waren höher als die der entsprechenden naiven NSCLC-Zelllinien, aber die Apoptoseraten der NSCLC/DDP-Zelllinien waren niedriger als die der entsprechenden naiven NSCLC-Zelllinien. Cisplatin und 740Y-P hatten beide eine fördernde Wirkung auf die zelluläre Funktion der naiven NSCLC-Zelllinien, während XL-765 eine hemmende Wirkung hatte. 2. Niedrige Konzentrationen von Cisplatin führten zu einer signifikanten Hochregulierung der Expression von PD-L1 an der Zellmembran und des Gesamt-PD-L1 in naiven NSCLC-Zelllinien, und die Expressionsniveaus tendierten dazu, mit steigenden Konzentrationen von Cisplatin zuzunehmen.3. die PD-L1-Expression war in NSCLC/DDP-Zellen signifikant höher als in naiven NSCLC-Zellen, wurde aber nach Stimulation mit XL-765 signifikant reduziert. In den Zelllinien H1975 und H1975/DDP konnte eine niedrige Konzentration von Cisplatin und 740Y-P die Expression von p-AKT, AKT, p-S6, S6 und PD-L1 erhöhen, während XL-765 die Expression verringern konnte. 5. Der Grad der Zunahme und Abnahme der Expression von p-AKT, AKT, p-S6, S6 und PD-L1 korrelierte mit der Behandlungszeit und der Behandlungskonzentration der Medikamente. Schlussfolgerung: Unsere Ergebnisse deuten darauf hin, dass niedrige Konzentrationen von Cisplatin die Expression von PD-L1 in NSCLC-Zellen fördern können, was zu Cisplatin-Resistenz und Immun-Escape in NSCLC-Zelllinien führt. Der überaktivierte PATSP war an der Cisplatin-induzierten Hochregulierung der PD-L1-Expression beteiligt. Die Hemmung dieses Signalwegs führte zu einer verringerten PD-L1-Expression und kehrte in gewissem Maße die durch Cisplatin-Resistenz verursachte Immunflucht in NSCLC-Zelllinien um

A Real-time SAR Echo Simulator Based on FPGA and Parallel Computing

This paper designs and implements a SAR (Synthetic Aperture Radar) real-time echo simulator based on multi-FPGA parallel computing. The one-dimensional frequency-domain Fourier transform algorithm is used in the simulator, and the echo signal model and the rapid calculation algorithm of impulse response function are introduced. The pipeline compute structure, multichannel parallel computing and procedure flow design are the key technologies of the simulator, which are also presented in details. And finally, the validity and correctness of the SAR echo simulator are verified through the imaging results of the point-array target and the nature scene target

Spatial Finite Element Analysis for Dynamic Response of Curved Thin-Walled Box Girder Bridges

According to the flexural and torsional characteristics of curved thin-walled box girder with the effect of initial curvature, 7 basic displacements of curved box girder are determined. And then the strain-displacement calculation correlations were established. Under the curvilinear coordinate system, a three-noded curved girder finite element which has 7 degrees of freedom per node for the vibration characteristic and dynamic response analysis of curved box girder is constructed. The shape functions are used as the interpolation functions of variable curvature and variable height to accommodate to the variation of curvature and section height. A MATLAB numerical analysis program has been implemented

Comprehensive Mapping of Common Immunodominant Epitopes in the West Nile Virus Nonstructural Protein 1 Recognized by Avian Antibody Responses

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex

Dual inhibition of AKT‐mTOR and AR signaling by targeting HDAC3 in PTEN‐ or SPOP‐mutated prostate cancer

Abstract AKT‐mTOR and androgen receptor (AR) signaling pathways are aberrantly activated in prostate cancer due to frequent PTEN deletions or SPOP mutations. A clinical barrier is that targeting one of them often activates the other. Here, we demonstrate that HDAC3 augments AKT phosphorylation in prostate cancer cells and its overexpression correlates with AKT phosphorylation in patient samples. HDAC3 facilitates lysine‐63‐chain polyubiquitination and phosphorylation of AKT, and this effect is mediated by AKT deacetylation at lysine 14 and 20 residues and HDAC3 interaction with the scaffold protein APPL1. Conditional homozygous deletion of Hdac3 suppresses prostate tumorigenesis and progression by concomitant blockade of AKT and AR signaling in the Pten knockout mouse model. Pharmacological inhibition of HDAC3 using a selective HDAC3 inhibitor RGFP966 inhibits growth of both PTEN‐deficient and SPOP‐mutated prostate cancer cells in culture, patient‐derived organoids and xenografts in mice. Our study identifies HDAC3 as a common upstream activator of AKT and AR signaling and reveals that dual inhibition of AKT and AR pathways is achievable by single‐agent targeting of HDAC3 in prostate cancer

PI3K and ERK-Induced Rac1 Activation Mediates Hypoxia-Induced HIF-1α Expression in MCF-7 Breast Cancer Cells

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1

A gene pathway enrichment method based on improved TF-IDF algorithm

Gene pathway enrichment analysis is a widely used method to analyze whether a gene set is statistically enriched on certain biological pathway network. Current gene pathway enrichment methods commonly consider local importance of genes in pathways without considering the interactions between genes. In this paper, we propose a gene pathway enrichment method (GIGSEA) based on improved TF-IDF algorithm. This method employs gene interaction data to calculate the influence of genes based on the local importance in a pathway as well as the global specificity. Computational experiment result shows that, compared with traditional gene set enrichment analysis method, our proposed method in this paper can find more specific enriched pathways related to phenotype with higher efficiency