In recent years, lots of studies have reported that m6A modification and m6A RNA methylation regulators were involved in cancer progression. However, the m6A level and its regulators in esophageal cancer (ESCA) remain poorly understood. In this study, we analyzed the expression of m6A regulators using The Cancer Genome Atlas data and found 14 of 19 m6A regulators are significantly increased in ESCA samples. Then we performed a univariate Cox regression analysis and LASSO (least absolute shrinkage and selection operator) Cox regression model to investigate the prognostic role of m6A regulators in ESCA, and the results indicated that a two-gene prognostic signature including ALKBH5 and HNRNPA2B1 could predict overall survival of ESCA patients. Moreover, HNRNPA2B1 is higher expressed in high-risk scores subtype of ESCA, indicating that HNRNPA2B1 may be involved in ESCA development. Subsequently, we confirmed that the level of m6A and HNRNPA2B1 was significantly increased in ESCA. We also found that HNRNPA2B1 expression positively correlated with tumor diameter and lymphatic metastasis of ESCA. Moreover, functional study showed that knockdown of HNRNPA2B1 inhibited the proliferation, migration, and invasion of ESCA. Mechanistically, we found that knockdown of HNRNPA2B1 inhibited the expression of de novo fatty acid synthetic enzymes, ACLY and ACC1, and subsequently suppressed cellular lipid accumulation. In conclusion, our study provides critical clues to understand the role of m6A and its regulators in ESCA. Moreover, HNRNPA2B1 functions as an oncogenic factor in promoting ESCA progression via up-regulation of fatty acid synthesis enzymes ACLY and ACC1, and it may be a promising prognostic biomarker and therapeutic target for human ESCA.N6-methyladenosine (m Esophageal cancer (ESCA) is one of the major malignant cancers that threatened human health worldwide , 2. Esop6A) modification is the most abundant modification involved in the progression of different cancers  or removed by m6A demethylases (erasers). In addition, the specific RNA-binding proteins (readers) could recognize and bind to m6A motif, regulating RNA metabolism, including RNA stability, degradation, splicing, transport, localization, translation, and others  dataset, as well as their association with the clinicopathological characteristics. After a comprehensive analysis, we found that HNRNPA2B1 may play a key role in ESCA development. Subsequently, we found that the levels of m6A and its regulator HNRNPA2B1 were significantly increased in ESCA, and HNRNPA2B1 acts an oncogenic role in the progression of ESCA cells, indicating that it may be a promising prognostic biomarker and therapeutic target for human ESCA.In the present study, we systematically analyzed the expression of 19 m6A-related genes in gastric cancer . All 19-gene expression data are downloaded via the R package “TCGA-Assembler.” The expression of 19 m6A-related genes in 160 ESCA tissues and 11 normal esophageal tissues was analyzed via limma package. Next, the violin map was used to visualize the expression of 19 genes in 160 ESCA tissues and 11 normal tissues. We then used the STRING database (http://string-db.org) to analyze the protein–protein interaction (PPI) among 19 m6A regulators. The correlation analysis of 19 m6A regulators was further analyzed by Pearson correlation analysis. To evaluate the association between the expression of m6A regulators and prognosis of ESCA patients, the ESCA cohort was clustered into different groups through consensus clustering analysis with “ConsensusClusterPlus” in R  Cox regression algorithm. The formula of the risk score for ESCA patients' prognosis prediction was as follows: risk score = the sum of each multivariate cox regression coefficient ratio of mRNA multiple each expression of mRNA. Based on the median risk score, we divided the patients into high- and low-risk subgroups. Each patient's survival status, death time, and gene expression profile in two subgroups were presented via “heatmap” and “survival” R packages , were obtained after signed informed consent. The ESCC tissue microarray  was obtained from servicebio . Immunohistochemistry (IHC) was performed according to standard procedures as described previously , 23. InsThe two specific HNRNPA2B1 siRNAs were designed and synthesized by RiboBio : the sequence of si-HNRNPA2B1#1: GGAGAGTAGTTGAGCCAAA and the sequence of si-HNRNPA2B1#2: GCTACGGAGGTGGTTATGA. The HNRNPA2B1 siRNAs and corresponding control siRNA were transfected into the ESCC cells by DharmaFECT4 .https://en.bio-protocol.org/e2095). The experiments procedures have been described previously . Reverse transcription (RT) was performed with HiScript Q RT SuperMix for qPCR . RT–polymerase chain reaction (PCR) was performed with an SYBR Green PCR Kit  on an Applied Biosystems 7900HT sequence detection system (Applied Biosystems), with triplicate reactions. The primers used are listed in For CCK8 assay, after the cells were transfected for 48 h, the cells were plated at a density of 2,000 cells per well in 96-well-plates. After 72 h, cell viability was determined using CCK-8 assay according to the manufacturer's instructions .For clonogenic assay, after the cells were transfected for 48 h, the cells were seeded at a density of 500 cells per well in 12-well-plates and incubated at 37°C for 10–14 days. Then, cells were fixed with methanol for 30 min and stained with crystal violet  for 30 min.For EdU assay, the cells were plated at a density of 10,000 cells per well in 96-well-plates, and the cells were transfected for 48 h. The cell immunofluorescence was determined by the EdU kit according to the manufacturer's instructions . Images of the cells were acquired with a Leica DMi8 system.For wound-healing assay, the transfected cells were grown to confluence in a 12-well-plate. Next, the cells were cultured in RPMI-1640 with 1% FBS for 12 h. The confluent monolayer was then disrupted with a cell scraper and filmed at the indicated hours via Leica DMi8 system. The rate of wound closure was calculated as the ratio of the average distance between the two wound edges and the total cell duration of migration.For Transwell assay, the transfected cells in 100 μL of serum-free medium were seeded in the upper chambers coated with or without 50 μL of Matrigel (BD Biosciences), and 600 μL of culture medium containing 10% FBS was placed in the lower chambers. After 12 h of incubation at 37°C, cells that migrated to the bottom of the membrane were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet  for 30 min, and imaged.The transfected cells were fixed in 4% paraformaldehyde solution on the 12-well-plates, stained with 0.05 μg/mL Nile red  for 10 min, washed with phosphate-buffered saline twice, and then stained with DAPI . Images of the cells were acquired with a Leica DMi8 system.6A-related genes in ESCA tissues and normal tissues from TCGA dataset was analyzed via one-way analysis of variance. Kolmogorov–Smirnov test was used to analyze the relationship between m6A-related genes and clinicopathological characteristics. The median risk score was used as a cutoff value to divide into a high- and a low-risk group. OS was analyzed between different groups through the Kaplan–Meier method. The relationship between the risk score and clinicopathological variables was analyzed through χ2-test. Functional experiments were performed at least three times. The representative data shown are means ± SD; P < 0.05 was considered statistically significant.The expression of 19 m6A RNA methylation regulators  in ESCA tissues and normal tissues from TCGA dataset  (P = 0.064)  . Moreove= 0.064) .6A regulators in the prognosis of ESCA patients, univariate Cox regression was used to analyze the expression of m6A-related genes associated with OS in ESCA TCGA dataset. The results demonstrated that high expression of ALKBH5 was significantly correlated with good OS , but high expression of HNRNPA2B1 was associated with poor OS  (http://gepia.cancer-pku.cn/). The results showed that the expression of HNRNPA2B1 was significantly positively correlated with the expressions of ALKBH5 (P = 0.05501)  than individual ALKBH5 or HNRNPA2B1 by univariate Cox regression, suggesting that the risk signature of them is more reliable for OS prognosis (P = 0.016), whereas whose with a high level of HNRNPA2B1 suffer a poor OS  . Further2–1.022) . Then, wf ALKBH5 . In orde0.05501) . Howeverrognosis . FurtherTo better understand the clinical relevance of two-gene signature in ESCA, we first selected the patients with clinical characteristic variables and divided these patients into low- and high-risk groups, which were assessed by two-gene expression and clinical characteristic variables . Intereshttp://ualcan.path.uab.edu/index.html). Compared with the normal subgroup, the HNRNPA2B1 expression was significantly up-regulated (P < 0.05) in cancer patients with different clinical characteristics . The results showed that the expression of HNRNPA2B1 was significantly positively correlated with the expressions of MKI67 and PCNA, which were the classic biomarkers of proliferative cancer cells  and nine readers  were significantly increased in ESCA tissues, whereas no significant difference was found for the two erasers (FTO and ALKBH5), and most of RNA m6A regulators in our study were overlapped with the previous study (6A regulators, which could be systematically and directly helpful to analyze the interaction between these regulators. Herein, we found there are direct or indirect interactions among the 19 m6A regulators, and the writers METTL3, METTL14, WTAP, and KIAA1429 and the erasers FTO and ALKBH5 may localized in the center of regulatory network. It was also demonstrated that the relationship between most of the m6A RNA methylation regulators is positively correlated, and the KIAA1429 and YTHDF3 genes are most relevant. Subsequently, we confirmed that the RNA m6A levels were significantly higher in 18 cancerous tissues than corresponding normal tissues in ESCC patients via a dot blot assay. These results suggest that RNA m6A modification may be involved in the ESCA development.The mg cancer , 34. Thethylases , 35. Mea cancers . Previouer types . Howeverus study . Meanwhi6A regulators and OS in ESCA via the consistent clustering analysis, and the results showed that cluster 1 had a shorter OS than cluster 2. In addition, the univariate Cox regression analysis and LASSO Cox regression data indicated that a two-gene prognostic signature including ALKBH5 and HNRNPA2B1 could predict OS of ESCA patients. Moreover, high expression of HNRNPA2B1 and low expression of ALKBH5 were indicated as the risk factor for the survival of ESCA, and the combination of these two factors showed more predictive potential than the alone, although the ROC curve did not show robust prediction within 4 and 5 years, which because of that there are too few patients in the fourth and fifth years, which may lead to the instability of the ROC curve. It is reported that HNRNPA2B1 could selectively bind to m6A-containing transcripts via the “m6A-switch,” a mechanism in which m6A weakens Watson–Crick base pairing to destabilize the RNA hairpin structure and thereby exposes the single stranded hnRNP binding motif (We then analyzed the relationship between RNA mng motif . HNRNPA2ng motif –40. HNRNng motif . It alsong motif . In thisde novo synthesis and oxidative metabolism of fatty acids, which provide the necessary raw materials for tumor cell proliferation (de novo fatty acid synthesis, fatty acid β-oxidation, and fatty acid uptake, revealing that de novo fatty acid synthetic enzymes ACLY and ACC1 were markedly positively regulated by HNRNPA2B1. However, the expression of FASN, fatty acid uptake, and fatty acid oxidation genes is inconsistent in the two ESCC cell lines with HNRNPA2B1 deficiency, which may be due to the heterogeneity of the two different ESCC cells. In addition, knockdown of HNRNPA2B1 suppressed cellular lipid accumulation. Collectively, the results reveal that HNRNPA2B1 could accelerate fatty acid synthesis via up-regulation of de novo fatty acid synthetic enzymes ACLY and ACC1.Further, we analyzed the KEGG enrichment of genes, which were correlated with HNRNPA2B1 expression in ESCA patients using TCGA data. The data indicated that HNRNPA2B1 may be involved in fatty acid metabolism of ESCA. We also found that the HNRNPA2B1 level was significantly higher in extreme obese subgroup than other subgroups. The abnormal lipid metabolism of tumor cells is mainly manifested in the activation of feration , 42. Theferation , 44. Sub6A and its regulator HNRNPA2B1 were significantly increased in cancerous tissues of ESCA, and overexpression of HNRNPA2B1 promotes ESCA progression via up-regulation of de novo fatty acid synthetic enzymes ACLY and ACC1. Therefore, HNRNPA2B1 may be a promising prognostic biomarker and therapeutic target for human ESCA.In summary, our findings reveal that the levels of mThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the Ethical Committee of Nanjing Drum Tower Hospital. Written informed consent to participate in this study was provided by the patients.SW, QW, and XZ provided the direction of this manuscript. HG, BW, and KX collected and analyzed the data. LN and YF analyzed and evaluated the IHC of TMA. KX and ZW performed the experiments. QW wrote the manuscript. SW revised this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Due to current insufficient therapies, evaluating novel therapeutic agents for late-stage PCa is of considerable interest for both species. PDA indolylmaleimides showed anticancer effects in several neoplastic cell lines. Herein, a comparative characterization of PDA-66 and PDA-377 mediated effects was performed in human and canine PCa cell lines, which is also the first detailed characterization of these agents on cells derived from solid tumors in general. While PDA-377 showed only weak growth inhibition on human PCa cell lines, PDA-66 inhibited proliferation and induced apoptosis in human and canine cell lines with concentrations in the low micromolar range. Morphological characterization and whole transcriptome sequencing revealed that PDA-66 induces mitotic death through its microtubule-depolymerizing ability. PDA-66 appears to be a worthwhile anti-mitotic agent for further evaluation. The similarities in cellular and molecular response observed in the cell lines of both origins form a solid basis for the use of canine PCa  Prostate cancer (PCa) is the most common malignancy diagnosed among males in almost all western countries . PCa emeCanine tumors are valuable naturally occurring models helping to reveal mechanisms in cancer development, behavior and treatment , 7. ProsVinca alkaloids, are used in various cancers  was cultivated in DMEM/Ham's F-12 medium . The human LNCaP cell line (RRID:CVCL_0395) was grown in RPMI 1640 medium (Biochrom GmbH). The canine cell line TihoDProAdcarc1258 (RRID:CVCL_W737) was established by our group and cultivated in medium 199 . All media were supplemented with 10% heat-inactivated fetal bovine serum  and 2% penicillin/streptomycin (Biochrom GmbH). All cells were cultivated at 37°C in a humidified atmosphere of 5% CO2.Two human and one canine prostate carcinoma cell line were used. The human PC-3 cell line   were applied (0.1% DMSO as control). The cells were observed for 72 h in 150 μl incubation medium under a live cell imaging microscope  at 37°C with 5% CO2. An image of each well was captured every 15 min during the incubation period and these single images were combined to create time-lapse movies.PC-3, LNCaP and CT1258 cells were seeded in 96-well plates with a cell density of 1 × 10Morphological changes mediated by PDA were analyzed by May-Grünwald-Giemsa staining.2 cell culture dishes and covered with 10 ml of culture medium. Per dish and per cell line, 1 x 106 cells were seeded. On the following day, the microscope slides were transferred to new dishes with incubation medium or 0.15% DMSO for the control cells. The slides were exposed to the PDA agents in two different settings. For the first setting, the slides were incubated with 15 μM PDA-66 or PDA-377 for 24 h, an application equal to the cells used in the transcriptomic analyses. For the second setting, the slides were incubated with 5 μM PDA-66 or PDA-377 for 72 h, a concentration that showed moderate effects in all cell lines in live cell imaging. Microscope slides were washed with PBS and air dried at room temperature. The slides were stained in May-Grünwald's eosine-methylene blue solution  (undiluted) for 5 min and rinsed with tap water. Afterwards, they were stained for 15 min in Giemsa's azur eosin methylene blue solution (Merck KGaA) (1:10 dilution) and rinsed thoroughly with tap water again. The slides were left to air dry before analysis.Microscope slides were placed in 60 cmProliferative index of cells in response to PDA-66 and PDA-377 exposure was evaluated using the assay Cell Proliferation ELISA, BrdU (colorimetric) . The assay measures the incorporation of the thymidine analog 5-bromo-2-deoxyuridine (BrdU) into newly synthesized DNA of replicating cells using an anti-BrdU monoclonal antibody.4 cells per well and incubated in 150 μl culture medium for 24 h to allow the cells to attach to the surface. Afterwards, the cells were exposed to different concentrations of the PDA derivatives. The human cell lines were treated with 0.25–10 μM of the derivatives and 0.1% (v/v) DMSO as control. CT1258 cells were exposed to 0.25–25 μM of both derivatives (0.25% DMSO as control) as no significant effect was displayed after 24 h with 10 μM PDA-66. The proliferation assay was carried out in accordance with the manufacturer's protocol for adherent cells with the exception that BrdU was added simultaneously with the PDAs. The reaction products were quantified by measuring the absorbance at 370 nm minus the absorbance at 492 nm over a 30 min period using the Multi-Mode Reader Synergy 2 . Each single experiment was performed 8-fold.All three cell lines were seeded in 96-well plates with a density of 1 × 10After this point, no further tests were performed with PDA-377 due to its limited effect on the human cell lines.The concentrations used for cell count analysis and apoptosis are based on the 72 h values of the BrdU assay. Concentrations were chosen, that lead to 50% inhibition (2.5 μM PDA-66 in PC-3 and 5 μM PDA-66 in LNCaP). These values were chosen to ensure that there would be some margin in both directions to calculate possible significances, even if the other tests would show higher or lower efficiencies. Based on this criterion, 5 μM PDA-66 should have been used for the CT1258 cell line. However, this concentration showed no effect after 24 h. As it was crucial to be able to detect effects early after exposure for the transcriptomic analysis, 15 μM was used for this cell line, the first concentration to show a significant effect in the BrdU assay after 24 h of exposure.4 cells per well in 1 ml culture medium and incubated overnight. Based on the proliferation assay, PC-3 cells were treated with 2.5 μM PDA-66 and 0.025% DMSO, LNCaP with 5 μM PDA-66 (0.05% DMSO), and CT1258 cells with 15 μM PDA-66 (0.15% DMSO). After 24 h, 48 h and 72 h, the cells were detached by TrypLE™ Express  and counted via automatic cell counter . The experiment was carried out in biological replicates three times.Cells were seeded in 24-well plates with a cell density of 5 × 10RRID:SCR_014489).Apoptosis rates were determined by staining cells with the fluorescence dyes Annexin V FITC and Propidium Iodide (PI)  in accordance with the manufacturer's protocol and determined by flow cytometry using a FACSCaliburTM . In addition to the cell specific concentrations used in the cell count analysis, two lower concentrations were used to demonstrate that the induction of apoptosis already starts at lower concentrations. The data were evaluated using BD CellQuest software , LNCaP to 1.0, 2.5, and 5.0 μM PDA-66 (0.05% DMSO), and CT1258 to 7.5, 10 and 15 μM PDA-66 (0.15% DMSO). After the drug exposure period, the medium was saved to collect the non-adherent cell fraction to count all treated cells regardless of their condition. Remaining adherent cells were detached by TrypLE™ Express  and combined with the medium cell fraction. In addition to the double stained samples, for each cell line and each incubation time point, single stained controls were measured. Exposure of the cells to PDA-66 and DMSO as reference was conducted in biological replicates three times.All three cell lines were seeded in 12-well plates with a cell density of 2 × 10As PDA-66 is a strong inducer of apoptosis, the transcriptomic analysis was performed on early time points (12 and 24 h). This early time points, before all the apoptosis signaling cascades are fully active and most transcripts would be cell death related, were chosen for the best chance to detect the direct effects of PDA exposure. As 15 μM was the first concentration to show significant effects in CT1258 after 24 h, this concentration was used for all cell lines to keep the transcriptomic data comparable (0.15% DMSO for the control groups). After 12 and 24 h, the cells were harvested and total RNA was extracted using the AllPrep DNA/RNA Mini Kit  in accordance with the manufacturer's protocol. For each treatment condition (DMSO control and PDA-66 exposed cells), three independent biological replicates were prepared and sequenced.RRID:SCR_010910). For each of the 24,581 annotated protein-coding canine genes (EMBL gene ID nomenclature), the aligned reads were counted using the R package GAGE (RRID:SCR_017067). For the human PC-3 and LNCaP, mapping and read counting were conducted using the RNA Express (version: 1.0.0) workflow within the Illumina basespace environment . In brief, after mapping to the human HG19 reference genome using the STAR (2.3.1s) aligner  read counts for 23,710 annotated RefSeq genes were generated.RNA integrity numbers (RIN) were determined using a Bioanalyzer 2100 . Sequencing libraries were prepared using 1 μg total RNA with RIN > 7. PolyA RNA was enriched and ligated to sequencing adapters using the NEBNext Ultra RNA preparation kit  in accordance with the manufacturer's protocols. Single-read sequencing (75 bp) was conducted on an Illumina NextSeq500 . Sequences were aligned to the canine genome  using the Burrows-Wheeler Aligner (BWA)   using SAS enterprise guide 7.1 . Differences were considered statistically significant for p < 0.05. IC50 values were calculated using GraphPad Prism 7.02 .Within each experiment, results were described using the mean values of the replicates. The graphs show the mean ± standard deviation. Significant differences between treatment and control were calculated by Dunnett's multiple comparison test for the confidence levels 95, 99, and 99.9%  or student's RRID:SCR_012802). The average total read counts mapped within annotated genes were 12.2M (STD: 0.7M), 15.8M (STD: 1.1M), 13.5M (STD: 0.9M) for CT1258, PC-3 and LNCaP samples, respectively. Each PDA-66 treatment group (12 or 24 h) was compared to the respective control cells treated with DMSO. After multidimensional scaling and plotting of the data, one PC-3 DMSO treated control sample was identified as outlier and was excluded from further analyses so that the PC-3-24 h-DMSO group consisted of only two independent samples. Genes with a false discovery rate adjusted p-value (FDR) of < 0.001 were considered to have significantly different expressions compared to the control samples. These genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for functional enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Tool (RRID:SCR_001881).Differential gene expression analyses for canine and human samples were conducted using the BioconductorR package edgeR 3.14.0   indicated the induction of cell death.Consistent with live cell imaging observations and the proliferation assay, a significant change in the total number of cells was observed 48 h after PDA-66 application in all tested cell lines compared to their tested control cells . While tPDA-66 significantly induced apoptosis in all three PCa cell lines compared to the DMSO-exposed controls at different concentrations . ApplicaAll cell lines displayed a higher number of differentially expressed genes  after 12 h compared to the 24 h treatment groups , correlaIn the present study, the effects of PDA-66 and PDA-377, two indolylmaleimide derivatives, were comparatively evaluated on three prostate carcinoma cell lines with a diverse genetic background. The human PC-3 and LNCaP cells are among the most used and best described PCa cell lines , 35, botBoth PDA derivatives caused two distinct morphological changes in all tested cell lines. Live cell imaging and May-Grünwald-Giemsa staining revealed clear apoptotic features through observed pyknosis and karyorrhexis in May-Grünwald-Giemsa staining as well as the formation of apoptotic bodies in live cell imaging. These features match the proposed mechanism of induced mitotic arrest and subsequent MD. Secondly, some enlarged multinucleated cells formed. Cells can overcome the mitotic arrest before MD is triggered; a process known as slippage. After DNA replication in the S phase and escaping the mitotic arrest, these cells fail cytokinesis and/or the equal distribution of chromosomes, leading to enlarged multi-nucleated cells similar to those we observed , 43, 44.Proliferation was measured via BrdU assay to quantify growth inhibition seen in live cell imaging. PDA-66 caused significant and dose-dependent anti-proliferative effects in all tested PCa cell lines. The herein demonstrated doses to inhibit proliferation significantly by at least 50% were between 5 and 10 μM PDA-66 within 72 h. The analyzed PCa cell lines were slightly less sensitive compared to other neoplastic cell lines like human neuroblastoma, human acute lymphoblastic leukemia or canine lymphoma –17. ConsPDA-377 also induced anti-proliferative effects in all tested cell lines. However, for both human cell lines, the inhibition of proliferation was limited and most likely dose-independent, as a comparable decrease in proliferation was measured for almost all tested concentrations after 48 h. The proliferative index did not drop below 50%. In the canine cell line, the proliferation inhibition was clearly dose-dependent. After a 72 h incubation period, the proliferative index dropped below 10% with an IC50 of 3.14 μM. PDA-377 may be suitable for the treatment of prostate cancer in dogs. Nevertheless, no further testing with this substance was conducted, as the limited effect on the human cell lines contradicts the cross-species approach with dogs as a model to generate transferable findings to the benefit of both species.Vinca alkaloids or colchicine-binding site drugs, lead to failures in mitotic spindle formation and therefore chromosome segregation, which triggers the spindle assembly checkpoint . SAC activation causes mitotic arrest in the metaphase of the M phase of the cell cycle. Within the enriched KEGG cell cycle pathway, several important genes involved in SAC are upregulated after PDA-66 exposure, e.g., BUB1B, CDC20, CDK1, and MAD2. These DEGs are commonly involved in MD , a gene that plays an important role in interphase checkpoint activation (Interestingly, there was also an upregulation of genes involved in G1/S phase cell cycle progression and DNA replication, especially within the two human cell lines. Given the M phase cell cycle arrest induced by SAC, an enrichment of S phase transcripts is unexpected. Moreover, G1/S phase transcripts were downregulated in canine lymphoma cell lines after PDA-66 exposure . Howevertivation .The observed morphological changes and the transcriptome analysis match other anti-mitotic drugs causing MD. While most cells die as a result of this intrinsic cell death, some cells remain in a polyploid state after mitotic slippage. These cells are usually senescent and therefore incapable of further proliferation. However, they bear the risk of reinforcing the tumor, as their potential proliferation can lead to aneuploid cells, a hallmark of cancer , 50, 52.PCa cell lines of human and canine origin showed comparable cellular and molecular reactions, including significant inhibition of proliferation and induced apoptosis after PDA-66 incubation. Furthermore, efficacy of PDA-66 was not influenced by the androgen status of the cell line. As PDA-66 mediates its effects in a hormone-independent manner through microtubule destabilization, the compound is suitable to be used in androgen-dependent as well as -independent PCa subtypes. Based on the findings of this study, PDA-66 appears to be a worthwhile agent for further evaluation and potential treatment of PCa in dogs and, if successfully introduced, also in humans.in vitro characterization of PDA-66 effects on PCa cells encourage, as a next step, to initiate an early clinical trial in dogs to evaluate the compound in naturally occurring PCa in the presence of an immune system. First, however, a pharmacokinetic study in dogs needs to be performed. An early study in mice revealed that i.p. application of 100 mg/kg PDA-66 once per day was well-tolerated (The cellular and molecular olerated . The valolerated –57. Treahttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162832.The original contributions presented in the study are publicly available. This data can be found at: JS carried out partial BrdU assay, May-Grünwald-Giemsa staining, cell treatment and RNA extraction for whole transcriptome analysis, performed data analysis, interpretation and visualization, and drafted the manuscript. IN provided the resources for all cellular analyses, supervised the study, and revised the manuscript. JB performed transcriptomic sequencing and revised the manuscript. DJ carried out partial BrdU assay, live cell imaging, cell count, and flow cytometry analyses. CR helped with primary study design. AP-D synthesized the PDA compounds. AR participated in initial compound generation. LH evaluated the May-Grünwald-Giemsa staining. MB supervised PDA compound design and synthesis. BB and ES provided the resources for the transcriptomic analysis and supervised the transcriptomic work packages. CJ and HM performed the primary study design. In addition, HM supervised the study and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors HM.


Granulosa cell tumor treatment is challenging as there are few effective options besides surgery. In this study, we obtained tumor tissue from patients at surgery and cultured tumor cells in the laboratory. After sufficient expansion, we tested the effects of current treatments, such as chemotherapy and anti-hormonal treatment, and novel anti-cancer treatment options on cell survival. Results were generated within three weeks after tissue collection. We found that all drugs were ineffective when used as single treatments; however, some combinations were very effective. The PI3K protein inhibitor alpelisib was effective in combination with chemotherapy and achieved 50% cell death at assumed tolerable patient plasma concentrations. In conclusion, this study shows an approach to rapidly establish patient-derived cell lines for drug screens. The effectiveness of combined treatment with alpelisib and chemotherapy in granulosa cell tumors should be further investigated and may be a promising novel treatment option in patients with a granulosa cell tumor.Adult granulosa cell tumors (AGCTs) arise from the estrogen-producing granulosa cells. Treatment of recurrence remains a clinical challenge, as systemic anti-hormonal treatment or chemotherapy is only effective in selected patients. We established a method to rapidly screen for drug responses in vitro using direct patient-derived cell lines in order to optimize treatment selection. The response to 11 monotherapies and 12 combination therapies, including chemotherapeutic, anti-hormonal, and targeted agents, were tested in 12 AGCT-patient-derived cell lines and an AGCT cell line (KGN). Drug screens were performed within 3 weeks after tissue collection by measurement of cell viability 72 h after drug application. The potential synergy of drug combinations was assessed. The human maximum drug plasma concentration (Cmax) and steady state (Css) thresholds obtained from available phase I/II clinical trials were used to predict potential toxicity in patients. Patient-derived AGCT cell lines demonstrated resistance to all monotherapies. All cell lines showed synergistic growth inhibition by combination treatment with carboplatin, paclitaxel, and alpelisib at a concentration needed to obtain 50% cell death (IC50) that are below the maximum achievable concentration in patients (IC50 < Cmax). We show that AGCT cell lines can be rapidly established and used for patient-specific in vitro drug testing, which may guide treatment decisions. Combination treatment with carboplatin, paclitaxel, and alpelisib was consistently effective in AGCT cell lines and should be further studied as a potential effective combination for AGCT treatment in patients. FOXL2 c.402C > G (C134W) mutation testing can be performed. This specific mutation is a hallmark of AGCTs and is present in 90–97% of patients . Su. Su® memeatments , includieatments . Next, t2 values were used to indicate how well the model fit the data  was plotted against the drug concentration range of each drug (in log10[μM]) using GraphPad Prism 8.3.1. Values that exceeded 100% survival  were normalized to 100% survival. Similarly, values below 0% cell survival  were normalized to 0% survival. Values exceeding this deviation were excluded. Dose–response curves were obtained using the model “log(inhibitor) vs. variable slope ” to fit the dose–response curves and Rthe data . We inteIn addition to the 11 monotherapies, 12 drug combinations were tested  simultanSimilar to monotherapies, the safety for in vivo use of drug combinations as assessed by comparison to Cmax or Css values of the individual drugs . The dosThis study shows that rapid, systematic, patient-specific AGCT drug screens are feasible and can be used to test individual responses to existing monotherapies and novel combinations. The findings from a set of 13 cell lines demonstrated synergistic growth inhibition of the PI3K inhibitor alpelisib combined with the current first-line chemotherapeutic agents carboplatin and paclitaxel at potentially tolerable concentrations in vivo. Therefore, alpelisib, carboplatin, and paclitaxel may be a promising novel combination for treatment of recurrent AGCTs.


Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer. Therefore, new therapeutic modalities are urgently required, particularly for tumors with high genetic heterogeneity and poor response to current treatments. Cancer is caused by the accumulation of dozens of mutations which lead to uncontrolled cell proliferation, but the precise biochemical changes and genetic background is unique to each individual cancer [Despite decades of research and an unprecedented advancement of therapeutic options , cancer l cancer . Even wil cancer . Cancer l cancer .MicroRNAs (miRNAs) are naturally occurring short (~22 nucleotide) double stranded RNA molecules with a unique nucleotide sequence (seed sequence) that binds, at least partially, to complementary mRNA sequences. This leads to mRNA degradation or suppressed translation which then silences gene expression , 5. The CCND1, KRAS, RASSF1, STMN1, and MCL1 [The therapeutic potential of miR-193a was identified in a high-throughput functional lentiviral-based screen of miRNAs in tumor cells and the 3p arm (miR-193a-3p) was subsequently demonstrated as the relevant active entity in screening assays . The tumand MCL1 , 24–26. in vivo. To overcome the challenge of delivering 1B3 to tumors upon systemic administration in experimental tumor models, it has been formulated in a novel lipid nanoparticle (INT-1B3). We recently evaluated the pharmacodynamic and pharmacokinetic profile of INT-1B3 and showed that it is efficiently delivered to tumor tissues and has a favorable safety profile (manuscript in preparation). In addition, RNA-sequencing and subsequent pathway analysis in a panel of cell lines confirmed the multi-targeted nature of 1B3 by revealing hundreds of target genes with key roles in tumor cell survival, proliferation, and migration [in vitro by silencing different 1B3 target genes. We also confirm the anti-tumor activity of INT-1B3 in experimental human tumor-bearing mouse models, indicating its potential as promising novel modality for therapeutic intervention in oncology.1B3 is a synthetic chemically modified miR-193a-3p mimic with 100% sequence homology to the mature miR-193a-3p guide (antisense) strand and a fully complementary passenger (sense) strand. The molecule incorporates chemical modifications on its passenger strand to improve strand-selection and prevent inflammatory pathway activation igration . In the TP53 deletion or loss of function  and KRAS activating mutations . The level of cell-associated oligonucleotide (1B3) measured after transfection with 10-nM 1B3 was 100- to 1,000-fold higher compared to mock-transfected cells , demonstrating significant transfection efficacy. As indicated in the Materials & Methods section, the 2-tailed RT-qPCR protocol detects both transfected 1B3 and endogenous miR-193a-3p. Notably, the increase after 1B3 transfection significantly exceeds the differences in basal expression among the tested cell lines. Further, 1B3 overexpression downregulated expression of the 1B3 target cyclin D1 (CCND1) by ~50% at the mRNA level  and completely abolished expression at the protein level , strongly demonstrating not only drug uptake, but efficient target engagement. These observed effects were 1B3-specific, as transfection of ‘negative’ miRNA control (3A1) did not lead to any significant changes in CCND1 mRNA or CCND1 protein expression. 3A1 is based on a commercially available random sequence miRNA that has been validated to not produce identifiable biological effects and is routinely used as a ‘negative’ miRNA control.To characterize the effect of 1B3 in cell-based assays, 1B3 was transiently transfected in a panel of human tumor cell lines from six different cancer types including colon (HCT116), liver , non-small cell lung carcinoma , skin (A2058), pancreatic (PANC-1), and breast (BT-549). The selected cancer cell lines had a variety of genetic backgrounds including Overexpression of 1B3 reduced cell number in every cell line by 25–93% compared to the mock control 96 h after transfection . The ‘neKRAS wild type, TP53 mutant, lung carcinoma cells (H460 and A549) had a very high caspase activation of 9- and 4.5-fold, respectively. Conversely, 1B3 induced only a mild caspase 3/7 activation (~1.3-fold) in the hepatocellular carcinoma (HCC) cancer cell lines SNU-449, HUH-7, and Hep3B. As with the effect on nuclei count, a milder effect was observed after treatment with 1-nM compared to 10-nM 1B3 . To validate the effect of 1B3 on apoptosis, 1B3 was overexpressed in the two cell lines with the highest caspase 3/7 activation  and the two cell lines with the lowest caspase 3/7 activation , and the level of cleaved poly (ADP-ribose) polymerase (PARP) was measured by Western blot. As expected, A549 and H460 had a strong increase in cleaved PARP compared to mock and 3A1-transfected cells, while no cleaved PARP was detected in SNU-449 or HUH-7 cells . Additionally, the presence of apoptotic cells after 1B3 overexpression in A549 and H460 cells was confirmed by flow cytometric analysis of annexin V/PI-stained cells (data not shown). These data show that 1B3 causes apoptosis in some, but not all, of the cancer cell lines tested, indicating that it is one of the anti-proliferative mechanisms of 1B3.As several biological processes can contribute to reduced cell number, including cell death, the effect of 1B3 on caspase 3/7 mediated apoptosis was investigated. Overexpression of 1B3 induced caspase 3/7 activity by more than 2-fold compared to the mock control in eight of the 11 cell lines tested . The effPremature cell cycle arrest can also reduce cell number over time, therefore the effect of 1B3 on the cell cycle was investigated using image based DNA-content analysis . As showBecause 1B3 overexpression reduced cell number but did not always lead to a strong caspase 3/7 induction (apoptosis), the effect of 1B3 on cell senescence (type of growth arrest that primarily occurs in aged cells) was investigated in a subset of cell lines. The cell lines selected represent a range of apoptotic response to 1B3 including SNU-449, Hep3B, A2058, and H460. To ensure that the effect of 1B3 on cell count did not bias the results, a total of 500 cells was counted for each condition. Overexpression of 1B3 increased the number of cells expressing the senescence marker, SA-β-gal, compared to mock and 3A1-transfected cells in all cell lines tested . A repreBoth G2/M cell cycle arrest and cell senescence are associated with an accumulation of DNA replication errors , therefoFinally, the effect of 1B3 on cell migration was investigated in a range of cancer cell types including A549, A2058, and SNU-449. The strongest effect on transwell cell migration was observed in A2058 and SNU-449 cells , while a 43% decrease was observed in A549 cells when compared to mock . AlthougTaken together, these data shows that transfection of 1B3 induces a range of anti-cancer phenotypes in a panel of cancer cell lines, supporting its potential as a novel cancer therapeutic by targeting multiple mechanisms .CCND1, CDK6), proliferation (KRAS), apoptosis , and cytoskeletal organization (STMN1). As the effects of 1B3 varied depending on cellular context, the effects were investigated in several human cancer cell lines representing three different cancer types.To further investigate the mode of action through which 1B3 exerts its effects on tumor cells, selected individual 1B3 target genes were silenced using siRNAs, and their effect on key phenotypes measured. MiRNAs suppress the expression of numerous different target mRNAs. We therefore tested siRNAs against several known and validated 1B3 mRNA targets which have been documented as critical for its tumor suppressive functions. These include regulators of cell cycle .Furthermore, the siRNAs tested had a diverse effect on cell cycle arrest in five different cell lines (data summarized in Supplementary Table 1). Again, no siRNA mimicked the exact effect of 1B3 in all cell lines. For example, siCCND1, siCDK6, siMCL1, and siYWHAZ phenocopied the effect of 1B3 by inducing a G0/G1 arrest, but only in one cell line (H1299). Nonetheless, and consistent with the effect of 1B3, siSTMN1 induced a G2/M arrest in three cell lines . These data suggest that although some of the 1B3 targets tested play a role in cell cycle arrest, this effect is cell line-dependent and does not always replicate the effect of 1B3.KRAS and MCL1 to induction of cellular senescence was measured. Neither siRNA mimicked the exact effect of 1B3 in all cell lines tested  (versus the 11-fold increase by 1B3). siMCL1 on the other hand did not induce senescence in any of the cell lines (data not shown). This shows that in some contexts, silencing of KRAS may be responsible for 1B3 induced senescence, although this effect was not observed in all cell lines.Next, the contribution of 1B3 targets nd H460) . AlthougKRAS and STMN1 in reduction of migration was assessed  and transfected them with combinations of siRNAs, then measured the effect on cell count  was observed between individual animal plasma AFP levels and tumor weights . Notably, INT-1B3 treatment resulted in a significant reduction in median AFP levels over time, highlighting its anti-tumor activity in the Hep3B model (p = 0.0005), reaching the same growth inhibition as the kinase inhibitor sorafenib  in the tumors .To evaluate INT-1B3 in the experimental human HCC Hep3B tumor model, Hep3B tumor fragments were implanted into the left liver lobe (orthotopic tumor model) of SCID/Beige mice. After tumor establishment, animals were treated intravenously with 10 mg/kg/administration INT-1B3 or vehicle control (PBS) for three consecutive days in the first week and maintained on a twice weekly treatment schedule for an additional three weeks . As it i3B model . INT-1B3orafenib  used as 3 treatment with 3 mg/kg/administration INT-1B3 or vehicle control (PBS) was administered intravenously daily for five days during the first week then twice weekly for the next two weeks (p = 0.0062) . This clearly indicates that 1B3 is effectively delivered and functional in experimental tumor models and leads to significant anti-tumor activity.To extend these findings, efficacy of INT-1B3 on tumor growth was further investigated in the human melanoma A2058 subcutaneous tumor model. Once tumors reached a median volume of ~150 mmwo weeks . Tumor vwo weeks . The eff 0.0062) . Four anin vitro and in vivo. 1B3 consistently reduced cancer cell proliferation, through a range of mechanisms, in a variety of cancer cell lines. This confirms our recent transcriptome-wide analysis of differential gene expression demonstrating 1B3 as a strong phosphatase and tensin homolog (PTEN) pathway activator which governs various cell processes including tumor cell survival, proliferation, and migration [Cancer utilizes an array of mechanisms to escape cell death and proliferate without control . MiRNAs igration . Our resin vitro are in agreement with data from a miR-193a-3p mimic screen in a panel of 122 human cancer cell lines from 13 different cancer types [The anti-proliferative effects of 1B3 er types . In thater types . In lineer types , 33 and CCND1, CCND2, CCNE2, CDK2, CDK4, and CDK6 [TP53 expression deficient repair could lead to further DNA damage accumulation and G2/M arrest. Nevertheless, the cell cycle is controlled by several factors and further investigation is required to dissect the specific role of 1B3 on this phenotype in different genetic contexts.MiR-193a-3p has been described as a cell-cycle regulating miRNA due to its silencing of several important cell cycle regulators, including and CDK6 , which wand CDK6 . As expeand CDK6  demonstrand CDK6  observedKRAS mutant cell lines HCT116, H460, A549, and PANC-1 cells were among those with the strongest response to 1B3, but proliferation was also reduced in wild-type KRAS expressing cells such as A2058, H1299, and SNU449. Expression of oncogenic KRAS is linked to aggressive disease and therapeutic resistance, yet despite years of effort no effective inhibitor has reached the clinic [KRAS-driven tumors, but also for tumors with wild-type expression of this oncogene. This broad application of 1B3 in different oncogene-driven tumors is supported by the 50% tumor growth inhibition of BRAF-mutated A2058-derived tumors after INT-1B3 treatment in vivo.An important observation is that some effects of 1B3 differ between human cancer cell lines. As illustrated by the different cell cycle arrest patterns and caspase 3/7 activation, the genetic background of the cancer cell may influence the exact phenotype induced by 1B3 overexpression. Despite different upstream events, 1B3 overexpression reduced proliferation in every cell line, regardless of genetic background. For example, the e clinic , 36. ConCCND1, cKIT, HMGB1, GRB7, PLAU, RASSF1, PAK4, S6K2, STMN1, and PTP1B [The exact mechanism through which miR-193a-3p induces an anti-cancer effect has been widely studied and although it is almost universally described as a tumor-suppressor miRNA, the genes which it targets are not always consistent. While some studies connect the anti-proliferative effect of miR-193a-3p mimics to a single gene target, our data suggests this effect is the result of unique, simultaneous, regulation of multiple mRNAs. In support of this, our recent transcriptome wide analysis of six human cancer cell lines uncovered activation of the PTEN tumor suppressor pathway as one of several key mechanisms behind the 1B3 mode of action . 1B3 exend PTP1B , 38–43. nd PTP1B . This cond PTP1B , 46.in vitro data suggest that in human cancer cells: 1) 1B3 overexpression leads to an anti-cancer effect, although the precise downstream mechanism varies between cell lines; 2) the anti-cancer effect of 1B3 is due to simultaneous targeting of multiple target genes, rather than one individual gene; and 3) the target genes responsible for the anti-cancer effect of 1B3 vary between cell lines. Thus, the broad anti-cancer effect of 1B3 lends it to a potentially useful and effective agent for therapeutic intervention in oncology. Cancer is a complex, multigenic, and heterogeneous disease, and a single patient will often carry cells with unique genetic origins. Therefore, a miRNA therapeutic which regulates multiple pathways, or multiple elements within a pathway, has the potential to destroy several different tumor subclones and might be less subject to drug treatment-acquired resistance [Overall, our sistance , 12. In sistance , 48.in vivo delivery remains a challenge. Indeed, most in vivo investigations of miR-193a-3p implant stably transduced tumor cell lines, rather than treating established experimental tumors. Our study extends the findings of several groups who have shown decreased tumor growth and metastasis of miR-193a-3p transduced cells in vivo [in vivo (manuscript in preparation). In the current study, we extend these findings by demonstrating that INT-1B3 significantly delays tumor growth in both HCC and melanoma mouse models. Interestingly, Seviour et al. [Despite growing evidence of the therapeutic value of miRNAs, effective  in vivo , 43, 49.r et al.  used a lin vitro. Some of these effects  can be broadly applied to all cancer cell lines, while others  are unique to specific cell lines and genetic backgrounds. Using both individual and combined siRNA(s) we showed that no individual 1B3 target reproduced the miRNA effect which suggests that multiple, simultaneous, gene repression by 1B3 is more effective at reducing tumor cell growth than single gene targeting. These data add to the growing body of evidence that miR-193a-3p has an anti-cancer effect through several mechanisms, and that this overall anti-cancer effect is conserved regardless of tumor origin or genetic background. Finally, in two different in vivo models we showed a significant reduction of tumor growth. Together, this evidence supports the further clinical development of INT-1B3 as an anti-cancer therapeutic molecule.In summary, we have shown that the miR-193a-3p mimic, 1B3, consistently suppresses several pro-tumorigenic phenotypes The miR-193a-3p mimic, 1B3, and miRNA control 3A1  were manufactured by BioSpring GmbH . The nucleotide sequences are as follows (with limited 2′-O-methyl nucleotide modifications on the passenger strand ):(3′)-TTUUGACCGGAUGUUUCAGGGU-(5′)1B3 passenger (sense) strand: (5′)-AACUGGCCUACAAAGUCCCAGU-(3′)1B3 guide (anti-sense) strand: (3′)-TTAAUGCAGCAGCGCAGCAAU-(5′)3A1 passenger (sense) strand: (5′)-UUACGUCGUCGCGUCGUUATT-(3′)3A1 guide (anti-sense) strand: in vivo. INT-1B3 represents 1B3 formulated in lipid nanoparticles [Small double stranded oligonucleotides, such as siRNAs or miRNAs targeting RNA, are easily degraded by nucleases and have low membrane permeability due to their high molecular weight and negative charges. Hence, an efficient drug delivery system is needed to improve their therapeutic efficacy articles  was manuIn addition, several siRNAs were used in the presented studies: siPOOL (negative siRNA control) and SMARTpool siRNAs  mRNA, respectively) were obtained from Dharmacon. Each SMARTpool contains four unique siRNA sequences targeting the same mRNA target.The human cell lines Hep3b (HCC), SNU-449 (HCC), H1299 (NSCLC), H1975 (NSCLC), H460 (NSCLC), A2058 (melanoma), BT-549 (TNBC), HCT116 (colon), PANC-1 (pancreatic), and A549 (NSCLC) cells were obtained from ATCC. Human HuH-7 (HCC) cells were obtained from the Japanese Cancer Resources Bank (JCRB). All cell lines were processed and cultured according to the vendor’s instructions. Cells were seeded at 80% confluency into 6-well culture plates 4 h before transient transfection. For single agent transfection the indicated concentrations of either 1B3, 3A1, siPOOL, or SMARTPOOL siRNAs were mixed with Lipofectamine RNAiMax (ThermoFisher) and added dropwise to the cell monolayer. For combination transfection, siRNAs were combined at the concentrations listed in For cell count and high content cell cycle analysis, transfected cells were seeded into triplicate wells of black-walled, clear bottom 96-well plates (Greiner) at the appropriate cell density to reach confluence 72 h later. Forty-eight, 72 and 96 h after transfection, media was removed and replaced with 100 μL staining mix containing 0.25% PFA, 0.075% saponin and 1 μg/mL Hoechst 33342 in PBS. Cell imaging was performed on the Thermo CellInsite Automated Imager using the inbuilt high content screening software to identify cell nuclei. Fifteen images/well using the 386_23 filter were captured at 10× magnification. High content cell cycle analysis was performed using nuclei intensity information as described previously . Taxol aFor Caspase-Glo 3/7 assay, transfected cells were seeded into triplicate wells of white-walled, clear-bottom 96-well plates (Greiner) at the appropriate cell density to reach confluence 72 h later. The Caspase-Glo 3/7 assay (Promega) was performed 48 and 72 h after transfection, according to the manufacturers’ instructions.For RNA extraction, 100–200 thousand transfected cells (dependent on the cell line) were seeded into 12-well tissue culture plates (Greiner). Twenty-four hours after transfection, media was aspirated from the 80% confluent cell monolayers, and frozen at –80°C. RNA was extracted using TRIzol (Thermo Fisher) according to the manufacturers’ instructions. RNA concentration was measured on NanoDrop One (Thermo Fisher).in vitro, the reverse transcription reaction of 20 ng of total RNAs was performed using 2-tailed RT primers designed by TATAA Biocenter and GrandScript cDNA synthesis Kit (TATAA Biocenter), according to the manufacturer’s instructions. Quantification of the miRNA expression levels were subsequently assessed by quantitative PCR (qPCR) using SYBR Green (Bio-Rad). Results were interpolated with a calibration curve to determine the amount of 1B3 per cell. It is of note that the guide (antisense) strand sequence of 1B3 is identical to the mature human miR-193a-3p. Therefore, primers cannot distinguish between endogenous miR-193a-3p expression and the level of exogenous (transfected) 1B3. All figures are labelled ‘1B3 level’ but it should be assumed that this also includes endogenous miR-193a-3p expression, which is determined in the mock-transfected conditions or PBS control treated animals.For the detection of miRNAs in vivo, the reverse transcription reaction of 100 ng of total RNAs was performed using in-house designed stem-loop RT primers (IDT). Quantification of the miRNA levels were subsequently assessed by quantitative PCR (qPCR) using SYBR Green (Bio-Rad). Cycle threshold (Ct) values were interpolated with a calibration curve to determine the amount of 1B3 per ng tumor tissue. The Stem loop primer sequences used in this study are provided in Supplementary Table 2. As indicated in the previous chapter, the primers used cannot distinguish between endogenous miR-193a-3p expression and the level of exogenous (transfected) 1B3. All figures are labelled ‘1B3 level’ but it should be assumed that this also includes endogenous miR-193a-3p expression, which is determined in the mock-transfected conditions or PBS control-treated animals.For the detection of miRNAs −ΔCt method. ΔCt was calculated by subtracting the geometric mean of the Ct of two reference genes from the Ct of the target genes. Values were normalized to mock to determine target downregulation.For the detection of target mRNAs, 100 ng of total RNA was first transcribed into single stranded cDNA. One μL synthesized cDNA was used in a 20 μL PCR amplification reaction using SYBR Green master mix (BioRAD) on CFX96 Real-Time qPCR machine according to the manufacturers’ instructions. Triplicate reactions were performed for each condition. The qPCR primer sequences used in this study are listed in Supplementary Table 3. Expression values were calculated using 2For protein extraction, cells were seeded into 6-well tissue culture plates at the appropriate cell density to reach confluence 72 h later. Cell pellets were collected 24, 48 and 72 h after transfection by trypsinization. One hundred to 200 μL RIPA buffer , supplemented with protease and phosphatase inhibitor cocktails, was added to harvested cells. Lysates were centrifugated at 15,000× g for 1 h at 4°C, and clarified by removing the cell debris pellet. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). Samples were separated at 120 V by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) on Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad). Proteins were transferred at 200 mA for 2 h to polyvinylidene difluoride (PVDF) membranes in transfer buffer . The membranes were blocked using 5% milk or 5% BSA in Tris-buffered saline with Tween  to prevent non-specific antibody binding. Blots were probed with primary and horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using enhanced chemiluminescence (ECL) reagents. Membranes were stripped by incubation in stripping buffer  for 30 min at 50°C and re-probed as appropriate. Band intensities were quantified by densitometry using ImageJ software and values normalized to the tubulin loading control and shown relative to the mock transfected cells.For the SA-β-gal assay, transfected cells were seeded in 6-well cell culture plates (Greiner) at the appropriate cell density to reach confluence 72 h later. One well of untreated cells was seeded in low-serum (0.1% FBS) media as a positive control for senescence. The SA-β-gal assay was performed 96 h after transfection using the SA-β-gal assay kit  according to the manufacturer’s instructions. Cells were imaged at 10x magnification using a Leica DMIL LED microscope. A total of 500 cells were counted from each well using the image analysis software Fiji .2 incubator. Forty-eight hours after transfection, unmigrated cells were removed from the chamber with a cotton swab and the chambers fixed with 70% ethanol then stained in a 1% Crystal Violet (Sigma) solution. Using a Leica DMIL LED microscope, 10 images per field at 20x magnification were obtained. The number of migrated cells was counted using the imaging software Fiji [For the migration assay, 16 h after transfection in 6-well plates, media was aspirated and replaced in low serum (0.2% FBS) media. After a further 6 h, cells were dissociated as described above and seeded into duplicate wells of 24-well cell culture migration inserts (Corning) at the appropriate cell density to reach confluence 24 h later. Complete media (10% FBS) was added to the bottom well. The cells were allowed to migrate for 24 h at 37°C in a humidified 5% COare Fiji . To cont3 piece of a subcutaneously grown Hep3B tumor into the left liver lobe (orthotopic tumor model). Tumor development was monitored by measurement of serum alpha-fetoprotein (AFP) which serves as tumor marker and indicator of liver tumor development [6 A2058 cells in 0.1-mL PBS. Once the mean subcutaneous tumor size reached ~150 mm3 , animals were randomized in the various experimental groups, and animals were treated with either vehicle (PBS) or 3 mg/kg/administration of INT-1B3 by repeated bolus injection (i.v) daily for 5 days during the first week, then maintained on a twice weekly (‘Monday/Thursday’) schedule for the next 2 weeks. Tumor volume was monitored by caliper-aided measurements twice a week throughout the study.Studies with the human HCC Hep3B and human melanoma A2058 experimental tumor models were performed at Crown Bioscience Inc  and Oncotest/Charles River , respectively. Study protocols were reviewed and approved by the Institutional Animal Care and Use Committee, and were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care. For the experimental Hep3B tumor model, Hep3B tumor cells were first injected into the flank of Balb/c immune-compromized (Nude) mice to form subcutaneous tumors. Then, 7- to 8-week old female SCID/Beige mice were implanted with a single 2-mmelopment . When in


Mastoid surgery is an aerosol-generating procedure that involves the use of a high-speed drill, which produces a mixture of water, bone, blood and tissue that may contain the viable coronavirus disease 2019 pathogen. This potentially puts the surgeon and other operating theatre personnel at risk of acquiring the severe acute respiratory syndrome coronavirus-2 from contact with droplets or aerosols. The use of an additional drape designed to limit the spread of droplets and aerosols has been described; such drapes include the ‘Southampton Tent’ and ‘OtoTent’.To evaluate the use of a novel drape ‘tent’ that has advantages over established ‘tent’ designs in terms of having: (1) a CE marking; (2) no requirement for modification during assembly; and (3) no obstruction to the surgical visual field.During mastoid surgery, the dispersion of macroscopic droplets and other particulate matter was confined within the novel drape ‘tent’. Use of this drape ‘tent’ had no adverse effects upon the surgeon's manual dexterity or efficiency, the view of the surgical field, or the sterility. Hence, our findings support its use during mastoid surgery in the coronavirus disease 2019 era. An aerosol can travel over longer distances, and refers to a suspension of fine solid or liquid droplet in a gas, with a diameter of 10 μm or less.13 given its connection with the viral-laden nasopharynx14 via the Eustachian tube. However, despite other coronaviruses having been found in the middle ear,15 there have been no reported investigations of SARS-CoV-2 in this anatomical location to date. Regardless, mastoid surgery is considered to be high risk for the transmission of Covid-19 from an infected patient to healthcare workers via the drill-induced dispersion of a mixture of water, bone, blood and tissue.6It is highly feasible that the middle ear of a Covid-19 positive patient contains SARS-CoV-2,6 the ‘Ototent’16 and other methods.18 Recent pre-clinical studies have suggested that the use of such a drape ‘tent’ could reduce the dispersion of particulate matter during temporal bone drilling.19 However, all of these established and previously evaluated ‘tent’ designs require modifications during the assembly process, or are associated with a significant amount of ‘spare’ drape that can interfere with surgical efficiency.19Infection prevention control measures that aim to reduce aerosolisation and contain the spread of droplets and other matter during mastoid surgery have been described. These include the use of an additional or multiple drape(s) to create a ‘tent’ over the surgical field, such as the ‘Southampton Tent’,We evaluated the use of a novel drape ‘tent’ that has advantages over other designs in terms of having: (1) a CE marking; (2) no requirement for modification during assembly; and (3) no significant ‘spare’ drape gathering around the surgical field.The evaluation of the novel drape ‘tent’ occurred during a mastoid surgical procedure performed on a two-year-old child.The microscope  was initially covered with a standard sterile microscope drape (Glass Lens Micro-Kover (product reference: 09-GL902); Advance Medical Designs, Marietta, Georgia, USA). The drape ‘tent’ used in this case consisted of a CE marked sterile polyethylene drape with a custom-made hole that fit securely over the lens cap of the standard microscope drape ; Vital Care Industries, Tinley Park, Illinois, USA ). The dr18 In order to access the surgical field, the operating surgeon was required to pass their hands underneath the drape ‘tent’.No modifications or alterations to the drape were required during assembly, and the visual field was unobscured by the drape (a hinderance of previous designs).The time taken to assemble the drape ‘tent’ was 3 minutes and 30 seconds, and the time taken to disassemble the drape ‘tent’ was 2 minutes; hence, each process had a negligible effect on the total length of the surgical procedure.The surgeon reported that the drape ‘tent’ did not affect their mobility, dexterity or tactility during the surgical procedure. No macroscopic droplet spread or particulate matter outside of the drape ‘tent’ was visualised by the surgeon at any point during the surgery.19The findings in this report align with evidence from pre-clinical studies, in which the use of a protective barrier in the form of a drape ‘tent’ substantially reduced the dispersion of particulate matter during temporal bone drilling.18 The ‘OtoTent’ utilises a 3M™ Steri-Drape™ 1060 that requires an incision to be made in the drape before it can be secured to the microscope lens mount.16 The Great Ormond Street Hospital drape ‘tent’ involves the customising and attaching of four overlapping panels of a 3M Steri-Drape 1060 to the side of a draped microscope.18 In contrast, the use of our novel drape ‘tent’ does not require any modifications for assembly, and no significant amount of ‘spare’ drape gathers around the surgical field. Furthermore, it is already CE marked for clinical use in its current form. We advocate the use of such a drape ‘tent’ during mastoid surgery in the Covid-19 era.We report substantial advantages for this novel drape ‘tent’ over other designs. Specifically, the ‘Southampton Tent’, which involves the use of a second microscope drape to create a ‘tent’ over the patient, is reported to have a significant amount of ‘spare’ tent; this subsequently impacts on surgical efficiency in terms of instrument use.


Background and Objectives: Intrahepatic cholangiocarcinoma (iCCA) is a pernicious tumor characterized by a dismal outcome and scarce therapeutic options. To substantially improve the prognosis of iCCA patients, a better understanding of the molecular mechanisms responsible for development and progression of this disease is imperative. In the present study, we aimed at elucidating the role of the maternal embryonic leucine zipper kinase (MELK) protooncogene in iCCA. Materials and Methods: We analyzed the expression of MELK and two putative targets, Forkhead Box M1 (FOXM1) and Enhancer of Zeste Homolog 2 (EZH2), in a collection of human iCCA by real-time RT-PCR and immunohistochemistry (IHC). The effects on iCCA growth of both the multi-kinase inhibitor OTSSP167 and specific small-interfering RNA (siRNA) against MELK were investigated in iCCA cell lines. Results: Expression of MELK was significantly higher in tumors than in corresponding non-neoplastic liver counterparts, with highest levels of MELK being associated with patients’ shorter survival length. In vitro, OTSSP167 suppressed the growth of iCCA cell lines in a dose-dependent manner by reducing proliferation and inducing apoptosis. These effects were amplified when OTSSP167 administration was coupled to the DNA-damaging agent doxorubicin. Similar results, but less remarkable, were obtained when MELK was silenced by specific siRNA in the same cells. At the molecular level, siRNA against MELK triggered downregulation of MELK and its targets. Finally, we found that MELK is a downstream target of the E2F1 transcription factor. Conclusion: Our results indicate that MELK is ubiquitously overexpressed in iCCA, where it may represent a prognostic indicator and a therapeutic target. In particular, the combination of OTSSP167  with DNA-damaging agents might be a potentially effective therapy for human iCCA. In early stage CCA, surgical resection and liver transplantation are the only effective treatment options, yet most of CCA patients are diagnosed at advanced stages [Cholangiocarcinoma (CCA) is a heterogeneous group of highly aggressive epithelial malignancies originating at any level of the biliary tract and showing features of cholangiocellular differentiation ,2. Over Dysregulation of multiple kinases has been described as one of the major mechanisms by which cancer cells escape normal physiological constraints on proliferation and survival. Maternal embryonic leucine zipper kinase (MELK), a member of the snf1/AMP protein kinase family of serine/threonine kinases, activates multiple signaling cascades driving oncogenic growth ,7. MELK Moreover, after binding and phosphorylation of FOXM1, the MELK–FOXM1 activated complex binds to the promoter and induces the transcription of the enhancer of zeste homolog 2 (EZH2) gene ,35. Uprewww.clinicaltrials.gov) [In light of these findings, suppression/inactivation of MELK may be an attractive therapeutic strategy in cancer. Currently, the only designated MELK inhibitor is OTSSP167, an orally administered drug, which is under evaluation in Phase 1 studies on various tumor types  ,45,46. Oals.gov) ,45,46. Ials.gov) . Also, Oals.gov) . Nonetheals.gov) ,50.In the present study, we sought to determine the function of MELK in human intrahepatic cholangiocarcinoma (iCCA) by evaluating the levels of MELK and some of its targets in a collection of human iCCA and corresponding non-tumorous surrounding livers. Subsequently, we assessed the effects of MELK modulation on proliferation and apoptosis of iCCA cell lines. Finally, we investigated the role of E2F1 as an upstream inducer of MELK. Overall, our results indicate that MELK is an E2F1 effector gene potentially important as a prognostic marker and therapeutic target for human iCCA.Five normal livers , 52 iCCA, and corresponding surrounding non-tumor liver tissues were used for the study. Patients’ clinicopathological features are summarized in Human liver specimens were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Sections were done at 5 μm in thickness. Liver lesions were assessed by two board-certified pathologists and liver experts (S.R. and F.D.). For immunohistochemical staining, slides were deparaffinized in xylene, rehydrated through a graded alcohol series, and rinsed in PBS. Antigen retrieval was performed either in 10 mM sodium citrate buffer (pH 6.0) by placement in a microwave oven on high for 10 min, followed by a 20-min cool down at room temperature. After a blocking step with the 5% goat serum and Avidin-Biotin blocking kit , the slides were incubated with the rabbit polyclonal anti-MELK , the rabbit monoclonal anti-EZH2 ; dilution 1:100), and the rabbit monoclonal anti-CK19  primary antibodies overnight at 4 °C. Slides were then subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and, subsequently, the biotin conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. The immunoreactivity was visualized with the Vectastain Elite ABC kit  and Vector NovaRed (Vector Laboratories) as the chromogen. Slides were counterstained with hematoxylin.MELK (ID# Hs01106438_m1), FOXM1 (Hs01073586_m1), EZH2 (Hs00544830_m1), E2F1 (ID # Hs00153451_m1), and β-Actin (ID # 4333762T) genes were purchased from Applied Biosystems . Quantitative values for each gene were calculated by using the PE Biosystems Analysis software and expressed as number target (NT). NT = 2−ΔCt, wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the target gene from the average Ct value of the β-Actin gene.Gene Expression Assays for human MELK  or E2F1 , following the manufacturer’s recommendations. In addition, HUCCT1 and HUH28 cells were seeded in 24-well plates and treated with the non-selective MELK inhibitor OTSSP167  at 40 and 100 nM concentration for 24–48 h. For the induction of DNA damage, cells were treated with 2 μmol/L doxorubicin  for 2 h, washed twice with phosphate-buffered saline (PBS), and returned to normal growth medium for the indicated time periods. Cell proliferation was assessed in the cell lines at 24- and 48-h time points using the BrdU Cell Proliferation Assay Kit . As concerns apoptosis, it was determined in the two iCCA cell lines using the Cell Death Detection Elisa plus Kit , following the manufacturer’ instructions. Cells were initially subjected to 24 h serum starvation. Subsequently, apoptotic cell death was assessed at 24- and 48-h time points. Finally, to assess DNA damage of cultured HCC cells, the DNA Damage Quantification Kit  was applied following the manufacturer’s instructions, and results were recorded at 24- and 48-h time points. All cell line experiments were repeated at least three times in triplicate.Mycoplasma-free, HUCCT1, and HUH28 human iCCA cell lines, after validation , were used for the in vitro studies. The two cell lines were kindly provided by Prof. Xin Chen . Cell lines were maintained as monolayer cultures in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 g/mL streptomycin . Cells were grown for 12 h, then serum-deprived for 24 h and treated with siRNA against t, and Mann–Whitney tests and linear regression analyses. Values of p < 0.05 were considered significant. Data are expressed as mean ± standard deviation for each group. Two-tailed unpaired t-test was used to compare the differences between two groups.GraphPad Prism version 6.0  was used to evaluate statistical significance by Tukey–Kramer, Student’s E2F1 putative transcription factor binding motifs on MELK gene promoters were predicted using the Eukaryotic Promoter Database New  that combines EPD promoters with promoter-specific high-throughput data [put data .MELK gene by real-time RT-PCR in an iCCA collection  and normal livers (NL) were detected  in human iCCA specimens.MELK mRNA in iCCA were related to the length of survival of the patients. Survival data were available for 39 of the 52 patients examined. Noticeably, we found that higher MELK gene expression correlates with lower iCCA survival rate, as assessed both by Kaplan–Meier and linear regression analysis  against MELK , a strong growth reduction and generation of DNA damage was induced when combining doxorubicin administration to MELK knockdown. As expected, at the molecular level  is a highly lethal tumor, characterized by rising incidence, clinical aggressiveness, high recurrence rate, and limited therapeutic options ,2,3,4,5.In the present investigation, we focused on the role in cholangiocarcinogenesis of MELK, a gene frequently activated in a variety of tumor types . We showIn in vitro experiments, we found that MELK suppression by way of the non-selective inhibitor OTSSP167 or specific siRNA significantly reduced the proliferation and augmented apoptosis in iCCA cells, suggesting that MELK contributes to cholangiocarcinogenesis via multiple mechanisms. Of note, the deleterious effects on cell growth were markedly amplified by the treatment with the DNA-damaging agent doxorubicin. These data suggest a major function of MELK in protecting the iCCA cells from DNA damage, in agreement with previous data obtained in glioma cells ,24. The MELK gene silencing) were able to reduce the growth of HUCCT1 and HUH28 cell lines, a stronger anti-neoplastic activity was observed when administering the non-selecting inhibitor to the cells. Since it has been proven that OTSSP167 does not selectively inhibit MELK [MELK suppression by siRNA did not recapitulate the mitotic effects of OTSSP167 treatment in MCF7 and Hela cancer cell lines. In these cells, the growth inhibitory activity of the drug in fact mainly depended on the inhibition of other kinases, such as BUB1 and Haspin [www.clinicaltrials.gov).Although both approaches aimed at inhibiting MELK activity (namely OTSSP167 administration and siRNA-mediated bit MELK ,50, we cbit MELK . Furtherd Haspin . Thus, td Haspin  and mighAt the molecular level, we identified the E2F1 transcription factor as the upstream inducer of MELK in iCCA. E2F1 is a major player in various physiological and pathological events, due to its ability to regulate a wide number of cellular events, including cell cycle, apoptosis, DNA repair, and metabolism ,57,58,59In summary, we revealed that MELK is an E2F1-responsive target presumably playing a crucial role in human iCCA development and progression. Also, we envisaged the possibility that MELK is a prognostic marker for this tumor type. Furthermore, we found that the anti-growth effects driven by MELK inhibition are strongly amplified by the concomitant administration of a DNA damaging agent. Thus, the administration of MELK inhibitors, together with DNA damaging agents, might represent a novel and promising therapeutic strategy against human iCCA.


Transporter inhibitors and several neuroprotective drugs inhibited [3H]l-tryptophan uptake by TR-BBB cell lines. Gabapentin and baclofen exerted a competitive inhibitory effect on [3H]l-tryptophan uptake. Additionally, l-tryptophan uptake was time- and concentration-dependent in both NSC-34 wild type (WT) and mutant type (MT) cell lines, with a lower transporter affinity and higher capacity in MT than in WT cell lines. Gene knockdown of LAT1 (l-type amino acid transporter 1) and CAT1 (cationic amino acid transporter 1) demonstrated that LAT1 is primarily involved in the transport of [3H]l-tryptophan in both TR-BBB and NSC-34 cell lines. In addition, tryptophan uptake was increased by TR-BBB cell lines but decreased by NSC-34 cell lines after pro-inflammatory cytokine pre-treatment. However, treatment with neuroprotective drugs ameliorated tryptophan uptake by NSC-34 cell lines after inflammatory cytokines pretreatment. The tryptophan transport system may provide a therapeutic target for treating or preventing neurodegenerative diseases.Tryptophan plays a key role in several neurological and psychiatric disorders. In this study, we investigated the transport mechanisms of tryptophan in brain capillary endothelial (TR-BBB) cell lines and motor neuron-like (NSC-34) cell lines. The uptake of [ Additionally, we aimed to examine the compensatory effects of neuroprotective drugs on both cell types under inflammatory conditions, thus, providing insight into a potential therapy for the prevention and treatment of ND.In this study, we hypothesized that ND pathology is correlated with the inflammatory state and oxidative stress, and that some common neuroprotective drugs may be effective in the prevention of ND. Therefore, we examined the transport characteristics of [3H]l-tryptophan (18.4 Ci/mmol) was purchased from PerkinElmer . Gabapentin, 3,4-dihydroxy-l-phenylalanine (l-dopa), baclofen, verapamil, phenylalanine, tyrosine, citrulline, glutamine, valine, cysteine, lysine, 2-aminobicyclo heptane-2-carboxylic acid (BCH), harmaline, α-(methylamino)isobutyric acid (MeAIB), homoarginine, dopamine, donepezil, riluzole, and amiloride were obtained from Sigma-Aldrich  and Tokyo Kasei Kogyo Co. . All chemicals and reagents were of reagent grade.Radiolabeled [wt and mutant type [MT]: NSC-34/hSOD1G93A; passages 10–19) cell lines were cultured using protocols described in our previous studies l-Tted time . To evalectively . After t® 2000 . For RNA interference analysis, total RNA (2 µg) was isolated from NSC-34 and TR-BBB cell lines using the RNeasy kit  according to the manufacturer’s instructions. RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit . After 48 h and 24 h, the transfected TR-BBB, and NSC-34 cell lines, respectively, were used for [3H]l-tryptophan uptake analysis, and relative gene expression was measured using GAPDH as a control. Real-time PCR was performed in 48-well plates using the StepOne Real Time-PCR system  l-tryptophan uptake analysis. Additionally, TNF-α/LPS-treated TR-BBB and NSC-34 cell lines were incubated for 24 h with LAT1 substrates/inhibitors or neuroprotective drugs, including baclofen (2 mM), gabapentin (0.5 mM), l-dopa (0.1 mM), and verapamil (0.1 mM), prior to [3H]l-tryptophan uptake analysis.TR-BBB and NSC-34 cell lines were pre-treated with LPS (20 ng/mL) and TNF-α (20 ng/mL) for 24 h ,30,31, fm), maximum uptake rate (Vmax), and first-order constant of the non-carrier-mediated process (Kd) of [3H]l-tryptophan were calculated using the following equation,3H]l-tryptophan at 5 min, C is the concentration of l-tryptophan. The initial uptake rate of [3H]l-tryptophan was estimated in the presence of compounds such as gabapentin and baclofen, using the following equation,i) and Ki is the inhibition constant. Statistical differences were calculated using the Student’s t-test and one-way ANOVA with Dunnett’s test, post hoc.For kinetic studies, Sigma plot ver. 12 (Sigma-Aldrich) was used for non-linear least-squares regression analysis. The concentration (C), Michaelis-Menten constant  of 121 ± 11 µM, maximum uptake rate (Vmax) of 4.69 ± 0.20 nmol/mg protein/min, and a Kd value of 3.66 µL/mg protein/min were obtained for [3H]l-tryptophan uptake by TR-BBB cell lines , ATA2 (MeAIB), and b°+  did not affect tryptophan uptake (3H]l-tryptophan by TR-BBB cell lines (l-Type amino acids (LAT1 substrates) such as phenylalanine, tyrosine, methionine, and citrulline exerted a strong inhibitory effect (>70%). Other l-type amino acids such as glutamine, valine, and cysteine induced >60% inhibition, while lysine induced >40% inhibition on tryptophan uptake, likely due to stereospecific effects. LAT1 is mostly stereoselective for l-type amino acids. In contrast, acidic amino acids such as aspartic and glutamic acids, and other amino acids such as taurine and choline, had no effect on tryptophan uptake  and baclofen (2 mM)  . The Linectively .l-tryptophan, TR-BBB cell lines were transfected with siRNA to knock down LAT1 and CAT1 expression. The relative mRNA expression of LAT1 and CAT1 was suppressed, confirming the presence of both transporters in TR-BBB cell lines  on [3H]l-tryptophan uptake. However, no effect on tryptophan uptake was observed in the presence of CNS-acting drugs such as dopamine, donepezil, riluzole, and amiloride,  and LPS (20 ng/mL)  . Pretrea control . To exam3H]l-tryptophan uptake by NSC-34 WT and MT cell lines, we conducted time- and concentration-dependent uptake assays. The time dependency uptake assay was carried out from 5 s to 60 min, revealing a linear uptake until 5 min in both WT and MT cell lines  and LPS (20 ng/mL) on NSC-34 WT and MT cell lines. To confirm the potential anti-inflammatory effects of neuroprotective drugs, NSC-34 cell lines (WT and MT) were then incubated with baclofen, gabapentin, l-dopa, and verapamil for 24 h. The tryptophan uptake was significantly decreased by 20–25% after pretreatment with TNF-α and LPS compared to that in the non-treated control , is implicated in a range of brain functions. Brain tryptophan concentration is correlated with that of serotonin and 5-hydroxyindoleacetic acid (5-HIAA), a major serotonin metabolite . Tryptop3H]l-tryptophan uptake by TR-BBB cells is stereospecific and dependent on sodium and tryptophan concentration, indicating a single saturable high affinity and low capacity transport system  inhibited the cellular uptake of [3H]l-tryptophan , choline , and glutamic and aspartic acids (acidic amino acids) had no effect on tryptophan uptake by TR-BBB cell lines [3H]l-tryptophan uptake by TR-BBB cell lines  inhibited tryptophan uptake by TR-BBB cell lines l-tryptophan uptake kinetic analysis, the transport system in MT cell lines had lower affinity with less sensitivity and higher capacity than that in WT cell lines  was suggested to be via the depletion of protein aggregation, which enhanced motor neuronal survival by ameliorating endoplasmic reticulum stress [The effect of pro-inflammatory cytokine (LPS and TNF-α) pretreatment for 24 h on TR-BBB and NSC-34 cell lines was determined by measuring [ve drugs . The eleytokines  (Figure ytokines . On the ytokines ,47. Up- ytokines ,30. Alonll lines . Baclofestage PD . The neum stress . Verapamm stress . NeuroprTryptophan is an amino acid precursor of serotonin, implicated in neuropsychiatric conditions such as depressive disorders, schizophrenia, and autism. The uptake of tryptophan was shown to be affected by kinetic parameters, its concentration, sodium, structural analogs, and inhibitors. Our findings demonstrated that tryptophan transport in TR-BBB and NSC-34 cell lines were primarily facilitated by the LAT1 transporter system, which was altered in NSC-34 cell lines. Under inflammatory conditions, treatment with neuroprotective/anti-inflammatory drugs attenuated inflammation in both cell types. We believe that the transport mechanisms of tryptophan could provide useful biomarkers for the target, design, and development of new therapeutic and preventative strategies for neurodegenerative and neuro-inflammatory disorders. Targeting the tryptophan transport system through the brain and neuronal cells may improve the efficacy and safety of therapeutics in NDs.