Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients. Ewing sarcoma (EWS) is an uncommon, aggressive and poorly differentiated tumor, most often occurring in children and young adults . MultimoMetastatic disease remains a huge challenge for researchers due to the high complexity of the tumor cell mechanisms controlling this process . AlthougEHMT2 (euchromatic histone lysine N-methyltransferase 2) and catalyzes the dimethylation of histone H3 lysine 9 (H3K9me2), which is associated with transcriptional repression [The histone methyltransferase G9a is encoded by pression . G9a plapression , 12. Prepression , lung capression , malignapression , breast pression  and colopression . This ovpression , 19. On pression . HoweverIn the present study, we revealed a remarkable role of G9a in the metastatic process in EWS. We first characterized and evaluated the expression of G9a in EWS tumors related to the clinicopathological features of EWS. We then investigated its role in the metastatic process in EWS cell lines and in a spontaneous metastases mouse model. We identified NEU1 (neuraminidase 1) a novel G9a target as well as one of the direct effectors of G9a activity in the metastatic process. Together, these findings indicate that G9a plays a relevant role in EWS metastatic behavior and is a potential therapeutic target for patients with metastatic EWS, the subgroup of patients carrying the worst prognosis.EHMT2 mRNA expression using the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). The overall survival and the disease-free survival rates of EWS patients were significantly worse in the group of patients with high levels of EHMT2 expression, in two independent series (Savola and Dirksen)  and disease-free survival (p = 0.0325)  Fig. , B. We nEHMT2 expression in primary tumors, recurrent tumors and metastases, in a public database (Savola) as well as in our EWS patient series. Our data showed that metastatic tumor samples had a significantly higher expression of EHMT2/G9a than primary or recurrent tumor samples, at both the mRNA (p = 0.0008) and the protein (p = 0.0005) levels , , Fig. . CollectRecently, we have suggested that the G9a histone methyltransferase could be a target for EWS therapy by using the specific and selective inhibitor BIX01294. Selected from the epigenetic drug library screening, BIX01294 was identified as one of the most effective inhibitors to repress the proliferation of EWS cell lines in vitro . HoweverWe first investigated how BIX01294 affects the growth of EWS cells lines. For this, we analyzed the proliferation IC50 values in twelve established EWS cell lines, seven cell lines of other tumor types and one non-tumor stem cell line (hMSC). Our results revealed that BIX01294 inhibited the proliferation of all EWS cell lines to a similar degree except for the CADO-ES cell line, which showed a higher resistance Fig. . StrikinWe then evaluated the effects of BIX01294 on G9a activity by the assessment of histone 3 dimethylation at lysine 9 (H3K9me2), as a specific target, at intermediate (IC50) and high (IC90) concentrations of BIX01294 at 24 and 48 h  significantly increased after treatment with an IC90 dose  Fig. . To testTo study the effects of G9a inhibition on cellular adhesion capacity, and taking into consideration that the interactions of cancer cells with the endothelium determines the metastatic spread , we analTo study whether BIX01294 treatment could also affect the final steps of the metastatic process, we performed a clonogenic assay to assess the ability of EWS cells to form colonies after exposure to intermediate or high doses of BIX01294. The percentage of colony formation was significantly reduced in a dose-dependent manner upon IC50 or IC90 doses of BIX01294, by 20% or 60% in RM82, and by 40% or 60% in CADO-ES cells, respectively, as compared to untreated cells Fig. G, H. ThuCD44, receptor tyrosine kinase (EphA2) and tissue factor pathway inhibitor 1 (TFP1) were upregulated in all cell lines when they formed VM  decreased significantly in the control cells (HUVEC and MUM 2B)  Fig. . Howeverine Fig. . ConsequAn analogous assay was performed under in situ treatment, by seeding all cell lines in a collagen matrix with medium containing BIX01294. We observed a higher inhibition of VM biomarker genes after BIX01294 pre-treatment than after in situ treatment in the HUVEC, MUM 2B and RM82 cell lines   by using two different anti-EHMT2 siRNA constructs. Indeed, we observed an increase of NEU1 expression after EHMT2/G9A inhibition at both the mRNA and protein levels in CADO-ES and RM82 cell lines  to the regulatory region of NEU1 promoter  by chromatin immunoprecipitation (ChIP). We confirmed that G9a was binding to NEU1 promoter in CADO-ES and RM82 cell lines  after IC50 or IC90 treatment with BIX01294 for 24 h in the CADO-ES or RM82 cell line (Supplementary Table T1) Fig. ; Fig. 5BTo investigate whether directly overexpressing NEU1 would impair the metastatic capacity and replicate the effects of G9a inhibition, we used the same experimental approaches as described above for BIX01294 functional studies in EWS cell lines see Fig. . We demoFinally, to determine whether the cell phenotype induced by G9a inhibition through treatment with BIX01294 was mediated by re-expression of NEU1, we raised rescue assays. NEU1 protein levels were depleted using esiRNA, and was compared to a negative control (EGFP) and the wild type cell lines. There were not differences between WT and the negative control . To confirm the drug-mediated effect, Ki67 nuclear expression was evaluated in tumor samples. The results revealed a significant decrease in Ki67 positive cells after treatment in BALB/c mice in accordance with the observed growth inhibitory effects Fig. . AdditioFollowing tumor resection, we monitored the animals for the appearance of lung metastases. At the end of the experiment, no macrometastasis, detected by luciferase activity or single-eye lung ex vivo evaluation, were observed in treated or untreated mice. Therefore, we evaluated the micrometastatic foci in the lungs (metastases detected by ex vivo and H&E), using lung sections stained with H&E , no toxicity was present in both models of experimental mice with BIX01294 treatment , and a negative correlation between NEU1 expression with G9a (p = 0.0242) and the number of metastatic foci (p = 0.0396). These in vivo results are in concordance with those obtained in vitro, and both revealed a role of G9a in the metastatic progression of EWS.Given the links between G9a and NEU1 expression and the metastatic process, we performed a multivariate analysis and a principal component analysis. G9a expression and the number of metastases had a positive relationship, and both of these variables had a negative relation with NEU1 expression Fig. . To confEWS is an extremely aggressive tumor with a clear tendency for local recurrence and distant metastases, often leading to a fatal outcome . The molNovel treatments currently use epigenetic drugs alone or in combination to improve the survival rate in different types of cancers , 37. TheEHMT2/G9a expression significantly higher than that of primary and recurrent tumor samples. These results are supported by many studies that have previously described G9a to be overexpressed in various tumors [We now report that high expression of G9a (both at the mRNA and the protein levels) in EWS defines a subset of patients with a worse prognosis. Moreover, we also show that metastatic samples had an s tumors –17, suggs tumors . We obses tumors ). In this tumors . Moreoves tumors , 41, ands tumors .The effectiveness of G9a inhibition was not restricted to proliferation, as we also demonstrated that the in vitro metastatic processes were impaired in EWS cell lines. We performed functional assays with BIX01294 treatment to evaluate the biological significance of G9a in the different steps of the metastatic process in vitro. EWS cell migration and invasion was impaired after G9a inhibition. As freely-circulating tumor cells must adhere to the endothelium, we studied the adhesion of tumor cells lines using different extracellular matrix components; notably, our results showed that G9a inhibition reduced adhesion of EWS cell lines. BIX01294 also impaired colony formation, which is essential to promote cell growth in a secondary location. Therefore, our results demonstrated the relevance and role of G9a in the metastatic process, in agreement with the results obtained in EWS patients. Accordingly, in other tumors, G9a has been shown to promote cancer invasion, migration, adhesion and metastases , 25, andCD44, TFPI-1 and EphA2 [In addition to the various steps evaluated in the metastatic process, EWS cell lines can form VM. This mechanism has been described as an enhancer of metastatic progression in cancer . Blood lnd EphA2 . Our expnd EphA2 . These dNEU1 as one of the targets involved. We confirmed that G9a inhibition (by either BIX01294 or siRNA anti-EHTM2) induced NEU1 overexpression in vitro. To date, however, the role of NEU1 in metastases is contradictory. NEU1 overexpression has been described to reduce metastases in vivo and to suppress migration, invasion and adhesion in vitro, yet its silencing results in the opposite effects in colon cancer [NEU1 siRNA knockdown inhibits cell invasion [NEU1 gene expression is direct in EWS cell lines, through dimethylation of Histone 3 in its promoter by G9a binding/activity. Therefore, we propose that NEU1 is one of the molecular direct targets of G9a that controls the malignant properties of EWS cells. This novel role of G9a/NEU1 represents a metastatic molecular mechanism that could be explored in other tumors. However, in light of other studies, it would be necessary to understand the specific role of NEU1 in each cellular context and its relationship with epigenetic disorders.Exploring the molecular mechanisms promoted by G9a in the metastatic process in EWS, we propose n cancer . On the n cancer . In pancn cancer ; in ovarinvasion . Our resTo evaluate our results in vivo, we used a metastatic EWS spontaneous metastases mouse model  in two mBIX01294 not only reduced tumor growth but also impaired the metastatic capacity of EWS cell lines in vivo. The percentage of mice with metastatic foci in lungs was statistically reduced in both mice models, regardless of the aggressiveness of the tumor growth. In agreement with our results, lung metastases were drastically reduced in SCID mice after injecting the tumor cells carrying the G9a knockdown into the lateral tail veins . Along tIn summary, our studies implicate G9a as having a central role in the metastatic process in EWS that involves the sialidase NEU1. Our findings contribute to understand the metastatic process and provide a new treatment option for inhibition of metastatic phenotypes in EWS patients. G9a pharmacological inhibition could be used as an adjunctive treatment to standard treatment for EWS patients, which is based fundamentally on cytotoxic action. Thus, this approach would combine a reduction of local tumor growth with an inhibition of the metastatic process.112 formalin‐fixed paraffin‐embedded (FFPE) EWS tumor samples, 44 of which corresponding to primary tumors without treatment, were obtained from the Department of Pathology at the Hospital Universitario Virgen del Rocío  and HUVR-IBiS Biobank. Approval of the Ethics Committee of our institution was obtained, and written informed consent was obtained before including samples and data in the HUVR-IBiS Biobank. Patients and samples characteristics are summarized in Supplementary Table Proteins were extracted from cell lines in RIPA buffer  NP40, 50 mM Tris-HCl pH 8.0, 0.1% (v/v) SDS, 1 mM EDTA, and 0.5% (w/v) deoxycholate), supplemented with 10 mM NaF and 2 mM NaOv protease inhibitors. Immunoblotting was performed using the primary antibodies listed in Supplementary Table Expression levels of selected genes were analyzed by RT-qPCR. RNA was isolated from cell lines using the miRVana miRNA Isolation Kit . The quantity and quality of the total RNA was determined with the Nanodrop ND-2000 Spectrophotometer (Thermo Scientific). Prior reverse transcription was performed using TaqMan Reverse Transcription Kit  in GeneAmp PCR 9700 thermocycler. All RT-qPCR measurements were obtained in a 7900HT Fast Real Time PCR System with ExpressionSuite Software v1.1 (Applied Biosystems). TaqMan probes are listed in Supplementary Table 5, 5 × 105, 1 × 104, or 15 × 103 cells per well in 24-well dishes coated by Basement Membrane Matrix . After 24 h, pre-treated cells were added and cultured in complete medium, and non-treated cells, in complete medium with BIX01294. Cell cultures on 24-well dishes without matrix were used as negative controls, and cultures of HUVEC and MUM 2B were used as positive controls. Tube formation were visualized and photographed under the Olympus BX61 microscope.EWS cell lines (RM82 and CADO-ES), MUM 2B and HUVEC were plated at 2 × 10EHMT2 transcript were used: #4392420_ID:s21469 for siRNA EHTM2_A and #4392420_ID:s21470 for siRNA EHTM2_B. Select Negative Control #1 siRNA  was included as a control for off-target effects. EWS cell lines were transfected using Lipofectamine® RNAiMAX Transfection Reagent  following the manufacturer’s instructions.Two pre-designed siRNA against the NEU1 cDNA of Human NEU1 Gene cDNA clone plasmid  was amplified using the follow primers: Fw −3′CAGGATCCGCCACCGATATGACTGGGGAGCGACC-3′ and Rv 5′-CTACGGAATTCGGCCGCACTAGTGATTCAGA-3′ . The amplicon was cloned into retroviral plasmid pBABE-puro  for EWS cell line transfection. Retroviral EWS cell line infection was performed using the protocol described previously [The eviously . At 24 hMann–Whitney U-test for two groups, and one-way analysis of variance test for more than two groups followed by Tukey’s multiple comparisons post-test, were used to evaluate differences between control and treatment conditions. The overall survival and disease-free survival time were analyzed using the Kaplan–Meier estimator and the Wilcoxon test. Biplots associated with principal component analysis was used to determine the relationship between different variables and Multivariate Spearman’s test was used to determine the correlations between different variables. Normal distribution of data was assessed before application of parametric tests. The variance was similar between groups that were being statistically compared. The data met the assumptions of the tests.p-values of ≤0.05 were considered statistically significant. Analyses were performed using the Prism 6.01 software (GraphPad) and multivariate analyses were performed using the JMP 10 statistical software . The average of at least three biological replicates performed in three technical replicates with SD is presented in all experiments. No data were excluded from the analyses.For all analyses, For cell experiments study, samples were no randomized and sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups, usually the number is three or more. For xenograft mice experiment, no statistical methods were used to predetermine sample size, which was determined based on previous experimental observations.Additional methods are described in Supplementary Material and Methods.Supplemental MaterialSupplementary Table1Supplementary Material & Methods


For patients with a variety of severe diseases, including primarily hematopoietic malignancies, immunodeficiency syndromes, and genetic disorders, allogeneic hematopoietic stem cell transplantation (aHSCT) represents a potentially curative therapeutic approach. In this context, despite significant progress in the optimization of aHSCT, the development of graft-versus-host disease (GVHD) remains a challenge to long-term transplant success after aHSCT. It is associated with significant morbidity and mortality and is the major cause of non-relapse mortality.GVHD occurs when donor T cells are primed by recipient antigens subsequently eliciting an inflammatory response against the host. Clinically, two types of GVHD are distinguishable: an acute form (aGVHD), and a chronic form (cGVHD). In brief, the main characteristics: aGVHD occurs in 30-50% of aHSCTs and is a multi-organ disorder resulting from inflammatory cytokines and donor T cells which primarily damage skin, liver, gastrointestinal tract, and eye. cGVHD, with a prevalence of 30-70% of aHSCTs, is induced by T and B cells resulting in a heterogeneous immunological complication affecting virtually every organ.Traditionally, broad immune-suppressive drugs (with considerable toxicities) including calcineurin inhibitors (CNI) (cyclosporin or tacrolimus), together with methotrexate or mycophenolate mofetil (MMF), and mTOR inhibitors (Sirolimus/Rapamycin) are used as GVHD prophylaxis. But despite first success reports, significant GVHD still occurs with these drugs. Other prophylaxis strategies like pre-transplant anti-thymocyte globulin (ATG) are effective in reducing severe GVHD but have no survival benefits and steroids have serious side effects.One of the most critical challenges in aHSCT is the development of less toxic and more targeted therapies that maintain the graft-versus-leukemia/tumor (GVL/T) effect but suppress GVHD while facilitating enhanced immune reconstitution relative to existing strategies. Recently, several prophylaxis strategies for GVHD have been developed and others are currently in development, including, for example in the case of haploidentical HSCT, post-transplant cyclophosphamide (PTCy), which seem to be very promising.In the frame of this specific Research Topic, we aimed to collect recent developments of innovative methods for both prevention and treatment of GVHD, without impairment of GVL. In 8 original research articles and 4 reviews, this edition provides a deep insight into the role of the microbiome and metabolism as well as recent advances of small molecule and cell therapy development.Mohamed et al., summarize metabolic pathways contributing to GVHD and discuss metabolic targets for acute and chronic GVHD in immune and non-immune cell as well as the immunomodulatory function of microbial metabolites. Furthermore, they examine the metabolic effects of co-inhibitory pathway blockade (PD-1) and cellular therapies (Tregs/MSCs/Bregs) in aHSCT. The mini review by Karl et al., provides an overview of metabolic T cell alterations in GVHD and illustrates the impact of conventional GVHD therapy on T cell metabolism.We are glad, that experts in the field highlight the recent progress in the broad field of immune cell metabolism with two comprehensive reviews. Ghimire et al., investigates the role of G-protein coupled receptors (GPR43 and GPR109A) which engage microbial derived metabolites, like short chain fatty acids, in the mitigation of GVHD in intestinal biopsies from patients after allo-HSCT. A second study by Heidrich et al., describes an association of dental biofilm microbiota dysbiosis with the risk of aGVHD.Recent studies have shown the association of microbiome dysbiosis and aGVHD. Here, primary research by Campe and Ullrich. Moreover, Agbogan et al., explore the immunomodulatory effect of adoptively transferred CpG-activated B cell progenitors to alleviate GVHD symptoms. In addition, Scheurer et al., describe an in vitro generated sub-population of CD11b+CD11c+ myeloid-derived suppressor cells (MDSCs) as potent immune modulators leading to the prevention of GVHD without negatively affecting tumor cytotoxicity. Another innovative and attractive strategy using CRISPR/Cas9 has been described by Majumder et al., for genetical engineering of naïve T cells pre transplant as a method for GVHD prevention in a major murine mismatch model.In the context of cell therapy development, the biological relevance of T helper cell lineage defining transcription factors as potential targets for GVHD therapy has been delineated in a review article by Braun and Zeiser thoroughly review the role of kinase inhibition as novel treatment strategies for acute and chronic GvHD after allo-HCT. Thangavelu et al., evaluate the efficacy of a novel agonist of the retinoic X receptor (RXR), IRX4204, to treat cGVHD in two complementary murine models with bronchiolitis obliterans or sclerodermatous manifestations. Primary research by Matos et al., analyzes a possible association of anti-thymocyte globulin (ATG) treatment and serum levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in 4 HSCT cohorts with different vitamin D3 supplementation. Lastly, Hadjis et al., characterize post-transplant cyclophosphamide as superior in ameliorating pre-clinical GVHD compared to five other optimally dosed chemotherapeutics  that vary in mechanisms of action and drug resistance.In addition, the recent therapeutic advances in the area of drug development, e.g. small molecules and antibodies, are also addressed. Finally, this Research Topic makes us again aware of how complex the regulation of GVDH is and in which fragile balance between GVHD and GVL patients after aHSCT find themselves. We are aware that this issue can only compile a first selection of innovative findings and treatment strategies that are currently being developed for the prevention and treatment of GVHD.GVHD biology and treatment remains a field that is always influenced by current research developments and new advances can be expected in a short time. Therefore, we will continue to monitor the field and provide updates to the authorship of Frontiers in Immunology.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.EU is a member of Phialogics and receives research support from BMS and Gilead. AB is a scientific cofounder of Aamuthera Biotech GmBH and Dualyx NV.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cancer cell lines have been widely used for decades to study biological processes driving cancer development, and to identify biomarkers of response to therapeutic agents. Advances in genomic sequencing have made possible large-scale genomic characterizations of collections of cancer cell lines and primary tumors, such as the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA). These studies allow for the first time a comprehensive evaluation of the comparability of cancer cell lines and primary tumors on the genomic and proteomic level. Here we employ bulk mRNA and micro-RNA sequencing data from thousands of samples in CCLE and TCGA, and proteomic data from partner studies in the MD Anderson Cell Line Project (MCLP) and The Cancer Proteome Atlas (TCPA), to characterize the extent to which cancer cell lines recapitulate tumors. We identify dysregulation of a long non-coding RNA and microRNA regulatory network in cancer cell lines, associated with differential expression between cell lines and primary tumors in four key cancer driver pathways: KRAS signaling, NFKB signaling, IL2/STAT5 signaling and TP53 signaling. Our results emphasize the necessity for careful interpretation of cancer cell line experiments, particularly with respect to therapeutic treatments targeting these important cancer pathways. Using a support vector machine learning approach and multi-omics data, dysregulation of key cancer driver pathways is revealed in cancer cell lines compared to primary tumors. Cancer cell line models grow quickly, are comparatively cost-effective, and are readily available. Their integration into preclinical research has led to remarkable advances in cancer characterization and treatment2. As mutation detection has become more accurate, multiple studies have reported that head and neck cancer cell lines tend to harbor more mutations than their tumors of origin4. A recent study showed that colorectal cancer cell lines recapitulate colorectal tumor subtypes, but that cell lines have more mutations than tumors5. In general, the current consensus concerning cancer cell lines as primary tumor models is that cell lines share many of the original tumor characteristics, but can harbor genetic changes of poorly characterized significance; and that some cancer cell lines may not even be molecularly appropriate or representative models for their tumor of origin6.However, additional genomic characterization of cancer cell lines has indicated that the transition from in vivo to in vitro may introduce key genomic alterations. One of the first groups to compare tumor and cell line used microarray gene expression profiles to identify breast cancer cell lines that seemed to be genetically inappropriate models for breast carcinoma6. It is essential that preclinical researchers know which biological pathways behave similarly in vivo and in vitro, and even more importantly, which pathways demonstrate altered activity as a result of alterations in environmental signals and stressors in the in vitro growth setting. If a key pathway behaves differently in cell lines as compared to primary tumors, preclinical testing of a drug targeting that pathway will not accurately predict patient tumor response.Nevertheless, cancer cell lines continue to be widely used in cancer research and therapeutic discovery. As the focus on molecularly targeted therapeutics grows, so does the need to thoroughly characterize how cancer cell lines diverge phenotypically from tumors due to their in vitro growth environmentIn order to characterize the specific pathway alterations that occur between primary tumors and tumor-derived cell lines, we analyze three types of high-throughput molecular data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). We perform transcriptomic analysis of bulk RNA sequencing of TCGA and CCLE samples and identify a set of differentially expressed genes. We integrate micro-RNA sequencing from the same projects and identify an interaction network of micro-RNA (miRNA), long non-coding (lncRNA) and protein-coding genes that is aberrantly expressed in cell lines compared to tumors. This network implicates four key cancer driver pathways that are often the subject of preclinical drug evaluation in cell lines, but whose activity in cell lines is not representative of original tumors. We use proteomic quantification data from the same studies to demonstrate that the aberrant cancer driver pathway expression observed in cell lines extends to the proteomic level. We also demonstrate similar findings in separate datasets of single-cell RNA sequencing from tumors and cancer cell cultures.7  which are the most useful and important for classifying a new tumor or cell line based on a trained model8. Feature identification using SVM allowed us to identify the set of genes that best differentiate between tumor and cell line regardless of whether each gene is more highly expressed in the tumor group or the cell line group. The SVM approach has been shown to eliminate noisy results in a high-dimensional gene expression comparison, by drawing out the greatest sources of variation across all samples10. In order to ensure robust results, we repeated the classification on fifty different random 80/20 test/training splits of the data. After sorting the genes by their SVM-assigned feature importance coefficients, we merged the top 10% of genes from all fifty classifications, resulting in 1854 genes that were in the top 10% of most important genes for each classification (Supplementary Data We then used a support vector machine (SVM) linear classifier within the Python 11. We found 27 gene sets with significant enrichment in the SVM-derived differentially expressed protein-coding genes  on the 1799 protein-coding genes using the Hallmark cancer pathway set from the Molecular Signatures Database (mSigDB v7.0)12. miRNet identified 227 miRNA with known interactions to the 54 lncRNA. In turn, these 227 miRNA had 580 known gene targets among the 1799 differentially expressed coding genes . GSEA of the 580 coding genes revealed 24 Hallmark gene sets with significant enrichment . Notably, our results . This pathway did not show differential expression between CCLE and TCGA . We also verified that this signal was not a disease-specific artifact by repeating the SVM after subsetting the data to the disease with the largest number of samples in TCGA (BRCA) and the smallest number of samples (DLBC). The same four cancer driver pathways were identified in both analyses  and lowest (PRAD) ESTIMATE scores . The four cancer driver pathways were identified as differentially expressed in both analyses  represents the protein counterpart of the STAT5 gene. The antibody NFKBP65_pS536 binds to phosphorylated p65, one of the two protein subunits of NFKB. Phosphorylation of p65 is one of several molecular mechanisms known to activate the NFKB pathway20.We next examined whether the downregulation of these pathways extended beyond gene expression into protein activity. Proteomics quantification of many CCLE and TCGA samples was performed using Reverse Phase Protein Array (RPPA) in the MD Anderson Cell Lines Project (MCLP) and The Cancer Proteome Atlas (TCPA)ata Fig. . PIK3R1 We noted significantly lower protein expression of PIK3R1, Cyclin D1, and STAT5 in the cell-line data, consistent with our gene expression results in the corresponding pathways. This carries important implications for the applicability of preclinical drug tests against these targets in cancer cell lines. Interestingly, the phosphorylation level of p65 is higher in cell lines than tumors, opposite the gene expression of the NFKB signaling pathway. This suggests that p65 phosphorylation may be playing a different role in cell lines, and underscores the importance of examining multiple types of data to elucidate complex molecular interactions.KRAS and TP53 genes22, we investigated whether there is a correlation between diseases with heavy mutation burden in these genes and diseases with higher correlation between tumor and cell line, with the assumption that cell lines derived from mutated tumors maintain those mutations. We found that there is no correlation between mutational burden and tumor-cell-line correlation; in fact, in the case of the TP53 pathway, there is a slight inverse correlation between the two factors . To further investigate the effect of KRAS and TP53 mutation status on the differentially expressed cancer driver pathways, we subset the TCGA and CCLE datasets to samples carrying a non-silent KRAS or TP53 mutation and repeated the SVM analysis (see “Methods”). In both the KRAS-mutant and TP53-mutant analysis, all four cancer driver pathways were again identified as differentially expressed in tumor compared to cell line. These results indicate that dysregulation of these pathways occurs in cancer cell lines regardless of the mutational status of the primary tumor, and is unrelated to the activating DNA mutations.Because activation of the KRAS and TP53 pathways is associated with mutations in the 25. miRNA directly affect gene expression by binding mRNA and targeting them for degradation28. In this method of expression control, lncRNA regulate miRNA, while miRNA regulate gene expression . With the exception of LINC00707, 10 of the 11 lncRNA are significantly underexpressed in CCLE  are more highly expressed in cell lines than tumors. Cytoscape was used to visualize the lncRNA-miRNA-coding gene network colored by gene type or by LFC  data from CCLE and TCGA. We used the ComBat method to correct for experimental batch effects mir-497, mir-195, mir-148a, and mir-152 directly inhibit genes in the KRAS/RAF/MEK/ERK pathway32. The TP53 pathway is repressed by mir-339, and the TP53-associated gene TP53INP1 is regulated by mir-9234. mir-519d directly represses STAT3, a key gene in the IL2/STAT5 signaling pathway35. The NFKB pathway is activated by mir-301a, which has lower expression in cell lines compared to tumors, in keeping with lower NFKB activity in cell lines36.In keeping with the lncRNA “sponge” regulatory model, the lncRNA are underexpressed in cancer cell lines, which could be involved with the observed overexpression of a majority of the miRNA whose expression is known to be kept in check by these lncRNA. The aberrant overexpression of inhibitory miRNA could explain the observed underexpression of key genes in four important cancer driver pathways in cancer cell lines. Consistent with this idea, we noted several miRNA with higher expression in cell lines that are known to play roles in regulation of the four cancer driver pathways. H19, a lncRNA strongly associated with the cancer stem cell state37, but show increased expression of mir-1 and mir-206, which promote cellular differentiation by blocking anti-differentiation signaling targets39. In addition, CCLE samples show reduced expression of CERS6-AS1, IQCH-AS1, and LINC00240, lncRNA implicated in mediating tight junctions or extracellular matrix interactions, which are features of epithelial and endothelial cells43. At the same time, CCLE samples have comparatively high expression of mir-9, which directly represses E-cadherin, a well-known epithelial marker44. E-cadherin repression is known to induce the epithelial-mesenchymal-transition, a process which plays a role in cancer progression from an epithelial state to a motile and invasive metastatic state45. CCLE samples display reduced expression of E-cadherin/CDH1, and higher expression of mesenchymal markers, including N-cadherin/CDH2, MUC1, and claudins CLDN1, CLDN2, CLDN346 .Because lncRNA and miRNA are known for cell-type-specific expression, we hypothesized that the observed dysregulation of lncRNA-miRNA expression networks is caused by biological selection for a subset of cancer cells which are more likely to survive the cell-line derivation process and thrive in a cell culture setting. Consistent with this hypothesis, both stem cell and epithelial cell-specific lncRNA and miRNA display reduced expression in cancer cell lines. Specifically, CCLE samples have reduced expression of The observed reduced epithelial and stem cell expression in cancer cell lines suggests that cancer cell culture conditions select for the subset of cancer cells with a mesenchymal, invasive and metastatic phenotype. Overall, these results indicate that selection against specific cancer cell types in tumor-derived cell lines may cause global downregulation of key cell-type-specific lncRNA, potentially allowing overexpression of a variety of miRNA, many of which play important roles in regulating cancer signaling pathways. However, more work is needed to fully investigate this hypothesis.7, we examined whether these cell lines show more representative expression of the 4 cancer driver pathways. We repeated the SVM after subsetting the CCLE dataset to the TCGA-110-CL and the TCGA dataset to the tumor types in the TCGA-110-CL  data from hepatocellular carcinoma (HCC) cell lines and patient samples. Within the scRNAseq patient tumor data, we differentiated the malignant cells from the normal cell infiltrate , very similar to the bulk RNA sequencing analysis . We again observe enrichment of the same four cancer driver pathways, with the exception of the KRAS signaling pathway in the first analysis.50. Two-dimensional cancer cell cultures have enabled researchers to discover how cancers arise, characterize cancer cell types and growth patterns, and identify effective pharmaceuticals through drug screens1. Today, personalized tumor-derived 2D and 3D cultures are increasingly in use for the identification of precision therapies for individual patients53. The rise of precision medicine and small-molecule inhibitor development brings an urgent need for molecular characterization of the cell culture models used widely for therapeutic development. Large-scale genomic efforts such as The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE) have enabled comprehensive comparison of cancer cell cultures and primary tumors.The ability to model and manipulate cancer cells has empowered therapeutic discovery since the derivation of the first cancer cell lineHere, we provide a comparative multi-omic analysis of cancer cell lines and primary tumors by leveraging several types of genomic data from large public compendia. Our gene expression analysis reveals reduced expression of key cancer driver pathways, including KRAS signaling, TP53 pathway, IL2/STAT5 signaling, and NFKB signaling in cancer cell lines. These results are recapitulated in a comparative analysis of protein levels for key proteins from each pathway. Our analysis indicates the need for caution when interpreting in vitro preclinical testing results of inhibitors targeting these pathways.54. Subsequent optimization of these compounds led to success in clinical trials, but initial results in cell lines did not predict in vivo results. Targeting both wild-type and mutant KRAS in cancer has been notably unsuccessful; in particular, several high-throughput screens of KRAS-mutant cancer cell lines identified compounds which subsequently only partially reduced tumor volumes in xenograft models56. In contrast, targeting the cholesterol biosynthesis pathway, which is not differentially expressed in cancer cell lines by our analysis, has been promising both in vitro and in vivo through the usage of the statin family of drugs58. In addition, targeted inhibition of cyclin-dependent kinases (CDKs) by agents such as PD-0332991/palbociclib has been promising in phase I and II clinical trials, and this drug was originally identified in cancer cell lines60. CDKs are active during cell cycle entry from G0 and during the G2M checkpoint pathway, which were not differentially expressed in cancer cell lines by our analysis. We note that apart from the four cancer driver pathways identified as dysregulated in cell lines, preclinical testing in cell lines can in many cases reliably predict in vivo responses.In fact, the consequences of preclinical testing in cell lines against these pathways have already been felt. For example, during preclinical testing several MDM2/TP53 small-molecule inhibitors displayed potent cancer cell-line inhibitory activity, but could only achieve partial tumor regression in xenograft modelsTo identify potential causes of dysregulation of cancer driver signaling in cell lines, we analyzed the expression of lncRNA and miRNA implicated in regulation of these pathways. In cell lines, we found reduced expression of a set of lncRNA predicted to regulate a downstream network of regulatory miRNA, which are in turn overexpressed. Several of these miRNA are directly involved in specific inhibition of these cancer driver pathways, linking their overexpression to the observed reduced expression of cancer driver pathways.61. It is well known that the presence of serum in culture media encourages cellular differentiation, and cancer cell lines grown in serum-free conditions contain larger populations of cancer stem cells63. This hypothesis is supported by the markedly lower expression of stem cell-specific lncRNA in cell culture, and higher expression of pro-differentiation miRNA. Because cancer stem cell populations are known for their chemoresistance, and even small populations are thought to be capable of tumor recurrence66, it is essential that preclinical models accurately model the response of cancer stem cells to potential therapeutics.We speculate that our results may indicate a partial loss of cancer stem cells in cancer cell culture due to the presence of serum in culture media. All CCLE cell lines were cultured in RPMI or DMEM media with 10% fetal bovine serum7. Our study provides additional information by identifying the set of genes which accounts for the highest variation between human tumors and cell culture models, and by identifying specific, clinically important cancer driver pathways which are poorly recapitulated in cancer cell culture models. Additionally, our finding of global cancer driver pathway dysregulation in cell lines may help to explain biologically the previous study’s finding of low correlation between some tumors and cell lines. We investigated the TCGA-110-CL cancer cell-line panel identified in the previous study, which has a higher overall cell-line-tumor gene expression correlation. The same four cancer driver pathways were identified by an SVM comparing these cell lines to disease-matched primary tumors, and in particular, the KRAS pathway had the lowest overall cell-line-tumor correlation. This indicates that even this subset of cancer cell lines does not fully recapitulate the cancer driver signaling of primary tumors, and studies on these pathways must be interpreted with caution.A recent study by Yu et al. also included a pan-cancer analysis of TCGA and CCLE data, finding that tumor type is not a large factor in overall tumor-cell-line correlation, but that some cell lines are poor models of their primary tumor type and tumor purity plays a large part in tumor-cell-line correlations7, we sought to assess the impact of tumor purity on our cancer driver pathway dysregulation findings by leveraging single-cell RNA sequencing data from HCC patients and cell lines. The TP53 and NFKB pathways remained significantly enriched in HCC tumor cells compared to cell lines when normal infiltrating cells were removed from the tumor-cell population, while the KRAS and IL6/STAT3 pathways were only significantly enriched with a less stringent statistical cutoff. These results indicate that the KRAS and IL2/STAT5 findings from the TCGA-CCLE gene expression comparison may be partially due to lower tumor purity. Moreover, this analysis indicates that normal cell infiltrate contributes a molecular signature to bulk gene expression data and highlights the utility of single-cell data. Finally, we found that the HCC cell lines most closely resemble the most invasive and metastatic HCC subtype, unlike the HCC tumor cells which resemble all three HCC subtypes, consistent with our hypothesis that cancer cell culture selects for the most invasive malignant cells. We saw similar results in an analysis of matched melanoma brain metastasis cell lines and tumors. A caveat of this analysis is that it focuses on only two types  of solid tumor; a broader investigation of single-cell RNA sequencing data in this context from multiple tumor types and cell lines will be informative when those data become available.In light of this finding that tumor purity impacts the correlation between tumor and cell lineTaken together, our results underscore the need for caution when interpreting preclinical cancer testing results in multiple model types, and point to specific signaling networks which can serve as litmus tests for the accuracy of past and future cancer laboratory models. We suggest several potential solutions to improve the efficacy of tumor-derived cell lines. Cancer cell culture in serum-free conditions may improve the maintenance of tumor stem cell populations and reverse the dysregulation of important regulatory gene networks. Specific efforts to model the immune microenvironment in cancer-derived organoids may improve cancer driver pathway expression related to the tumor microenvironment. A potential solution may be genetic manipulation of tumor-derived models with an emphasis on preserving or rescuing the intrinsic cancer driver pathway expression which is most at risk for dysregulation. Overall, this study provides much-needed genomics-based considerations for future preclinical cancer model development and result interpretation.n samples = 10,535) and CCLE (n samples = 933) were downloaded from the UCSC Xena browser. These data were processed uniformly through the TOIL UCSC RNA sequencing data processing pipeline  to remove technical batch effects67. Both datasets were normalized by log2(TPM + 1) and duplicate genes were averaged. Genes not expressed in 80% of samples were removed, and 20% of the lowest varying remaining genes were removed, leaving 46865 remaining genes. Both datasets were subset to the 19 overlapping cancer types for subsequent analysis . All heatmaps use a random subset of samples from each dataset with equal numbers from each disease. The random subset method is used because the CCLE data holds approximately 1/10 the number of samples as the TCGA data, making it impossible to visually detect gene expression differences in heatmaps containing all samples from both datasets.Gene expression transcripts per million (TPM) matrices from TCGA (TCGA somatic mutation data were downloaded from UCSC Xena (xena.ucsc.edu). CCLE somatic mutation data were downloaded from the Broad Institute CCLE Database .68. CCLE Nanostring probe miRNA quantification data were downloaded from the Broad Institute CCLE database: https://portals.broadinstitute.org/ccle/data69. For dataset comparability, miRNA naming formats were harmonized, and duplicates were averaged. Because different miRNA sequencing methods were used in each dataset, ComBat was used to batch-correct the data29. Pre- and post-batch effect correction data were then log2(count + 1) normalized for downstream visualization and analysis. Supplementary Fig. 5 shows pre- and post-batch effect correction expression distributions of several housekeeping genes to validate successful correction71.TCGA micro-RNA (miRNA) Illumina sequencing read counts data were downloaded from the Genomic Data Commons Data Portalhttps://tcpaportal.org/tcpa/download.html and http://tcpaportal.org/mclp/#/download). Both datasets were subset to the 16 overlapping cancer types for subsequent analysis .Level 4 Reverse Phase Protein Array (RPPA) data for the TCGA and CCLE samples were downloaded from the The Cancer Proteome Atlas (TCPA) portal . HCC tumor 10x Genomics single-cell RNA sequencing datasets from seven samples from two patients were downloaded from the Gene Expression Omnibus (accession GSE112271). All single-cell gene expression data analysis was performed using scanpy (v1.6.7) in Python (v3.6.8). The datasets were combined, and cells expressing >5000 genes and/or >30 mitochondrial genes were excluded, leaving 52,630 cells. Leiden clustering was performed using the top 40 Principal Components, cell types assigned based on cell-type markers  containing 10% fetal bovine serum (FBS) and 1% (v/v) antibiotic/antimitotic. Early passage cells (<P3) were grown to confluency and then harvested for single-cell RNA sequencing analysis as described above.Hepatocellular carcinoma (HCC) circulating tumor cell and cell line 10x Genomics single-cell RNA sequencing datasets were downloaded from the Gene Expression Omnibus (accession GSE103867). The cell-line cells were selected based on the expression of In Python (v3.6.8), the sklearn module (v0.21.2) was used with linear kernel to train a support vector machine (SVM) on 50 random 80/20 splits of the merged TCGA-CCLE gene expression dataset, in which 50 different training sets (and corresponding test sets) were resampled from the same data. The top 10% of genes from each training run based on the magnitude of their feature weights coefficients were merged in a non-duplicate manner, to account for variation in prediction results based on the training and corresponding test set, resulting in 1858 genes. Gene set enrichment from this analysis revealed 26/100 enriched pathways were immune-related, so a non-redundant immune gene list was created by merging all genes from the 26 enriched immune pathways (Supplementary Data P value < 0.05. Significantly enriched gene sets were identified using gene set enrichment analysis11 with FDR q-value < 0.05. For bulk RNA-seq, heatmaps are generated using seaborn clustermap method in Python with z-scores calculated per gene. For single-cell RNA-seq, heatmaps are generated using scanpy heatmap method in Python with values scaled between 0 and 1. Figures were produced using pandas v0.25.3, matplotlib v3.0.3, and seaborn v0.9.0 in Python v3.6.8. Numerical data used to generate figures are available here: https://drive.google.com/drive/u/1/folders/16pqRgDIxS0Wj_H9rIwyjB11a0IE6TAXH All data were used for each figure, except when otherwise indicated.Expression comparisons of individual genes between datasets were performed using a two-sided Mann-Whitney significance test with significance defined as Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


The newly synthesized enoxacin derivatives have shown a magnified cytotoxic effect with an emphasis on prooxidative, proapoptotic and microRNA interference actions. The mentioned mechanisms seem to contribute to a safer, more selective and more effective anticancer therapy. Enoxacin is a second-generation quinolone with promising anticancer activity. In contrast to other members of the quinolone group, it exhibits an extraordinary cytotoxic mechanism of action. Enoxacin enhances RNA interference and promotes microRNA processing, as well as the production of free radicals. Interestingly, apart from its proapoptotic, cell cycle arresting and cytostatic effects, enoxacin manifests a limitation of cancer invasiveness. The underlying mechanisms are the competitive inhibition of vacuolar H+-ATPase subunits and the c-Jun N-terminal kinase signaling pathway, causing a decrease in cell invasiveness. Interestingly, the prooxidative nature of the subjected fluoroquinolone enhanced the cytotoxic effect. Crucial for the anticancer activity were the carboxyl group at the third carbon atom, fluorine at the seventh carbon atom and nitrogen at the eighth position of naphyridine. Modifications of the parent drug improved the induction of oxidative stress, cell cycle arrest and the dysregulation of microRNA. The inhibition of V-ATPase–microfilament binding was also observed. Enoxacin strongly affected various cancer but not normal cells, excluding keratinocytes, which suffered from phototoxicity. It seems to be an underestimated anticancer drug with pleiotropic action. Furthermore, its usage as a safe antibiotic with well-known pharmacokinetics and selectivity will enhance the development of anticancer treatment strategies. This review covers articles published within the years 2000–2021, with a strong focus on the recent years (2016–2021). However, some canonical papers published in twentieth century are also mentioned.Enoxacin as a second-generation synthetic quinolone is known for its antibacterial action; however, in recent years there have been studies focusing on its anticancer potential. Interestingly, it turns out that compared to other fluoroquinolones, enoxacin exhibits uncommon cytotoxic properties. Besides its influence on apoptosis, the cell cycle and cell growth, it exhibits a regulatory action on microRNA biogenesis. It was revealed that the molecular targets of the enoxacin-mediated inhibition of osteoclastogenesis are vacuolar H Fluoroquinolones are synthetic antibacterial agents, representing the first set of anti-infective agents that were not modeled knowingly after any natural antibiotics ,2. Over Enoxacin (ENX)  is an orFluoroquinolones are a group of substances that have been thoroughly tested in terms of their anticancer activity. Only in recent years have a number of reviews describing the cytotoxic properties of fluoroquinolones been published ,12,13,14Like most fluoroquinolones, enoxacin has been tested for its anticancer activity. However, distinct activities of enoxacin, such as anti-ALS, antidepressant, antiaging and antiviral activities, are reported in literature . In thisThe miRNA biogenesis begins with transcribing a gene into a large primary transcript (pri-miRNA). The transcription is typically mediated by RNA polymerase II . The priIt has been clear for almost twenty years that miRNA expression is dysregulated in human malignancies. The underlying mechanisms include chromosomal abnormalities ,26,27,28Small molecules can influence miRNA biogenesis. Enoxacin was reported as the first and unique small-molecule enhancer of microRNA (SMER) maturation . Jin et al. found that the increase in miRNAs was associated with high levels of their own precursors, thereby suggesting that enoxacin could promote the DICER processing activity without influencing the miRNA precursor expression . The samThe role of the TRBP in enoxacin-mediated cytotoxicity was confirmed in the three colorectal cancer cell lines  and their mutants with impaired TRBP expression. TRBP-impaired cells were more resistant to enoxacin, resulting in a 2-fold increase in the effective concentration (EC50). Mutant cells also did not undergo cell cycle arrest. Additionally, enoxacin induced a two-fold expression increase in 24 miRNAs in RKO and HCT-116 cells and from 1.5-fold up two five-fold increases in the expression of those miRNAs in RKO and HCT-116 xenograft mouse model. Enoxacin treatment also significantly decreased the number of lung and liver metastases in the HCT-116 xenograft . InteresThe role of enoxacin as an enhancer of DNA damage response (DDR) signaling was confirmed in cervical cancer HeLa cells. This antibiotic augmented the DDR mediator factors pATMS1981, 53BP1, MDC1 and pS/TQ without affecting their expression, whereas the activity of γH2AX levels was not affected. Based on the knowledge of the activation of TRBP by enoxacin, it is not surprising that TRBP silencing diminished ENX’s stimulation of DDR. The knockdown of PACT or GW182 proteins , which are effectors for miRNA-guided gene silencing, did not affect ENX-mediated DDR stimulation. These results seem to strengthen and confirm enoxacin’s specificity towards TRBP .Regarding the molecular target recognized by enoxacin, an additional protein has been proposed that merits mentioning. In 2017, Abell et al. determined that the Piwi-like protein 3 (PIWIL3) is a potential enoxacin target . PIWIL3 Additionally, ENX affected RNA helicase DHX9, a member of the RISC. The expression of DHX9 was much higher in the small-cell lung cancer cell line H446 than in the non-small-cell lung carcinoma cell lines A549 and PC9. Enoxacin inhibited the proliferation of A549 in a dose-dependent manner. Similarly, it decreased the expression of DHX9 in A549 cells. However, silencing DHX9 impaired the cytotoxic effect of the drug . A strong inhibitory effect of enoxacin on the proliferation of human melanoma A375, Mel-Juso and Mel-Ho cell lines was observed. It dysregulated a set of 55 miRNAs in A375 cells . Two upregulated miRNAs, miRNA-3154 and miRNA-4459, control the p53-Mdm2-MdmX network . InteresA different ENX antiproliferative mechanism without affecting apoptosis has been noted in 4T1 murine breast cancer cells. An increased level of GW/processing bodies, which are considered the surrogate markers for both the microRNA-mediated repression of translation and the extracellular vesicle (EV) packaging sites, was observed. MiRNA expression levels in the 4T1 cells’ cytosol and their EV were compared. Enoxacin significantly increased only miRNA-214-3p and slightly increased miRNA-146a-5p, miRNA-290, miRNA-689 and let-7b-5p cytosolic levels. In contrast, significant decreases in let-7b-5p, miRNA-146a-5p and miRNA-689 expression and an enormous 22-fold increase in miRNA-214-3p were observed. All mentioned miRNAs are involved in the regulation of bone remodeling and osteoclastogenesis. Additionally, EVs from enoxacin-treated 4T1 cells enhanced the proliferation of murine macrophage cells . The effAlthough it has been clear that enoxacin did not affect all existing miRNAs  CD8 cells from an autoimmune cholangitis mouse model. Despite the fact that enoxacin did not change the amount of CD8 T cells, it significantly decreased their proliferative response. Enoxacin also significantly decreased the level of interferon γ in mouse serum . There iEnoxacin is also capable of affecting artificial miRNAs (amiRNAs), especially by enhancing the amiRNA-mediated reversible inhibition of the CRISPR-Cas9 system in both in vitro and in vivo studies. Interestingly, amiRNA alone did not show an inhibitory effect towards the CRISPR-Cas9 system, indicating the crucial role of enoxacin. In contrast, some of the naturally occurring miRNAs were able to inhibit CRISPR-Cas9 activity by binding to single-guide RNA (sgRNA), a part of the sgRNA/Cas9 complex, in the absence of enoxacin. Surprisingly, the presence of enoxacin in concentrations up to 50 µM did not affect the natural miRNAs’ influence on the CRISPR system. The proposed explanation of these differences in the impacts of enoxacin on the inhibition of natural and artificial miRNAs is based on the low binding capacity of amiRNAs towards RISC and the high binding capacity towards the RISC of natural miRNAs. Another factor could be the difference in the amounts of amiRNAs vs. natural miRNAs. The amiRNAs are believed to outnumber natural miRNAs. Taking the above into account, the amiRNA/enoxacin system turned out to be specific and reversible, making it a convenient tool for CRISPR-Cas9 regulation .In 2008, Shan et al. investigated the ability of microRNA processing to enhance some fluoroquinolones, including enoxacin and its three derivatives. They identified the structural elements of enoxacin responsible for its activity, such as a carboxyl group at the 3rd carbon atom, fluorine at the 7th carbon atom, as well as nitrogen at the 8th position of naphyridine. .Enoxacin affected cancer cells via various mechanisms . It indu+-ATPase (V-ATPase) and microfilaments. The affected subunits of V-ATPase were B2 subunit ..69].LZ-106 induced apoptosis via activation of the ROS-dependent DNA damage response in NSCLC  in both cultured cells and in a xenograft mouse model. The authors demonstrated that LZ-106 notably induced ROS overproduction and DDR. Interestingly, additional evidence in their findings revealed that DDR and apoptosis could be alleviated in the presence of an ROS scavenger, N-acetyl-cysteine (NAC), indicating ROS-dependent DDR involvement in the LZ-106-induced apoptosis. This research not only offered a new therapeutic candidate for NSCLC, but also gave new insights into the pharmacological research of quinolones .In 2019, Yang et al. continued to investigate the mechanism of the cytotoxic activity of LZ-106. They substantiated the involvement of P53 activation in intracellular ROS generation upon LZ-106 treatment and related P53 to the ROS-induced viability inhibition and apoptosis, which was presented in previous research. P53 was shown to play an indispensable role in the upregulation of intracellular ROS in LZ-106-treated NSCLC cells through ROS detection. The authors further identified the antiproliferation effect of LZ-106 in NSCLC cells exhibited through G1 phase cell cycle arrest with a cell cycle analysis, with an expression analysis of the key proteins, and discovered that the cell cycle arrest effect is also mediated by the induction of ROS in a P53-dependent manner. In addition, the tumor-suppressive effect exhibited in vivo was demonstrated to be similar to that in vitro, which requires the participation of P53. Thus, LZ-106 is a potent antitumor drug possessing potent proliferation inhibition and apoptosis induction abilities through P53-dependent ROS modulation both in vitro and in vivo .In 2018, Vracar et al. showed that enoxacin and bis-enoxacin  stimulatBis-enoxacin (BE) did not induce apoptosis or inhibit 4T1 murine breast cancer cell proliferation; however, it altered the release of extracellular vesicles and altered the miRNA expression. It also increased the number of GW/P bodies. Both enoxacin and bis-enoxacin at a concentration of 50 µM increased the number of GW/P bodies, but there were minimal changes in microRNA levels. Extracellular vesicles (EVs) released from 4T1 cells treated with 50 µM enoxacin or 50 µM bis-enoxacin stimulated the proliferation of RAW 264.7 cells, and both drugs significantly inhibited osteoclastogenesis in calcitriol-stimulated mouse marrow. EVs from 4T1 cells treated with enoxacin and bis-enoxacin displayed small reductions in the amounts of miRNA-146a-5p and let-7b-5p. In marked contrast, miRNA-214-3p, which has been shown to regulate bone remodeling, was increased 22-fold and 30-fold, respectively. We conclude that enoxacin and bis-enoxacin trigger in 4T1 cancer cells the release of EVs that inhibit osteoclastogenesis . SimilarThe introduction of 2-(5-chlorothiophen-2-yl)ethyl into the piperazine ring of enoxacin  increase50 range for NOH of ca. 3–10 µM and a IC50 range for NOMe of ca. 3–20 µM (depending on the cell line). A similar IC50 range of 2–14 µM for NOBn derivatives was observed in melanoma, epidermoid, cervical and bladder carcinoma cells. The IC50 values of NOBn derivatives in other cell lines and those of oxygen in all tested cell lines were much higher, but still significantly lower than the IC50 of unmodified enoxacin. To sum up, the introduction of the 2-(5-chlorothio-phen-2-yl)ethyl moiety into the 4′ position of enoxacin increased the cytotoxicity of the compound; however, the strength of the effect depended on the structure of the ethyl spacer. Introducing another nitrogen atom  led to a significant increase in antitumor activity [The synthetized derivatives differ from each other in the modification of the structure of an ethyl spacer. The introduction of oxygen into the ethyl spacer increased the cytotoxic activity from 137–196 µM for enoxacin to 137–131 µM for the oxygen derivative, which was a relatively minor change. The introduction of NOH or NOMe led to the strongest cytotoxic activity vs. all tested cell lines, with a ICactivity . A series of enoxacin derivatives was formulated via the transformation of the carboxyl group in the skeleton of N-methylenoxacin (position 3) to an aldehyde group, followed by a direct reaction with N-arylhydrazinecarbothioamide (thiosemicarbazide) (CN106674220) or subjected to a condensation reaction with methyl hydrazinodithioformate and next to a nucleophilic substitution reaction with some N-alkylamines ,105. TheThe obtained derivatives were tested for antitumor activity on cancer cell lines, including Hep-3B (human liver cell line containing integrated hepatis B virus genome), PANC-1 (human pancreatic cancer cell line) and HL-60 (human leukemia cell line). The cytotoxic activity against cancer cell lines was compared to that against a normal cell line—Vero . 50 6.0–20.5 µM). The most active derivative was the derivative with the cyclopropyl substituent in the tiosemicarbazide part. Extending the alkyl substituent, especially above four carbon atoms, resulted in lower cytotoxic activity. All derivatives were characterized by significantly lower cytotoxicity against the normal Vero cell line (over 66.5 µM) [The alkyl derivatives showed cytotoxicity within limits (IC66.5 µM) .50 3.2–6.2 µM). In this case, all derivatives were also characterized by significantly lower cytotoxicity compared to the normal Vero cell line (above 73.4 µM) [The aryl derivatives, similar to the alkyl ones, were characterized by much higher cytotoxicity compared to enoxacin, in the range of 2.7 to 33.5 µM. Derivatives 1a–5a and 7a are characterized by more than two times lower cytotoxic activity compared to derivatives 6a and 8a–11a. The introduction of fluoro substituents to the phenyl ring or the replacement of the phenyl substituent with pyridyl, furanyl and thiophenyl substituents increases the cytotoxic activity. The most active compound was the derivative containing a pyridyl substituent in the tiosemicarbazide part (IC73.4 µM) .50 1.8–21.5 µM. The derivatives numbered 1–4 are characterized by more than two times lower cytotoxic activity compared to the derivatives numbered 5–10. The most active derivative was the derivative containing a benzenesulfonamide substituent in the rhodanine part (10). In this case, all derivatives were also characterized by a significantly lower cytotoxicity compared to the normal Vero cell line (above 58.3 µM) [Chinese scientists have tested the cytotoxic activity of some rhodanine derivatives of N-methylenoxacin . The met58.3 µM) .Another patented derivative was a dimeric form of enoxacin. The 1-(N-enoxacin amide)-enoxacin derivative  was obtaEnoxacin is a multifaceted drug. It is not only an effective antibiotic but also a promising anticancer agent. Unlike other fluoroquinolones, ENX exhibits extraordinary cytotoxic mechanisms of action. The presence of a carboxyl group at the third carbon atom, fluorine at the seventh carbon atom, as well as nitrogen at the eighth position of naphyridine is crucial for the regulation of miRNA abundance. ENX has the potential for the dysregulation of miRNA biogenesis by interfering with the PIWIL3 protein or DHX9 helicase, a part of the RNA-induced silencing complex (RISC), as well as the RISC-loading complex proteins DICER and TRBP. Additionally, it induced an elevated number of GW/P bodies. The alteration of those pathways was confirmed in the enoxacin-induced cytotoxicity towards colorectal, breast, prostate, thyroid and Ewing’s sarcoma family (ESFT) cancer cells. Changes in miRNA biogenesis resulted also in a decrease in inflammatory response in mice, affecting the neurobiology, including in neuronal differentiation and the inhibition of osteoclastogenesis. Another confirmed mechanism of osteoclast bone resorption inhibition was the suppression of the JNK signaling pathway and binding with V-ATPase subunits. Moreover, it has been described as a convenient tool for the regulation of CRISPR-Cas9 activity. Another anticancer mechanism was the induction of DNA damage by ROS stress generation. Enoxacin caused an induction of oxidative stress by singlet oxygen but not by superoxide radicals. Interestingly, it induced morphological changes, cell cycle arrest, growth inhibition, a decrease in invasiveness and apoptosis in cancer but not in normal cells. The above findings give evidence for the use of enoxacin, especially in bladder cancer therapy, where the achievable urine concentrations are far higher than IC50 and can be maintained constantly for a long time period. Furthermore, this type of cancer could be harnessed using two sources of enoxacin—urine and blood.The modification of the piperazine ring or the carboxylic group resulted in enhanced cytotoxicity comparing to free enoxacin. Some of the derivatives were proven to cause cell cycle arrest and the induction of oxidative stress, while others dysregulated the levels of miRNA. Interestingly, the LZ-106 derivative caused the formation of superoxide radicals, in contrast to the parent drug. Unfortunately there are no clinical studies on enoxacin’s anticancer activity, while the results from in vitro studies are scarce compared to other fluoroquinolones. Taken together, enoxacin has anticancer potential, and its modifications may bring additional therapeutic benefits.


Pinus koraiensis), using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. We found that exogenous GSH promoted cell proliferation in both cell lines, while exogenous BSO inhibited proliferation in both cell lines. At 35 d with exogenous GSH treatment, the fresh weight of F and S cell lines increased by 35.48% and 48.39%, respectively, compared with the control. The exogenous application of GSH increased the intracellular levels of GSH, total GSH (T-GSH), oxidized glutathione (GSSG), ascorbic acid (ASA), total ASA (T-ASA), and the ratios of GSH:T-GSH and ASA:T-ASA in both F and S cell lines. Furthermore, exogenous GSH increased the activity of both glutathione reductase (GR) and dehydroascorbate reductase (DHAR) while decreasing the activity of ascorbate peroxidase (APX) in both cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is conducive to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low proliferation rates, hindering the production of improved plant materials. One way of improving proliferation rates is achieved by improving the redox status of the culture medium. In this study, we investigated the effects of exogenous glutathione (GSH) and L-buthionine sulfoximine  on the EC proliferation rate in Korean pine ( Somatic embryogenesis (SE) is the artificial formation of embryo-like structures from somatic cells without the need for gametic fusion . SE is oPinus koraiensis Sieb. et Zucc.) is a dominant species in mixed-broadleaved Korean pine forests in Northeast China [Korean pine  [Picea glauca), regulation of the redox status of the SE medium by application of exogenous GSH or GSSG promoted a dramatic increase in both EC proliferation (25% by weight) and the number of early filamentous embryos, accompanied by an increase in ATP and nucleotide production. Inhibiting GSH synthesis through the application of L-buthionine sulfoximine (BSO) has been shown to induce a pro-oxidative environment, thereby reducing the cellular GSH: total glutathione (T-GSH) ratio [Cellular antioxidants such as glutathione (GSH)/oxidized glutathione (GSSG) and ascorbic acid (ASA)/dehydroascorbic acid (DHA) play an important role in many biological processes, including cell division and differentiation, and can impact the process of SE through changes in cellular reduction oxidation (redox) status ,19,20,21ylifera) . In whitH) ratio . Both ASThe proliferative capacity of EC in conifers is essential for the large-scale production of high-quality seedlings. GSH promotes EC proliferation in white spruce, but it is unclear whether GSH has an effect on EC proliferation in Korean pine. The effect of exogenous GSH on the accumulation of GSH and ASA in EC cells of Korean pine and the cycling between them is not clear. In this study, we investigated the effects of exogenous GSH and BSO on the EC proliferation rate in Korean pine, using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. Furthermore, we examined redox regulation, as well as intracellular GSH and ASA synthesis, in these cell lines. This work will serve to optimize large-scale breeding efforts in woody plants by improving SE efficiency, particularly in Korean pine and other conifer species.p < 0.05) (The growth status of the two cell lines is shown in  < 0.05) . Exogeno < 0.05) a, wherea < 0.05) b. The FW < 0.05) a. At 35 p < 0.05) (The effects of exogenous application of GSH and BSO on EC dry weight (DW) of the two cells lines were different ( < 0.05) . Overall < 0.05) a, while  < 0.05) b. The DW < 0.05) a. The DWp > 0.05)  .The exogenous application of GSH promoted T-GSH synthesis in both cell lines a,b. In tp < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular GSH content of the two cell lines were different ( < 0.05) c,d. The  < 0.05) c. The ex < 0.05) d. The inp < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular GSSG content of the two cell lines were different ( < 0.05) e,f. Exog < 0.05) e. The ex < 0.05) f. The inThe exogenous application of GSH increased the intracellular GSH:T-GSH ratio in F and S cell lines g,h, withThe exogenous application of GSH promoted the synthesis of intracellular T-ASA in F and S cell lines a, with ip < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular ASA content of the two cell lines were different ( < 0.05) c,d. The  < 0.05) c. The in < 0.05) d, with tp < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular DHA content of the two cell lines were differed ( < 0.05) e,f. The  < 0.05) e, with t < 0.05) f, with tThe exogenous application of GSH tended to increase the intracellular ASA:T-ASA ratio in F cell lines g, althoup < 0.05) (The effects of exogenous application of GSH and BSO on intracellular GR activity of the two cell lines were differed ( < 0.05) a,b. Intr < 0.05) a. Intrac < 0.05) a. The ex < 0.05) b. Intracp < 0.05) (The effects of exogenous application of GSH and BSO on intracellular APX activity of the two cell lines were differed ( < 0.05) c,d. Intr < 0.05) c. At day < 0.05) d, with ip < 0.05) (The effects of exogenous application of GSH and BSO on intracellular DHAR activity of the two cell lines were differed ( < 0.05) e,f. In t < 0.05) e. Simila < 0.05) f. IntracTen differentially expressed Korean pine genes were selected for qRT-PCR validation . Of thes. Healthy EC tissue and a fast proliferation rate are essential during SE. To that end, GSH has been shown to regulate cell division and differentiation in many organisms, and has been found in high concentrations in rapidly proliferating cells [P. glauca) SE have shown that exogenous application of GSH promotes the proliferation of EC, increasing EC FW by as much as 25%. In this work, we examined the effect of exogenous application of GSH and BSO on Korean pine SE and found that GSH promoted EC proliferation, while BSO inhibited EC proliferation, compared to CK.EC material allows for efficient genetic transformation, makes an ideal system for studying single-cell differentiation and allotropic expression, and can form the basis for large-scale production of woody plants . Healthyng cells . More spng cells . StudiesGSH is present in most prokaryotic and eukaryotic cells  and playNicotiana tabacum), exogenous application of ASA during cell division has been shown to increase intracellular soluble protein content, and decrease both malondialdehyde (MDA) content and cell membrane permeability [ASA is the primary component of the ASA-GSH cycle and plays an important role in cellular growth, elongation, and division frequency, and also in protecting the cell membrane . In tobaeability . ASA is GSH is an electron receptor for DHA and plays an important role in scavenging intracellular ROS and maintaining redox homeostasis in plants . In bothEupatorium adenophora) have been found to upregulate GR activity when exposed to low temperatures, but not high temperatures [Eupatorium odoratum) show the opposite trend. In white spruce (P. glauca), the exogenous application of BSO during SE increased intracellular GR activity [Brassica napus), the exogenous application of BSO during SE reduced intracellular GR activity [GR is a flavinase which catalyzes the formation of GSH from GSSG. GR also plays a crucial role in maintaining cellular redox homeostasis by scavenging intracellular ROS. Previous studies have found that GR activity increases when plants are subjected to stressful environmental conditions, including exposure to ozone, drought, heavy metals, bright light, salt, and low temperatures. Additionally, the GR activity of stress-tolerant plant species remains high during stress exposure, but drops in susceptible species . Plants eratures . Contraractivity , while iactivity . In thisCasuarina equisetifolia) [Pisum sativum) [Citrus reticulata) [Oryza sativa) [Solanum tuberosum) varieties, while APX activity decreased in salt-sensitive varieties. In oilseed rape (B. napus), APX activity was higher in embryos treated with exogenous BSO during the globular embryo period, and APX activity could be modified by exogenous GSH [In the ASA-GSH cycle, ASA is converted by APX to monodehydroascorbic acid (MDHA) . Changestifolia) , pea (Pisativum) , citrus iculata) , and ric sativa) . Additionous GSH . These rnous GSH . In thisDHAR is a key enzyme in the ASA-GSH cycle, which promotes ASA regeneration and serves to protect cells against oxidative damage. Specifically, DHAR catalyzes the production of ASA from GSH, thus helping to regulate the ratio of intracellular ASA to DHA . In thisTwo Korean pine cell lines, one with high proliferative potential 001#-001 (F) and one with low proliferative potential 001#-010 (S), were revived and cultured after ultra-low temperature preservation. EC induction was performed using mLV ,49 mediuAll culture media in this study had a pH of 5.8 and were autoclaved  after pH adjustment. After the medium was sterilized and cooled to approximately 55–60 °C, Gln and ABA were added by filter sterilization (filter membrane pore size 0.22 μm). The medium was inoculated with MG as the explants and the cultures were cultured in the dark at 23 ± 2 °C.http://www.njjcbio.com/, accessed on 1 August 2022). Intracellular DHA content and DHAR activity were determined using Beijing Solarbio assay kits . The absorbance values of GSH and GSSG were determined by an enzyme marker, and the absorbance values of DHAR, ASA, DHA, GR, and APX were determined by spectrophotometry, according to kit instructions. Samples were prepared for each assay according to the following paragraphs. Methods were as follows:The intracellular GSH, GSSG, and ASA content, and GR and APX activity, were determined using Nanjing Jiancheng assay kits  for 10 min. The GSSG content was calculated according to the formula method. Three replicates per treatment were carried out. The concrete method please refer to the introductions.The concrete method please refer to the instructions.To determine ASA content: 0.2 g of EC was mixed with 0.5 mL of PBS buffer, homogenized in an ice bath, and centrifuged at 5000 rpm for 10 min. To determine DHA content: 0.1 g of the sample was mixed with 0.5 mL of extract, homogenized in an ice bath, and centrifuged at 16,000 rpm for 20 min at 4 °C. The absorbance value ‘A1’ was recorded at 10 s, and the absorbance value ‘A2’ was recorded at 130 s. The change (Δ) was calculated as A2-A1, and 0.5 μmol/mL DHA was used as the standard tube. Four replicates per treatment were carried out. The concrete method please refer to the introductions.To determine GR activity: 0.2 g of EC was mixed with 1.8 mL of PBS buffer, homogenized in an ice bath, and centrifuged at 2500 rpm for 10 min. Absorbance was measured at 340 nm with a 1 cm optical diameter cuvette. The absorbance value ‘A1’ was recorded at 30 s and the absorbance value ‘A2’ was recorded at 2 min at 37 °C. Double-distilled water was used as the standard tube. Four replicates per treatment were carried out. The concrete method please refer to the introductions.To determine APX activity: 0.3 g of the sample was mixed with 0.6 mL of buffer, homogenized in an ice bath, and centrifuged at 10,000 rpm for 10 min at 4 °C. The absorbance value ‘A1’ was recorded at 10 s, and the absorbance value ‘A2’ was recorded at 130 s. The change (Δ) was calculated as A2-A1. Double-distilled water was used as the standard tube. Three replicates per treatment were carried out. The concrete method please refer to the introductions.To determine DHAR activity: 0.6 g of the sample was added to 0.6 mL of extraction solution, homogenized in an ice bath, and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was added to the reagent according to the method of the kit, and the absorbance value at 412 nm of the enzyme-labeled instrument was measured. Three replicates per treatment were carried out introductions.Transcriptomic sequencing revealed the presence of 10 differentially expressed genes between the F and S cell lines, and these genes were used for qRT-PCR validation. The Korean pine AAE7 gene  was used as an internal reference gene. The AAE7 gene was selected because it is stable, with gene activity being largely independent of external interference. Transcriptomic sequencing revealed the presence of 10 differentially expressed genes between the F and S cell lines, and these were used for qRT-PCR validation. Primer sequences were synthesized by Beijing Bemec Biotechnology Co., , and can be found in Experimental calculations, including proliferation parameters, were performed using Excel 2003 . A one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons tests were performed using SPSS 19 . Data concerning percentages were arcsine-transformed before analysis. Graphs were constructed in Sigma Plot 12.0 . Proliferation parameters were calculated according to the following equations:The study confirmed that exogenous GSH promotes the proliferation of Korean pine EC. This is because the exogenous application of GSH increased the intracellular contents of T-GSH, GSH, GSSG, T-ASA, and ASA and the ratios of GSH: T-GSH and ASA: T-ASA in both F and S cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is beneficial to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.