Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library  covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large. Metabolic reprogramming contributes to cancer development and progression. Here, the authors show the utility of a metabolic drug library to uncover metabolic vulnerabilities and obtain functional insights into myeloid leukemia biology. The first observations that transformed cells exhibit a distinct metabolic program came from Otto Warburg almost hundred years ago. The Warburg effect depicts the phenomenon of cancer cells preferentially undergoing glycolysis and converting carbon to lactate even in high oxygen conditions3. In cancer, genetic events activate signaling pathways that subsequently modulate cellular metabolism to satisfy the increased bioenergetic, biosynthetic and redox demands6. Moreover, this supports cancer initiation and progression and is typically accompanied with changes in expression of metabolic enzymes and transporters, which has implications for nutrient uptake, distribution of nutrients to pathways for biomass generation ultimately affecting therapy response8. Therefore, cancer-specific metabolic changes confer a selective advantage for survival, but also introduce metabolic liabilities that provide a unique opportunity for therapeutic targeting.Cellular metabolism represents a dynamic network of regulated pathways that is frequently reprogrammed in cancer and has been recognized as an emerging hallmark10. Hence, despite glycolysis many more metabolic dependencies are altered to support cancer cell proliferation8. A greater insight in the metabolic differences between cancer and nonmalignant cells may lead to development of improved cancer therapies and ultimately patient outcomes. However, normal cells often physiologically activate metabolic pathways that are upregulated in cancer. Thus, identifying metabolic processes that offer sufficient therapeutic window and patients most likely to respond to a given therapy remains a challenge11.Considering the matter closer, it becomes apparent that there are different individual metabolic signatures of cancer cells. Transformed cells display greater flexibility in catabolite utilization, contingent on nutrient accessibility in the environment, cell lineage or tissue of origin, and genetic events13. While the hematologists toolbox has increased, the survival of AML patients remains poor14. Moreover, prior studies have illustrated metabolic pe3culiarities in myeloid leukemias that can be exploited for either the development of novel therapies or for particular stratification rationales21. For instance, the development and clinical utility of IDH1/2 inhibitors for IDH1/2 mutant AML25 as wells as BCL-2 inhibitors26 illustrates that untangling metabolic changes could provide therapeutic avenues in AML. Here, we profiled the metabolic drug library phenotypically in a panel of 15 diverse myeloid leukemia cell lines for drug-induced effects on cell growth and survival.Obtaining a collection of chemical agents affecting different aspects of cancer metabolism would represent a toolbox allowing to interrogate cell lines, primary samples and animal models in a versatile mode and may suggest therapeutic strategies. Here, we assembled a custom metabolic drug library covering 243 compounds allowing to systematically identify metabolic dependencies in high-throughput phenotypic screens. We focused on myeloid leukemias as a model disease, as they are heterogeneous clonal malignancies for which there is a need for comprehensive understanding of disease-specific molecular mechanisms. Even though, there has been substantial advancement in mapping the genetic landscapes of AML, the first approvals for targeted agents came only in the last few yearsWe functionally grouped the cell lines and drugs based on metabolic drug efficacy patterns and associated them with distinct genomic and metabolic attributes. Moreover, we identified a number of differential vulnerabilities, such as the sensitivity to the monocarboxylate transporter (MCT) SLC16A1  inhibitor AZD3965, that could be rationalized by differential expression as well as target essentiality. Our data provides a primer for the use of a focused metabolic drug library in phenotypic screening platforms, thereby identifying metabolic vulnerabilities and pinpointing that targeted metabolic perturbations may provide promising new therapeutic strategies for myeloid leukemias and beyond.27. CLIMET was compiled in a stepwise fashion, starting from 8000 candidate compounds, after a survey of public drug-target databases, and ending with 243 highly curated compounds after extensive crosschecking for approval status, structural information, compound’s potency and selectivity for the intended target, pathway/target redundancy, and commercial availability . While probing cellular metabolism could also be achieved with genetic screening technologies such as CRISPR, compound libraries are more readily applicable to challenging cell and tissue samples including primary patient cells and provide rapid result outputs. Furthermore, they can easily be combined with other interventions  for higher-order perturbationsity Fig. .Fig. 1Won = 84), protein and amino acid (n = 43), and glycolysis/sugar metabolism (n = 39)  compounds are clinically approved for various disease indications allowing for rapid translation, roughly half  are chemical probes, 10 in clinical trials and 25 either withdrawn or their clinical development discontinued Fig. . CLIMET 39) Fig. .To assess the potency of CLIMET compounds, we screened the full collection against a panel of myeloid leukemia cell lines . Each compound was tested for its effect on cell growth and survival over a 10,000-fold concentration range (1 nM to 10 µM) enabling the generation of dose–response curves and area under the curve (AUC) calculation Fig. . From thr = 0.78), with the exception of CML cells being more sensitive to Torin1 (mTOR inhibitor), cytarabine , and rotenone (complex I inhibitor)  was selectively sensitive to a cluster of compounds targeting processes and enzymes capable of producing reactive oxygen species such as cytochrome P450 oxidases (econazole), NADPH oxidases (GKT-136901), mitochondrial complex II (3-nitropropionic acid), and phosphodiesterases (PF-02545920)  and extracellular acidification rate (ECAR) as proxies for mitochondrial respiration and glycolysis, respectively, by performing the Seahorse mitochondrial stress test  approach was applied on 225 metabolites in 928 cell lines from more than 20 different cancer types. Data were available for 13 of the 15 cell lines and the levels of 57 metabolites were found to be significantly different between at least two groups using a two-way ANOVA analysis  in comparison to largely glycolytic cell lines (Groups III and V) , revealed statistically significant novel and previously known correlations between FLT3 mutations and sensitivity to 5-fluorouracil, lestaurtinib32, and PF-02545920. Moreover, CREBBP mutations linked to sphingosine kinase inhibitor  sensitivity, whereas RAS and TP53 mutations conferred a resistance phenotype especially to energy and lipid metabolism inhibitors such as PI3K/mTOR and mitochondrial respiration inhibitors  and dorsomorphin (AMPK inhibitor). These findings imply that the activity of these compounds in the setting of myeloid malignancies could be functionally related.Co-occurring and mutually exclusive drug sensitivities were detected by pair-wise Spearman’s correlation analysis of the 77 active drugs Fig. . InhibitTo investigate the identified drug interactions further we performed drug combinatorial screens in four different cell lines  that best captured the significant drug–drug correlations. The results showed that in majority of cases combinatorial effects were observed as hypothesized  Fig. . For insC2A Fig. . This isers Fig. . Thus, wPIK3CA, PIK3CB or PIK3CD in comparison to HL-60 cells lentivirally transduced with an empty vector 29, and drug sensitivity data36 in HL-60 cells and cluster I cell lines (MV4-11 and MOLM-13) revealed that HL-60 cells exhibit higher dependency and activity on the MAPK pathway and mTORC2 complex signaling resulting in reduced sensitivity to AKT and mTOR inhibitors . Varied gene expression analysis identified 49 most differently expressed genes in relation to the gene expression profile of the remaining 13 cell lines  and SLC16A3 (low/no) may serve as an actionable biomarker for AZD3965 response in hematological malignancies. To explore the potential clinical value of this biomarker, we turned to The Cancer Genome Atlas (TCGA) gene expression dataset accessed via the UCSF Xena project45. Utilizing the gene expression profiling interactive analysis server (GEPIA2)46, we assessed the expression pattern of SLC16A1 and SLC16A3 in cancer patients in comparison to normal samples (TCGA-matching and the Genotype-Tissue Expression project47)  and downregulated in AML, while the opposite is true for SLC16A3. Moreover, the expression of SLC16A1 and SLC16A3 is negatively correlated on a pan-cancer and AML-specific level  Fig. . For insvel Fig. , indicatTo determine whether metabolic vulnerabilities can be influenced by drug exposure we re-tested the entire drug library in HL-60 cells in the presence of vehicle (DMSO) or 100 nM AZD3965, pictilisib, or GSK2194069 for 72 h Fig. . Even thn = 15; Supplementary Table r = 0.5716) with ~64% of compounds not exhibiting a differential response  were predominantly glycolytic and displayed lack of sensitivity to mTOR and mitochondrial respiration inhibitors in line with previous reports that RAS-mutated cancers display a Warburg effect phenotype exemplified by increased glucose utilization and lactate production, and upregulation of the glucose transporter SLC2A154. While some genotype to phenotype links were identified, for the majority of drug responses no clear genotype associations could be established, in accordance to prior studies showing a better association between multiple molecular datasets and drug sensitivities56.The tested cell lines were functionally grouped in five taxonomic groups that could further be associated with somatic mutational patterns as well as cellular metabolic profiles. For instance, cell lines harboring activating mutations in the 42. A recent study found that signaling models based on multiplex single-cell mass cytometry data were more predictive of pictilisib sensitivity than gene expression alone in breast cancer cell lines57. We, further, detected a significant positive correlation between the FASN inhibitor, GSK2194069, and pictilisib, which also showed a combinatorial effect. This finding suggests a functional link between PI3K signaling and de novo FA synthesis. In breast cancer cells synergistic responses between rapamycin and FASN inhibition have been reported58. The PI3K/AKT pathway could stimulate the expression of enzymes necessary for FA synthesis and promote activation of ATP-citrate lyase, an enzyme that catalyzes the production of acetyl-CoA from cytoplasmic citrate60. PI3K signaling in known to induce lipogenic enzymes, including FASN61. Cancer cells with upregulated PI3K signaling have been linked to increased FASN expression levels and enhanced glucose uptake thus fulfilling the increased need for membrane synthesis of rapidly dividing cells63.The sensitivity of the PI3K inhibitor pictilisib could be associated with lower activation of the mTORC1 complex, as mTORC1 activation has been described to be a crucial event in the resistance to PI3K inhibitors in many tumor typesSLC16A1 in sensitive cell lines due to near or complete absent expression of the functionally related transporter SLC16A3. Hence, inhibition of SLC16A1 in cells lacking SLC16A3 led to cell death due to the synthetic lethal relationship of these transporters and functional redundancy64. Similar observations have been made in small cell lung cancer, DLBCL, and Burkitt lymphoma66. The MCTs are bidirectional and execute proton-linked transport of lactate, pyruvate, and ketone bodies across the plasma membrane thereby playing a crucial role in preventing intracellular acidification68. Importantly, several cancers use lactate as a metabolic energy source70 and MCTs have been reported to impact cancer cell growth and survival71. Thus, as our data also show, MCTs represent attractive potential therapeutic cancer targets. Several SLC16A1-specific inhibitors have been developed72 with AZD3965 being in phase I clinical trials for advanced cancers (NCT01791595). The structural basis for its specificity and mode of action was also recently identified73. Our study illustrates that a relatively small percentage of myeloid malignancies (10–13%) would be susceptible to SLC16A1 inhibition, but highlights phenotypic drug screening as attractive means of identifying responding cells/patients harboring metabolic dependencies.We also observed a differential sensitivity pattern to AZD3965, which could be rationalized by dependency on 74, that could guide the exploitation of novel metabolic vulnerabilities by alternative chemical agents. Nonetheless, any therapy success of metabolic inhibitors would be contingent on an adequate integrative patient stratification. Several limitations of our study should be contemplated for future metabolic drug profiling efforts. First, the inactivity of roughly 70% of CLIMET compounds might be due to the concentration range used as some compounds are known to have activity in higher concentrations. Second, we may have missed out on interesting compounds that affect cellular metabolism by not impacting cell viability. Third, we envisage a wider collection of metabolic inhibitors would create insights into larger array of metabolic pathways/processes. Lastly, the composition of cell culture media is likely distinct from in vivo settings and in future, this parameter would need to be assessed more carefully. Nonetheless we uncovered functional and clinically relevant determinants of metabolic drug response. In-depth characterization of cancer metabolic dependencies and differences between normal and cancer cells will be a crucial step toward translating metabolic inhibitors to the clinic.While the genetic diversity of cancers is substantial, changes in numerous oncogenes and tumor suppressor genes stimulate a limited number of metabolic changes converging on a metabolic phenotype. In fact, previous studies have linked lack of drug response or the presence of drug resistance to specific intrinsic molecular and/or metabolic cellular propertiesMOLM-13, MV4-11, Mono-Mac-6, KG-1, SHI-1, ML-2, NOMO-1, HEL, LAMA-84, KU-812, KCL-22, and BV-173 were obtained from DSMZ, K-562 and HL-60 from NCI 60 panel and THP-1 from ATCC. All cell lines were maintained in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cell lines were authenticated with STR profiling and mycoplasma tested by PCR prior to the study.D or IC50) together with PubChem commercial chemical vendor records, which led to initial 8000 compound candidates  information. The target annotation was performed by collecting information from publicly available databases  and additional manual curation. The compounds were ordered via the Sigma-Aldrich Marker Select service and sourced at 10 mM concentration in DMSO directly on Echo 384-well plates . In addition, we obtained 1 mL of 10 mM stock in vials for future use. The detailed description of the compounds is depicted in Supplementary Data Information on metabolic enzymes and drug-target annotations were integrated from publicly available databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG), the Small Molecule Pathway Database (SMPDB) and ChEMBL target database  after 72 h incubation at 37 °C according to the manufacturer’s instructions. The highest cell number at which the growth curve was in linear range was chosen for drug testing. The compounds were printed on tissue culture-treated 384-well plates (Corning) using Echo 550 (Labcyte Inc.) in five different concentrations in duplicate in tenfold dilutions encompassing a 10,000-fold concentration range . Twenty-five microliters of single-cell suspension were seeded to each well with a Multidrop Combi peristaltic dispenser (Thermo Scientific). The plates were incubated at 37 °C and 5% CO2 for 72 h after which cell viability was measured using CellTiter-Glo in a multilabel plate reader . Data were normalized to DMSO wells serving as negative controls and 10  μM bortezomib wells  serving as a positive control. A four-parameter log-logistic model was fit for each compound’s dose–response data using the drc75 R package and corresponding AUC values calculated for each compound using the compute AUC function of the PharmacoGx76 R package. Unsupervised hierarchical clustering of the active compounds  was performed with Gene Cluster 3.0 software (complete linkage method and Spearman (drugs) and Euclidean (cell lines) distance measures). The resulting heatmap was visualized with Java Tree View software Version 1.1.6r4. Reproducibility of the clustering was assessed with the bootstrap resampling method using the Pvclust R package77. Similarity of drug sensitivity was evaluated by SD of AUC values for a particular compound within a cluster with SD ≤ 0.05 indicating concordance. The results were visualized with Venn diagrams with the Venn webtool from the University of Gent (http://bioinformatics.psb.ugent.be/webtools/Venn/). For detecting selective drug sensitivities in each cell line, the average AUC per drug was subtracted from the AUC value of the drug in each individual cell line and visualized with a spider plot using R statistical environment.The optimal cell number per well for each cell line was assessed for cell viability analysis in 384-well plates (Corning 3764). Briefly, cells were suspended in RPMI 1640 and seeded in increasing concentrations in triplicate  and cell viability was assessed with CellTiter-GloEvaluation if metabolic inhibitors can alter metabolic vulnerabilities was performed by rescreening the entire drug library as described above (each drug tested in five different concentrations in duplicate) in HL-60 cells in the presence of either DMSO, 100 nM AZD3965, 100 nM pictilisib, or 100 nM GSK2194069. Data analysis was the same as for the original screen with overall drug response profiles being compared with a Spearman correlation analysis performed in GraphPad Prism 9.To assess basal cellular metabolic activity, we utilized the Seahorse XF Cell Mito Stress Test Kit (Agilent) according to the manufacturer’s instructions. Briefly, cells suspended in Seahorse XF base medium, supplemented with glucose (10 mM), sodium pyruvate (1 mM) and L-glutamine (2 mM), were plated at 150,000 cells per well in XF 96-well cell culture microplates (Agilent). After 1 h of incubation at 37 °C the plate was loaded into the XF96 Extracellular Flux Analyzer (Agilent) and the Seahorse XF Cell Mito Stress Test was performed using oligomycin (1 µM), FCCP (1 µM) and rotenone (0.5 µM).28 was utilized for assessment of the metabolic profiles of the cell lines included in this study. A LC–MS approach was used to measure 225 metabolites in 928 cell lines from more than 20 cancer types. For analysis the imputed batch-corrected dataset with log10-transformed values (clean data) was used. To determine metabolite levels that were significantly different between at least two groups, a two-way ANOVA analysis and multiple testing correction with false discovery rate (FDR) of 10% was performed with GraphPad Prism version 9. The results were normalized with a Z score for a heatmap visualization. Metabolite enrichment and pathway analysis was performed with MetaboAnalyst79.Publicly available targeted metabolomics dataset from the CCLE80. For each recurrent mutation in at least three cell lines an unpaired two-sided parametric Student’s t test was performed to identify significant associations between mutations and drug response. FDR was computed using the Benjamini–Hochberg method over all drugs .Somatic mutation data for the cell lines included in this study was obtained from The Cancer Dependency Map portal 81 R package. The analysis was applied for the 77 active drugs and the Spearman’s correlation coefficient of each drug was plotted onto a clustered heatmap visualized with a correlogram. Positive correlations are showed in red and negative in blue. The intensity of the color and size of the circles are proportional to the correlation coefficients. The significant drug–drug correlations were identified after correcting for multiple testing with Benjamini–Hochberg at FDR of 10% and highlighted with thick black border.Co-occurring and mutually exclusive drug sensitivities were identified by pair-wise Spearman’s correlation analysis using the corplot83 web portal (https://synergyfinder.fimm.fi) using the Bliss independent model.The significant drug–drug interactions identified in Fig. 80 as log2 (TPM+1). Data were available for 14 of the 15 cell lines included in this study. Genes that were not expressed in any of the cell lines of interest (expression level log2 (TPM) < 1) were excluded from further analysis. Most variedly expressed genes in HL-60 cells were identified by assessing the SD between gene expression levels in HL-60 in relation to the average gene expression across the remaining 13 cell lines. Moreover, genes for which the SD was above 1.7 were considered and additionally filtered for unique expression pattern in HL-60 cells. Pathway and process gene set enrichment analysis was performed with Metascape84.Gene expression data for the cell lines included in this study were obtained from The Cancer Dependency Map portal t-test for each expressed gene. The resulting P values were adjusted with a Benjamini–Hochberg correction with FDR of 5% deemed significant. A volcano plot was generated with Prism 8. Biological process and molecular function gene annotation was retrieved from the gene ontology resource86. An SLC16A1/SLC16A3 expression ratio of 3 or higher was considered predictive of AZD3965 sensitivity, as K-562 cells were tested to be sensitive in this study but had the lowest SLC16A1/SLC16A3 expression ration of 3.1 among the 44 cell lines deemed sensitive to AZD3965. Thus, the threshold cutoff was placed at SLC16A1/SLC16A3 expression ratio of 3. Lineage and disease subtype of the cell lines was retrieved from the DepMap portal (20Q2 data release).Differentially expressed genes in AZD3965 sensitive and nonsensitive cell lines were identified by performing an unpaired two-sample, two-sided SLC16A1 and SLC16A3 deficient cells were generated by CRISPR/Cas9 technology as previously described87 using the pLentiCRISPRv2 vector (Addgene plasmid #52961)88. In brief, guide RNAs (sgRNA) were designed using CHOPCHOP89 and sgRNA designer tools90, oligonucleotides harboring BsmBI restriction site-compatible overhangs were annealed, phosphorylated and ligated into pLentiCRISPRv2 by standard cloning methods and sequences were confirmed by Sanger sequencing. An sgRNA targeting the Renilla luciferase coding sequence:Ren.208  was used as a negative control. Three different sgRNAs per gene were used and the sequences are provided bellow:sgSLC16A1.530 sgSLC16A1.777 sgSLC16A1.1291 sgSLC16A3.381 sgSLC16A3.384 sgSLC16A3.1059 .sgSLC16A1, SLC16A3, PIK3CA, PIK3CB, PIK3CD, and PIK3CG was performed by lentiviral cDNA delivery experiments. SLC-encoding cDNAs were obtained as codon-optimized versions from Genscript and transferred into pDONR221 entry vector using BP reaction gateway cloning (Invitrogen). PI3K isoform cDNAs were ordered from Addgene .Generation of cells overexpressing 87 or LE3FgwIP (pRRL-EF1a-3xFLAG-gw-IRES-PuroR) using LR reaction-based gateway cloning (Invitrogen). Cells infected with a corresponding empty lentiviral expression vector LEIB (pRRL-EF1a-IRES-BlastR) or LEIP (pRRL-EF1a-IRES-PuroR) served as negative control.In the case of Addgene plasmid #81843 the stop codon was removed by site directed mutagenesis to enable C-terminal tagging . Constructs were subsequently transferred into lentiviral expression vectors LEgwSHIB (pRRL-EF1a-gwSH-IRES-BlastR)For lentivirus production, HEK293T cells were transiently transfected with psPAX2 and pMD2.G packaging plasmids (Addgene) and corresponding expression vectors. Media were changed 24 h post transfection and replaced with the target cell line-specific media. After 48 h the supernatant (containing virus) was collected, filtered through a 0.45 μm filter supplemented with 8 μg/mL protamine sulfate (Sigma-Aldrich) and added to subconfluent target cells. Cells were then spinfected at 2000 rpm for 45 min at room temperature. After 24 h the media were exchanged with fresh media and 24 h later the media were complemented with selection antibiotic for 5–7 days to select infected target cells. Knockout and overexpression efficiency was evaluated by immunoblot analysis.Cells were centrifuged, washed once with cold PBS and subsequently lysed in Nonidet-40 lysis buffer  per 50 mL) for 10 min on ice. Lysates were then centrifuged  and proteins were quantified with the Bradford assay using γ-globin as a standard (Bio Rad). Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes Protran BA85 . The membranes were incubated with α-SLC16A1 , α-SLC16A3 , α-FLAG , α-HSP90  or α-AKT  and visualized with goat-anti-mouse IgG (115-035-003) or goat-anti-rabbit IgG (111-035-003) horseradish peroxidase-conjugated secondary antibodies  utilizing the ECL western blotting system (Thermo Fisher Scientific).n = 13) and CML (n = 3) after written informed consent were used for this study. Ethical approval was granted by the Ethics Commission of the Medical University of Vienna (Ethik Kommission 1676/2016). Patient characteristics are described in Supplementary Table Vitaly frozen mononuclear cells (MNCs) from patients with AML n = 3 and CMLFASN mRNA in response to pictilisib treatment and GADPH expression in HL-60 cells was performed using high-capacity RNA-to-cDNA kit and SYBR qPCR Green PCR Master Mix (Applied Biosystems). The following primers were used:Total RNA was extracted using Trizol according to the manufacturer’s protocol (Gibco). Quantification of Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting summary


Evidence bearing on the role of statins in the prevention and treatment of cancer is confounded by the diversity of statins, chemotherapeutic agents and cancer types included in the numerous published studies; consequently, the adjunctive value of statins with chemotherapy remains uncertain.Saccharomyces cerevisiae. Cell density (OD600) data were analyzed for synergism and antagonism using the Loewe additivity model implemented with the Combenefit software.We assayed lovastatin in combination with each of ten commonly prescribed chemotherapy drugs in highly reproducible in vitro assays, using a neutral cellular substrate, Four of the ten chemotherapy drugs – tamoxifen, doxorubicin, methotrexate and rapamycin – exhibited net synergism with lovastatin. The remaining six agents  compiled neutral or antagonistic scores. Distinctive patterns of synergism and antagonism, often coexisting within the same concentration space, were documented with the various combinations, including those with net synergism scores. Two drug pairs, lovastatin combined with tamoxifen or cisplatin, were also assayed in human cell lines as proof of principle.The synergistic interactions of tamoxifen, doxorubicin, methotrexate and rapamycin with lovastatin – because they suggest the possibility of clinical utility - merit further exploration and validation in cell lines and animal models. No less importantly, strong antagonistic interactions between certain agents and lovastatin argue for a cautious, data-driven approach before adding a statin to any chemotherapeutic regimen. We also urge awareness of adventitious statin usage by patients entering cancer treatment protocols.The online version contains supplementary material available at 10.1186/s12885-021-07963-w. From antiquityIn 1971 Akira Endo isolated the first statin progenitor, citrinin . Soon itWithin a few years of the introduction of statins, however, concerns regarding their safety emerged, notably an associated increase in non-cardiac mortality . Animal Several lines of evidence have suggested that statins might also have value in the treatment of cancer: statins modulate the mevalonate pathway , which ualone have not proven effective as anticancer therapy; however, there is evidence that statins might potentiate the effects of anti-cancer drugs [Disappointingly, statins er drugs . A recener drugs  , in comSaccharomyces cerevisiae [S. cerevisiae has been utilized in numerous pharmacologic studies and high throughput screens [S. cerevisiae essential genes. Barcoded pools [Combenefit dual-drug interaction software [Combenefit package.Because our objective was to compare the ten drug pairings with each other, it was important to use a neutral cellular substrate, rather than a particular human cancer cell line which because of its inherent cellular origin and genetic mutations might favor or disadvantage a particular drug pair. We therefore chose as the cellular substrate the exceptionally well-studied model organism, revisiae . There irevisiae –28. Manyrevisiae  and many screens –40, incl screens ). In anted pools  were emped pools , 34, 36 ed pools .  Wesoftware ; cell cosoftware  which isS. cerevisiae but with variations in the patterns of synergism and antagonism between individual cell lines, even of the same cancer type.Our data demonstrate that combining lovastatin with conventional chemotherapeutic agents results in drastically different interactions, ranging from strong synergism to profound antagonism, sometimes within the same concentration space. Of the ten chemotherapeutic drugs, four  exhibited net synergism with lovastatin; two drugs (gemcitabine and 5-fluorouracil) had neutral scores; and four  displayed net antagonism. As proof of principle, two of the drug combinations, tamoxifen/lovastatin and cisplatin/lovastatin, were further evaluated in human cancer cell lines. The results in cell lines were generally accordant with the data obtained with S. cerevisiae heterozygous diploid essential gene deletion strains was obtained from ThermoScientific. A balanced pool was created, aliquoted and frozen : cisplatin (cis-Diamminedichloroplatinum), cyclophosphamide (hydrate), epothilone B, etoposide, 5-fluorouracil, lovastatin , rapamycin, and tamoxifen. Compounds obtained from Millipore Sigma included: doxorubicin hydrochloride, gemcitabine hydrochloride, and methotrexate.S. cerevisiae from the NetwoRx data base [In order to determine suitable concentration ranges for the dual drug dilution assays, dose response curves were compiled for each of the 11 drugs, using a range of concentrations based upon experimental data in ata base  and/or pata base , 47, 48 600), after two and 3 days of incubation. Each experiment was conducted a minimum of 3 times; the numbers of biologic replicates are given in the figure legends.Dual drug assays were performed in 96-well tissue culture plates using a uniform format compatible with the input requirements of the Combenefit software package (see later). In each assay, lovastatin was paired with a single chemotherapeutic drug. A schematic diagram of the plate configuration is shown in Additional file Crosswise, dual-dilution format assays were also performed using human cancer cell lines as substrate. The cell lines, obtained from ATCC, included: A549 , HCC827 , HT-29 , MCF7 , MDA-MB-231 , and SK-BR-3 . The cells were cultured according to the corresponding ATCC protocols.S. cerevisiae experiments and likewise included a plate blank, individual drug dose responses, and cells-only positive controls , according to the manufacturer’s protocol.The dual drug crosswise dilution matrix assays were performed in a similar format as the S. cerevisiae assays, each plate was scrutinized for outlier values by the Grubb’s test, implemented with the XLSTAT software package. When an outlier was identified, it was removed and replaced, as described in Additional file In the http://sourceforge.net/projects/combenefit/) calculates and displays the synergism-antagonism distributions and computes a variety of metrics from the distributions [experimental concentration space to a reference concentration space. The latter is calculated from the individual dose-response curves for the two drugs in accordance with the assumptions of the Loewe model; thus, these must be included in each assay  to a given cell, the cell is colored appropriately to highlight its synergy or antagonism  doxorubicin (+ 5.43), methotrexate + 2.93) and rapamycin + 2.16) each achieved net synergism when paired with lovastatin , yet a further 0.70 dilution of lovastatin (108 μM) at the same concentration of tamoxifen resulted in significant antagonism , Figs. Tamoxifen, which earned the highest composite score (+ 9.36) on day two Fig. a, also eThe interactions of doxorubicin with lovastatin were statistically neutral over more than 80% of the concentration space Fig. d; howeveThe SUM_SYN_ANT score for the methotrexate-lovastatin pairing was a modest, perhaps misleading, + 2.93 Fig. a: statis50 for rapamycin increased by about 3.5-fold on day three  positive, MCF7, and two ER negative, MDA-MB-231 and SK-BR-3 cell lines (see Methods). The results obtained with the ER positive MCF7 cell line are shown in Fig. When tamoxifen and lovastatin were assayed in the lung adenocarcinoma cell line, HCC827  SUM_SYN_ANT scores, with weak maximum synergy and stronger maximum antagonism .Together, the results with lovastatin in combination with cisplatin in three human cell lines and tamoxifen with MCF7 breast cancer cells are concordant with the findings in the Saccharomyces cerevisiae; a balanced pool of heterozygous deletions of all essential genes was created for this study. Highly reproducible dilution assays were performed in microplates; cell densities were compiled with a plate reader. The data from the assays were submitted to rigorous statistical analysis to identify synergistic and antagonistic interactions, according to the Loewe additivity model, implemented with the Combenefit software package [The fundamental question we sought to answer in this study is whether statin-chemotherapy interactions are a manifestation of a global effect of statins or are, as we hypothesized, specific to individual chemotherapeutic drugs. To that end, we assayed a single statin, lovastatin, paired with each of ten commonly prescribed chemotherapeutic agents, with various mechanisms of action Table . The cel package .The Combenefit program compiles the data from the plate replicates for a given drug pair and computes various global metrics according to assumptions dictated by the chosen model. The metric “SUM_SYN_ANT”, which is as summation of all interactions within the concentration space, proved most useful. In accordance with our hypothesis, the ten chemotherapeutic agents exhibited a spectrum of global interactions, ranging from synergism to neutrality to antagonism Fig. a. Four cS. cerevisiae, which is devoid of estrogen receptors.  The efng yeast , 53–58, S. cerevisiae, devoid of estrogen receptors (ERs), yet displayed a similar pattern of strong synergism only in a breast cancer cell line, MCF7, which possesses ERs. 50 of lovastatin showed substantially greater variability between breast cancer cell lines than did the EC50 of tamoxifen. A possible resolution of these seeming contradictions is suggested by the reports of Radin and Patel [Curiously, the ECnd Patel  and Tan nd Patel ; higher nd Patel .S. cerevisiae-based experimental design by testing the combination of cisplatin and lovastatin in three human cell lines: A549 (lung adenocarcinoma); HT29 (colon adenocarcinoma); and MCF7 (breast carcinoma). This combination was chosen as a surrogate for the six neutral or antagonistic pairings because it so commonly prescribed for a variety of malignancies. In the yeast model, the combination exhibited strong antagonism  The balanced pool of heterozygous deletion strains of all essential genes, which we created in order to have a cellular substrate with consistent genetic diversity, proved to be only modestly more sensitive to the various drugs than the cognate wildtype strain. However, because each deletion is bar-coded, the pool provides a useful resource for the analysis of genetic targets and resistance mechanisms of drug treatments, studies beyond the scope of this report.Two features of the experimental design merit further discussion. Because our intent was to compare the ten statin-chemotherapeutic drug pairs with each other, an important consideration was that the cellular target be a neutral one, free of the biologic and genetic biases inherent in every cancer cell line; choosing any one human cancer cell line for the assays risked biasing the assays for or against one or another drug pair. In the Background we set forth our rationale for choosing the model organism  studies , 34, 36  studies , which iA second important consideration in experimental design was the choice of synergy model. The Combenefit software package  renders Undue reliance upon global metrics, as for example in high throughput studies, risks overlooking potentially useful synergistic interactions which are confined to portions of the concentration space, often with coexisting antagonism . In Additional file 1.Additional file 2: Legends Figure S1. Schematic diagram of the 96-well plate for the dual-drug assays (see Methods). The dose-responses (Columns 9 and 10), which are required for the computation of Loewe additive synergy, are performed at the same starting concentrations and with the same dilutions as in the dual-dilution portion of the plate. The format shown is compatible with the Combenefit software. BL = Plate blank (synthetic complete media + dextrose only). Figure S2. Tamoxifen plus lovastatin data on day 3. The comparable data for day two are depicted and described in Figure Figure S3. Doxorubicin and lovastatin interactions on day three. The data are quite similar to the results on day two, shown in Figure Figure S4. Methotrexate and lovastatin on day three. The patterns are quite similar to those recorded on day two   and MCF7 (breast cancer) , exhibited significant antagonism (and no significant synergism) on day two; the antagonism was more pronounced on day three. The HT29 colon adenocarcinoma cell line exhibited synergism and antagonism, both statistically significant, on day two (B) and to a lesser degree, on day three (E). The data shown here are comparable to that obtained with the S. cerevisiae assays (see Table


A combination of the lactones with glucocorticoids confirmed their synergy in anti-tumor activity in vitro. Conclusions: Methoxy-substituted flavanone-derived lactones effectively kill canine lymphoma/leukemia cells in vitro and, thanks to their synergistic action with glucocorticoids, may potentially be applied in the treatment of hematopoietic cancers.Background: The study investigated four flavanone-derived γ-oxa-ε-lactones: a parent unsubstituted compound and its three derivatives with the methoxy group in positions 2′, 4′ and 8. Our objective was to find out if the introduction of the methoxy group into the aromatic ring affects in vitro anti-tumor potency of the investigated lactones. Methods: Cytotoxic and pro-apoptotic effects were assessed with cytometric tests with propidium iodide, annexin V, and Western blot techniques. We also investigated potential synergistic potency of the tested lactones and glucocorticoids in canine lymphoma/leukemia cell lines. Results: The tested flavanone-derived lactones showed anti-cancer activity in vitro. Depending on its location, the methoxy group either increased or decreased cytotoxicity of the derivatives as compared with the parent compound. The most potent lactone was the one with the methoxy group at position 4′ of the B ring (compound  Compounds with a lactone ring in their structure are the subject of numerous studies due to their common occurrence in nature as well as a broad spectrum of biological activity. Lactones are secondary metabolites of various plants, insects, microorganisms and marine organisms and exhibit biologically important properties such as antifeedant, antibacterial, antifungal and cytostatic properties . Lactone1 (lactone with unsubstituted phenyl rings) and its three derivatives  with methoxy groups in positions 2′, 4′ and 8, respectively , and on the other hand, the dogs are excellent animal models for studying human non-Hodgkin’s lymphomas  . Researc1 by an addition of methoxy substituents increased or decreased the cytotoxic activity of the new derivative, depending on the position of the group. Excluding the resistant CNK-89 cell line, the introduction of the methoxy group into position 8 of the A ring significantly lowered the activity in comparison with the parent compound, and γ-oxa-ε-lactone 4 was also less effective than other derivatives. However, the presence of the methoxy group in the B ring increased the cytotoxic activity of the resulting compounds, which was particularly noticeable in the case of lactone with the methoxy group at position 4′ (3) when tested on the CLB-70 cell line.The study began with checking the cytotoxic activity of the tested compounds by means of propidium iodide staining and flow cytometry analysis. All tested compounds at the concentration range up to 50 µg/mL exhibited cytotoxic activity. The differences in their strength were visible for individual derivatives  and indi2 in the highest concentration induced marked cell death in this cell line. Detailed results are shown in In addition, the cytotoxic effect in the noncancerous canine cell line was evaluated and it was found that in this cell line none of the compounds was able to kill 50% of the cells and only compound 50 for this cell line, except for the most potent derivative—lactone 3. Detailed differences in the sensitivity of each cell line to individual compounds are shown in Not only tested γ-oxa-ε-lactones differed in their activity but the cell lines also showed different sensitivity to their action. While the cell lines representing typical B-cell proliferation turned out to be highly sensitive to the anti-proliferative effect of the examined lactones, the GL-1 cell line representing B/T-cell leukemia seemed slightly less sensitive, and the cell line formed by neoplastic NK cells was clearly resistant. Within the range of concentrations tested, it was not possible to determine ICSince the assay with propidium iodide alone showed the cytotoxic effect of the tested compounds, we decided to check the type of cell death (apoptosis or necrosis) induced by the tested compounds. To distinguish apoptosis from necrosis the cells were additionally stained with annexin V, which binds with phosphatidylserine externalized in apoptotic cells.3, but we observed no differences in the type of cell death induced by the tested compounds. Similarly, in the two less sensitive cell lines, GL-1 and CNK-89, the populations of necrotic and late apoptotic cells were of comparable size. Detailed results along with representative dot-plots showing apoptotic and necrotic cells are presented in The experiment revealed both types of cell death ensuing from the treatment with the tested compounds. A particularly high percentage of necrotic cells was found in CLB70 and CLBL-1 cell lines, which were the most sensitive to the tested lactones. The population of necrotic cells was accompanied by the population of cells in late apoptosis also at the lowest (12.5 µg/mL) concentration of the tested substances. The strongest effect was again shown for lactone Weak apoptosis induction was also confirmed by the Western blot, which showed no statistically significant changes in the expression level of the most important anti-apoptotic protein, i.e., Bcl-2. As the imbalance between pro- and anti-apoptotic proteins occurs only in living cells, relatively low concentrations of tested lactones were used in the study: 3.125 and 6.25 µg/mL for the sensitive cell lines (CLBL-1 and CLB70) and 6.25 and 12.5 µg/mL for the resistant ones (CNK-89 and GL-1). Detailed results are shown in 2 and 3, were selected for these tests with two cell lines (CLBL-1 and CLB70) most sensitive to the lactone action. The experiments clearly showed that the combination of glucocorticosteroids (both dexamethasone and prednisolone) with the flavonoid derivatives induced stronger cell death than either of the substances alone. Synergy evaluation confirmed the synergistic effect for the combinations of dexamethasone and prednisolone with lactones 2 and 3 in both cell lines (synergistic effect was identified by combination index (CI) values less than 1). The effect was most pronounced at low concentrations of the tested lactones (3.125 µg/mL) in combination with 2.5 µg/mL of steroid drugs. In these concentrations, in the CLB70 cell line, CI was about 0.4 for combinations of lactone 2 and both glucocorticosteroids while for lactone 3 it was about 0.62 and 0.76 for dexamethasone and prednisolone, respectively. In the CLBL-1 cell line, the CI for lactone 2 in combination with dexamethasone or prednisolone was about 0.5, while for lactone 3 it was about 0.75. At the higher concentration the lactones were so effective in inducing cell death that the effect of their combination with steroids could not be observed. Detailed results are shown in As our research demonstrated the anti-tumor potential of the tested compounds, we decided to check whether the obtained derivatives could be used in the treatment of lymphomas/leukemias in dogs. To this end, we resolved to verify whether the tested compounds act synergistically with common anti-cancer drugs used in canine therapy. As each therapeutic regimen used in the treatment of hematopoietic neoplasms in dogs contains glucocorticosteroids, we investigated the lactones in combinations with dexamethasone and prednisolone. The two most potent methoxy substituted lactones, Despite years of intensive research into the causes and treatment of cancer, it still remains a huge and unsolved problem for mankind. According to the data provided by the World Health Organization (WHO) in 2018, the global annual number of new cancer cases is estimated at about 18.1 million. The same source states the specific cancer-related mortality at about 10 million yearly. Moreover, the WHO estimates that the global cancer incidence could increase by more than 63% in 2040 as compared with 2018 . Such a The search for new compounds with potential anti-cancer activity among agents of natural origin is still a popular field of research. In fact, the treatment of cancer has started with the use of natural substances. An example here could be basic cytostatic drugs such as irinotecan, vincristine, etoposide and paclitaxel sourced from plants, actinomycin D and mitomycin C produced by microorganisms or marine-derived bleomycin . FlavonoConsidering the above, we decided to combine our knowledge of the anti-cancer properties of flavonoids and natural compounds containing lactone moiety to create new compounds with the advantages of both these groups. Since poor bioavailability penetration into the cells through membranes may significantly limit the activity of the flavanone-derived lactones, we decided to modify the structure of our compounds by synthesis of methoxy derivatives. We also resolved to determine if the location of the methoxy group in the tested compounds would affect their activity.Our previous research demonstrated that introduction of lactone function increased anti-microbial activity of a tested compound as compared with its flavonoid precursors . Studies4) with lower activity than that of the parent compound 1  [2 and/or 4′-methoxylactone 3 on the balance between pro- and anti-apoptotic proteins in the cell that would facilitate the course of apoptosis  ,22 than ctively) , or NK cctively) . The rempoptosis . It can Artocarpus heterophyllus heartwoods on the anti-cancer activity of cisplatin against h460 and mcf-7 cell lines [At the final stage of the study, we assessed the potential use and role of the obtained lactones in the treatment of hematopoietic cancers. A combination treatment strategy, which serves as an important direction for the development of natural products in cancer therapy, seems an interesting research direction. Adjuvant treatment with natural products supplementing current treatment regimens could be beneficial in multiple aspects, including mitigation of adverse effects, overcoming drug resistance and improving therapeutic response. The idea of a combination therapy with flavonoids is already being researched. Firstly, numerous studies confirmed that flavonoids show better anti-tumor activity when used in complex preparations containing a mixture of several compounds from this group ,29,30,31ll lines , synergill lines , or querll lines . More anll lines  or immunll lines .2 and 3. We found that non-toxic concentrations of both glucocorticosteroids (2.5 µg/mL) markedly potentiated the killing effect of these derivatives. The effect of the combination was so strong that cell viability could only be assessed at the lowest concentration of lactones used in the study (3.125 µg/mL). The scope and strength of the cytotoxic effect of the proposed combination as well as its mechanism of action require further research but we established the obtained compounds as useful adjuvants in standard therapeutic protocols and possibly as an interesting alternative for classic chemotherapy.To initially assess the usefulness of the lactones in the adjuvant therapy of lymphomas and leukemias, we examined their synergistic action with basic drugs used in the treatment of hematopoietic neoplasms, i.e., glucocorticosteroids. This preliminary study, involving two canine cancer cell lines, confirmed the synergistic effect of both dexamethasone and prednisolone with the two lactones with the highest cytotoxic activity, 1–4) obtained in a three-step synthesis from corresponding flavanones as described earlier [The study investigated four flavanone-derived γ-oxa-ε-lactones ( earlier . The strAll tested compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare 10 mg/mL stock solution. The stock solutions were stored at room temperature. Tested dilutions were obtained by dissolving the stock solution in a complete culture medium.The study involved a panel of canine cancer cell lines: CLBL-1 (B-cell lymphoma), GL-1 (B/T-cell leukemia), CLB70 (B-cell chronic lymphocytic leukemia) and CNK-89 (NK-cell lymphoma), and a normal canine cell line (MDCK). CLBL-1 was obtained from Barbara C. Ruetgen from the Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria ; GL-1 frThe cell lines were maintained in RPMI 1640   or Advanced RPMI  (CLB70 and CNK-89) culture medium supplemented with 2 mM L-glutamine , 100 U/mL penicillin, 100 μg/mL streptomycin  and 10–20% heat-inactivated fetal bovine serum—FBS .4 cells per well in a 96-well plate . The cells were incubated in the medium alone or in the medium containing either the vehicle control or increasing concentration of the tested substance for 48 h. After the incubation, the cells were harvested and washed twice in PBS, transferred into cytometric tubes and stained with propidium iodide . Flow cytometry analysis was performed immediately, using a flow cytometer FACS Calibur . CellQuest 3.lf. software  was used for data analysis on the basis of histograms of FL2-H showing the population of alive (PI negative) and dead (PI positive) cells. The values presented as means with standard deviations were obtained from three independent experiments and shown as a concentration-dependent curve. IC50 for the tested compounds was calculated as a mean concentration inhibiting cell viability by 50% in three independent experiments.To evaluate and compare the effects exerted by γ-oxa-ε-lactones 1–4 on the cultures of canine lymphoma  and leukemia  cell lines, the cells were treated with increasing concentrations of each compound  for 48 h. This incubation time was chosen after preliminary studies showed that 48 h of treatment of the cells with the tested compounds allows both the evaluation of the cytotoxic effect and the observation of late signs of apoptosis. The cells were seeded at a concentration of 1 × 102 and 3, and two cell lines (CLBL-1 and CLB70) most sensitive to the activity of the tested lactones, were selected. Prednisolone and dexamethasone, frequently used in the treatment of lymphomas and leukemias in dogs, were tested as glucocorticoid compounds. The cells were incubated with individual compounds or their combinations for 48 h. The range of the tested concentrations for a single treatment was 3.125, 6.25 and 12.5 µg/mL for lactones and 2.5, 5 and 10 µg/mL for glucocorticoids. Dexamethasone and prednisolone concentrations of 2.5 µg/mL were chosen to evaluate the synergistic effect, and these drugs were added together with the lactones in the entire range of concentrations tested . Synergy was estimated using CompuSyn software and the Chou-Talalay method [1.The method of staining cells with propidium iodide was also used to evaluate a synergistic effect of the tested derivatives with glucocorticosteroids. For this study, the two most potent derivatives y method . The syn6 cells per 25 cm2 cell culture flasks and the compounds were added at two selected concentrations (3.125 and 6.25 µg/mL for the resistant cell lines and 6.25 and 12.5 µg/mL for the sensitive cell lines). After 48 h incubation the cells were harvested, rinsed with cold PBS, suspended in a lysis buffer  and incubated for 20 min on ice. The suspensions were centrifuged at 10,000 rpm at 4 °C for 12 min. Then, a sodium dodecyl sulfate (SDS) sample buffer was added to clear supernatants, and the samples were boiled at 95 °C for 5 min and subjected to SDS-PAGE on 12% gel. The resolved proteins were transferred to a PVDF membrane , using Semidry Transfer Cell . After the transfer, the membrane was blocked for 1 h with 3% bovine serum albumin (BSA) in TBS at room temperature, and then incubated with primary antibody (dilution 1:1000)  at 4 °C overnight, followed by secondary horseradish peroxidase-labeled antibody  for 1 h at room temperature. The bound antibodies were visualized using ChemiDoc Touch Instruments . The anti-β actin (C-4) and anti-Bcl-2 (C-2) antibodies from Santa Cruz Biotechnology  were used in this study. For Bcl-2 expression quantification, Western blot normalization using a single protein  was performed using Image LabTM software .For Western blot analysis the cells were seeded in a total of 5 × 10Early apoptotic events were detected by means of annexin V FITC/PI staining. Briefly, the cells were treated with the tested compounds at 12.5 and 25 µg/mL for 48 h. The concentrations of the compounds were the same as for determination of cell viability. After harvesting the cells and washing them twice with cold PBS, they were suspended in a binding buffer and stained with annexin V-FITC for 10 min at room temperature or PI . Immediately after, the incubation flow cytometric analysis was performed using a FACS Calibur analyzer . CellQuest 3.lf. software was used for data analysis.p < 0.05.All data are shown as means with standard deviations (SD). Statistical differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical analysis was performed with STATISTICA version 13.3 software . The results were considered significant at 1. The most potent lactone was obtained by the introduction of the methoxy group at position 4′ of the B ring (compound 3), whereas the presence of this group at C-8 in the A ring resulted in its lower activity. The substitution pattern of methoxy-substituted flavanone-derived lactones did not affect the mechanism of the cytotoxic action of the investigated compounds but influenced the strength of their anti-cancer activity. In vitro examination of the potential synergistic anti-tumor activity of the tested lactones with glucocorticoids showed that this combination was more potent than either of the compounds alone. This may indicate a potential for the application of the lactones in the adjuvant treatment of hematopoietic neoplasms in the future. However, further studies on the mechanism of action and possible in vivo use of the obtained flavanone-derived lactones are required.The tested flavanone-derived lactones show anti-cancer activity in vitro. Depending on its location, the presence of the methoxy group in the molecules can both increase or decrease their cytotoxicity in comparison with the parent compound


Cardiovascular disease (CVD) and cancer are two major causes of death worldwide. The question is, “Could there be a link between these two pathologies in addition to their shared, common risk factors?” To find some answers, we studied the effect of oxidized low-density lipoproteins (oxLDL) on head and neck cancer (HNC) cell lines, since oxLDL is a major contributor to atherosclerosis and the principal cause of CVD. In this study, we exposed three HNC cell lines  to oxLDL. We investigated two oxLDL receptors, CD36 and Lox-1, using immunofluorescence. Cancer cell migration was evaluated using Boyden chambers and the Wnt/β-catenin pathway was investigated using Western blotting. We demonstrated that the expression of CD36 and Lox-1 significantly increases after exposure to oxLDL. Moreover, we found that oxLDL reduces the migration of HNC cell lines, an observation that is in line with an increased degradation of β-catenin under oxLDL. Finally, the inhibition of CD36 with sulfosuccinimidyl oleate (SSO) reverses the inhibition of cell migration. In conclusion, we report that oxLDL seems to induce an increase in CD36 expression on HNC cell lines, enhancing the uptake of these lipids in cells to finally decrease cancer cell migration via the CD36/β-catenin pathway. Cardiovascular disease (CVD) and cancer are the two main causes of death worldwide. Indeed, the World Health Organization (WHO) has estimated that the global burden of cancer rose to 18.1 million new cases and 9.6 million deaths in 2018, with an estimated 5-year prevalence of 43.8 million, placing cancer as the second leading cause of death worldwide  vs. 0.58 × 106 µm2 [oxLDL], t-test, p = 0.001), -B , -C ). To understand this result, we explored the Wnt/β-catenin pathway, which is involved in cell motility and migration [In order to assess whether oxLDL impacts HNC cell migration, we used Boyden chambers and showed that oxLDL decreased cell migration in all HNC cell lines (igration . Western6 µm2 (oxLDL) vs. 4.7 × 106 µm2 (oxLDL + SSO), t-test, p = 0.04). The involvement of CD36 in the decreasing cell migration induced by oxLDL is reflected in the significant increase in the surface area covered by cells under oxLDL and SSO treatment.To investigate the mechanisms involved in the cell migration decline after oxLDL exposure, we explored the implication of CD36 in Detroit 562 cells after its inhibition with 1 µM of SSO. No change in cell migration appeared between the control cells (NT) and the SSO-treated cells, but oxLDL exposure led to a decrease in cell migration that was significantly overcome by the SSO treatment  (another oxLDL receptor) in macrophages, where the oxLDL uptake by CD36, but not by SR-BI, is dependent on dynamin . Howeverbright cells were described in oral cancer as initiating metastasis. They did not overexpress the mesenchymal genes, were slow cycling and expressed high levels of the fatty acid receptor CD36 and lipid metabolism genes [Regarding CD36, we have demonstrated that its expression increases under oxLDL exposure, which is not demonstrated elsewhere in cancer. In ovarian and cervical cancer, it was observed that CD36, as Lox-1, leads to cancer progression and metastasis ,30. Indesm genes . They alsm genes . Howeversm genes . In factsm genes . This reRegarding the Wnt/β-catenin pathway, it is well known that without the stimulation of this pathway, β-catenin is phosphorylated by the destruction complex, which is composed of adenomatous polyposis coli, Axin 1/2, casein kinase I and glycogen synthase kinase 3β . Wnt ligOLR1 gene coding for the Lox-1 receptor [Another aspect of this study is the use of HPV-negative HNC cell lines, which represent the major HNC population. However, HPV infection is estimated to cause 22% of oropharyngeal cancer and 47% of tonsillar cancer cases . A studyreceptor . In thisIn conclusion, oxLDL seems to induce an increased expression of both the Lox-1 and CD36 receptors on HNC cell lines, enhancing their uptake of oxidized LDL and decreasing cancer cell migration via the CD36/β-catenin pathway. However, more clarifications are needed to verify the exact contribution of CD36 and Lox-1 in cancer progression. Indeed, studies demonstrated that the upregulation of CD36 is associated either with tumor progression or tumor repression ,32. Fina


Helleborus species have been found to be useful in various chronic diseases. This has brought Helleborus to the attention of medical researchers. H. purpurascens is a plant characteristic of the Carpathian area, known since ancient times for its beneficial effects. The aim of the study was to evaluate the flavonoids composition of a hydroalcoholic extract of H. purpurascens, as well as to assess its antioxidant activity and antitumor potential at the level of two healthy cell lines and four tumor cell lines. In addition, the expression of the genes involved in the apoptotic process  were evaluated. The results indicated that the extract has a high concentration of flavonoids, such as epicatechin, quercetin, and kaempferol. The extract has an increased antioxidant activity, very similar to that of the standard, ascorbic acid and cytotoxic effects predominantly in the breast cancer cell line, being free of cytotoxic effects in healthy cell lines. Underlying the cytotoxic effect is the induction of the process of apoptosis, which in the present study was highlighted by decreasing the expression of anti-apoptotic genes (Bcl-2) and increasing the expression of pro-apoptotic genes (Bad and Bax). In conclusion, the hydroalcoholic extract of H. purpurascens can be considered an important source for future medical applications in cancer therapy.In the field of oncology, the plant kingdom has an inexhaustible supply of bioactive compounds. Phytochemical compounds isolated from  The threat of cancer continues despite scientific advancements in the medical field. According to data provided by the World Health Organization, cancer was the leading cause of death globally in 2020. The main types of cancer diagnosed, except skin cancer, are breast, lung, and colorectal cancers . Skin caIn recent years, remarkable progress has been made in the field of oncology. Thus, the neoplastic process has been more closely studied and understood and, implicitly, the antitumor treatment has shown considerable improvements. Conventional cancer therapy consists of chemotherapy, radiation therapy, and surgery. However, the main disadvantage of classical antineoplastic therapy is the non-selective mode of action which results in the appearance of toxic reactions at the systemic level, as well as the low response rate to treatment . For thiHelleborus species represent a new treatment opportunity. The genus Helleborus is part of the Ranunculaceae family and includes over 20 different species with a predominant distribution in Europe and West Asia [Helleborus, namely, H. purpurascens and H. odorus. Although H. purpurascens has a high toxic potential, it has been used since ancient times to treat various diseases such as psychiatric and cardiovascular disorders or various pains [H. purpurascens include muscle relaxation and analgesic properties. However, the exact phytocomponents responsible for these biological activities are not known [H. purpurascens root extract caused an immunostimulatory effect in vivo by stimulating lymphocytes and neutrophils, [H. purpurascens, it was determined that this plant has a rich composition of natural compounds with biological effects, such as polyphenols, tannins, and glycosides [H. purpurascens extracts [Recent research in the field of phytocompounds has shown that est Asia . Numerouest Asia . In the us pains . Multifaot known . Additiorophils, . Followiycosides ,10. In lextracts . Regardiextracts . One of the modern directions of antitumor therapy is based on the elimination of cancer cells through the process of apoptosis . ApoptosH. purpurascens (HPex) in terms of the composition of polyphenols. In addition, the antioxidant activity of the extract was determined and the potential cytotoxic effect were assessed on four different cancer cell lines: squamous carcinoma—A431; murine melanoma—B164A5; and breast cancer—MCF-7 and MDA-MB-231. For a complete evaluation of the cytotoxic effect, two healthy cells line were selected: human keratinocyte cell lines (HaCaT) and murine epidermal cells (JB6). Finally, the expression of the main genes involved in the apoptosis process was determined, thus providing a possible explanation for the cytotoxic effect of H. purpurascens extract.For this reason, the present study focused primarily on evaluating the composition of a hydroalcoholic extract of The extract obtained was subjected to LC-MS quantification and in The  TAOxA of the four different extract concentrations was evaluated by DPPH radical scavenging assay. Ascorbic acid, positive control, exerts a TAOxA of 97.79% as can be observed in Cell viability was determined by the Alamar blue method and expressed as a percentage of viable cells compared to control cells.According to To determine the in vitro cytotoxicity effect, four tumor cell lines were chosen: MCF-7, MDA-MB-231, B164A5 and A43, at which the five concentrations previously tested on healthy cell lines were evaluated.In At the first four concentrations tested, the viability of the murine melanoma cells, B164A5, showed a slight decrease, being around 89%. However, at the highest concentration, cell viability dropped to about 68%. A plateau value of cell viability was also recorded in the case of squamous cell carcinoma, A431, where at the lowest concentration tested cell viability was similar to that of control cells, and at concentrations of 250, 500, and 1000 µg/mL, the values of cell viability were similar, approximately 85%, 84%, and 83%, respectively .Following the results obtained in the cell viability test, it was observed that HPex causes a marked decrease in the cell viability of breast cancer cells—MCF-7. In order to provide a more detailed picture of the mode of action of HPex at the level of this cell line, it was decided to determine the expression of the genes involved in the apoptosis process, namely: Bax and Bad—pro-apoptotic genes; Bcl-2—anti-apoptotic gene. H. purpurascens is a widespread plant in Eastern Europe, especially in the Carpathian Mountains. Although the plant has a high toxic potential, it is used in traditional medicine for a wide range of pathologies [Helleborus species are currently being considered a promising antitumor therapy [Rannuculaceae family, including the species of H. purpurascens, they were studied for their content rich in active compounds and for antitumor activity [H. purpurascens has been studied for its antitumor effect in vitro, but the biological mechanisms associated with this therapeutic activity have not been fully elucidated [hologies . Due to  therapy . Regardiactivity . In addiucidated .H. purpurascens. This therapeutic effect has been studied in conjunction with the evaluation of phytocomposite content, as well as with the evaluation of the beneficial antioxidant effect in antitumor therapy.Based on existing scientific considerations, the present study aimed to provide innovative information on the potential antitumor effect of a hydroalcoholic extract of To elucidate the cytotoxic effect of HPex, four tumor cell lines were chosen, two of breast cancer (MCF-7 and MDA-MB-231) and two of skin cancer (B164A5 and A431). Additionally, the effects exerted by HPex were studied in two healthy cell lines (HaCaT and JB6), to provide a more accurate picture of the cytotoxic effect. Cell viability was determined using the Alamar blue method, after 24 h of stimulation. Results of the study indicated that HPex has a selective cytotoxic effect, meaning healthy cells are not affected by its use. In contrast, in tumor cells there was a decrease in cell viability depending on the concentration tested. The most visible cytotoxic effects were observed in the breast cancer cell line (MCF-7), where cell viability decreased by up to 24%. The cytotoxic effects were also observed in the other cells studied, but the effect on cell viability was moderate. Helleborus species have been tested for their antitumor potential. In the preliminary study by Lindholm et al. [H. cyclophyllus extract showed antitumor effects, but the mechanism of action has not been fully elucidated. Another example is represented by H. caucasicus which has proven its cytotoxic effects on various tumor cell lines such as lung cancer (A549) or colorectal cancer (DLD-1) [H. niger on several tumor cell lines, including melanoma and breast cancer. The results revealed that H. niger has a concentration-dependent cytotoxic and antiproliferative effect on all cell lines used [H. niger extract on the melanoma cell line due to its ability to induce cell apoptosis [H. odorus, H. multifidus, and H. hercegovinus have been tested in vitro on Burkitt’s lymphoma B cells (BJAB). The results revealed that the strongest antiproliferative effect was observed in H. multifidus [H. purpurascens species, previous studies have shown that the alcoholic extract has an antiproliferative effect on the tumor cell line of cervical cancer (HeLa), causing a decrease in the number of mitoses [H. purpurascens, an aqueous extract and a hydroalcoholic extract. They determined that both extracts have cytotoxic effects on the HeLa tumor cell line, but the hydroalcoholic extract has a significantly more intense activity than the aqueous extract [H. purpurascens. Many m et al. , more th (DLD-1) . A similnes used . In addipoptosis . Other sltifidus . Regardi mitoses . Voichit extract . Thus, tH. niger on the effect on genes involved in apoptosis, mainly the Bcl-2 gene, noting that it induces Bcl-2-dependent cellular apoptosis [H. cyclophyllus, Yfanti and collaborators determined that it has pronounced cytotoxic effects in the lung adenocarcinoma cell line, A549. In addition, the group of researchers observed that H. cyclophyllus extract induces morphological changes characteristic of the apoptosis process, and in addition, causes a decrease in procaspase-3 levels and cleavage of PARP1 [The B-cell CLL/lymphoma 2 (Bcl-2) family plays a major role in the process of mitochondrial-mediated apoptosis in breast cancer. This family of proteins is divided into two categories: anti-apoptotic members such as Bcl-2 and pro-apoptotic members such as Bax and Bad . A featupoptosis . Regardiof PARP1 .H. purpurascens, the studies showed a correlation between its polyphenol content and the antitumor effect exerted at the level of different tumor cell lines [H. purpurascens extract, namely epicatechin, rosmarinic acid, quercetin, and kaempferol, have also been studied for their potentially beneficial role in the treatment of cancer. Epicatechin has previously been studied for its effect in the treatment of triple negative breast cancer using a murine model. It has been found to have strong antitumor activity in combination with doxycycline, inhibiting cell proliferation by modulating adenosine monophosphate-activated protein kinase-mediated pathways [p53 gene, known as a tumor suppressor protein [p53 gene, quercetin has found its utility in the treatment of skin cancer. In addition to this mechanism, quercetin also increases the activity of the tyrosine kinase enzyme, thus causing the antioxidant effect [As a means of better understanding the cytotoxic effect, this study used the LC-MS method to quantitatively determine the phenolic composition of the HP extract. Thus, it was found that the hydroalcoholic extract has a high content of epicatechin, rosmarinic acid, quercetin, and kaempferol, the latter having the highest concentration (approximately 68 µg/g). A wide range of evidence suggested that polyphenolic compounds could be therapeutic in diseases such as cardiovascular disease ,29,30 anpathways . Similarpathways . An in-dpathways . Rosmaripathways . The ben protein . Additiot effect . Kaempfet effect . H. purpurascens has many phytoconstants that underlie its antitumor activity. Thus, Franz et al. discussed the composition of aqueous and organic extracts of H. purpurascens. Thus, they identified the fact that in terms of aqueous extract, it has a high amount of amino acids, especially asparagine [H. purpurascens extract in more detail, providing information on its rich composition in carbohydrates, glycosides, saponins, and tannins [Helleborus species is hellebrin, which is found in the largest amount in the species of H. purpurascens [H. caucasicus, studies have shown that these natural compounds are involved in reducing the viability of tumor cells by inducing apoptosis due to decreased expression of the GRP78 gene [H. niger, it was determined that due to the composition of bufadienolide and ecdysteroid, the extract has antitumor capacity by inducing apoptosis [However, in addition to its rich flavonoid composition, studies have shown that paragine . Similar tannins . Probablurascens . Studiesurascens , pancreaurascens , breast urascens , and othurascens . In addiurascens , but alsurascens . RegardiP78 gene . Similarpoptosis .As for the MCF-7 breast cell line, it differs from the other MDA-MB-231 breast cancer cell line, mainly due to the fact that MCF-7 is hormone dependent, having positive estrogen and progesterone receptors, while MDA-MB-231 is triple negative. For this reason, MDA-MB-231 does not respond to antiestrogen therapy . Over tiH. purpurascens. A 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was applied to evaluate the antioxidant activity of four extract concentrations . The results obtained were related to the antioxidant activity exerted by ascorbic acid and considered 100%. Consequently, the antioxidant activity was higher than 50% in all concentrations, while the most intense antioxidant activity was recorded at the concentration of 100 µg/mL, approximately 78%. Its remarkable antioxidant properties are due to the compounds that were found in H. purpurascens extract, which have been associated with the neutralization of oxygen-free species [H. purpurascens extracts, one aqueous and one alcoholic, were tested. The obtained results showed that the alcoholic extract has a higher antioxidant activity than the aqueous one, with a value of 78% [Helleborus species. Thus, Öztürk et al. evaluated the flavonoid content and antioxidant activity of an H. orientalis extract. They determined that the extract has a significant antioxidant activity, observing an increase in the antioxidant activity and the protective effect against oxidative alterations with increasing concentration of the tested extract [H. vesicarius. In this study it was determined that this species has an intense antioxidant activity as well as an increased oxidizing potential [Helleborus, H. odorus, H. multifidus, and H. hercegovinus. In this study, it was determined that the extract made from the leaves has a more intense antioxidant activity than that obtained from the root [In light of the fact that flavonoids are antioxidant compounds  and that species . The ant species . Similare of 78% . These r extract . Mohammeotential . Čakar ethe root .Helleborus purpurescens) used in this study, was harvested from Timis County, Western Romania, and certified at Pharmaceutical Botany Department (voucher herbarium specimen no. HP/S12/2020), Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy. The plant was prepared for extraction according to European Pharmacopoeia (Ph. Eur.) 10th Edition.The plant material (Helleborus purpurescens extract (HPex) the maceration method was used. Plant material (5 g), crushed and homogenized, was placed in the flask and 25 mL of ethanol (EtOH) 70% were used to sequentially extraction for seven days. The final extract was filtered through filter paper, the solvent was removed by a rotary evaporator (Heidolph Hei-VAP Advantage Rotary Evaporator package) under vacuum, the pellet being lyophilized and stored in a dark glass tube at 2–8 °C until further analysis.To obtain The hydro-alcoholic hellebore extract, was subjected to a LC-MS analysis, and the following steps were accomplished: (i) extract homogenization  and centrifugation for 120 s at 10,000 rpm ; (ii) LC-MS analysis on a Shimadzu chromatograph ; (iii) chromatographic conditions, extraction time 60 min, room temperature, EC 150/2 NUCLEODUR C18 Gravity SB 150 mm × 2.0 mm × 5 μm column , flow rate 0.2 mL/min, two mobile phases , wavelengths 280 and 320 nm; (iv) gradient elution—0–20 min 5% B, 20–50 min 40% B, and 50–60 min 95% B; (v) calibration curves between 10 and 50 µg/mL and limit of quantification 0.3 µg/mL.−4 M) was prepared in ethanol and 103 µL was added to 3 × 103 µL of hydro-alcoholic HP extracts, at four different concentrations: 10, 25, 50, and 100 μg/mL; (ii) the absorbance was measured continuously for 15 min at 516 nm T70 UV/VIS Spectrophotometer, PG Instruments Ltd., UK); (iii) ascorbic acid was used as positive control, while distilled water as negative control. The final TAOxA was presented as percentage obtained after applying the formula presented in our previous studies [DPPH  free-radical scavenging assay was selected to evaluate the total antioxidant activity (TAOxA) of hydro-alcoholic HP extract, according to the method described in the literature ,77. Brie studies ,77.®, Manassas, VA, USA, CRL-1555™); murine melanoma cell line—B164A5 ; breast cancer cell lines—MCF-7  and MDA-MB-231 . Healthy cell lines were represented by human keratinocyte cell lines—HaCaT ; murine epidermal cells—JB6Cl41-5a . HaCaT, B164A5, A431, and MDA-MB-231 were cultured in the specific culture medium represented by Dulbecco’s Modified Eagle’s Medium high glucose in which were added 10% fetal bovine serum  and 1% penicillin/streptomycin solution . JB6Cl41-5a and MCF-7 were cultured in Eagle’s Minimum Essential Medium, which was supplemented with 5% fetal bovine serum  and 0.1% non-essential amino acids for JB6Cl41-5a and 10% fetal bovine serum  for MCF-7. To prevent microbial contamination, 1% penicillin/streptomycin solution mixture  was added. During the experiments, the cells were maintained in constant conditions of temperature and humidity (5% CO2 and 37 °C). Countess II Automated Cell Counter , was used to determine the number of cells in the presence of Trypan blue. The tumor cell lines selected in the present study were squamous cell carcinoma cell line—A431 . The treated cells were incubated for 24 h. After this interval, cell viability was determined using the Alamar Blue method using the protocol described above [In order to determine the cell viability following the treatment with the hydroalcoholic extract of ed above . Thus, aed above .6 cells/well. After reaching a confluence of approximately 90%, the cells were stimulated with the sub-cytotoxic concentration of HPex (100 µg/mL) for a period of 24 h. After this time, RNA was isolated using Trizol reagent and the Quick-RNA ™ purification kit and its amount was determined using a DS-11 spectrophotometer . Finally, RNA transcription was completed using Maxima® First Strand cDNA Synthesis Kit , and quantitative real-time PCR analysis was performed using Quant Studio 5 real-time PCR system  in the presence of Power SYBR-Green PCR Master Mix.Given that, following the cell viability test, the most affected cell line was breast cancer MCF-7, it was decided that the influence of HPex on gene expression should be established by applying the reverse transcription–polymerase chain reaction (RT-PCR) method to this cell line. To determine the expression of the Bax, Bcl-2  and Bad , the cells were cultured in 6-well plates in a number of 1 × 10H. purpurascens, a medicinal plant with therapeutic effects applied in traditional medicine since ancient times. LC-MS analysis revealed that the extract contains high levels of flavonoids, especially quercetin, kaempferol, and epicatechin. First of all, the hydroalcoholic extract exerted a strong antioxidant effect, close to that of ascorbic acid, mainly at a concentration of 100 µg/mL. Further, starting from the beneficial role played by antioxidants in antitumor therapy, the hydroalcoholic extract proved its selective cytotoxic effects in the four tumor cell lines used, the proliferation of healthy cells not being affected by stimulation with H. purpurascens extract. Finally, the influence of HPex on increasing the expression of pro-apoptotic genes (Bad and Bax) and decreasing the expression of anti-apoptotic genes (Bcl-2) was highlighted in the breast cancer cell line—MCF-7. In conclusion, the extract has an important therapeutic value for the future of oncological therapy, but, nevertheless, additional studies are needed to unravel the mechanism of action and identify the key phytoconstituents which explain these biological effects.The present study was designed to determine the chemical composition and biological properties of a hydroalcoholic extract of


Stable cell lines derived from primary tissues and tumors are widely used in medical research. This study presents interesting findings from evaluation of publicly available gene expression profiles (GEPs) of primary tissues derived from the normal esophagus, Barrett’s esophagus (BE), esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), as well as available esophageal cell lines. We observed that the GEPs of currently available BE cell lines deviate from the GEPs of primary BE tissues (columnar) and EAC tumors (glandular), and are unexpectedly similar to the GEPs of normal esophageal mucosa and ESCC tumors. In vitro exposure to an acid and bile environment was not sufficient to reverse this “squamous like” GEP adopted by a BE cell line, BAR-T. This incomprehensible change in the GEP may result in ambiguous changes in the phenotype of the BE cell lines, and needs careful consideration during experimental design.hi) had considerably lower expression in EAC cell lines than EAC tumors. However, more surprisingly, unlike various normal cell lines  which were very similar to various tissues from normal esophagus, BE cell lines  were extremely different from primary BE tissues, as BE cell lines had substantially lower levels of EAChi and substantially higher levels of ESCChi gene expression. This ESCC-like profile of the BAR-T remained unaltered even after prolonged exposure to an acidic bile mixture in vitro resulting in malignant transformation of this cell line. However, primary BE tissues had EAC-like gene expression profiles as expected. Only one EAC case from the Cancer Genome Atlas resembled BE cell lines, and while it had the clinical profile and some mutational features of EAC, it had some mutational features, the copy number alteration profile, and the gene expression profile of ESCC instead. These incomprehensible changes in gene expression patterns may result in ambiguous changes in the phenotype and warrants careful evaluation to inform selection of appropriate in vitro tools for future studies on esophageal adenocarcinoma.Esophageal adenocarcinoma (EAC) is strongly associated with Barrett’s esophagus (BE), a pre-malignant condition resulting from gastric reflux. Esophageal squamous cell carcinoma (ESCC), the other major subtype of esophageal cancer, shows strong association with smoking and alcohol intake and no association with gastric reflux. In this study, we constructed and validated gene expression signatures of EAC vs. ESCC tumors using publicly available datasets, and subsequently assessed the enrichment levels of these signatures in commonly used EAC and ESCC cell lines, normal esophageal tissues and normal esophageal cell lines, and primary BE tissues and BE cell lines. We found that unlike ESCC cell lines which were quite similar to primary ESCC tumors, EAC cell lines were considerably different from primary EAC tumors but still more similar to EAC tumors than ESCC tumors, as the genes up in EAC vs. ESCC (EAC Esophageal cancer has two distinct histologic subtypes  with difDue to asymptomatic disease progression in esophageal cancers, the diagnosis is delayed until later stages of disease, leading to increased mortality and only 20% 5-year survival after diagnosis . Therefohi) show substantially lower expression in BE cell lines than primary BE tissues, but also the genes up in ESCC vs. EAC (ESCChi) had substantially higher expression in BE cell lines than primary BE tissues. We validated this “squamous like” GEP of BE cell lines in a previously published in vitro model of BE carcinogenesis (BEC) [In this study, we constructed and validated gene expression signatures of EAC vs. ESCC tumors, and subsequently quantified the enrichment of these signatures in the established EAC and ESCC cell lines, normal esophageal tissues and normal esophageal cell lines, and primary BE tissues and BE cell lines. We found that unlike ESCC cell lines which were quite similar to primary ESCC tumors, EAC cell lines were considerably different from primary EAC tumors. The normal cell lines  were very similar to various tissues from normal esophagus, but the BE cell lines  were extremely different from primary BE tissues. This was because not only did the genes up in EAC vs. ESCC (EACis (BEC) ,21,22,23is (BEC) , chromosis (BEC) , and malis (BEC)  of a benis (BEC)  but did is (BEC)  resemblehttps://gdc.cancer.gov/about-data/publications/pancanatlas) (accessed on 1 March 2020). The list of EAC (n = 72) and ESCC (n = 91) tumors and the contributions of COSMIC17 and APOBEC mutation signatures [Gene expression data of tumor and adjacent normal samples were obtained from the TCGA Pan-Cancer Atlas website , a list of ESCC cell lines was obtained from https://depmap.org/portal/context/esophagus_squamous (accessed on 4 April 2021), and gene expression data of these cell lines were obtained from https://www.cbioportal.org/study/summary?id=ccle_broad_2019 (accessed on 17 June 2021). For the Genotype-Tissue Expression (GTEx) dataset [https://www.gtexportal.org/home/datasets (accessed on 17 June 2021). Gene expression data of 13 datasets were obtained from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=) (accessed on 17 June 2021): GSE2144 [https://www.ebi.ac.uk/gxa/experiments/E-MTAB-2706/Downloads) (accessed on 17 June 2021), E-MTAB-3983 (https://www.ebi.ac.uk/gxa/experiments/E-MTAB-3983/Downloads) (accessed on 17 June 2021), and E-MTAB-4054 [https://www.ebi.ac.uk/gxa/experiments/E-MTAB-4054/Downloads) (accessed on 17 June 2021).For the Cancer Cell Line Encyclopedia (CCLE) dataset , a list  dataset , gene ex GSE2144 , GSE9768 GSE2144 , GSE1337 GSE2144 , GSE3720 GSE2144 ,33,34,35 GSE2144 , GSE5896 GSE2144 , GSE1300 GSE2144 , GSE1652 GSE2144 , GSE1731 GSE2144 , and GSE GSE2144 . Gene ex GSE2144   tables from GEO, and subsequently matched with Entrez IDs from the TCGA pan-cancer atlas. For genes with multiple probes, the probe with highest mean of log2 transformed expression was chosen.https://www.ensembl.org/biomart/martview/) (accessed on 17 June 2021). In datasets where Ensembl IDs were available, they were converted to Entrez IDs using Ensembl BioMart, and subsequently matched with Entrez IDs from the TCGA pan-cancer atlas. In a dataset where RefSeq IDs were available instead, they were first converted to Ensembl IDs using Ensembl BioMart, and then processed as described above. In datasets where only gene symbols were available, they were first matched with gene symbols from the TCGA pan-cancer atlas, and then corresponding Entrez IDs were used as input in Ensembl BioMart to retrieve gene length (if necessary).For RNA-sequencing datasets, if normalized data was unavailable, then read count data was converted to RPKM using gene lengths from Ensembl BioMart (hi) were included in the EAC signature (hi) were included in the ESCC signature (hi) were included in a longer EAC signature (hi) were included in a longer ESCC signature (hi and ESCChi genes (100 and 500) in each sample of each dataset. A two-sided Wilcoxon ranksum test was used to compare the enrichment levels in different types of samples, and statistical significance was assessed at p < 0.05.A total of 9485 genes were common between the 22 datasets analyzed in this study; therefore, all datasets were restricted to these 9485 genes. To create gene expression signatures of EAC and ESCC, differential expression analysis was performed on the rank-normalized TCGA dataset. A two-sided Wilcoxon ranksum test was used for pairwise comparison, and the Benjamini–Hochberg method was used for multiple hypothesis testing correction. The genes that differed a full quartile  between EAC and ESCC were sorted by false discovery rate, and the top 100 genes up in EAC  and ESCC (n = 91) cases from TCGA (hi) (shown in red in hi)  and ESCC cases  from TCGA, and samples from other datasets .By comparing EAC  from the ESCC cases (green) in TCGA , except for one EAC case (see result section below). Thus, the gene expression signatures of EAC and ESCC passed the consistency check.Enrichment levels of the 100 EACn = 23) from GSE130078 [n = 17) [n = 15) [n = 51) [n = 77) [To validate these gene expression signatures, we tested ESCC cases    ,33,34,35[n = 51)     (accessed on 29 July 2021). As a representative example, the ESCC cell line OE21 from GSE57130 [hi genes compared with ESCC cases from TCGA, they are quite similar to primary ESCC tumors and therefore ESCC-like.First, we investigated ESCC cell lines A–D and EGSE57130   from GTEx  show tisWhile the normal esophageal cell line EPC2 from GSE173169  Figure G was ESChi and 500 ESCChi genes, respectively).Similar results were observed  for longhi genes and low expression of EAChi genes (or vice versa).It should be noted that the classification of normal mucosa and EPC2 as ESCC-like does not mean they are tumor-like, it means they are “squamous like” instead of “columnar/glandular like”. Since we are plotting the enrichment levels of genes differentially expressed between EAC and ESCC , normal samples can cluster with ESCC (or EAC) tumors from TCGA if they have high expression of ESCCPrimary BE tissues with specialized intestinal metaplasia from GSE58963 A, non-dyhi genes than primary BE tissues , but also a substantially higher enrichment level of the 100 ESCChi genes than primary BE tissues (which is not true for EAC cell lines vs. EAC tumors).Unlike primary BE tissues, the BE cell line CP-A from GSE62909  Figure G and GSEhi and 500 ESCChi genes, respectively).Similar results were observed  for longThe notable observation that (unlike primary BE tissues) BE cell lines are ESCC-like (instead of EAC-like) was subsequently validated in a previously published in vitro BEC model ,21,22,23hi genes.We sequenced 10 samples with various levels of ABS exposure obtained from the BEC model  and 8 ABS unexposed samples in total at 3 different centers, and the three RNA-sequencing datasets  confirmehi and 500 ESCChi genes  but were low in expression in the BE cell lines CP-A and BAR-T , while most of the ESCChi genes showed the opposite trend (hi genes and low expression of ESCChi genes (except for SK-GT-4 which had high expression of some ESCChi genes) and may be considered EAC-like. The majority of EAChi and ESCChi genes were low in expression in normal muscularis, GEJ, and Het-1A, which are neither EAC-like nor ESCC-like  clustered with/near BE cell lines B–D, so w2 transformed gene expression data clustered this case with ESCC cases instead of other EAC cases  had a high contribution from COSMIC17 mutation signature , even hiThe mechanism of CDKN2A loss in this case—oncogenic mutation accompanied by heterozygous deletion—is consistent with the EAC diagnosis, as the mechanism of CDKN2A loss in ESCC is homozygous deletion D. HoweveThus, the only EAC case in TCGA that had a GEP similar to BE cell lines was an unusual case that had features of both EAC and ESCC: the clinical profile of EAC, and some mutational features typical of EAC, but the copy number alteration profile and the gene expression profile of ESCC, and some mutational feature typical of ESCC.hi and ESCChi genes that could reliably discriminate between primary EAC and ESCC tumors, this study found that unlike the normal cell line EPC2 that had a GEP similar to normal mucosa, the GEPs of representative BE cell lines (BAR-T and CP-A) showed notable deviation from the expected primary BE/EAC-like profile to ESCC-like profile , ESCC-like (low enrichment of EAChi genes and high enrichment of ESCChi genes), or neither (low enrichment of both EAChi and ESCChi genes) based on the top 100 genes selected from each histological type. Similar results were observed for the top 500 genes which confirmed that the similarities or differences observed are global with respect to gene expression and may be associated with the tissue/cells of origin (squamous or glandular epithelium), particularly in case of the primary tumors, and normal and primary BE tissues. However, this was not true for BE cell lines: while EAC cell lines had considerably lower enrichment of EAChi genes compared with EAC tumors  if RNA-sequencing data of those samples are available. While a set of 100 EAChi genes  were usehi genes B. Howevehi and ESCChi genes may be useful in selecting appropriate cell lines for an experiment. Our results suggest that EPC2 may be suitable for studying normal mucosa, while Het-1A may be more suitable for experiments studying normal muscularis or GEJ. Similarly, while some EAC cell lines such as KYAE1 and ESO26 are very similar to EAC tumors, certain EAC cell lines such as OACM51, FLO1, and SK-GT-4 may not be suitable for studying EAC tumors. While most ESCC cell lines are quite similar to ESCC tumors, certain ESCC cell lines such as OE19 and TE-4 may not be suitable for studying ESCC tumors. Commonly used BE cell lines  were extremely different from primary BE tissues and the only EAC case from TCGA (TCGA-IN-A7NT) that was comparable to BE cell lines was an unusual case that had features of both EAC and ESCC  (accessed on 4 April 2021), clustered with EAC tumors from TCGA. This cell line is indeed labeled as EAC cell line in Cellosaurus (https://web.expasy.org/cellosaurus/) (accessed on 29 July 2021), suggesting the possibility that it may be an EAC cell line accidentally misclassified as an ESCC cell line, and warrants further investigation.Another potential utility of our method could be identification of misclassified EAC and ESCC cases, which may have clinical relevance. While validating the gene expression signatures of EAC and ESCC, we observed that a small number of EAC and ESCC cases clustered with ESCC and EAC tumors from TCGA, respectively. While some of these may be rare cases of EAC that resemble ESCC  or vice-versa, most of these are likely ESCC and EAC cases, respectively, that were accidentally misclassified. A similar example was observed in case of cell lines, where OE19, a cell line labeled as ESCC cell line in DepMap (This study points to notable changes in the gene expression profiles of established Barrett’s esophagus cell lines that deviate significantly from primary Barrett’s esophagus tissues thus making them more “squamous like”. These incomprehensible changes in gene expression patterns may result in ambiguous changes in the phenotype and warrant careful evaluation to inform selection of appropriate in vitro tools for future studies on esophageal adenocarcinoma.