GATA2, GATA1, and RUNX1 as candidateinducers of the expression of gangliosides and sialylation via regulationof the GTs ST3GAL2 and ST8SIA1.In conclusion, we show that GSL glycan expression levels are associatedwith hematopoietic AML classifications and TF and GT gene expression.Further research is needed to dissect the regulation of GSL expressionand its role in hematopoiesis and associated malignancies.Aberrant expressionof certain glycosphingolipids (GSLs) is associatedwith the differentiation of acute myeloid leukemia (AML) cells. However,the expression patterns of GSLs in AML are still poorly explored becauseof their complexity, the presence of multiple isomeric structures,and tedious analytical procedures. In this study, we performed anin-depth GSL glycan analysis of 19 AML cell lines using porous graphitizedcarbon liquid chromatography-mass spectrometry revealing strikinglydifferent GSL glycan profiles between the various AML cell lines.The cell lines of the M6 subtype showed a high expression of gangliosideswith α2,3-sialylation and Neu5Gc, while the M2 and M5 subtypeswere characterized by high expression of (neo)lacto-series glycansand Lewis A/X antigens. Integrated analysis of glycomics and availabletranscriptomics data revealed the association of GSL glycan abundanceswith the transcriptomics expression of certain glycosyltransferases(GTs) and transcription factors (TFs). In addition, correlations werefound between specific GTs and TFs. Our data reveal TFs  In mammals, GSLs can be classifiedinto three subgroups on the basis of common neutral core structures,namely, gangliosides, (neo)lacto-series, and globosides.7 GSLs play essential roles in cellularprocesses including adhesion, proliferation, differentiation, andrecognition.8 AbnormalGSL expression is associated with the development of many types ofcancers including leukemia.9 A subtypeof leukemia, AML, is a heterogeneous clonal disorder of hemopoieticprogenitors of the myeloid lineage which gives rise to red blood cells(RBCs), several different white blood cells, and platelets.10 AML is the second most common type of leukemiadiagnosed in adults and children, with most cases occurring in adults.The mortality rate of AML in the United States is estimated at 11,400for 2021, accounting for approximately 48% of all leukemia cases.11 AML can be classified into eight subtypes (M0–M7),according to the French–American–British (FAB) classificationsystem which was established in 1976.12 The subtypes are defined based on morphological and cytochemicalcharacteristics of the leukemia cells. Basically, subtypes M0 to M5are all derived from certain immature white blood cells called myeloblasts,M6 AML cells are initiated from an immature form of RBCs (erythrocytes),and M7 AML cells are from immature forms of cells which make platelets(megakaryocytes).Alterations of glycosylation including changes of sialylation and fucosylation  have been describedfor various hematological malignancies for both glycoproteins andGSLs.13 Previousstudies have indicated that certain GSLs play a critical role in thedifferentiation of AML cells.13 For example,the incorporation of GSL GM3 into HL60 cells resulted in the growth inhibition and monocytic differentiationof the cells.14 During the differentiationof monocytic THP-1 cells into macrophages, α2,6-sialylationslightly decreased and GM3 expression increased which correlated directlywith the overexpression of ST3GAL5.15 Theconcerted upregulation of ST3GAL5 and GM3 resulted from the activationof the PKC/ERK pathway during the differentiation of HL60 cells.16 In addition, the decrease of GSL Lc3 expression in HL60 and NB4 was induced by treatment with either all-transretinoic acid which can stimulate the differentiation of cells intothe neutrophil lineage or phorbol 12-myristate 13-acetate which inducedthe differentiation of cells along the monocyte lineage.It is suspected that GSLs, which are presenton the cell surface,might participate in myelopoiesis.Inthis study, we characterized the expression of GSL glycans in19 different AML cell lines belonging to different FAB subtypes. TheGSL glycans were profiled, identified, and quantified using porousgraphitized carbon nanoliquid chromatography hyphenated with massspectrometry via electrospray ionization (PGC-nanoLC–ESI-MS/MS).Overall, a striking diversity of GSL glycan expression was found betweencell lines. In addition, the association of GSL glycan expressionwith GT and TF gene expression and with AML classification was explored.4, Dowex cation-exchange resin (50W-X8), HCl, and ammonium bicarbonate were purchased from Sigma-Aldrich. KOH and 100% glacial acetic acid were from HoneywellFluka . A Sep-Pak tC18 reverse-phase (RP) solid-phaseextraction (SPE) cartridge (50 mg) was obtained from Waters . Ziptip C18 was obtained from Millipore ,and SPE bulk sorbent Carbograph was purchased from Grace DiscoverySciences . TopTip  wasobtained from Glygen Corporation , and 2-propanol waspurchased from Biosolve Chemie . Acetonitrile (MeCN)of LC–MS grade was purchased from Biosolve . Endoglycoceramidase I (EGCase I), 1× EGCase I reactionbuffer, α2–3 neuraminidase S, α1–3,4 fucosidase,α1–2,4,6 fucosidase O, purified bovine serum albumin(BSA), and 10× GlycoBuffer 1 were purchased from New EnglandBiolabs . Iscove’s modified Dulbecco’smedium (IMDM) was purchased from Gibco . PenStrep was obtained from Invitrogen(Thermo Fisher Scientific). Fetal calf serum (FCS) was purchased fromBodinco . The granulocyte-macrophage colony-stimulatingfactor (GM-CSF) was obtained from Cellgenix . Insulinand transferrin were purchased from Sigma .Ultrapure water was generated using a Q-Gard 2 system and used forall preparations and washing steps.Chloroform and trifluoroacetic acid (TFA)were purchased from Merck . Methanol (MeOH) ofultra LC–MS grade was obtained from Actuall Chemical . NaBH2. The AML193 medium was supplemented with 5 μg/mL insulin,5 μg/mL transferrin, and 5 μg/mL GM-CSF, and the mediaof cell lines M07e and TF1 were supplemented with 20 ng/mL GM-CSF.For more information regarding each cell line, see Supplementary Information, Table S-1.AML cell lines were grownat the Department of Immunopathology of Sanquin Research . Briefly, all cells were cultured in IMDM supplementedwith heat-inactivated FCS  and 1% PenStrep at 37 °C and 5% CO17 Briefly, prior tocell lysis, two million cellswere suspended in 200 μL of H2O and vortexed followedby a 30 min sonification at room temperature (RT) in a glass vial.Next, 550 μL of chloroform and 350 μL of MeOH were addedto the suspension. The sample was again briefly vortexed, sonicatedfor 30 min, and incubated for 4 h by horizontally shaking at 1000rpm. After incubation, the sample was centrifuged for 15 min at 3000× g at 20 °C. The upper phase (containingGSLs) was collected, and sequentially 400 μL of chloroform/MeOH(2:1) and 400 μL of MeOH/ H2O (1:1) were added tothe sample. After overnight incubation with shaking at RT, two additionalextraction steps were performed by collecting the upper phase (centrifugationat 3000 × g at 20 °C for 15 min) and adding400 μL of MeOH/ H2O (1:1) to the sample followedby sonication for 10 min before centrifugation. The collected upperphases  were combined into a single vial and evaporatedto dryness with vacuum. The tC18 RP-SPE cartridge was preconditionedby the sequential passing of 1 mL of chloroform/MeOH (2:1), 1 mL ofMeOH, and 2 mL of MeOH/H2O (1:1) over the cartridge. Theextracted GSLs were resuspended in 200 μL using a mixture ofMeOH and H2O (1:1) and loaded on the tC18 RP-SPE cartridgeby passing the sample three times over the cartridge. The loaded cartridgewas washed with 2 mL of MeOH/H2O (1:1) followed by a sequentialelution of the GSLs using 2 mL of MeOH and 2 mL of chloroform/MeOH(2:1). The samples were dried under vacuum in an Eppendorf Concentratorat 30 °C.The cells were processed as describedin a previous study.Rhodococcus triatomea recombinantlyproduced in Escherichia coli was usedto release the glycans from the extracted GSL samples according tothe manufacturer’s instruction with some slight modifications.Briefly, 36 μL of H2O, 4 μL of EGCase I reactionbuffer, and 2 μL of EGCase I enzyme were added to each lyophilizedsample. The final mixture (42 μL) was incubated at 37 °Cfor 36 h. After incubation, the GSL glycans were retrieved using atC18 RP-SPE cartridge, which was preconditioned with 2 mL of MeOHand 2 mL of H2O prior to applying the sample onto the cartridge.The purified GSL glycans were eluted using 500 μL of H2O. The mixture, consisting of the elution and flowthrough, was lyophilizedunder vacuum.EGCaseI from 18 Briefly, GSL glycanswere reduced by adding 40 μL of 1 M NaBH4 in 50 mMKOH to each sample followed by incubation at 50 °C for 3 h. Thereduction mixture was quenched with 4 μL of glacial acetic acid.The RP C18 Ziptips containing 50 W-X8 resin were preconditioned byapplying sequentially 3 × 60 μL of 1 M HCl, 3 × 60μL of MeOH, and 3 × 60 μL of H2O ontothe column. Subsequently, the solution, containing glycan alditols,was loaded, and the column was washed with 100 μL of H2O. The flowthrough and washes were collected and dried under vacuum.To remove residual borate, 150 μL of MeOH was added twice tothe samples during the drying step.Reductionand desalting of the purified GSL glycans were performed as describedin a previous study.2O followed by the addition of either2 μL of α2–3 neuraminidase S from Streptococcuspneumoniae recombinantly expressed in E. coli or 4 μL of α1–2,4,6 fucosidase O from Omnitrophica bacterium recombinantly expressed in E. coli. For α1–3,4 fucosidase  treatment, 5 μLof the pooled sample, 11 μL of H2O, and 2 μLof 10× glycobuffer 1 were mixed followed by addition of 2 μLof 10× BSA and 2 μL of α1–3,4 fucosidase.Combined enzymatic digestion was performed by adding 2 μL ofα2–3 neuraminidase S and 4 μL of α1–2,4,6fucosidase O or 2 μL of α1–3,4 fucosidase to 6μL of the pooled sample with the addition of 2 μL of 10×glycobuffer 1 and 13 μL of H2O. As a control, theenzyme was replaced with 10× glycobuffer 1. Each enzymatic digestionwas performed twice, and all samples were incubated overnight at 37°C followed by RP-SPE and drying of the purified glycans by vacuumcentrifugation.A pool of glycan alditolsreleased from cell lines Molm13, HL60, ML1, ME1, HEL, PLB985, EOL-1,and AML 193 was used for exoglycosidase treatment. In brief, 5 μLof the pooled sample was added to 2 μL of 10× glycobuffer1 and 13 μL of H2O with 0.1% TFA (v/v) followedby a shaking step for 30 min. PGC SPE clean-up was performed as describedpreviously19 with some slight modifications.Briefly, columns were packed with 50 μL of PGC resin slurry followed bya conditioning step by loading 3 × 60 μL of 80% MeCN with0.1% TFA (v/v) followed by 3 ×60 μL of H2O with 0.1% TFA (v/v). After sample loading, the columns were washed with 2× 60 μL of H2O with 0.1% TFA (v/v). Subsequently, the GSL glycans were eluted byadding 2 × 40 μL of 60% MeCN with 0.1% TFA (v/v). The two elution fractions were combined anddried under vacuum.Lyophilized GSL glycans were reconstitutedin 40 μL of H2O. A volumeof 1 μL of the sample was loaded onto a Dionex Ultimate 3000nanoLC system equipped with a Hypercarb PGC-trap column  and a HypercarbPGC nanocolumn . The separation platform was coupled to an amaZon speedion trap MS . Buffer A consistedof 10 mM ammonium bicarbonate and buffer B of 60% MeCN in 10 mM ammoniumbicarbonate. The separation of GSL glycans was conducted over a linearnanoLC gradient of solvent B from 1 to 65% in 80 min at a flow rateof 0.4 μL/min. After analysis, the column was washed with 85.5%MeCN in 10 mM ammonium bicarbonate for 10 min. MS spectra were acquiredwithin a mass to charge ratio (m/z) of 340–2000 in negative ion mode with a target mass of thesmart parameter setting at m/z 1200.The glass capillary voltage, dry gas temperature, dry gas flow, andnebulizer gas were set at 1000 V, 280 °C at 5 L/min, and 3 psi,respectively.Prior to MS analysis, the purified GSL glycanalditols were resuspended in 20 μL H22 and general glycobiology knowledge about the biosynthetic pathwaysof GSLs from a KEGG database.23 In addition,exoglycosidase digestion was used to further confirm specific linkages.Extracted ion chromatograms (EICs) were generated by extracting thetheoretical mass of GSL glycans of the observed singly and doublycharged species using the first three isotopes. Peaks were manuallyevaluated and automatically integrated using the Data analysis software(version 5.0). The peak area-under-the-curve was obtained to integrateeach peak of individual glycans if the signal-to-noise ratio was ≥6.Relative quantification was calculated on the total area of all GSLglycans within one sample normalizing to 100%.The GSL glycan structureswere assignedon the basis of the known MS/MS fragmentation patterns in negativeion modeTable S-2). For further data analysis and visualization,the packages “ggplot2”, “tidyverse”, “mixOmics”,24 “devtools”, “MASS”,“lattice”, “BiocManager”, “tidyHeatmap”,and “complexUpset” were used in “R” software(version 4.0.5). In addition, rcc and cim functions of the “mixOmics”package were used for canonical correlation analysis and clusteredimage mapping in which the colored block indicates the correlationbetween glycosylation features and relevant gene expression data extractedfrom the Cancer Cell Line Encyclopedia (CCLE) database, obtained bynonstrand specific RNA sequencing using the large-scale, automatedvariant of the Illumina TruSeq RNA sample preparation protocol.25Glycans wereassigned into different glycosylation features . To reduce the complexity of each GSL glycomic profile per cellline and define technical variability of the workflow used in ourstudy, the single mass spectrometry average composition (MSAC) wascalculated  from relative abundances.27 The low technical variability of the workflow is illustrated bythe clustering of the two technical replicates of each cell line ina principal component analysis (PCA) .To characterizestructural features of GSL glycans in 19 AML celllines, GSLs were extracted from the cell lines followed by glycanhead group release and analysis by PGC-nanoLC-ESI-MS/MS.21 Known diagnostic ions such as 0,2A-type and 2,4A-type were used to characterize C-4 substitution of theGlcNAc and Glc residues, respectively.22 Blood group and Lewis antigens were defined among the GSL glycansbased on the presence of specific D and A diagnostic fragment ions. The subtypes M2 and M4 revealed thehighest diversity, showing 9 and 18 unique GSL glycans, respectively,and most of the unique glycans expressed in M4 belong to (neo)lacto-series.These glycans carried blood group H or Lewis B/Y antigens . However, LewisA/X antigens were more broadly expressed in the M2/4/5 and 7 subtypes.Furthermore, three types of gangliosides and two kinds of (neo)lacto-seriesglycans were found across all subtypes, for more details see the SupplementaryInformation, Figure S-4 and Table S-5. Globoside glycans were present atlow abundance in most AML cell lines but were relatively abundantin the cell lines THP1 (32%), TF1 (17%) and M07e (20%) . As shown in Figure S-5). In contrast, higher expression ofgangliosides as well as globosides including SSEA-3 was found in theglycan profile of cell line M07e. Interestingly, N-glycolylneuraminic acid (Neu5Gc) was detected in most of the AMLcell lines .A high variation was observed in the GSL glycomic profilesbetweenthe 19 AML cell lines as exemplified in Table S-2). Variability in the loadings plot was assessedto elucidate which glycosylation features drive the separation inthe PCA model.PCA was performed todiscover possible associations between GSLglycosylation traits and the AML classification of the cell lines.For this, GSL glycans were grouped based upon the composition andspecific structural features  and sialylation,including α2,3-sialylation and glycans occupied with Neu5Gc. The expression levels of gangliosides,sialylation, α2,3-sialylation, and Lewis A/X antigen in AMLcell lines can be found in the Supplementary Information, Figure S-6. A similar glycosylation profile withadditional high expression of gangliosides with GalNAc was observedfor the M3 subtype cell line NB4, which is the only M3-classifiedcell line in our panel. Cell lines belonging to the M5 subtype  are mainly grouped inthe bottom region of the PCA scores plot because of the higher expressionof (neo)lacto-series, sialyl-Lewis A/X, and Lewis A/X lacto-series expressionof M5 cell lines vs 44.7% for the other 16 AML celllines . In contrast, cell line U937 – albeit also of the M5 class– showed very low expression of (neo)lacto-series with highexpression of gangliosides, and cell line THP1 indicated a specificallyhigh expression of globosides. No consistency was found for the M4subtypes with regard to GSL glycan feature expression. However, itshould be noted that a uniquely high expression of blood group H-typeantigens was found in the ML1 cell line. Two cell lines belongingto subtype M2 (PLB985 and HL60) also show high (neo)lacto-series expression, notably accompanied by high expressionof Lewis A/X type antigen  . HL60 has a highexpression of α2,6-sialylation while the third M2 cell line,Kasumi1, appears to have a distinct profile from the other two, revealinga high abundance of gangliosides, sialylation, and α2,3-sialylation.The M07e cell line, the only M7-classified cell line in our panel,shows specifically high expression of sialyl-Lewis A/X antigens togetherwith higher expression of globosides as shown in As shown in 31 To explore the associations between the expressionof GSL glycans and the expression of a preselected set of genes includingGTs involved in GSL biosynthesis and TFs involving the differentiationof the blood cells, the transcriptomic data of the same AML cell lineswere extracted from the CCLE.25 The selectionof hematopoietic TFs was based on their key roles in the regulationof the process of blood cell differentiation and maturation and theirdysregulation involved in the development of AML.35 Canonical correlation analysiswas performed between expression of GSL glycan traits as listed in B3GNT5 expression, a gene that codes for theonly enzyme that drives (neo)lacto-series biosynthesis (r = 0.38). Likewise, the relative abundance of globosides correlatedwith A4GALT gene expression coding for the firstenzyme specific for the globoside biosynthetic pathway (r = 0.38). A subtle correlation was found between the gene that isinvolved in the ganglioside series, B4GALNT1 (r = 0.21) and the expression of gangliosides. The expressionof the fucosyltransferases FUT7 and FUT9 encoding genes correlated positively with the fucosylated LewisA/X antigen  andα1,3/4-fucosylation .Similarly, a positive correlation was found between the α2,3-sialyltransferase encoding gene ST3GAL2 and α2,3-sialylation (r = 0.24) and between ST8SIA1 and sialylation (r = 0.30) andgangliosides (r = 0.32).Cellular glycosylation signaturesare known to be largely determined by GSTs on the one hand, and variousTFs on the other hand, as indicated by various studies using celllines as model systems, with glycosylation changes often reflectingcellular differentiation.GATA1, GATA2, RUNX1, and MECOM showed positivecorrelations with gangliosides  and sialylation . Meanwhile, GATA1 presented strong positive correlations with ST3GAL2 and ST8SIA1 , In addition, high positive correlations were foundbetween MECOM with ST3GAL2 (r = 0.75) and ST8SIA1 (r = 0.85) as well as between RUNX1 with ST3GAL2 (r = 0.40) and ST8SIA1 (r = 0.40) . These findings revealed that GATA2, GATA1, MECOM, and RUNX1 might play parts in the high expression of sialylation within theM6 subtype by regulation of corresponding GTs ST3GAL2 and ST8SIA1.To elucidate the potential regulationof GSL glycan expression,the correlations between GSL glycosylation features as listed in CEBPA (r = 0.14) and MYB (r =0.22), showed a moderate positive correlation. Interestingly, B3GNT5 responsible for production of (neo)lacto-series exhibitedstrong positive correlations with CEBPA (r = 0.54) and MYB (r =0.39), respectively . Additionally, TFs CEBPA and MYB showed positive correlations with FUT7  which were positivelycorrelated with the Lewis A/X antigen structure and α1,3/4-fucosylationas shown above. Thus, our in-depth analyses show that several relationsexist between the expression of specific GTs and TFs and the biosynthesisof GSL glycans.For the (neo)lacto-series, theTFs, 36 The structural and functional classificationof GSLs is mainly based on the glycan part. Little is known aboutthe expression and regulation of GSLs in AML cells because isolationand characterization of the GSLs is a challenging task due to theheterogeneity and isomeric complexity present in the glycan constituents.In this study, a PGC-nanoLC-ESI-MS/MS platform was employed to detect,identify, and quantify the glycan head group of GSLs extracted fromAML cell lines. The broad specificity enzyme EGCase I was used forreleasing GSL head groups, allowing to profile all human GSL seriesin a single analysis.37 In total, 79 GSLglycans were detected and characterized. An overall high diversityin GSL glycan profiles was found for the 19 AML cell lines.GSLs are involved inmany cellular processes including cellularinteraction, differentiation, signal transduction, and oncogenesis.38 In line withthese reports, we found associations between gene expression and GSLexpression in 19 AML cell lines. The expression of (neo)lacto-seriescorrelated positively with the expression of B3GNT5 which encodes the key enzyme involved in the biosynthesis of (neo)lacto-seriesGSLslacto-series GSLs in the inhibitionof immune recognition.39 Accordingly, onemay speculate about the potential immunosuppressive roles of (neo)lacto-seriesGSLs in AML. Meanwhile, both globosides and SSEA-3, which is a surfacemarker of human embryonic stem cells40 andhuman-induced pluripotent stem cells,41 showed positive correlations with A4GALT whichencodes the corresponding GT responsible for the biosynthesis of globosideglycans. Similarly, the expression of B4GALNT1 encodingfor a key enzyme in ganglioside biosynthesis positively associatedwith ganglioside levels.Previous studies indicated that incomplete biosynthesis and neosynthesisare the two main mechanisms that underlie aberrant glycan expressionin hematological malignancies because of the alteration of transcriptionof GTs and/or glycosidase gene(s).riesGSLs4 and 5. 7FUT7 and FUT9 are known for their involvement in the biosynthesis of α1,3-and α1,4-linked fucosylation, which is in agreement with ourfindings that FUT7 and 9 show positivecorrelations with the levels of α1,3/4-fucosylation . Our data imply that the expressionof FUT7 may play a role in the synthesis of Lewis A/X antigens ratherthan sialyl-Lewis A/X. In addition, the finding of a previous studyof increased Lewis A/X antigens on N- and O-glycome in the M2 and M5 subtypes is consistent with ourcurrent result.5 In contrast, blood groupH antigens were detected in cell lines originating from myelocytes/erythrocytes/monocytes.These H antigens are highly expressed in ML1 and ME1 (M4 subtype belongingto myeloblasts). Additionally, Lewis B/Y antigens which are potentialtherapeutic targets due to the high expression in various cancerswere only detected in ML1 and ME1 cell lines .44 Therefore, Lewis B/Y antigen mightbe a potential target for M4 subtype AML. To further corroborate this,AML cells obtained from AML patients with the M4 subtype could beinteresting study objects for glycomics analysis.Fucosylation, which is a nonextendablemodification, plays a criticalrole in the homing of hematopoietic cells to the bone marrow.sylation 4. Of notcmah, Neu5Gcis absent in human glycans.45 However,GSL glycans with Neu5Gc were found in most AML cell lines in thisstudy, including GM3 (Neu5Gc), GD3 (Neu5Gc), GM1a (Neu5Gc), and twoglycans in the (neo)lacto-series. The abundance of glycans with Neu5Gcvaried between cell lines with an average Neu5Gc expression of 2.3%in M6 cell lines vs only 0.8% in the rest of theAML cell lines . Several studies have suggested that increased levelsof Neu5Gc may be attributed to the higher metabolic rate in malignantcells,46 the induction of sialic acid transportersunder hypoxic conditions, and dietary incorporation.49 We suspect that the Neu5Gc found in the studied AML cell lines mayhave been incorporated from the culturing medium or its biosynthesismight originate from the mutation of relevant genes in AML cell lines.Previous studies revealed the expression of Neu5Gc in humans beingassociated with malignant transformation, becoming, therefore, anattractive target for cancer immunotherapy.50 Therefore, the glycans with Neu5Gc may be potential therapeutictargets for M6-stage AML. For a better understanding of the underlyingmechanism of expression of glycans with Neu5Gc in AML cell lines,more studies are needed.Because ofa deletion of the gene 51 Iantigens were first identified on human RBCs and have a reciprocalrelationship with i antigens.52 Adult humanRBCs fully express I antigens and contain only few i antigens, andalteration of expression patterns of I and i antigens has been foundduring oncogenesis.52 In this study, theexpression of I antigens was also found in most of the cell linesbelonging to the subtypes M2, M4, and M5. Interestingly, all the celllines expressing I antigens – except for U937 – wereinitially derived from peripheral blood . In addition, the cell lines HEL92.1.7,KG1, KG1a, TF1, and NB4 clustered well in the scores plot in PCA analysisand were all obtained from bone marrow , which indicates that the expressionof certain GSLs may reflect the origin of the cell lines.A recent study revealed that I bloodgroup antigens (I-branchedglycans), which are initiated by the addition of a GlcNAc to internalresidues of poly-LacNAc chains in β1,6 linkage, played a criticalrole in cancer progression through the regulation of malignancy-associatedadhesive and metastatic activities.34 Accordingly,certain hematopoietic TFs such as CEBPA,53MYB,54 and RUNX1 and the expressionof GATA2 correlated positively with sialylation,α2,3-sialylation, and gangliosides (GATA2 correlated with ST3GAL2 (r = 0.66) responsible for additionof sialic acid to terminal galactose of gangliosides and with ST8SIA1 (r = 0.66) gene expression encodingfor an α2,8-sialyltransferase which acts onsialic acid residue by addition of another sialic acid . Therefore, GATA2 might be involved in the upregulation of gangliosideexpression and sialylation in the M6 subtype. We speculate that poorprognosis of AML might be linked to the high expression of α2,3-sialylation,sialylation, and gangliosides regulated by GATA2 whichis highly expressed in M6-subtype cell lines. Our data point towarda shared effect of certain TFs on different types of glycoconjugates,as GATA2 likewise shows positive correlation withthe total levels of sialylation on N- and O-glycome in AML cell lines.5 Meanwhile, erythrocyte/megakaryocyte-related TF GATA1 and the megakaryocyte-derived M07e cellline . Interestingly, GATA1, MECOM, and RUNX1 expression positively correlated withsialylation .Strong positive correlations were observed for GATA1 with ST3GAL2 and ST8SIA1 , for MECOM with ST3GAL2 and ST8SIA1  as well as for RUNX1 with ST3GAL2 and ST8SIA1 (r = 0.40 and 0.40) . These findings indicate that GATA2, GATA1, MECOM, and RUNX1 may be involved in the high expression of sialylationin the M6 subtype by regulation of the corresponding GTs ST3GAL2 and ST8SIA1. Taking into account the previous findings,TF GATA2 may play an essential role in the regulationof sialylation of various glycoconjugates in M6 cell lines. To confirmthis, knockout, knockdown, and overexpression experiments targeting GATA2 in M6 cell lines assessing the effect on various glycoconjugateswould be informative.5High expression of CEBPA participates in the differentiation of commonmyeloid progenitors into basophils,32 andits mutation is associated with AML.58 Additionally, MYB plays a key role in the hematopoietic system and hasbeen recognized as an attractive therapeutic target for the treatmentof leukemia.59 Both TFs (CEBPA and MYB) are mainly expressed in subtypes M2 andM5 of the AML cell lines which are characterized by high expressionof (neo)lacto-series and Lewis A/X antigens . Our study revealed a modest positivecorrelation for TFs MYB and CEBPA with (neo)lacto-series structures  (B3GNT5 (r = 0.39 and 0.54) . As mentioned above, the Lewis A/Xantigen structure and α1,3/4-fucosylation were positively associatedwith FUT7 (CEBPA (r = 0.84) and MYB (r = 0.31) . The results indicate that the hematopoieticTFs CEBPA and MYB may play criticalroles in expression of GSL glycan structures ((neo)lacto-series, LewisA/X and α1,3/4-fucosylation) within AML classifications M2 andM5 by regulation of the expression of GTs B3GNT5 and FUT7 lacto-series, respectively) positively correlated with theexpression of gangliosides, globosides, and (neo)lacto-series, respectively(ST3GAL2/3/4 positively correlated with α2,3-sialylation andsialylation. Lewis A/X and α1,3/4-fucosylation showed positivecorrelations with FUT7 and FUT9, respectively. Moreover, certain hematopoieticTFs may play essential roles in the association of GSL glycans withAML classifications by regulating the related GTs as shown in In summary, the expression of genes ectively5. In addOur currentwork is fully descriptive in nature, providing a detailedmap of GSL glycan structures in AML cell lines. The multiple, reasonablystrong correlations between glycan species and GT and TF transcriptomicdata lead to new hypotheses regarding the regulation of the GSL repertoirewhich will benefit from experimental confirmation. This will be thesubject of future studies, with implications for cancer biology. Likewise,the analysis of patient materials using a similar approach is warranted,the main challenge being to obtain patient AML cells of sufficientpurity for deriving disease glycomics signatures.GATA2, GATA1, MECOM, and RUNX1 potentially play a role in the high expression of gangliosides,sialylation, and α2,3-sialylation within the M6 subtype viaregulation of GTs ST3GAL2 and ST8SIA1. The hematopoietic TFs CEBPA and MYB may play decisive roles in determining the expression of GSL glycanswithin AML classifications M2 and M5 by regulating the related GTs B3GNT5 and FUT7. Finally, the explorationof expression and regulation of GSL glycans in AML cell lines pavesthe way for future studies on the functional role of such GSL glycans.The enzymes in control of their production may provide novel therapeutictargets for specific AML subtypes.In this study, a striking diversity in expression of GSL glycanswas found between different AML cell lines. The biosynthesis of GSLglycans is regulated by specific GTs, some of which were confirmedat a transcriptional level. Additionally, we found new associationsof GSL glycans with AML classifications. These are potentially generatedby GTs whose expression is under the control of a subset of TFs. Ourstudy shows that the hematopoietic TFs


For decades, various plants have been studied as sources of biologically active compounds. Compounds with anticancer and antimicrobial properties are the most frequently desired. Cruciferous plants, including Brussels sprouts, broccoli, and wasabi, have a special role in the research studies. Studies have shown that consumption of these plants reduce the risk of lung, breast, and prostate cancers. The high chemopreventive and anticancer potential of cruciferous plants results from the presence of a large amount of glucosinolates, which, under the influence of myrosinase, undergo an enzymatic transformation to biologically active isothiocyanates (ITCs). Natural isothiocyanates, such as benzyl isothiocyanate, phenethyl isothiocyanate, or the best-tested sulforaphane, possess anticancer activity at all stages of the carcinogenesis process, show antibacterial activity, and are used in organic synthesis. Methods of synthesis of sulforaphane, as well as its natural or synthetic bifunctional analogues with sulfinyl, sulfanyl, sulfonyl, phosphonate, phosphinate, phosphine oxide, carbonyl, ester, carboxamide, ether, or additional isothiocyanate functional groups, and with the unbranched alkyl chain containing 2–6 carbon atoms, are discussed in this review. The biological activity of these compounds are also reported. In the first section, glucosinolates, isothiocyanates, and mercapturic acids (their metabolites) are briefly characterized. Additionally, the most studied anticancer and antibacterial mechanisms of ITC actions are discussed. Aliphatic GSLs covering more than 50% of glucosinolates are divided into: alkenyl (1–3), hydroxyalkenyl (4–5), and those containing sulfur on II (6–7), IV (8–10), and VI (11) oxidation states ..33) -L-homoserine lactones (3-oxo-C12-HSL) and lasR, genes encoding transcription regulators. The rhl system consists of the pair RhlI/RhlR, which respond to N-butyryl homoserine lactones (C4-HSL) pyridine (b), 6-methoxy-1H-benzo[d]imidazole (c), 5-phenyl-1H-tetrazole (d), or benzo[d]thiazole (e). The sulfides 81a–e were transformed into amines 82a–e, and then were converted into sulfide derivatives of ITCs 83a–e with thiophosgene (34), in good and very good yields. In the next step, compounds 83d–e were oxidized with MCPBA into sulfoxide analogues of SFN 84d–e with good yields  to sulfoxides 85a–c and sulfones 86a–c and then transformed to amines 87a–c and 88a–c using methylamine. In the final step, isothiocyanation of amines 87a–c and 88a–c with thiophosgene afforded ITCs 84a–c and 89a–c in good yields . Among all synthesized analogues, tetrazole analogues 83d, 84d, and 89d were generally the most potent—and significantly more active—than SFN against the tree cancer cell lines  involved in the antioxidant defense in the neuronal cell line. All hybrids produced a significant decrease in ROS production elicited by pro-inflammatory stimulus compared to the rivastigmine, which completely lacks antioxidant activity. The new hybrids were also able to reduce NO release in BV-2 cells, whereas rivastigmine showed no effect. Moreover, the most active compounds 99 and 100 increased the GSH level in the human neuroblastoma cell line SH-SY5Y.Studies on the murine microglia cell line (BV-2) showed that all synthesized ITC hybrids 48) as a desulfurizing agent. Sulfide derivatives of ITCs 28, 128–147, were prepared, in the presence of Et3N, from the corresponding ω-alkanamines 61, 108–127, carbon disulfide (35), and mesyl chloride (48) in 43–65% yields. The key intermediates, the appropriate ω-alkanamines 61, and 108–127, were obtained from ω-bromoalkylphthalimides 59 and 105–107 in a standard procedure. Oxidation of sulfides 28, and 128–147 with MCPBA, led to sulfoxide analogues 24 and 148–167, with very good yields , human lung adenocarcinoma (A549), woman breast cancer ER+ (MCF-7), human colon cancer cell line (HCT-116) and human neuroblastoma cell line (SH-SY5Y). All tested compounds exhibited more potent inhibitory against five cancer cell lines than SFN. The IC50 for the most active ITCs are presented in All synthesized ITCs 151 with the IC50 value 2.05 μM. ITC 161 had the strongest inhibition against A549 with IC50 5.64 μM, and ITC 155 possess significant cytotoxicity for MCF-7 with IC50 3.3 μM. Derivative 152 against HCT-116 had an IC50 value of 2.06 μM, and isothiocyanate 157 against SH-SY5Y ITC had an IC50 value 2.79. Based on the tested ITCs 28, 24, 148–167, it was found that compounds with sulfanyl moiety 135 showed weaker inhibitory effects than most derivatives; however, compound 168 with a sulfone group showed a higher inhibitory activity against all cancer cell lines than SFN. It may indicate that replacing the sulfoxide group with a sulfone group results in higher biological activity. The studies on anticancer mechanisms examined on the HepG2 cancer cell line showed that SFN, as well as model ITC 155, could induce the S or G2/M phase cycle arrest, and 155 has more potent inhibitory activity than SFN. Moreover, ITC 155 exhibited greater induction of apoptosis upon treatment than SFN. ITC 155 presented a time- and dose-dependent activation on the Nrf2 transcription factor. Moreover, 155 acted as a more potent Nrf2 inducer than SFN.For HepG2, the most active was ITC 24) and its analogue erucin (28) via the tandem Staudinger/aza-Wittig reaction. Thus, the starting 4-bromo-butanol (169) was transformed using a standard procedure into azido thioether 170. Then, this compound, in the reaction with triphenylphosphine and carbon disulfide, was converted into erucin (28) in an 81% yield. SFN (24) was obtained by oxidation of erucin (28) with MCPBA in 77% yield (Meijer and co-workers  synthesi7% yield .P. aeruginosa show that SFN and erucin strongly inhibit quorum sensing (QS) and virulence (biofilm formation and pyocyanin production). The assays on P. aeruginosa and in E. coli strongly indicate that SFN and erucin effectively bind LasR, resulting in inhibition of QS activation at concentrations that can be found in broccoli.Studies on R)-SFN ((R)-24) pyrimidines-2-amine from ribociclib as CDK9 pharmacophore, and analogues of SFN with alkyl chains of different lengths as targeting CSC pharmacophore. Briefly, the key N-Boc amines 462a–e and 463aa–de were converted into the final ITCs 464a–e and 465aa–de after prior deprotection of the amino group by trifluoroacetic acid, followed by the reaction with carbon disulfide and N,N’-dicyclohexylcarbodiimide   . The sam and 471 .464a–e, 465aa–de, 469, 470a–g, and 471 were evaluated regarding inhibition activity against CDK4, CDK6, and CDK9, with ribociclib as the positive control. Some compounds showed high potent activity against CDK9 as well as high selectivity for CDK9. The inhibition of tested ITCs was similar or higher than inhibition of ribociclib. In particular, ITC 470e with an 8-isothiocyanatooctanamide linker, exhibited high enzymatic inhibition (IC50 = 11 nM vs. IC50 of ribociclib = 197 nM). ITC 470e was tested against several tumor cell lines, including NSCLC, breast cancer, hepatocarcinoma, cervical cancer, leukemia, and lymphoma, using CCK8 assay and SFN as positive controls. ITC 470e had the highest activity against NSCLC cell lines, especial A549 and H1299, with IC50 values less than 0.5 μM. For comparison, IC50 of SFN on A549 was more than 10 μM and IC50 of ribociclib was 7.455 μM. For other cell lines, ITC 470e exhibited lower anti-viability activity. These results demonstrated good cellular selectivity of ITC 470e. Additionally, studies on A549 and H1299 showed that ITC 470e dose-dependently inhibited the cell cycle at G2/M phase and induced apoptosis of A549 cells in a concentration-dependent manner. Moreover, ITC 470e decreased the formation of colonies in the two NSCLC cell lines. In vivo studies on H1299 xenograft mouse models showed that ITC 470e displayed potent anti-tumor activities.All ITCs P. aeruginosa. Briefly, etherification of phenol 472, followed by amino group deprotection with TFA and a two-step reaction with carbon disulfide and molecular iodine, afforded ether-linked isothiocyanates 473 and 474 in low yields  with no effect on bacterial growth.Perez and co-workers  synthesiw yields . A LasR 2S donors with the isothiocyanate group. For this purpose, the starting N-Boc-glycoconjugates 475a–b were deprotected with TFA, followed by the reaction of amino derivatives 476a–b with an excess of carbon disulfide, triethylamine, and hydrogen peroxide to afford final, fully acetylated ITCs 478a–b, with low yields. On the other hand, deacetylation of O-acetylated amines 476a–b with sodium methanolate, followed by the isothiocyanation of amines 477a–b, performed in the same manner as above, gave isothiocyanate glycopyranosides 479a–b in 21% and 22% yields, respectively . Studies showed that glucose-derivative 479a was totally ineffective at inhibiting cell viability. However acetyl-galactosamine-derivative 478b exhibited a marked cytotoxicity against AsPC-1 tumor cells in a concentration-dependent manner with a pIC50 value of 4.45 ± 0.02. ITC 478b efficiently released H2S intracellularly with desirable slow kinetics and inhibition of cell cycle progression G0/G1 phase.ITCs 481a–c were obtained in good yields from amines 480a–c in a two-step reaction with carbon disulfide and triethylamine used as a base, followed by desulfurization with acyl chloride (He and co-workers  designedchloride .481a–c showed higher anti-tumor effects in vitro than dihydroartemisinin (DHA) against human glioblastoma U87 cells. The most active was ITC 481b (IC50 = 7.41 ± 1.56 μM) (IC50 DHA = 118.95 ± 12.39 μM). ITC 481b reduced the viability of glioblastoma multiforme (GBM) in a concentration-dependent manner. ITC 481b also inhibited migration and induced apoptosis in U87 cells. Pyknosis and nuclear shrinkages were observed in ITC 481b-treated cells. Additionally, caspase 9 and cytochrome c were induced in U87 cells treated with ITC 481b and cleaved caspase 3 was increased. On the other hand, the anti-apoptotic protein Bcl-2 was downregulated, and pro-apoptotic protein BAX was upregulated.All ITCs N-acetyl cysteine. Both isothiocyanate functional groups in diITCs were separated with unbranched alkyl linkers containing three and four carbon atoms (484 and 485). DiITCs 484 and 4825 were obtained from the corresponding diamines 482 and 483 in a two-step procedure with carbon disulfide and triethylamine as a base, followed by the reaction with HBTU as desulfurizing reagents  than Gram-negative . Studies on wide selections of Gram-negative as well as Gram-positive strains showed that the most active were diITCs 492 and 493, which, on Gram-positive strains, such as Streptococcus, Staphylococcus, and Bacillus, exhibited high activity (MIC = 1–2 μg/mL). DiITCs 492 and 493 presented high activity against mycobacteria. The most active were against M. bovis and M. tuberculosis (MIC = 2–4 μg/mL) than against nontuberculous mycobacteria. DiITC 492 efficiently inhibited the growth of the pathogens, such as Candida albicans or Candida glabrata, for which MIC < 1 μg/mL.The growth-inhibitory activity of synthesized diITCs The described above methods of the synthesis of SFN and its bifunctional analogs are presented in 2) is dominant. The next often used method is the tandem Staudinger/aza-Wittig reaction, and the least frequently used is the two-step method using a desulfurizing agent. Another group involves phosphorus analogues of SFN (P-SFN) with phosphonate, phosphinate, or phosphine oxide moiety with aliphatic or aromatic substituents. For this class of compounds, both the tandem Staudinger/aza-Wittig reaction and the method using a desulfurizing agent were equally frequently used. The reaction with CSCl2 for synthesis P-SFN was used less frequently. Carbonyl and amide analogs of SFN were synthesized by all three methods. The synthesis of ether-linked analogs of SFN was completely performed by the two-step methodology with a desulfurizing agent, while the synthesis of diisothiocyanates was also performed with the desulfurizing agent and thiophosgene. The tandem Staudinger/aza-Wittig reaction, to the synthesis of ether-linked analogs of SFN and diisothiocyanates, was not used.Based on the 75, 80d), heterocyclic (84d), or aliphatic  (152) substituents result in increases of the biological activities of analogs of SFN on different cancer cell lines, such as Malme-3M, MCF-7, or A549. For phosphorus analogs of SFN (P-SFN), the presence of phosphine oxide moiety with methyl substituents (191) does not affect biological activity. However, replacement of the methyl sulfinyl group by phosphonate moiety with aliphatic (298) or aromatic (407) substituents or phosphinate moiety (372) results in an increase in the anticancer activity on LoVo and LoVo/DX cancer cell lines. Carbonyl or amide analogs of SFN (419 and 455) can by characterized by biological activities similar to SFN, but on the other hand, for some analogs , biological activity could be higher than for SFN. The presence of the second isothiocyanato group (484) also results in an increase in the biological activity on LoVo and LoVo/DX cancer cell lines.Based on There are many examples describing the synthesis of SFN and its natural or synthetic difunctional analogues, with methyl sulfinyl group replaced by sulfinyl, sulfanyl, sulfonyl, phosphonate, phosphinate, phosphine oxide, carbonyl, ester, amide, ether, or a second isothiocyanate group. The most common strategies involve the use of thiophosgene as a thiocarbonyl transfer reagent, utilization of a carbon disulfide/desulfurizing agent system (dithiocarbamates approach), or use of the tandem Staudinger/aza-Wittig reaction (triphenylphosphine/carbon disulfide system). In the first two approaches, primary amines are utilized as substrates; the last comprises azides as starting materials. The choice of method depends on the availability of starting substrates and sensitivity of functional groups present in the substrates to the reaction conditions. Several ITCs are characterized by high biological activity , usually higher than parental SFN. Which modifications of the structure of SFN seem to have important impacts on the biological activities of the new analogues of SFN.As SFN is biologically active, natural ITC is found in cruciferous vegetables, and it is non-toxic—it has been selected for phase I and II clinical trials, where it is administered in the form of an extract or broccoli sprouts. The results of these studies are promising and indicate which SFN, in the future, may be considered as an anticancer drug. The situation is different for synthetic analogs of SFN, which have been described in this review, having sulfinyl, sulfanyl, sulfonyl, phosphonate, phosphinate, phosphine oxide, carbonyl, ester, amide, ether, or a second isothiocyanate group. The vast majority of these compounds have only been tested for anticancer or antibacterial activity in vitro, and a few analogs, additionally in vivo, on mice or Zebrafish. None of the synthetic analogs of SFN described in this review qualified for clinical trials. This is likely due to the toxicity of these compounds in higher doses, often seen in in vitro or in vivo studies. SFN, which is a natural product, does not show toxicity, even in higher doses. Another reason is the financial constraints of many research teams.One interesting solution would be to synthesize a conjugate—a combination of SFN and a drug or peptide, with anticancer properties, characterized by no (or low) toxicity. This conjugate could separate, in cancer cells, on two drugs, causing increased anticancer activity.


Mature T-cell lymphomas (MTCLs) represent a heterogeneous group of aggressive non-Hodgkin lymphomas comprising different entities. Anthracycline-based regimens are considered the standard of care in the front-line treatment. However, responses to these approaches have been neither adequate nor durable, and new treatment strategies are urgently needed to improve survival. Genomic instability is a common feature of cancer cells and can be caused by aberrations in the DNA damage response (DDR) and DNA repair mechanisms. Consistently, molecules involved in DDR are being targeted to successfully sensitize cancer cells to chemotherapy. Recent studies showed that some hematological malignancies display constitutive DNA damage and intrinsic DDR activation, but these features have not been investigated yet in MTCLs. In this study, we employed a panel of malignant T cell lines, and we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Moreover, we report the efficacy of targeting the apical kinase ATM using the inhibitor AZD0156, in combination with standard chemotherapy to promote apoptotic cell death. These findings suggest that DDR is an attractive pathway to be pharmacologically targeted when developing novel therapies and improving MTCL patients’ outcomes. Nodal T-cell lymphomas (TCL) are a rare and aggressive subgroup of lymphoid malignancies, accounting for 10–15% of non-Hodgkin lymphomas (NHL). According to the latest World Health Organization (WHO) classification, they comprise several entities, such as angioimmunoblastic T-cell lymphoma (AITL), anaplastic large T-cell lymphoma (ALCL), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), which is the most common subtype including all the TCL cases that still fail to be categorized . AnthracIn recent years, several efforts have been made by us and others to elucidate the genetic features of TCLs, reporting a broad range of copy number alterations (CNAs) and structural variations (SVs) rather than point mutations ,7,8, thuHere, we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Additionally, we provide evidence that targeting the apical kinase ATM in combination with CHOEP is effective in promoting apoptotic cell death.We first explored whether T-cell lymphomas as well as solid tumors and some hematological malignancies are characterized by intrinsic DNA damage, a feature of genomic instability. The presence of γH2AX and 53BP1 foci, well-recognized markers of DNA damage ,27, was We then assessed whether the presence of intrinsic DNA damage was associated with constitutive activation of DDR signaling pathways. To this aim, we studied the expression of activating post-translational modifications of several DDR players including ATM, Chk2, Chk1, and KAP1 before and after 3 h treatment with increasing concentrations of CHOEP and with 20 µM etoposide alone, which is well known to activate DDR in human cells ,32. As sTaken together, these data indicate that in vitro models of T-cell lymphoma show intrinsic DNA damage and constitutive activation of the DDR signaling pathways, despite some degree of heterogeneity observed in the analyzed cell lines.As we have shown that the DDR signaling cascade is basally activated and gets further triggered in response to chemotherapy, we hypothesized that targeting the DDR could block the possible attempts made by the cell exposed to CHOEP to repair the induced DNA lesions, thus leading to augmented apoptosis. For this purpose, we took advantage of a recently developed inhibitor, AZD0156, that targets the apical kinase ATM  and is b50 range: 0.55–2.3 µM) (20 (50 (not shown) doses of AZD0156 with no significant changes detected among untreated and AZD0156-treated cells.First, we exposed cell lines to increasing concentrations of AZD0156 (range 1 nM–500 µM) for 48 h and observed a dose-dependent reduction in viable cells in all cell lines, even though sensitivity to AZD0156 was heterogeneous A. The on–2.3 µM) . The dec–2.3 µM) B. This w µM) (20  and IC5020 CHOEP (defined in our previously reported study [20 AZD0156 (p < 0.05)  A. Consis < 0.05) B. Import < 0.05) C.ATM and/or TP53, we performed targeted sequencing analysis. As expected, all cell lines bear pathogenic TP53 alterations but no ATM mutations. ATM is mutated only in JURKAT cell line, but we were unable to define a clear inactivating effect of the detected alterations  (p: ns)  A, with a 0.0001) B. As exp (p: ns) . The oth (p: ns) A.To conclude, we confirmed the efficacy of ATM inhibition in potentiating CHOEP effects using another ATM inhibitor, KU-55933, widely used in in vitro studies.We tried to titrate KU-55933 in our cell lines, but in a range of concentrations between 16 nM and 16 μM, we observed a reduction in cell viability of 10–20%. Cell viability was significantly reduced when we reached higher concentration (100 μM–200 μM) (data not shown). As KU-55933 has been administered to cell lines at 10 μM to inhibit ATM kinase activity in other studies ,36, we d20 CHOEP, and we observed a significant reduction in cell proliferation J recombination, which expose cells to high levels of DNA damage. For this, in recent years, several inhibitors of DDR proteins—including ATR, DNA-PK, PARP, Chk1, and WEE1, administered alone and in combination with other agents—have been preclinically tested . Consistn status .In the present study, we report encouraging preclinical data describing the benefits of ATM inhibition in promoting CHOEP-induced cell death in preclinical models of T-cell lymphoma using two different chemical compounds—AZD0156 and KU-55933. Despite the presence of basal DDR activation and intrinsic DNA damage observed in all the cell lines included in this study, their response to AZD0156 and AZD0156–CHOEP treatment was heterogeneous, further confirming the heterogeneity of the pathology.Interestingly, we observed a reduction in γH2AX foci upon combined treatment with AZD0156 and CHOEP because this serine is directly phosphorylated by ATM. Nonetheless, 53BP1 foci formation was not impaired by AZD0156 addition to CHOEP in our models. Thus, the decrease in γH2AX foci is not suggestive of the absence of DNA damage, but rather of dysfunctional DDR signaling caused by ATM inhibition. Consistently, DNA damage is not efficiently repaired, leading to apoptotic cell death, as in The AZD0156 inhibitor is currently clinically tested in advanced solid tumors alone or in combination with olaparib and irinotecan. Moreover, recent preclinical studies reported AZD0156 capability to enhance the genotoxic effects of olaparib and radiation in preclinical models of different solid tumors ,25. In aGiven alone, AZD0156 does not induce cell cycle perturbations in malignant T cells, as observed in solid tumors ,25. NoneIn recent years, first-line regimens built on a CHOP-like backbone have been studied, but none, including the one combining the histone deacetylase (HDAC) inhibitor romidepsin with CHOP (Ro-CHOP), significantly improved the survival of patients affected by PTCL , thus suTP53 mutations, suggesting that AZ0156 is effective independent of TP53 mutational status. On the contrary, we did not observe ATM putative inactivating mutations. Nonetheless, consistent with the fact that mutations have been reported in the ATM gene in TCL patients [ATM.The in vitro models of T-cell lymphoma employed in this study are characterized by pathogenic patients ,42, addiNotably, our in vitro data suggest that suboptimal concentrations of the ATM inhibitor are not detrimental for healthy T lymphocytes. Nonetheless, we cannot rule out the possibility that the combined treatment with AZD0156 and CHOEP could cause adverse effects in humans. Preliminary data from the phase I clinical trial aimed at assessing efficacy and tolerability of AZD0156 in combination with olaparib reported minor toxicities in about 40% of patients enrolled in the study. However, hematologic toxicities were observed when higher doses of both drugs were used . Thus, pKARPAS-299 (ALCL), SUP-T1 (lymphoblastic TCL), HH (cutaneous TCL), HD-MAR-2 (T-cell leukemia), and JURKAT (T-cell leukemia) were purchased from DSMZ–Leibniz Institute . OCI-Ly12 cell line (PTCL-NOS) was a kind gift from Dr. Leandro Cerchietti  . Cells w50) were determined upon 48 h of chronic exposure, as the concentration of drug able to reduce 50% of cell growth.All drugs  were purchased from Selleck Chemicals . CHOEP was prepared as described in [Upon treatment, cells were transferred on glass slides using a cytospin centrifuge (5 min at 500 rpm). Glass slides were dried overnight at room temperature, then fixed with 2% paraformaldehyde and permeabilized with PBS 0.2% Triton X-100. Saturation was performed in PBS 5% BSA 0.2% TWEEN 20. Primary antibodies used were γH2AX #A300-081A  and 53BP1 #NB100-305 ; secondary conjugated antibody used was Alexa Fluor 488 #A11034 . At the end, nuclei were counterstained with DAPI . Images were acquired with a Nikon Eclipse E1000 fluorescence microscope equipped with a DSU3 CCD camera, using a 100× magnification objective, as previously described . In eachCell viability, cell death, mitochondrial membrane potential, and cell cycle were studied by flow cytometry as previously described . BrieflyCell lysates, SDS-PAGE and Western blot were performed as described in . AntibodGenomic DNA was extracted using the Nucleospin Tissue kit  and quantified using Qubit 2.0 and the Qubit DNA HS Assay kit . DNA was run on the Oncomine Comprehensive Assay (OCA) Plus targeted panel of 1.7 Mb  with an AmpliSeq-based enrichment. All library preparation was performed manually according to manufacturer’s instructions (MAN0018490). Multiplex PCR amplification was conducted using 20 ng of DNA as input. Purified libraries were quantified with real-time PCR with the Ion Library TaqMan™ Quantitation Kit . The 50 pM libraries were pooled and loaded onto Ion 550™ Chips  according to manufacturer’s instructions (MAN0017275) and prepared for sequencing using the Ion Chef™ System . Sequencing was performed using the Ion Gene Studio S5 Sequencer .https://opencravat.org (accessed on 14 February 2022)) and the Ion Reporter™ Software . Variants were classified using ClinVar (available online: https://www.ncbi.nlm.nih.gov (accessed on 14 February 2022)), cBioPortal ), and dbSNP databases. Variants categorized as neutral/benign and variants with a frequency >0.00001 in the population (not pathogenic) were considered as single nucleotide polymorphisms (SNPs) and were thus excluded.For the analysis, data were initially processed using Ion Torrent Suite Software™ , and variant calling was performed using the Variant Caller plugin. Variants were filtered for coverage greater than 40 reads, frequency greater than 5%, quality value greater than 30, and coverage depth greater than 500X. Resulting variants were annotated using the OpenCravat tool  of independent experiments. GraphPad Prism 9 software  was used to perform graphs and statistical analyses. Specifically, one-way ANOVA test was used to calculate significance of cell viability, cell death, and TMRE assays. Tukey post hoc test was applied. Two-way ANOVA followed by Bonferroni posttest was used to calculate the significance of cell cycle experiments. For γH2AX and 53BP1 foci enumeration,


WT) and mutant type  amyotrophic lateral sclerosis (ALS) models. The uptake of [3H]L-carnitine was dependent on time, temperature, concentration, sodium, pH, and energy in both cell lines. The Michaelis–Menten constant (Km) value as well as maximum transport velocity (Vmax) indicated that the MT cell lines showed the higher affinity and lower capacity transport system, compared to that of the WT cell lines. Additionally, LC uptake was inhibited by organic cationic compounds but unaffected by organic anions. OCTN1/slc22a4 and OCTN2/slc22a5 siRNA transfection study revealed both transporters are involved in LC transport in NSC-34 cell lines. Additionally, slc22a4 and slc22a5 was significantly decreased in mouse MT models compared with that in ALS WT littermate models in the immune-reactivity study. [3H]L-Carnitine uptake and mRNA expression pattern showed the pretreatment of LC and acetyl L-carnitine (ALC) attenuated glutamate induced neurotoxicity in NSC-34 cell lines. These findings indicate that LC and ALC supplementation can prevent the neurotoxicity and neuro-inflammation induced by glutamate in motor neurons.L-Carnitine (LC) is essential for transporting fatty acids to the mitochondria for β-oxidation. This study was performed to examine the alteration of the LC transport system in wild type (WT, NSC-34/hSOD1 Amyotrophic lateral sclerosis (ALS), which is a devastating neurodegenerative disease, causes motor neuronal loss in the brain stem and spinal cord, resulting in paralysis, muscle wasting and respiratory failure, which ultimately leads to death. The demand for extending the quality of life in ALS patients is increasing, mainly due to the lack of an effective cure for this disease ,2. FamilCarnitine plays a major role in the transportation of long chain fatty acids to the mitochondria for β-oxidation, a process which generates ATP. Under physiological conditions, oxidization of fatty acids primarily involves mitochondrial β-oxidation, which is an important energy production mechanism found in mammals ,9. The mOrganic cationic/carnitine transporters (OCTN) are unique zwitter-ionic transporters expressed in mice. These are categorized under the solute carrier family (SLC) and its members 4, 5 and 21 are represented by OCTN1/Slc22a4, OCTN2/Slc22a5, and OCTN3/Slc22a21 ,13. AppaG93A mutation were used to examine the neuroprotective effects of LC and ALC. In this study, we hypothesized that carnitine and ALC pretreatment may provide neuroprotective effects against glutamate-induced excitotoxicity in ALS model cell lines. Similarly, we investigated the effects of altering carnitine transportation, as well as carnitine pretreatment, on [3H]L-carnitine uptake in motor neurons, such as NSC-34 cell lines.Previous studies have demonstrated that concentrations of various compounds, such as amino acids and monocarboxylates, are altered in the mutant ALS model. Therefore, investigating the transportation and supplementation of LC is important for acquiring a better understanding of mammalian mitochondrial energy metabolism and the prevention of neurodegenerative diseases, such as ALS ,16. Howe3H] (77.0 mCi/mmol) was purchased from Perkin-Elmer . All chemicals and reagents were high-grade commercial products purchased from Sigma-Aldrich .L-Carnitine hydrochloride L-Carnitine uptake in NSC-34 cells was studied based on a previously described method [3H]L-carnitine (32.5 nM) labeled compound was then added in the presence or absence of inhibitors for a designated time period at 37 °C -L-carnitine at 15 min and the concentration of unlabeled carnitine, respectively.For kinetic studies , the Mici) was calculated using Equation (3).For the competitive inhibition study ,20, low-® 2000 (Invitrogen). After the designated time interval, mRNA expression level and [3H]L-carnitine uptake in siRNA-transfected NSC-34 cell lines were evaluated  in a blocking solution at 4 °C for 24 h. After washing three times with PBS, the tissue slides were processed with Vector ABC Kit . The Slc22a4 and Slc22a5 signals were developed with DAB chromogen . Images were then analyzed via using an Olympus microscope system . The semi-quantification of immunoreactivity was analyzed by NIH ImageJ. Especially, the intensity of Slc22a4 and Slc22a5 signals was counted from 5~10 motor neurons per section.3H]L-carnitine uptake, and slc22a4 mRNA expression level were examined  assay was performed, following which [examined .Statistical analyses were conducted via the unpaired 2-tailed Student’s t-test or one-way ANOVA followed by Dunnett’s post-hoc test. Sigma Plot  was used to create figures.3H]L-carnitine transport system on WT and MT NSC-34 cell lines. The uptake of [3H]L-carnitine was time-and temperature-dependent until 60 min in both NSC-34 WT and MT cell lines (3H]L-carnitine transporter in NSC-34 cell lines (m1) and the maximum uptake rate (Vmax1) were 1.94 ± 0.34 µM and 0.295 ± 0.059 pmol/mg protein/min, respectively, for the WT cell line  values for WT and MT cell lines, were 0.149 (µL/(min/mg protein)) and 0.168 (µL/(min/mg protein)), respectively , and rotenone was used to deplete ATP , γ-butyrobetaine (GBB), acetyl L-carnitine (ALC) and tetraethyl ammonium (TEA) (To examine the transporter(s) responsible for transporting carnitine into NSC-34 cell lines (ALS model cell lines), cellular uptake of [um (TEA) . Similarum (TEA) .3H]L-carnitine uptake intersected at the ordinate axes, showing competitive inhibition in the presence or absence of metformin, DPH, quinidine and pyrilamine in both WT , mOCTN2 (slc22a5) and control siRNA (negative control) then using the transfected cells, the mRNA expression levels and L-carnitine uptake was evaluated . mOCTN1 n = 7) compared to normal subjects (n = 7) (n = 7) compared to normal subjects (n = 7), indicating the altered expression of carnitine transporters in human ALS patients ] mice. Interestingly, the immunoreactivity of Slc22a4 and Slc22a5 carnitine transporters were decreased in the spinal cord gray matter and motor neurons of SOD1(G93A) mice compared to the WT littermates A,B. Simi (n = 7) a. SLC22Apatients b. These patients .An MTT assay was performed on the ALS model of WT and MT cell lines following glutamate pretreatment to examine glutamate-induced cytotoxicity as well as the neuroprotective effect of LC and ALC under glutamate-induced cytotoxicity. Glutamate pre-incubation induced a significant decrease in cell viability compared to the control, but LC and ALC ameliorated glutamate-induced neuronal cell death in both NSC-34 cell lines A,B.3H]L-carnitine uptake and slc22a4 mRNA expression levels in both WT and MT cell lines. Pre-incubation with glutamate significantly increased the [3H]L-carnitine uptake rate, which was recovered of control level or decreased by pretreatment of LC and ALC in both NSC-34 cell lines (Similarly, the transport effects of LC and ALC pretreatment against glutamate excitotoxicity were examined by evaluating L-Carnitine uptake in NSC-34 cell lines was shown to be time-, temperature-, concentration-, ion-, energy-, and pH-dependent. The characteristics of its transport system were similar to those described in previous studies [3 and rotenone act by interfering with the electron transport chain in mitochondria and inhibition of the ATP synthase/production [3H]L-carnitine uptake was strongly inhibited in the presence of organic cationic compounds, including inhibitors as well as the substrates of the OCTN1 and 2 transporters, such as PLC, GBB, ALC, quinidine, verapamil, metformin, pyrilamine, TEA, clonidine, and DPH, in both NSC-34 cell lines [[ studies ,26,27. L studies  (Figure  studies ,17. As moduction ,26. Kineoduction . In addioduction . Furtherll lines ,32,33,34ll lines ,30,35. Nll lines .Amino acids, neurotransmitters and monocarboxylic acids as well as many other low-molecular-weight polar compounds can be transported to various cell lines using multiple transport systems that enable these substances to cross plasma membranes ,36. Simi+ and Cl−, which destabilizes intracellular Ca2+ homeostasis, thereby disrupting neuronal energy status and causing excitotoxic neuronal death, which constitutes the major root of neurodegenerative diseases, such as ALS [G93A mutant models [On the other hand, glutamate is an important neurotransmitter, its pretreatment overstimulates its receptors, leading to the activation of glutamate receptors and an influx of Nah as ALS . Additioh as ALS ,39. Howeh as ALS ,39. In oh as ALS ,41. As sh as ALS ,11,42. St models . These oOur results suggest that the energy-metabolizing compound carnitine can be transported to motor-neuron like cell lines using OCTN1 and OCTN2 transporters. LC transporter systems in the NSC-34 cell lines of mouse MT ALS models were altered compared to those of the WT control. Thus, LC and ALC show potential as good candidates for the prevention of neurodegenerative diseases, including ALS caused by the glutamate-induced motor neuronal cell death.


CCR1, CCR2, CCR3, and CCR5, the EBV DNA load and expression of EBV latent genes in nine EBV-carrying and four EBV-negative BL cell lines. We revealed that CCR1 is expressed at high mRNA and protein levels in two CD10-negative BL cell lines with co-expression of the EBV latent genes EBNA2, LMP1, and LMP2. Low levels of CCR2 transcripts were found in three BL cell lines. CCR3 and CCR5 transcripts were hardly detectable. Our data suggest that in vivo, CCR1 may be involved in the dissemination of BL cells and in the selection of BL cells with restricted EBV gene expression programs.Chemokines and their receptors regulate the migration of immune cells and the dissemination of cancer cells. CCR1, CCR2, CCR3, and CCR5 all belong to a single protein homology cluster and respond to the same inflammatory chemokines. We previously reported that CCR1 and CCR2B are induced upon Epstein-Barr virus (EBV) infection of B cells in vitro. EBV is present in almost all cases of endemic Burkitt lymphoma (BL); however, the contribution of EBV in the pathogenesis of the disease is not fully understood. Here, we analyzed the relation of the expression of  The chemokine-receptor network controls immune responses and directs the migration of immune cells. Chemokines transmit signals via chemokine receptors, which are G protein-coupled, cell-surface receptors. The C-C chemokine receptors, namely CCR1, CCR2, CCR3, and CCR5, belong to the same protein sequence homology cluster. They share responsiveness to multiple inflammatory chemokines. Inflammatory chemokines and their receptors are induced during inflammation and under many pathophysiological conditions. However, the roles of the chemokine receptors CCR1, CCR2, CCR3, and CCR5 in B lymphocytes remain unknown (reviewed in [CCR1 and CCR2B (an isoform of CCR2) mRNA and protein expression levels are upregulated in peripheral blood (PB) B cells upon EBV infection in vitro and in established lymphoblastoid cell lines (LCLs) [CCR1 or CCR2 nor their proteins were detected in the EBV-negative and EBV-carrying BL cell lines with latency I. Enhanced transcription of CCR2, CCR7, and CCR9 was previously detected in tonsillar B cells upon EBV infection in vitro [CCR2 starting from day 4 and after [In our previous work, we showed that s (LCLs) . We alsoin vitro . A recennd after .c-MYC oncogene is a major growth-promoting event. Endemic BL classically presents as a monoclonal cell tumor, in which every cell carries the c-MYC translocation and EBV. Most EBV-carrying BL tumors express only one EBV protein, EBNA1, from the alternative promoter Qp (a latency I type program) [EBV, a human B lymphotropic γ-1 herpesvirus, is associated with a number of human B cell lymphoproliferative disorders (LPDs), including BL, posttransplant lymphoproliferative disease (PTLD), lymphomas in immunodeficient patients, and diffuse large B cell lymphomas (DLBCLs). In actively proliferating PTLD and LCLs generated by a viral infection of resting B cells in vitro, EBV expresses the full set of latency type III (latency III) proteins: six nuclear antigens  and latent membrane proteins  . EBprogram) ,10,11 (rprogram) ,7,8). Deprogram)  and geneprogram)  indicateprogram) ,17,18,19program) ,20.CCR1, CCR2, CCR3, and CCR5 in BL cell lines with different EBV latency programs aiming to find a putative link between the expression of particular EBV latent genes and the expression levels of the chemokine receptors.Up to now, not much is known about the impact of EBV on eBL disease progression. To answer the question of whether the inflammatory chemokine receptors could be implicated in the selection of the EBV-infected BL cells in vivo, we analyzed the expression pattern of the known inflammatory chemokine receptors CCR1, CCR2, CCR3, and CCR5 in 13 BL cell lines, four EBV-negative cell lines , and in nine EBV-carrying cell lines. The EBV-carrying BL cell lines included three type I cell lines with latency I , four type II cell lines , and two type III cell lines (Mutu cl.99 and BL18). The Burkitt lymphoma cell lines used in the present study and references to the reported characteristics are shown in We analyzed the mRNA expression levels of the inflammatory chemokine receptors In the type II BL cell lines in our study, we did not detect LMP2A or LMP2B transcripts D. HoweveCCR2 mRNA expression levels we detected in three BL cell lines, with the following values normalized to TBP mRNA expression: 0.022 ± 0.001 in BL18; 0.040 ± 0.006 in Mutu cl.99; and 0.066 ± 0.002 in Jijoye P79. By comparison, in LCL-2mon cells, the value was 0.111 ± 0.031 (CCR2 (isoform CCR2B) was expressed and that the receptor was functional in LCLs and in three endemic BL cell lines , in which the EBV-encoded latent protein EBNA2 is abundantly expressed [Low  ± 0.031 A. We alsxpressed   or were hardly detectable in LCLs as in the type III and type II BL cell lines. The levels of the CCR5 normalized mRNA expression were as follows in the respective cell lines: 0.00082 ± 0.00003 in LCL-2mon; 0.00170 ± 0.00070 in LCL-1year; 0.00047 ± 0.00007 in BL18; 0.00161 ± 0.00001 in Mutu cl.99; 0.00228 ± 0.00054 in Jijoye P79; and 0.00006 ± 0.00000 in Akuba.In most BL cell lines in this study, CCR1 mRNA expression, with normalized expression values of 0.40 ± 0.07 and 0.46 ± 0.05, were detected in two CD10-negative type III BL cell lines, BL18 and Mutu cl.99 cells, respectively, in which the EBV latent genes EBNA2, LMP1, and LMP2A/2B were also expressed  [CCR1 mRNA expression levels and quantification of CCR1 transcript copy number were carried out using RT real-time PCR and primers targeting exons 1 and 2. The presence of the CCR1 ORF transcripts in the type III BL cell lines was confirmed using RT-duplex PCR  . Determiplex PCR G.Immunofluorescent staining definitively detected CCR1 protein in only two BL cell lines, Mutu cl.99 and BL18 cells, which is consistent with mRNA expression data E. The meExpression of the CCR1 protein (CD191) on the cell surface and percentages (%) of CD191-positive (CD191+) and CD10-positive (CD10+) cells were determined using flow cytometry; annexin V-stained (apoptotic) cells were excluded from the analysis. The percentages of CD191+ cells were assessed after recycling of the receptor to the cell surface, as a functional response to serum deprivation, after the cells were cultured in serum-free medium for 3 h . Serum dHowever, in Akuba cell line (the early passage type II BL cell line), only a small fraction of cells (~8.4%) carried CCR1. Cell surface expression of CCR1 was not detected in the CD10-positive and the EBV-negative , EBV-positive type I , or EBV-positive type II  BL cell lines . We did CCR1 or CCR2 . Howeiewed in ,19 and biewed in ,17. A priewed in . The autiewed in . Subsequpression .The majority of BL cell lines generated from tumors with EBV latency I maintained their original cell phenotype and latency I. However, some of the BL cell lines, upon the establishing, after 20–50 passages in culture, shifted to a lymphoblastoid phenotype and expressed EBV latency III proteins  ,11,20. FCCR1, CCR2, CCR3, and CCR5, in 13 BL cell lines with different expression patterns of the EBV latent genes EBNA2, LMP1, LMP2A, and LMP2B in comparison with LCLs. While only trace amounts of the CCR3 and CCR5 transcripts were found, and low levels of CCR2 mRNA expression were detected in type III BL cell lines and LCLs, CCR1 mRNA expression was high in all three LCLs and in all two type III BL cell lines. Neither CCR1 mRNA nor the CCR1 protein was detected in the EBV-negative and the latency I BL cell lines with the GC phenotype.In our previous work, we demonstrated that infection of PB B cells with EBV upregulated two inflammatory chemokine receptors, namely CCR1 and CCR2, and the expression of these chemokine receptors persisted in established LCLs . In the CCR1 and CCR2 mRNA expression levels as the transcript copy number per 1000 copies of the housekeeping gene TBP transcript (relative copy number). While the CCR1-transcript relative copy number in LCLs and two type III BL cell lines ranged from 969 in LCL-1year to 2670 in LCL-1mon and from 1080 in Mutu cl.99 to 1130 in BL18, the CCR2 mRNA expression levels, as in LCLs and in BL cell lines, were low compared to the CCR1. Specifically, we detected the CCR2 relative copy numbers of only 24, 50, and 26 in LCL-1year, LCL-2mon, and LCL-1mon, respectively, and of 10 in Mutu cl.99, and 23 in Jijoye P79 . ConseqConcluding, we demonstrated that only one CCR1 out of four known chemokine receptors in the family of inflammatory chemokine receptors was highly upregulated in the EBV-carrying LCLs and BL cell lines with co-expression of the EBV latent genes EBNA1, EBNA2, LMP1, and LMP2A/2B. In the CD10-positive BL cell lines, EBV-negative and EBV-positive with latency I program, none of the inflammatory chemokine receptors was expressed, which suggests that in vivo CD10-positive BL cells escape chemokine ligand-expressing immune cells, such as macrophages, T- and NK-lymphocytes. Our results may help to reveal mechanisms of endemic BL lymphomagenesis, contributing to understanding the selection of EBV-restricted latency BL-cell subsets within a tumor mass. We also speculate that in vivo endemic BL cells expressing CCR1 are prone to migrate toward ligand-expressing immune cells and ligand-rich blood organs, and therefore, in an immunocompromised state, dissemination of malignant cells may contribute to the pathogenesis of this cancer.The BL cell lines analyzed in this study along with the references to their origin, expression of EBV latent genes, and their reported characteristics are listed in CCR1, CCR2, CCR3, and CCR5 genes and the EBV genes, EBNA2, LMP1, LMP2A, LMP2B, and BZLF1, were assessed using real-time RT-PCR with the PerfeCTa SYBR Green FastMix  and the CFX96 Touch Real-Time PCR detection system . The mRNA expression levels of the genes of interest were normalized to the mRNA expression levels of the housekeeping gene TBP (encoding TATA box-binding protein). The presence of the CCR1 open reading frame (ORF) transcript was determined using RT-duplex PCR performed with two pairs of primers flanking the CCR1 ORF and matching the housekeeping gene GAPDH . We previously described the real-time RT-PCR procedure, the RT-duplex PCR protocol, and the corresponding primers in prior publications [The mRNA expression levels of the ications . The EBVications ; the EBVications ; and theications .CCR1 and TBP transcripts were determined by quantitative real-time RT-PCR. For copy number quantification, we applied standard curves based on serial dilutions of the plasmids with the inserted CCR1 or TBP transcripts derived from the established LCL-2mon reference line. The transcripts were cloned into the pCR-TOPO vector  and sequenced for verification. The EBV DNA copy number per cell for each cell line was quantified using the EBV Real-TM Quant kit .The copy numbers of the https://www.ncbi.nlm.nih.gov/igblast/, accessed on 1 January 2021).The analysis of the immunoglobulin heavy-chain variable region (IGHV) gene mutations was carried out according to the described protocol  using siWestern blot analyses were performed, using whole-cell lysates, with a mouse monoclonal anti-EBNA2 antibody  and a mouse monoclonal anti-LMP1 antibody . CCR1 immunostaining was performed using goat polyclonal anti-human CCR1 antibodies  and donkey anti-goat IgG, F(ab’)- fragment antibody conjugated with FITC . DNA in the cells, fixed with a mixture of methanol and acetone (1:1), was stained using Hoechst 33258 .For the three-color flow cytometry analysis, cells were stained with a mouse monoclonal anti-CD191 antibody  targeting the cell surface CCR1 or with a mouse monoclonal anti-CD192 antibody  targeting the cell surface CCR2 and a mouse monoclonal anti-CD10 , according to a standard protocol [


Breast cancer is a multifactor disease, and many drug combination therapies are applied for its treatment. Selenium derivatives represent a promising potential anti-breast cancer treatment. This study reports the cytotoxic activity of forty-one amides and phosphoramidates containing selenium against five cancer cell lines  and two nonmalignant cell lines (184B5 and BEAS-2B). MCF-7 cells were the most sensitive and the selenoamides I.1f and I.2f and the selenium phosphoramidate II.2d, with GI50 values ranging from 0.08 to 0.93 µM, were chosen for further studies. Additionally, radical scavenging activity for all the compounds was determined using DPPH and ABTS colorimetric assays. Phosphoramidates turned out to be inactive as radical scavengers. No correlation was observed for the antioxidant activity and the cytotoxic effect, except for compounds I.1e and I.2f, which showed dual antioxidant and antitumor activity. The type of programmed cell death and cell cycle arrest were determined, and the results provided evidence that I.1f and I.2f induced cell death via autophagy, while the derivative II.2d provoked apoptosis. In addition, Western blot analysis corroborated these mechanisms with an increase in Beclin1 and LC3-IIB and reduced SQSTM1/p62 levels for I.1f and I.2f, as well as an increase in BAX, p21 and p53 accompanied by a decrease in BCL-2 levels for derivative II.2d.  Cancer is considered a serious public health burden because it affects millions of people worldwide. Cancer is the second lethal disease globally, and about one out of six deaths are due to cancer . WorldwiIn this context, researchers worldwide have focused their attention on the synthesis of selenium-containing compounds due to their promising results against different types of cancer . SeveralN-acetylcysteine [In spite of the controversy about the use of antioxidants for cancer treatment, different clinical trials have demonstrated their efficacy . If we fcysteine , garlic cysteine  or flavocysteine  have beecysteine ,18. In tAccumulating evidence in the literature has illustrated that among the different selenated scaffolds, selenenocyanate ,20 and dIn this study, we provide an evaluation of the synthesized selenium derivatives as cytotoxic and antioxidant agents. The antiproliferative effect was tested in vitro using five human cancer cell lines as well as two nonmalignant cell lines. The antioxidant activity was determined by DPPH and ABTS assays. To better understand the potential mechanisms of action, the most active and selective compounds were further evaluated for their effect on the cell cycle distribution, cell death induction and alteration of different proteins related to autophagy or apoptosis processes.Synthesis, purification and characterization of the compounds have been previously described . Chemica2.Cell lines were provided by the European Collection of Cell Cultures (ECACC) or by the American Type Culture Collection (ATCC). Seven cell lines were used: MCF-7 (breast adenocarnicoma), 184B5 , CCRF-CEM (lymphoblastic leukemia), HT-29 (colon carcinoma), HTB-54 (lung carcinoma), BEAS-2B  and PC-3 (prostate carcinoma). MCF-7, CCRF-CEM, HT-29, HTB-54, BEAS-2B and PC-3 cell lines were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum , 100 units/mL penicillin and 100 mg/mL streptomycin (Gibco). 184B5 cells were grown in DMEM/F12 medium supplemented with 5% FBS, 1 × ITS (Lonza), 100 nM hydrocortisone (Aldich), 2 mM sodium pyruvate (Lonza), 20 ng/mL EGF (Sigma-Aldrich), 0.3 nM trans-retinoic acid (Sigma-Aldrich), 100 units/mL penicillin and 100 mg/mL streptomycin. Cells were maintained at 37 °C and 5% COThe cytotoxic effect of each substance was tested by the MTT method. Each compound was initially dissolved in DMSO at a concentration of 0.01 M, and serial dilutions were prepared with nonsupplemented medium. The cytotoxic effect of each compound was tested at 50 and 10 µM as a first screening. Compounds with a cell growth percentage under 50% at 10 µM in at least one cell line were selected and tested at five different concentrations ranging between 0.01 and 100 µM.4 cells/well in 96-well plates were treated with increasing concentrations of the corresponding compounds for 48 h at 37 °C in a humidified atmosphere containing 5% CO2. Then, cells were incubated with 50 µL of MTT (2 mg/mL stock) for 4 h. The medium was then removed by aspiration, and the formazan crystals were dissolved in 150 mL of DMSO. Results are expressed as GI50, the concentration that reduces by 50% the growth of treated cells with respect to untreated controls (0.1% DMSO); TGI, the concentration that completely inhibits cell growth; and LC50, the concentration that kills 50% of the cells. Data were obtained from at least 3 independent experiments performed in quadruplicate. The standard error of the means (SEM) for the cytotoxic parameters was calculated applying the standard deviation formula to the mean values of each parameter for the three independent experiments performed.A total of 1 × 102 flasks  treated with DMSO (negative control), 6 µM of camptothecin (positive control) and different concentrations of I.1f, I.2f and II.2d.For the MCF-7 cell line, both cell cycle analysis and the cell death percentage were determined using the Apo-Direct kit (BD Pharmingen) based on the TUNEL technique, according to the manufacturer’s instructions. Cells were seeded in 25 cmAfter treatment, cells were collected and fixed with 1% paraformaldehyde (Sigma) in PBS (pH = 7.4), incubated in ice for 40 min, collected by centrifugation, washed with PBS and incubated with 70% ethanol for 30 min at −20 °C. After fixation, cells were washed twice with PBS and incubated for 1 h at 37 °C with FITC dUTP-DNA Labeling Solution. Cells were then rinsed and incubated in the dark for 30 min at room temperature with PI/RNase staining buffer before being analyzed by flow cytometry . Then, cells were treated with compounds I.1f (80 µM), I.2f (30 µM) and II.2d (5 µM) at different times (from 8 to 72 h) following the same methodology.For autophagy and caspase inhibition assays, cells were pretreated with 100 nM of autophagy inhibitor  and 50 µM of pan-caspase inhibitor  for 1 h. The cells were treated with the compounds I.2f (30 µM) and II.2d (20 µM) for 24 h and I.1f (80 µM) for 72 h. Samples were processed following the same methodology stated above. DMSO was used as a negative control for both inhibitors.g for 15 min at 4 °C to remove cell debris. The nonprotein fraction and supernatants were stored at −80 °C before use. Protein concentration was determined with the BCA Protein Assay. Cell samples (20–40 μg) were placed in SDS-sample buffer and 2% 2-β-mercaptoethanol, boiled for 5 min and subjected to SDS-PAGE on 12% Tris-glycine gels. Separated proteins were transferred onto 0.22 μm nitrocellulose membranes at 100V for 1 h. The membranes were incubated in blocking solution (5% nonfat dry milk-TBS-Tween-20) for 1 h at room temperature. Primary specific antibodies were incubated in 5% milk-TBS-Tween-20  to detect LC3B, Beclin-1 (D40C5), SQSTM1/p62, AMPK, JNK, BCL-2, BAX, p-53 and p21  and actin (Santa Cruz Biotechnology). After incubation with the HRP-conjugated secondary antibody  in 5% milk-TBS-Tween-20 , a chemiluminescence kit was used for visualization.After treatment, cells were lysed in cell lysis buffer consisting of: 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% Triton X-100, supplemented with protease inhibitors and phosphatase inhibitors (10 mmol/L sodium fluoride and 10 mmol/L sodium orthovanadate) for 30 min on ice. Lysates were centrifuged at 13,200× control refers to the absorbance of the negative control, and Asample refers to the absorbance of the tested compounds. Results are expressed as a percentage of DPPH radical scavenged ± SEM. The DPPH  assay measures the hydrogen donation ability of the antioxidant to convert the stable DPPH free radical into 1,1-diphenyl-2--hydrazine. After radical reaction with the compounds, a decrease in the absorbance was detected at 517 nm, which is accompanied by a change of color from violet to light-yellow. The target compounds were dissolved in methanol to make 1 mg/mL stock solutions, which were diluted to five concentrations as test samples. Ascorbic acid was used as a standard, and the experiment was carried out on 96-well plates. The DPPH solution was prepared at a 100 µM concentration. For each test, 100 µL of DPPH solution was added to 100 µL of methanolic solution containing the tested derivatives, and the absorbance was determined at different time points. All the measurements were carried out in triplicate. Results were expressed as the percentage of the radical scavenged, calculated using the following formula:•+ with potassium persulfate . This cocktail was kept overnight away from light at room temperature. Then, this ABTS•+ reaction mixture was diluted with a 50% ethanolic solution in order to achieve absorbance values of 0.70 ± 0.02 at 734 nm for measurements. Finally, 1 mg/mL stock solutions for the selected derivatives were formed in absolute ethanol and 20 µL of these solutions were added to 180 µL of the diluted ABTS•+ cocktail. After 6 min of incubation, absorbances at 734 nm were registered. A 50% ethanolic solution was used as blank along with Trolox (TROL) and ascorbic acid (Asc) as positive controls. All of the determinations were performed in triplicate using 96-well plates. The same time intervals as in the DPPH assay were also measured. The ability to scavenge ABTS•+ was calculated using the following formula:control refers to the absorbance of the negative control and Asample refers to the absorbance of the tested compounds. The results are expressed as percentage of ABTS radical scavenging ± SEM.ABTS radical scavenging was additionally assayed with a colorimetric assay following a previous methodology . Brieflyt-test. * Statistical significance was defined as p < 0.05 (*); p < 0.01 (**) or p < 0.001 (***).Data were expressed as the mean ± SEM and analyzed by the Student’s The route adopted for the synthesis of the novel forty-one compounds presented in this work has been previously reported ,31. StruThe synthesis of both series of compounds followed similar synthetic routes. Briefly, 4-aminophenylselenocyanate (0A) and bis-(4-aminophenyl)diselenide (0B) were achieved as previously reported . For serFor series II, 0A and 0B were treated dropwise with the corresponding phosphoryl chlorides under different temperatures, atmospheric conditions and reaction times .Herein, two series of compounds based on amides (series I) and phosphoramidates (series II) derived from 4-aminophenylselenocyanate and bis-(4-aminophenyldiselenide) were tested in vitro against cell lines derived from breast adenocarcinoma (MCF-7), lymphoblastic leukemia (CCRF-CEM) and colon adenocarcinoma (HT-29). Evaluation was performed at 48 h of treatment following the MTT -2,5-diphenyltetrazolium bromide) methodology as previously described . The in 50, TGI and LC50 values were calculated from the curves and are shown in 50 (184B5)/GI50 (MCF-7) and GI50 (BEAS-2B)/GI50 (HTB-54) . The presence of electron-withdrawing groups  in the phenyl ring favored the cytotoxic activity in the amides, but in the phosphoramidates, the best results were obtained for aliphatic chains methoxy  and ethoxy . Additionally, the replacement of oxygen  by sulfur  resulted in the abrogation of the activity. In relation to selenium entity, amides with selenocyanate motif were more active than the corresponding diselenides, and this is particularly evident in PC-3 . However, no significant differences were observed for phosphoramidates. Twelve compounds: 1b, 2b, 1e, 2e, 1f, 2f, 1h and 2h (series I) and compounds 1b, 2b, 1d and 2d (series II) showed cell growth below 50% at a 10 µM concentration. Hence, they were tested at five concentrations between 0.01 and 100 µM against five cancer cell lines, MCF-7, CCRF-CEM, HT-29, HTB-54 (lung carcinoma) and PC-3 (prostatic adenocarcinoma), and two nonmalignant cells, 184B5 and BEAS-2B. GI(HTB-54) . CisplatThe lack of selectivity toward cancerous cells is one of the major issues during the discovery and development of new anticancer agents. Therefore, we evaluated the cytotoxic activity of our compounds against two cell lines derived from nonmalignant breast tissue (184B5) and no malignant bronchial epithelium (BEAS-2B) . Data anCollectively, considering all the results, compounds I.1f, I.2f and II.2d showed the most significant cytotoxicity against all the screened cell lines along with acceptable safety and were picked for further pharmacological research in the breast adenocarcinoma cell line MCF-7.In a first approach, the radical scavenging activity for all the synthesized compounds was characterized by the DPPH colorimetric assay at a dose of 0.03 mg/mL of each compound and six different time points . Ascorbic acid and Trolox were used as radical scavenger gold standards. Three diselenides  and three selenocyanates  from series I showed DPPH activity inhibition values greater than 35% after 30 min A of treaIn a second approach, the antioxidant activity of the six compounds that showed DPPH activity inhibition was also further confirmed using the ABTS colorimetric assay C. SurpriApoptosis and cell cycle arrest are typical mechanisms of action for many anticancer drugs, including selenocompounds ,35,36. IAs seen in To further understand the mechanism of action of these potent compounds, their effects on cell cycle distribution in MCF-7 were studied by flow cytometry. This assay was carried out with different concentrations  and diffAfter treatment with I.1f, I.2f and II.2d at various time points (24–72 h for I.1f and 8–48 h for I.2f and II2d), the results suggested that these compounds arrested cell cycle in a time-dependent fashion .Apoptosis and autophagy are considered as two recognized pathways for anticancer agents due to their effects on cell survival ,38. In tAs illustrated in Conversely, preincubation with wortmannin A could nTo further confirm the programmed cell death pathway, the expression levels of several markers of autophagy  and apopBax is an important proapoptotic protein implicated in apoptosis induction. On the contrary, Bcl-2 is an important antiapoptotic protein that suppresses apoptosis. The balance between Bax and Bcl-2 is very important for judgmental cell apoptosis . The exp50 values < 1 µM . These compounds were approximately three-fold more potent than cisplatin, a reference drug used clinically. Additionally, in vitro radical scavenging studies have shown that only series I compounds (amide derivatives) present antioxidant activity. Surprisingly, no phosphoramidate showed this effect. No correlation was found for either structure- or cytotoxicity-antioxidant activity, except for compound I.2f. Moreover, compounds I.1f, I.2f and II.2d showed the highest SI values when comparing the cytotoxic activity against 184B5 nonmalignant cell lines and were selected for further biological studies. Flow cytometry studies suggested that amides I.1f and I.2f induced cell cycle arrest at the S phase and cell death in a time- and dose-dependent manner and cell death through an autophagy process. Meanwhile, phosphoramidate II.2d flow cytometric studies showed an early caspase-dependent cell death at low doses and cell cycle arrest at G0/G1 phase at a 5 µM dose. However, as the dose increases, the number of subdiploid cells in the SubG1 phase also increases significantly. Both autophagy processes caused by I.1f and I.2f and apoptosis induction caused by II.2d were confirmed by Western blot analysis.Forty-one new amides and phosphoramidates bearing selenocyanate and diselenide scaffolds were prescreened against three tumor cell lines at two doses. Twelve compounds were selected and tested against a panel of five tumor cell lines and two nonmalignant cell lines. The breast tumor cell line MCF-7 was the most sensitive, with six compounds showing GI