Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs.Protein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the ‘The Cancer Genome Atlas’.A total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs . Western blotting confirmed regulation of key resistance-associated proteins . GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines.High resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.The online version contains supplementary material available at 10.1007/s00345-022-03936-1. Testicular germ cell tumours (GCTs) are the most common malignant solid neoplasms in men between the age of 15 and 40 with an increasing incidence seen over the last 4 decades . NSGCT cIn this study, we compared cisplatin-resistant and cisplatin-sensitive cell lineages of pluripotent NTERA-2 and NCCIT as well as the nullipotent 2102EP cells  , 14 by hl-glutamine . The incubation temperature was 37 °C in a humid atmosphere with 5% carbon dioxide in the air.The two human GCT cell lines NTERA-2 (CRL 1973) and NCCIT (CRL 2073) were supplied by ATCC, USA. 2102EP cells were a kind gift from PD Dr. Dr. Friedemann Honecker. All cell lines were cultured in HEPES-buffered RPMI-1640  supplemented with fetal calf serum , penicillin , streptomycin  and For all parental GCT cell lines , isogenic cisplatin-resistant sublines  were established through repeated cisplatin-exposure to increasing sublethal cisplatin concentrations (0.01–0.5 µg/ml) over a time period of 9–12 months, as described previously . Cisplatl-arginine-13C6 (Arg + 6) and 0.275 mM l-lysine-2D4 (Lys + 4) or equimolar amounts of l-arginine-13C615N4 (Arg + 10) and 0.275 mM l-lysine-13C6,15N2-Lys (Lys + 8)  for at least ten cell cycles. Labelled cells were lysed in 0.5% Nonidet P-40 buffer containing 50 mM Tris/HCl, pH 7.8, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 0.2% lauryl maltoside and protease inhibitors . After isolation, the protein amounts were determined by a colorimetric BCA assay. Equal amounts of SILAC-labelled proteins , were mixed and separated by SDS-PAGE . Proteins were visualized with Coomassie brilliant blue stain and each lane cut into 11 equidistant slices irrespective of staining. Gel slices were reduced with 10 mM DTT for 55 min at 56 °C, alkylated with 55 mM IAA for 20 min at 26 °C and digested with modified trypsin  overnight at 37 °C. Resulting peptides were concentrated on a C18 precolumn  for 5 min at a flow rate of 10 μl/min and separated on a C18 capillary column  at a flow rate of 300 nl/min, with a gradient of 5–35% acetonitrile 0.1% formic acid over 90 min using a Proxeon nano LC coupled to a Q Exactive mass spectrometer . MS conditions were as follows: spray voltage, 1.8 kV; heated capillary temperature, 270 °C; and normalized collision-energy (NCE) 28. The mass spectrometer was operated in data-dependent acquisition mode. Survey MS spectra were acquired in the Orbitrap (m/z 350–1600) with a resolution setting of 70,000 at m/z 200, a target fill value of 1e6 and a maximum fill time of 60 ms. The 15 most intense ions were sequentially isolated for HCD MS/MS fragmentation and detection at a resolution setting of 17,500, a target fill value of 2e5 and a maximum fill time of 60 ms. Raw data were analysed with MaxQuant software version 1.3.0.5  against the UniprotKB human reference proteome revision 02–2017 containing 92,928 sequence entries. Up to two missed cleavages of trypsin were allowed. Oxidized methionine, N-terminal protein acetylation and the respective isotope-labelled arginine and lysine residues were searched as variable modifications and cysteine carbamidomethylation as fixed modification. The false discovery rates at the protein and peptide levels were both set to 1%, respectively. Missing values in individual experiments are denoted by ‘Not a Number (NaN)’. No further data imputation or similar was used.We performed protein expression profiling by mass spectrometry in combination with stable isotope labelling by amino acids in cell culture (SILAC) as described before –18. NTER2 ratios of quantification to the intensity, Log10 values were calculated for intensities of each mean value. Afterwards, significance B analysis using a Wilcoxon-Mann–Whitney test with a Benjamini–Hochberg FDR < 5% was performed by Perseus software to calculate the p values of outlier proteins from the 1/1 ratio [10 intensity was plotted against Log2 ratios of quantified resistant/parental ratios. Proteins were coloured according to their p values where blue colour means > 0.05, red color 0.05–0.01, yellow between 0.01 and 0.001 and light green coloured proteins mean that they have p values less than 0.001.Perseus Software version 1.5.2.6  was used for statistical evaluation. The mean of the biological replicates was calculated for each cell line . In order to compare Log/1 ratio . Log10 i® CA-630) with protease inhibitors. Protein concentration was quantified by the Bio-Rad DC Protein Assay . The Western blot analyses were performed using the following primary antibody dilutions: monoclonal rabbit anti-CBS , polyclonal rabbit anti-LDHA , monoclonal rabbit anti-ANXA1 , monoclonal rabbit anti-CTH  monoclonal mouse anti FDXR . Polyclonal immunoglobulins/HRP secondary antibodies  were used for the detection of primary antibodies. Membranes were developed using the ECL system .Cell lines were lysed in RIPA buffer  [p values < 0.05 and FDR q < 25% as suggested by GSEA. For visualization in a bar graph p- and q-values were − log2 transformed. Enrichment plots were saved from the GSEA reports.Functional annotations were predicted using gene set enrichment analysis (GSEA) software downloaded from the home page (dex.jsp) , 22. Loghttps://string-db.org) [https://david.ncifcrf.gov/home.jsp) [https://www.cbioportal.org/) [The STRING algorithm was used to predict protein–protein interaction by confidence using standard settings (-db.org) . The DAVome.jsp) . The proal.org/) , 26.2 SILAC ratios were seen in all three cell lines  and NADPH-adrenodoxin oxidoreductase (FDXR) were increased in all three resistant cell lines . In each cisplatin-resistant cell line individually, we found a heterogenous set of processes affected. The analysis of NTERA-2-R detected ‘Interferon alpha and gamma’ signalling and ‘epithelial to mesenchymal transition’ (EMT) as mainly enriched gene sets . Two sets related to ‘Myc targeting’ and ‘DNA repair’ were significantly affected in NCCIT-R cell line . Furthermore, GSEA revealed seven significantly affected gene sets including ‘P53 signalling’, ‘hypoxia’, ‘fatty acid metabolism’, ‘glycolysis’, ‘late estrogen response’, ‘oxidative phosphorylation’ and ‘IL2 STAT5 signalling’ for 2102EP-R . The combined analysis of the three cell lines highlighted ‘DNA repair’, ‘oxidative phosphorylation’ and ‘early estrogen response’ as the three most prominently enriched gene sets  of all genes coding for proteins altered in each cell line individually or commonly. GSEA determines, if a defined set of genes shows a statistically significant difference in enrichment between two biological states (cisplatin resistance vs. sensitivity) , 22. We First, the DAVID Gene ontology tool was used to predict molecular functions of all proteins altered in the resistant cell lines . Proteins increased in amount in the resistant cells compared with the parental cell lines were involved in oxidoreductase activity, acetylation and metal binding were among others commonly deregulated in all three cell lines. Next, the STRING algorithm was used to predict protein–protein interactions of the enriched proteins . Proteins involved in the molecular processes found by the DAVID analysis were highlighted by colour. In contrast, proteins decreased in amount in resistant cell lines compared with the parental cell lines were involved in phosphatidyl-inositol binding and DNA methyltransferase activity .IFITM3 (enriched in 2102EP-R) harboring a deep deletion in 2.1% of samples, GDF3 being amplified in 7% (enriched in NCCIT-R) and STK38L (enriched in NT2/D1-R) being amplified in 8% as most affected genes by mutation.Furthermore, using cBioPortal, we screened the TCGA GCT tissue cohort for mutational footprints and expression of all genes coding for affected proteins in resistant cell lines individually . In general, 149 GCT (seminoma and nonseminoma) samples were included. The majority of the samples were highly aneuploid, harbored a 12gain, were mainly in the typical age of 14–44 years and were isolated from testis. Indeed, expression of analyzed genes was mainly associated with a nonseminomatous cell character . Nevertheless, expression of a subset of genes could also be related to seminomas. Mutations in the analyzed genes were overall rare, with Cisplatin resistance in GCTs rarely occurs in primary diseases, but in up to 15% of metastatic diseases with poor prognosis for the affected patient . Differe2S) [2S production might activate the intrinsic apoptotic pathway via release of mitochondrial cytochrom C [S-transferase (GST) [Cystathionine beta-synthase (CBS) was significantly decreased in amount in all three cisplatin-resistant cell lines in comparison to their cisplatin-sensitive parental lineage. CBS is an enzyme that regulates homocysteine metabolism and catalyzes the formation of cystathionine . Further2S) . CBS is 2S) , 31. Its2S) . For exa2S)  as well 2S) , colorec2S)  and pros2S)  have shoochrom C . Accordiochrom C , testicuse (GST) . These f2S. It plays a key role in the bodies H2S production [2S in metastatic prostate cancer [Another enzyme of the cysteine metabolism, namely cystathionine gamma lyase (CTH), showed decreased levels in two of the three resistant cell lines (NTERA-2-R and NCCIT-R). CTH catalyzes the breakdown of cystathionine to cysteine, alpha-ketobutyrate and Hoduction , 30. Itse cancer , similare cancer , 39. InhThe Annexin A1 (ANXA1) levels were significantly increased in resistant NTERA-2 and NCCIT cell lines. ANXA1 is a calcium-dependent phospholipid binding protein that belongs to the Annexin superfamily –42. ANXAIn addition, the protein levels of L-lactate dehydrogenase A chain (LDHA) were elevated in NTERA-2-R and 2102EP-R cells. LDHA plays an important role in anaerobic glycolysis , 49. In Furthermore, a significantly increase of COL1A1 and COL1A2 levels was detected in cisplatin-resistant NTERA-2 cell lines in comparison with the parental cell line, with decrease in resistant NCCIT cell line. Both proteins resemble extracellular matrix proteins for that increased levels have been detected in stressed cells . They haGSEA revealed that DNA repair is the only significant gene set deregulated in all three cell lines, which has been considered for cisplatin resistance in GCT before . In NTERDAVID Gene Ontology and STRING analyses showed a deregulation of proteins involved in oxidoreductase activity, acetylation and metal binding. The upregulation of NADPH-adrenodoxin oxidoreductase (FDXR) in resistant cell lines could be validated by Western blot. Oxidoreductase activity, acetylation and metal binding have been described in connection to p53 –57. A reScreening of the TCGA GCT cohort demonstrated that most genes/proteins found increased in the resistant situation, show already basal expression in GCT tissues (mostly nonseminomas). Thus, rather an overshooting/upregulation of expression than an induction of gene expression is associated with acquisition of therapy resistance. The mutational burden was overall low, nevertheless some genes were frequently mutated in GCT tissues, putatively affecting their molecular function. During development of cisplatin resistance, these mutated genes might be induced as well, further contributing to cisplatin resistance by their altered function or increased expression turnover.In summary, high resolution mass spectrometry in combination with SILAC quantification is a powerful tool to detect differences of protein levels in cisplatin-resistant and cisplatin-sensitive cell lines. We detected 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. The findings of mass spectrometry could be validated by Western blot analysis. With this study, we therefore provide a large proteomic resource in vitro library for studying proteomic alterations contributing to acquired cisplatin resistance in testicular germ cell tumour cell lines. The detected and analysed proteins need further investigations to unravel their putative role in cisplatin resistance of GCT and to determine possible new treatment approaches.Supplementary file1 (PPTX 277 KB) Supplementary Figure 1: Distribution of proteins in all cell lines. This figure shows the normal distribution of proteins from SILAC analysis in all cisplatin-resistant and cisplatin-sensitive cell lines.Supplementary file2 (XLSX 695 KB)Supplementary file3 (PPTX 1041 KB) Supplementary Figure 2: GSEA analysis of all three GCT cell lines separated. (a) The top ten affected gene sets of NTERA-2 represented as the − log2 of the NOM q-value and the FDR q value. Significant enrichment plots of the resistant vs native ratios of NT2: (b) INTERFERON ALPHA RESPONSE , (c) INTERFERON GAMMA RESPONSE  and (d) MESENCHYMAL TRANSITION . (e) The top ten affected gene sets of NCCIT represented as the − log2 of the NOM q value and the FDR q value. Significant enrichment plots of the resistant vs native ratios of NCCIT: (f) MYC TARGETS_V1 , (g) DNA REPAIR  and (h) MYC TARGETS V2 . (i) The ten top affected gene sets of 2102EP represented as the − log2 of the NOM q value and the FDR q value. Significant enrichment plots of the resistant vs native ratios of 2102EP: (j) P53 PATHWAY , (k) HYPOXIA , (l) FATTY ACID METABOLISM , (m) GLYCOLYSIS , (n) ESTROGEN RESPONSE_LATE , (o) OXIDATIVE PHOSPHORYLATION  and (p) IL2 STAT5 SIGNALLING Supplementary file4 (PPTX 10786 KB) Supplementary Figure 3: Summary of the results of STRING analysis and DAVID annotation.  Increased proteins in NTERA-2-R cell lines: green – phosphoprotein, blue – acetylation, yellow – oxidoreductase, red – PcG protein complex.  Increased proteins in NCCIT-R cell lines: blue – oxidoreductase, red – mitochondrial biogenesis, green – cholesterol biosynthesis, yellow – lipid biosynthesis, pink – glutamine family amino acid biosynthetic process.  Increased proteins in 2102EP-R cell lines: blue – RNA N6-mehtyladenosine methyltransferase complex, red – glycogen storage disease.  Decreased proteins in NTERA-2-R cell lines: red – metabolism, green – phosphatidylinositol binding.  Downregulated proteins in NCCIT-R cell lines: red – metabolism, biosynthesis of amino acids, green – phosphatidylinositol binding.  Decreased proteins in 2102EP-R cell lines: green – Ubl conjugation, red – DNA methyltransferase activity, blue – microRNAs in cancer, yellow – kinase, pink – signal recognition particleSupplementary file5 (PDF 1232 KB) Comparison of data to TCGA GCT cohortBelow is the link to the electronic supplementary material.


WT  and hSOD1G93A  cell lines. [3H]L-Arginine uptake was concentration-dependent, voltage-sensitive, and sodium-independent in both cell lines. Among the cationic amino acid transporters family, including system y+, b0,+, B0,+, and y+L, system y+ is mainly involved in [3H]L-arginine transport in ALS cell lines. System b0,+ accounted for 23% of the transport in both cell lines. System B0,+ was found only in MT, and whereas, system y+L was found only in WT. Lysine competitively inhibited [3H]L-arginine uptake in both cell lines. The nNOS mRNA expression was significantly lower in MT than in WT. Pretreatment with arginine elevated nNOS mRNA levels in MT. Oxidizing stressor, H2O2, significantly decreased their uptake; however, pretreatment with arginine restored the transport activity in both cell lines. In conclusion, arginine transport is associated with system y+, and neuroprotection by L-arginine may provide an edge as a possible therapeutic target in the treatment of ALS.L-Arginine, a semi-essential amino acid, was shown to delay dysfunction of motor neurons and to prolong the lifespan, upon analysis of transgenic mouse models of amyotrophic lateral sclerosis (ALS). We investigated the transport function of arginine and neuronal nitric oxide synthase (nNOS) expression after pretreatment with L-arginine in NSC-34 hSOD1 Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with most cases being sporadic; however, 5–10% are familial .G93A) and wild-type  cell lines [3H]L-tryptophan was lower in the MT than in the control cell lines L-arginine uptake under neuroinflammatory conditions.Our study aimed to evaluate the transport characteristics of arginine in ALS cell lines and to investigate nNOS expression. Using quantitative real-time polymerase chain reaction (qPCR) analysis, we further sought to investigate nNOS regulation after treating NSC-34 cell lines with arginine and citrulline. In addition, we also investigated the effect of [3H]L-arginine was purchased from PerkinElmer . Other reagent-grade chemicals and drugs were purchased from Sigma Aldrich .A total of 45.1 Ci/mmol of L-arginine uptake study in NSC-34 cells was conducted according to previously described methods [3H]L-arginine  to each well, cell lines were incubated for 5 min at 37 °C. Next, ice-cold ECF buffer was used to terminate [3H]L-arginine uptake. Thereafter, 750 µL of 1 N NaOH was used to solubilize the cells overnight at room temperature. Radioactivity was measured using a liquid scintillation counter . The cell-to-medium ratio (µL/mg protein) was calculated as follows: [3H] radioactivity (dpm/µL) in the sample per milligram of cellular protein (dpm/mg protein) L-arginine at 0–1 mM was calculated using the Michaelis–Menten constant (Km) and maximum uptake rate (Vmax) as shown in the following equation 1:max1 and Vmax2 are the maximum uptake rates for high- and low-affinity processes, respectively; Km1 and Km2 are the Michaelis–Menten constants for the high- and low-affinity processes, respectively; and Kns1 and Kns2 are first-order constants for the non-saturable components in the high- and low-affinity processes, respectively.For saturation kinetics, the initial uptake of L-Arginine uptake was found to be concentration-dependent in NSC-34 cell lines; however, its uptake in the MT cell lines was lower than that in the WT cell lines  and maximum uptake rate (Vmax) were fitted to the Equation (1), and at the high-affinity site, the affinity (Km) was 23-fold lower and the capacity (Vmax) was approximately 40-fold higher in the MT cell lines than in the WT cell lines. However, at the low-affinity site, both affinity and capacity in the MT cell lines did not markedly differ from those in the WT cell lines , a system y+ specific inhibitor, significantly inhibited the uptake in both cell lines, showing sensitivity to system y+. N-methylmaleimide (NMM), which is system y+L inhibitor, also showed significant inhibition in both cell lines. However, 2-aminobicyclo--heptane-2-carboxylic acid (BCH), a system Bo,+ inhibitor, showed no inhibition in either cell line  strongly inhibited [ell line .3H]L-arginine transport was scrutinized in NSC-34 cell lines. Quinidine, an antiarrhythmic drug, significantly inhibited [3H]L-arginine uptake at a concentration of 2 mM. Notably, [3H]L-arginine uptake was inhibited by more than 50% in the presence of gabapentin in both cell lines, whereas verapamil showed 68 and 73% inhibition in MT and WT cell lines, respectively. Clonidine and donepezil did not inhibit [3H]L-arginine uptake in either cell lines  in the presence or absence of 500 µM lysine, quinidine, and gabapentin. In the case of lysine, the two lines of the plot intersected at the ordinate, indicating that lysine competitively inhibited L-arginine a,b.Quinidine showed non-competitive inhibition in the WT cell lines, with a Ki value of 2.2 mM, and competitive inhibition in the MT cell lines, with a Ki value of 0.64 mM c,d. In aTo assess the pattern of nNOS levels, we measured mRNA expression using qPCR in WT and MT cell lines, with or without the addition of arginine and citrulline. nNOS mRNA expression was lower in the MT cell lines than in the control; however, nNOS relative gene expression significantly increased in MT cell lines treated with arginine and citrulline, in comparison to untreated MT cell lines. In the WT cell lines, there was no statistical difference in the mRNA expression between the untreated and arginine-treated cells. In contrast, citrulline treatment resulted in a significant increase in nNOS expression compared with that in untreated WT cell lines .3H]L-arginine uptake upon preincubation of NSC-34 cell lines with 100 µM H2O2 are shown in Changes in [2O2 significantly decreased [3H]L-arginine uptake in both cell lines compared with that in control. Additionally, 10 mM of arginine and citrulline increased [3H]L-arginine transport rate in the MT cell lines. The Slc7a1 mRNA expression was markedly decreased in the presence of H2O2 alone in the MT cell lines; however, in addition to co-treatment with H2O2 and arginine, co-treatment with H2O2 and citrulline also resulted in a significant increase in the [3H]L-arginine uptake rate. These results suggest the restorative and neuroprotective roles of arginine and citrulline in ALS cell lines.Hwt) and SOD1 (G93A) gene mutation and are widely used for the study of ALS disease mechanisms and also to discover the therapeutically effective drug regimen in motor neurodegeneration L-Arginine uptake occurs in a Na+-independent manner, ruling out the idea of a Na+-dependent transport system such as system B0,+ in NSC-34 cell lines. In addition, [3H]L-arginine uptake was voltage-sensitive  and polyamines and is involved in various biochemical pathways, such as protein synthesis and the urea cycle .For the neration ,35. Argill lines , suggestll lines . [3H]L-Aensitive , consist+, b0,+, B0,+, and y+L. Previous studies have also shown that system y+ is an arginine-specific transporter associated with several cell types, including neurons L-arginine uptake was significantly inhibited by typical CAT-1 substrates, including arginine, lysine, histidine, and ornithine, in both cell lines  [+ and our data revealed significant inhibition of [3H]L-arginine transport due to NEM in both cell lines, leading to the hypothesis that system y+ was primarily involved in arginine transport in these cell lines  . NEM, isll lines . However3H]L-arginine uptake in ALS model cell lines. Earlier studies have shown the effectiveness of quinidine combined with dextromethorphan in treating pseudobulbar affect, a consequence of neurological damage caused by ALS, AD, and other traumatic brain injuries, overall the combination has shown to improve the quality of life of ALS patients [3H]L-arginine uptake in ALS model cell lines (3H]L-arginine was competitively inhibited by lysine, which is also a substrate of CAT-1, suggesting that the high-affinity uptake of arginine and lysine both shared the same transport process which is system y+. Therefore, showed competitive inhibition in both cell lines. Similar results have been reported previously on arginine, in which it was competitively inhibited by ornithine, another CAT-1 substrate L-arginine uptake was markedly reduced in the presence of H2O2 in NSC-34 cell lines, which might be attributed to the reduced expression of the transporters involved in arginine transport in ALS cell lines. However, arginine and citrulline pretreatment significantly increased arginine uptake in the MT cell lines, indicating that arginine and citrulline restored arginine uptake by restoring the expression of transporters. Our qPCR analysis revealed a similar pattern of results (2O2 and was recovered by lysine. L-a results . Previou lysine. . Overall lysine. , and out lysine. .+ in NSC-34 cell lines and that two saturable transport processes were involved in both NSC-34 cell lines. Additionally, arginine exhibited regulatory effects, restoring nNOS expression in the disease model cell lines, and a neuroprotective effect against the oxidizing stress-inducing agent H2O2. Therefore, by understanding the transport system involved in arginine transport using motor neuron models, such as NSC-34 cell lines, L-arginine may act as a potential candidate for improving the pathological conditions in ALS and other neurodegenerative diseases.In conclusion, our study revealed that arginine transport was mainly mediated by system y


The compound 1a was highly anti-proliferative and reached IC50 values in the nanomolar concentration range. 1a was superior to the known anti-tumorally active curcuminoid EF24 (2) and its known N-ethyl-piperidone analog 1b in all tested tumor cell lines. Furthermore, 1a induced a noticeable increase of intracellular reactive oxygen species in HT-29 colon adenocarcinoma cells, which possibly leads to a distinct increase in sub-G1 cells, as assessed by cell cycle analysis. A considerable activation of the executioner-caspases 3 and 7 as well as nuclei fragmentation, cell rounding, and membrane protrusions suggest the triggering of an apoptotic mechanism. Yet another effect was the re-organization of the actin cytoskeleton shown by the formation of stress fibers and actin aggregation. 1a also caused cell death in the adherently cultured glioblastoma cell lines U251 and Mz54. We furthermore observed that 1a strongly suppressed the stem cell properties of glioma stem-like cell lines including one primary line, highlighting the potential therapeutic relevance of this new compound.A new and readily available pentafluorothiophenyl-substituted N-methyl-piperidone curcuminoid  Though displaying promising anticancer activities, its potential as a drug candidate is limited by its rapid metabolic degradation, its low water solubility and its insufficient bioavailability both in plasma and in tissues. , 540 (42), 513 (41), 512 (41), 414 (11), 310 (22), 296 (23), 270 (45), 242 (8), 143 (7), 115 (13), 43 (14); HRMS (ESI) C20H18F10NOS2 [M + H]+ calcd. 542.06709, found 542.06612. 1-Methyl-4-piperidone  was dissolved in MeOH (5 mL) and 4-(pentafluorothio)-benzaldehyde  was added. NaOH  and HVbl (ACC-149) cervix carcinoma, MCF-7Topo (ACC-115) breast carcinoma, HT-29 (ACC-299), HCT-116 (ACC-581), DLD-1 (ACC-278), SW-480 (CCL-228) colon carcinoma, were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, and 1% antibiotic-antimycotic at 37 °C, 5% CO2, and 95% humidity. To keep MCF-7Topo and KB-V1Vbl cells resistant, the maximum-tolerated dose of topotecan or vinblastine were respectively added to the cell culture medium 24 h after every passage. The cells were used in passages between 40 and 47 (518A2), 102 and 110 (Panc-1), 223 and 229 (KB-V1Vbl), 41 and 46 (MCF-7), 34 and 51 (HT-29), 67 and 73 (HCT-116), 51 and 56 (DLD-1), as well as 43 and 48 (SW-480). U251   A A2) can  3 and 7 , suggestd 7  from the characteristic cortical filaments (shown by the untreated control) to cell rounding and actin clustering.  which effectively blocks proliferation and stemness in GSCs -4-piperidinone (EF24), which evokes promising antitumour properties such as cell cycle arrest, apoptosis induction, and inhibition of HIF-α and NF-κB. ,42,43,44ctivity.  Due to tuminoids . Comparioids [1a . 1a effectively reduces the viability of numerous cancer cell lines derived from multiple cancer entities. Similarly, the lead compound Curcumin (3) has been shown to be an effective anticancer agent across several cancer cell lines as well as cancer models in vitro and in vivo. [1a exerts comparable pleiotropic effects by acting on similar processes than Curcumin, albeit at much lower concentrations. As such, we observed potent inhibition of angiogenesis, induction of ROS, cell cycle inhibition and an increase of effector caspases 3 and 7. Similarly, Curcumin has been shown to inhibit angiogenesis for example in models of pancreatic cancer [We could previously demonstrate that Curcumin effectively targets GBM cells both in vitro and in vivo ,47 and win vivo.  Based onc cancer  and breac cancer . Furtherc cancer  Addition1a not only decreases viability of GSCs, but also reduces their stem-cell frequency using sub-lethal drug concentrations indicating that 1a has the potential to target the stem-like phenotype of cancer cells. In fact, it is well-established that GBM cells can obtain a stem-like phenotype in vivo and that these cells are largely responsible for treatment resistance and finally disease recurrence. [Importantly, we next focused on a more complex model system by employing three GSC spheroid cell lines, which are known to more closely mimic the original tumor and as such are a better preclinical model system . We coulurrence.  Althoughurrence.  Similarlurrence. ,54 Thus,1a was prepared in one step and tested against a panel of tumor cell lines. Our working hypothesis was that 1a is a superior anticancer drug compared to 1b and 2 and our primary endpoint of analyses was after assessment of this anticancer activity across several cancer cell lines derived from multiple cancer entities. The compound showed a notable higher anti-proliferative activity than close congeners such as EF24, Curcumin, and 1b. Cell death and apoptosis induction by 1a occurred at lower doses than by 1b. This was further corroborated by a strong increase in ROS production and the Sub-G1 phase of the cell cycle of HT-29 adenocarcinoma by 1a could be determined. Also, the effect on the actin cytoskeleton in treated cells was clearly improved compared to 1b. Additionally, 1a showed similar growth-inhibiting efficacy when tested against several GSC cultures, including the primary culture 17/02, which is derived from a heavily pre-treated GBM patient. 1a was also able to effectively inhibit stemness in all three cultures in low nanomolar doses. Hence, 1a appears to be an optimized anti-tumor curcuminoid and further investigation of this promising compound is warranted.A new Curcumin derivative


Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors. Many forms of cancer have developed mechanisms to prevent mitochondrial apoptosis by altering the equilibrium between pro- and antiapoptotic proteins2. Expression levels and functional importance of individual antiapoptotic proteins are considered to be different in the different types of tumors1.Apoptosis or programmed cell death is a process which eliminates old cells or cells that are otherwise no longer needed or detrimental. Decisive for the fate of the individual cell is the interaction between pro- and antiapoptotic members of the BCL2 family4. Mimicking proapoptotic BH3-only proteins, BH3 mimetics antagonize the function of antiapoptotic BCL2 family proteins, providing a new opportunity for treatment of these diseases4. Antagonizing BCL2, venetoclax was the first BH3 mimetic approved for CLL and then—in combination with hypomethylating drugs—also for AML5. Various new drugs targeting BCL2 family members are currently evaluated in clinical trials1.The aberrant expression of BCL2 and of other members of the BCL2 family is well-described in follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and in acute myeloid leukemia (AML)6.Aim of our study was to identify hematologic entities not yet in the focus of broad interest, but with a conspicuous expression pattern of antiapoptotic BCL2 family members, indicating a potential for the application of BH3 mimetics. To delineate disease-specific expression patterns of antiapoptotic genes in hematologic tumors, we screened cell lines from the leukemia-lymphoma 100 (LL-100) panel. In 2019, we had developed this panel of cell lines covering 22 entities of leukemia and lymphoma malignancies as novel platform for the development of targeted therapies9. BH3 mimetics targeting MCL1 are promising options for the therapy of both diseases8. We describe the effects of the MCL1 inhibitor AZD-5991 on growth and apoptosis in ALCL and PEL cell lines. Furthermore, we discuss under which circumstances the combination of AZD-5991 with other BCL2 family inhibitors might be of clinical benefit. Inhibitors against the antiapoptotic AKT pathway are an alternative for combination treatment with AZD-5991.RNA-seq analysis of the LL-100 panel showed that cell lines derived from two rare lymphomas, anaplastic large cell lymphoma (ALCL) and from primary effusion lymphoma (PEL) stood out by high expression of MCL1. MCL1 had already been described as essential mediator for the survival of multiple myeloma (MM) and of AML11. Deregulation of BCL2 in hematopoietic tumors could be traced back to the translocation t, amplification or hypomethylation of the gene2.Overexpression of antiapoptotic BCL2 family genes promotes survival of cells in hematological malignancies12. To find potential targets of BH3 mimetics, we examined the expression of BCL2 family genes in the LL-100 cell lines panel. Remarkably, all ALCL and PEL cell lines showed an MCL1high/BCL2low expression pattern  cells15.MCL1 is the dominant antiapoptotic BCL2 family member in MM, more critical than BCL2 and BCLXLMCL1 gene is located on chromosome 1 (q21.3), in a region which is often amplified in human cancer16. According to our quantitative genomic PCR, 6/7 ALCL cell lines and 6/8 PEL cell lines carry gains of MCL1 (Suppl. Table MCL1 locus (9n), suggesting that focal gain can play a role in the aberrant overexpression of MCL1  are emerging agents for the treatment of BCL2 dependent tumors. Different compounds are in clinical trials, with the BCL2 inhibitor Venetoclax (ABT-199) being the first BH3 mimetic approved by the FDA for CLLpos/BCL2neg) and RI-1 (MCL1low/ BCL2pos) , AZD-5991 efficiently inhibited growth of the MCL1pos cell line (U-2946)  and of one BCL2/BCLXL inhibitor (ABT-263) on the diffuse large B cell lymphoma (DLBCL) cell lines U-2946 . Therefore, we assessed BCLXL as candidate antiapoptotic mediator in ALCL.ABT-263 inhibits not only BCL2, but also BCLXL and BCL-W. Therefore, expression of the latter two genes might explain responsiveness of a BCL2pos/BCLXLpos cell line L-82  RNA oligonucleotides was verified by qRT-PCR  and ABT-263 (300 nM) yielded synergistic antiproliferative and proapoptotic effects when compared to the effects of each inhibitor alone  expressed BCLXL   encoding the NPM-ALK fusion gene.MCL1 had been described as the main antiapoptotic mediator in ALCLhigh), but not in L-82 (BCL2A1low)  to test whether this drug would repress MCL1 in our cell lines. We speculated that a double hit against MCL1, reducing expression (BEZ-235) and inhibiting its function (AZD-5991), might create cooperative effects.The rationale for these experiments lies in earlier studies reporting that apoptosis in ALCL and DLBCL models was induced by downregulation of MCL1 following inhibition of the AKT/mTOR pathway29  resulted in a dramatic drop of MCL1  and induced apoptosis (12 h) in a dose-dependent manner  induced expression of several dozen genes in cell lines L-82 and SU-DHL-1 . Cell lines were authenticated by DNA profiling and cytogenetics. Detailed references and cultivation protocols have been described previously6. RNA-seq data were deposited at ArrayExpress E-MTAB-7721. For the heatmap visualization 0.1 was added to normalized expression levels prior to log2 transformation.Total RNA was extracted via miRNeasy Mini Kit  including DNase digestion. Library preparation and sequencing were commissioned to GATC Biotech . RNA expression analysis was performed as described previously4 cells (in 50 µl) were seeded in triplicate in 96-well flat bottom microtiter plates. Effectors were added as 2 × concentrated solutions in a 50 µl volume. Growth and apoptosis were determined with the IncuCyte S3 Live-Cell analysis System . We used the Caspase 3/7 reagent (Essen Bioscience #4704) to detect apoptotic cells.About 2 × 1039. Gene-specific siRNA oligonucleotides and AllStars negative control siRNA were obtained from Qiagen. The siRNA (80 pmol) was transfected into 1 × 106 cells by electroporation using an EPI-2500 impulse generator  at 350 V for 10 ms. Treated cells were harvested after 24 h or 48 h.Transfection with siRNA oligonucleotides was performed as described previouslyBAX (Hs00180269_m1), BCL2 (Hs00153350_m1), BCL2A1 (Hs00187845_m1), BCLW (Hs00187848_m1), BCLXL (Hs00236329_m1), CDKN1A (Hs00355783_m1), MCL1 (Hs03043899_m1), MDM2 (Hs01066930_m1) and NOXA1 (Hs00611456_g1) using TBP as endogenous control. DNA was isolated with the High Pure PCR Template Preparation Kit . Quantitative genomic PCR was performed with SYBR-green reagents (Applied Biosystems). Primers and PCR conditions for genomic PCR are shown in Table RNA was prepared using the RNeasy Mini kit (Qiagen). For mRNA quantification, reverse transcription was performed using the SuperScript II reverse transcriptase kit . PCR was performed on a 7500 Applied Biosystems . TaqMan probes (Applied Biosystems) were used to quantify human 40. Anti AKT (#4691), pAKT (Ser473) (#4060), BAD (#9239), pBAD (Ser136) (#4366), BCL2 (#4223), BCL2A1 (#14093), BCL-W (#2724), BCLXL (#2764), pGSK3B (Ser9) (#9323), MCL1 (#39224), pMCL1 (Ser159/Thr163) (#4579), S6 (#2317) and pS6 (Ser235/236) (#2211) antibodies were purchased from Cell Signaling . Anti GAPDH (ab8245) antibody was from Abcam . Specific bands on nitrocellulose membranes were visualized with the biotin/streptavidin–horseradish peroxidase system  in combination with the “Renaissance Western Blot Chemoluminescence Reagent” protocol .Samples were prepared as described previouslyCell lines L-82 and SU-DHL-1 were stimulated with BEZ-235 . RNA was isolated using the RNeasy Mini kit (Qiagen). Quality and integrity of total RNA was controlled on Agilent Technologies 2100 Bioanalyzer . For biotin labelling 2–10 ng of total RNA were used according to GeneChip Pico Kit (Affymetrix). Then, 5.5 µg of biotinylated cDNA were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls  as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix Clariom S (400 Format) for 17 h at 45 °C. Hybridization was done for 16 h at conditions recommended by the manufacturer. Clariom S chips were washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the Affymetrix GCS 3000. Image analysis was done by Affymetrix GeneChip Command Console Software (AGCC) and Affymetrix Expression Console Software. Clariom S arrays were analyzed via R/Bioconducor using the package oligo including multi-array average (RMA) background correction, quantile normalization and log2 transformation, whereas the annotation file was retrieved from Thermofisher . Genes were filtered to at least log2FC < -1 in control versus 6 h after BEZ-235 treatment. The data were deposited with ArrayExpress under E-TAB-10903.Supplementary Information.


Recent studies on cancer cell metabolism have achieved notable breakthroughs that have led to a new scientific paradigm. How cancer cell metabolic reprogramming is orchestrated and the decisive role of this reprogramming in the oncogenic process and tumor adaptative evolution has been characterized at the molecular level. Despite this knowledge, it is essential to understand how cancer cells can metabolically respond as a living whole to ensure their survival and adaptation potential. In this work, we investigated whether different cancers and different subtypes display different metabolic phenotypes with a focus on breast cancer cell models representative of each clinical subtype. The potential results might have significant translational implications for diagnostic, prognostic and therapeutic applications.Metabolic reprogramming of cancer cells represents an orchestrated network of evolving molecular and functional adaptations during oncogenic progression. In particular, how metabolic reprogramming is orchestrated in breast cancer and its decisive role in the oncogenic process and tumor evolving adaptations are well consolidated at the molecular level. Nevertheless, potential correlations between functional metabolic features and breast cancer clinical classification still represent issues that have not been fully studied to date. Accordingly, we aimed to investigate whether breast cancer cell models representative of each clinical subtype might display different metabolic phenotypes that correlate with current clinical classifications. In the present work, functional metabolic profiling was performed for breast cancer cell models representative of each clinical subtype based on the combination of enzyme inhibitors for key metabolic pathways, and isotope-labeled tracing dynamic analysis. The results indicated the main metabolic phenotypes, so-called ‘metabophenotypes’, in terms of their dependency on glycolytic metabolism or their reliance on mitochondrial oxidative metabolism. The results showed that breast cancer cell subtypes display different metabophenotypes. Importantly, these metabophenotypes are clearly correlated with the current clinical classifications. In recent years, our concepts and knowledge of cancer cell metabolism have experienced a profound breakthrough that has led to a new scientific paradigm ,2,3. SinIn this context, a foundational scientific proposition is the concept of tumor metabolic reprogramming. This proposition was based on crucial experimental observations, such as the apparently paradoxical catalytic inefficiency of the glycolytic enzyme pyruvate kinase M2 (PKM2), which is expressed only in cancer and proliferative cells, relative to the more efficient PKM1, which is expressed in nontransformed somatic cells. PKM2 catalyzes the last glycolysis reaction; therefore, glycolytic flux is modulated by its lower catalytic velocity, which acts as a limiting factor. Consequently, many glycolytic intermediaries accumulate and can be used in branching anabolic routes, thus guaranteeing the high demand of biomass to sustain cell proliferation ,9,10,11.Metabolic reprogramming represents an orchestrated network of evolving adaptations during oncogenic progression. Cancer cells can adopt different metabolic landscapes in response to their own oncogenic evolution, changes in the cellular niche and environment, and resistance responses to chemo- and radiotherapy ,19. ImpoIn line with this research demand, we recently developed a novel metabolic nanosensor for robust measurements of mitochondrial pH. Interestingly, we found significant variations in intramitochondrial pH among different breast cancer cell lines that correlated with the current clinical subtype classification. Moreover, the variations in pH also correlated with differential responses to some metabolic drugs . In a seNevertheless, there are still relevant open questions from a functional perspective of breast cancer metabolic reprogramming. In this context, it is particularly interesting to investigate whether distinct metabolic profiles could correlate well with breast cancer clinical subtypes. The potential results from this type of investigation might have significant translational implications at different levels. Accordingly, the correlation between the current classification criteria for breast cancer subtypes and well-defined metabolic profiles could profoundly enhance the diagnostic and prognostic capacities. In addition, such work could pave the way for the development of novel therapeutic strategies based on “antimetabolic” subtype-specific drugs as well as novel technical applications with diagnostic and therapeutic purposes.Recently, foundational contributions initiated the investigation of key metabolic features in different breast cancer cell models in vitro and in vivo. In this context, the characterization of glutamine dependence tested by differential sensitivity responses to transaminase inhibitor aminooxyacetate (AOA) was of particular relevance. Interestingly, these results were in correlation with the oncogene c-Myc expression levels between breast cancer cell lines representative of clinical subtypes . In addiMoreover, there have been important contributions aiming to address whether breast cancer cell subtypes might feature different metabolic phenotypes. Most of these elegant works were performed by unlabeled metabolomic analysis to obtain quantitative data about total metabolite content. Some noticeable results were obtained but the data were not totally concordant displaying some discrepancies ,50,51,52Under these premises, as already proposed by the aforementioned authors, further steps to address metabolic phenotypic differences between breast cancer cell subtypes need to be performed by dynamic metabolic flux analysis. In this sense, isotope labeling experiments are required to identify dynamic flux differences in central metabolic routes, such as glucose preferential usage and TCA reliance, as well as to decipher key enzymatic activities promoting those preferential pathways.Notwithstanding the foundational and valuable information accounted by the aforementioned works, further steps are required in breast cancer cell metabolism research, particularly to accomplish the investigations about phenotypic and molecular metabolic differences among breast cancer cell subtypes.Accordingly, the present work was designed to gain further insights in that direction. First, breast cancer cell lines were profiled by data combined from different treatments with five metabolic inhibitors: AOA, EGCG, phenformin, the PI3K inhibitor BKM120, and the mitochondrial membrane uncoupler BAM15. This profiling approach, testing the five selected inhibitors rather than profiles based on a single drug-single metabolic question design, was accomplished as a combinatorial profile aiming to evaluate systemic functional responses of breast cancer cell lines representative of each clinical subtype. Thus, following some insights provided by previous works, where single experimental treatments with AOA, EGCG or Phenformin were performed, in the present study the degree of glycolytic flux dependence was tested by sensitivity responses to phenformin and BKM120 treatments. In addition, the reliance on tricarboxylic acid cycle (TCA) activity and its connection with the potential preferences for cytosolic versus mitochondrial energy supply was studied combining data by AOA, BAM15 and EGCG treatments. Secondly, potential dynamic metabolic flux differences between breast cancer cell lines were studied by stable-isotope dynamic labeling for key targets, such as pyruvate, based on preferential pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) activities to quantitatively analyze the degree of TCA reliance, and other central pathways as glutamine/glutamate metabolism preferences based on anaplerotic input to TCA or their preferential use by reductive carboxylation.The results confirmed that there are differential dependences on glycolitiyc flux activity and TCA and anaplerotic activity reliance between hormone receptor-positive and triple-negative breast cancer subtypes. In addition, the cell lines representative of these clinical subtypes showed different dependencies on transaminase or glutamate dehydrogenase activity for recycling nonessential amino acids. Interestingly, the metabolic phenotypes were in correlation with the different breast cancer subtypes. Finally, as a mechanistic metabolic proof of principle, differences were found between hormone receptor-positive and triple-negative representative cell lines for preferential flux through pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) by isotope labeling dynamic flux analysis. In addition, different preferences were also found in glutamine/glutamate paths between mitochondrial anaplerotic oxidative TCA recycling or cytosolic reductive carboxylation.Five breast cancer cell lines were selected, with each representative of a clinical subtype: MCF-7 (ER+PR+ve/HER2-negative); ZR751 (ER+PR+/HER2-negative); SKBR3 (ER/PR-negative/HER2+ve); MDA-MB-231 (ER/PR/HER2-negative or triple-negative); MDA-MB-468 (ER/PR/HER2-negative or triple-negative) ,53,54. M4 cells/cm2 for 72 h of culture or 1.46 × 104 cells/cm2 for 96 h. The SKBR3 cell line was cultured with McCoy’s 5A medium for optimal growth (Sigma). Culture media was reconstituted with 10% FBS, 1% GlutaMAX and 1% P/S. SKBR3 cells were routinely subcultured at a density of 1.8 × 104 cells/cm2 for 72 h of culture or 1.46 × 104 cells/cm2 for 96 h. The MDA-MB-231 and MDA-MB-468 cell lines were cultured with Dulbecco’s modified Eagle’s medium with GlutaMAX . DMEM was reconstituted with 10% FBS and 1% P/S. MDA-MD-231 cells were subcultured at a density of 1.8 × 104 cells/cm2 for 72 h of culture or 1.46 × 104 cells/cm2 for 96 h. MDA-MD-468 cells were subcultured at a density of 1.6 × 104 cells/cm2 for 72 h of culture or 1.2 × 104 cells/cm2 for 96 h. The ZR751 cell line was cultured with a slightly different protocol. This cell line has a longer doubling time; thus, ZR751 cells were regularly subcultured once a week, although the culture medium was changed twice between each cell seeding. ZR751 cells were cultured with RPMI 1640 (Sigma) for optimal growth. RPMI was reconstituted with 10% FBS, 1% GlutaMAX, 1% sodium pyruvate , and 1% P/S. The cells were habitually subcultured at a density of 2.4 × 104 cells/cm2 in T75 flasks.The cell lines were subcultured every 72–96 h. Cell culture medium was refreshed the day before subculture. Cells were routinely plated in T75 flasks. The MCF7 cell line was cultured with minimum essential media  for optimal growth. MEM media was reconstituted with 10% fetal bovine serum , 1% GlutaMAX (GIBCO), 1% nonessential amino acids  and 1% penicillin/streptomycin . Cells were routinely subcultured at a density of 1.8 × 10Each cell line was cultured strictly following the directions and protocol provided by the commercial brand suppliers to guarantee their optimal growth. The goal of this work was to study differential responses to metabolic inhibitors, and to perform dynamic flux analysis of metabolic activities and paths by isotope labeling approaches. Accordingly, the culture of all cell lines under the same media formulation have would affect their optimal proliferative and growth capacity, and thus possibly masking the results by an undesired challenging effect. Nonetheless, the original cell culture formulations provided by brand indications are supplied within optimal molar ranges for glucose, glutamine, pyruvate, amino acids, among other metabolites.The transaminase inhibitor aminooxyacetate (AOA), glutamate dehydrogenase inhibitor epigallocatechin gallate (EGCG), mitochondrial complex I inhibitor phenformin hydrochloride (Phem), and mitochondrial membrane uncoupler BAM15 were obtained from Sigma-Aldrich. The PI3K inhibitor BKM-120 was obtained from Caymanchemic. All the drugs were prepared following brand-protocol instructions. Concentrated stock solutions were routinely aliquoted and stored at −20 °C. Working solutions were freshly prepared for each set of experiments.4 cells/well, whereas the cell lines ZR751, SKBR3, MDA-MB-231 and MDA-MB-468 were seeded at 8 × 103 cells/well. All the cell lines were maintained in the wells with 100 μL of their corresponding culture medium. After plating, the metabolic drugs were added, and the plates were incubated for 96 h at 37 °C.Experiments were performed in real time by directly seeding the cells in flat bottom black 96-well plates for further fluorescence analysis. Each cell line was seeded in the wells with their corresponding culture medium. Specifically, MCF7 cells were seeded at 1 × 10® cell viability assay (Promega) under the conditions described in each experiment. CellTiter was assessed according to the manufacturer’s protocol, and the fluorescence amount obtained was proportional to the number of viable cells. Viability was expressed with respect to the percentage of untreated cells (100%). Blank wells were included on each plate to measure the fluorescence from serum-supplemented culture medium in the absence of cells. Then, the cell plates were incubated for 20 min at 37 °C, and the fluorescence was measured in a GloMax®-Multi+ Detection System (Promega).After 96 h of treatment, cell viability was monitored using a CellTiter-BlueAll experiments were performed five independent times in quadruplicate replicas for each experimental condition. Data analysis was performed by calculating the average value for all replicas of each experimental condition, and then media-blank values were subtracted from averages. Finally, data were expressed as percentages of viability relative to control untreated cells (100%).Cells were pulsed with heavy isotopes  dissolved in the corresponding routine media for MCF7 and MDA-MB-231 cell lines. Individual wells of a 6-well plate were washed with ice-cold PBS and then extracted in 0.5 mL extraction buffer . Extracts were centrifuged at maximum speed and stored at −80 °C.LC-MS was carried out using a Thermo Ultimate 3000 HPLC in line with a Q Exactive mass spectrometer. A 32 min gradient was developed over a 100 mm × 4.6 mm ZIC pHILIC column with a guard column (Merck-Millipore) from 10% buffer A (20 mM ammonium carbonate) and 90% buffer B (acetonitrile) to 95% buffer A and 5% buffer B. Samples were acquired in positive-negative switching mode, and a standard ESI source and spectrometer settings were applied . Metabolites were identified by standard metabolite matching to m/z and retention time. Integrated peak areas and label incorporation were quantified using AssayR .We have recently found that breast cancer cells representative of the clinical subtypes featuring positive expression to hormone receptors or triple-negative expression displayed significant differences in mitochondrial pH . MitochoChanges in breast cancer cell metabolism have already been documented, and foundational works have already shown differential responses to single metabolic inhibitor treatments, metabolite availability selective conditions, as well as by metabolomic studies primarily using unlabeled total metabolite content analysis. Accordingly, following some insights provided by previous works, in the present study the degree of glycolytic flux dependence was tested by sensitivity responses to phenformin and BKM120 treatments ,57. In aThe breast cancer cell lines showed the same pattern of response for the two experimental strategies selected for examining their glycolytic dependence . For theCollectively, these data initially indicate two differential phenotypes for glycolytic dependence between the tested breast cancer cell lines. Eventually, the SKBR3 cell line was found to show intermediate behavior between the other two phenotypes.Consistent with the findings described above, we further investigated whether these two phenotypes coincide with the cellular reliance on TCA and mitochondrial oxidative phosphorylation activities .Treatments with the transaminase inhibitor AOA short circuits the mitochondria-cytosol transitions of reducing power conducted by the aspartate-malate shuttle. Consequently, mitochondrial TCA activity is collapsed by AOA treatment. Again, the breast cancer cell lines showed two clearly different responses to AOA A. The MCSince TCA activity inherently requires a level of mitochondrial oxidative phosphorylation over a threshold, breast cancer cell lines that present sensitivity to TCA collapse should similarly be sensitive to mitochondrial membrane uncoupling. Accordingly, breast cancer cell lines were tested for the presence of the mitochondrial membrane uncoupler BAM15. In a recently published study, we tested BAM15 collateral toxicity in control experiments and did not observe side effects on breast cancer cells. The results of the BAM15 treatments corroborated the two phenotypic differences already found for the AOA treatments B. The MDThe results described above point to two potential metabophenotypes. Interestingly, the metabophenotypes correlated well with the proliferative capacity already reported for breast cancer cell lines. Moreover, the less proliferative MCF7 and ZR-751 cell subtypes displayed the same responses to the different metabolic inhibitors tested and the more proliferative MDA-MB-231 and MDA-MBD-468 cell subtypes displayed a common response between them that was clearly different from that of the other cell lines.Glutamine and glutamate present several metabolic destinations and are key metabolites that sustain the production of nonessential amino acids (NEAA) that are crucial for cell proliferation. It has been recently demonstrated that glutamate is preferentially metabolized by transaminase or dehydrogenase activity depending upon the proliferative capacity of the cells A. SimilaAccordingly, to test whether breast cancer cells also showed a differential response to glutamate and NEAA requirements, the cells were treated with the GDH inhibitor epigallocatechin-3-gallate (EGCG). Breast cancer cell lines displayed the expected behavior depending upon their proliferative degree, i.e., the less proliferative MCF7 and ZR751 cells were sensitive to EGCG treatment, thus displaying almost a total loss of cell viability, whereas MDA-MB-231 and MDA-MB-468 cells showed a clearly milder decrease in cell viability of 42% and 45% respectively B.As already shown in Among these two metabophenotypes, the SKBR3 cell line interestingly seemed to confirm a different behavior in response to metabolic inhibition. On the one hand, it showed a milder dependence on glycolysis than the cell lines MCF7 and ZR751, as indicated by its intermediate level of sensitivity to phenformin and BMK120 treatments between the other two cellular metabophenotypes . On the In summary, breast cancer cell lines showed two clearly differentiated metabophenotypes . Of note13C-pyruvate isotope cell labeling experiments were performed to decipher whether the major dependence on glycolysis or TCA activity already observed was supported by a predominant metabolic route and/or enzymatic activity.Next, further experiments were performed to investigate the metabolic mechanisms, in terms of dynamic metabolic flux, that potentially generated the metabophenotypes found in this work. Consequently, 13C-pyruvate pulses were performed to investigate whether the breast cancer cell lines that already pointed to display different metabophenotypes might also show a preferential pyruvate destination via PDH or PC activities.Pyruvate labeling pulses were chosen to perform metabolic flux dynamics to directly analyze the preferential mitochondrial destination of pyruvate. TCA reliance and the preferential biosynthetic pathways are determined by pyruvate metabolic destinations that are regulated by PDH and PC enzymatic activities. Hence, to primarily focus on these two pathways, 13C-pyruvate labeled in its three carbon atoms can be metabolized in mitochondria by two main pathways that are regulated by pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) activities. Pyruvate is decarboxylated by PDH to provide acetyl-CoA into TCA, whereas pyruvate is converted to oxalacetate (OAA) by PC, which in turn is mainly redirected to anabolic routes (As illustrated in c routes A. Acetylc routes A.Cancer cells with a higher energetic and biosynthetic demand (via TCA anaplerotic routes) predominantly exhibit high PDH activity ,61. The In contrast, hormone-positive MCF7 breast cancer cells, which were more dependent on glycolysis than on TCA activity, showed noticeably predominant PC activity A. The prImportantly, these results visibly support the presence of two different metabophenotypes: the PDH predominant phenotype displaying stronger reliance on TCA activity in MDA-MB-231 triple negative breast cancer cells and the phenotype showing larger PC activity that is associated with a stronger dependence on glycolysis in MCF7 hormone receptor-positive breast cancer cells.13C-glutamine isotope cell labeling experiments were conducted to further investigate metabolic mechanisms underlying the metabophenotypes found in this work  was of particular relevance. Interestingly, these results were in correlation with the oncogene c-Myc expression levels between breast cancer cell lines representative of clinical subtypes . In addiMoreover, there have been important contributions performed by unlabeled metabolomic analysis to obtain quantitative data about total metabolite contain breast cancer cell models. Some noticeable results were obtained but the data were not totally concordant displaying some discrepancies ,50,51,52Under these premises, as already discussed by the aforementioned authors, further steps to address metabolic phenotypic differences between breast cancer cell subtypes should be performed by dynamic metabolic flux analysis for concrete pathways, starting by central metabolic routes, such as glucose preferential usage and TCA reliance. Accordingly, the present work was designed to gain insights in that direction. First, to identify different functional responses (sensitivity or resistance primarily) to alterations provoked by the selected inhibitors that might be in concordance with breast cancer subtypes. Secondly, to investigate possible metabolic flux preferences by stable-isotope dynamic labeling for key targets, such as pyruvate, based on preferential PDH or PC activities to quantitatively analyze the degree of TCA reliance, and other central pathways as glutamine/glutamate metabolism.Thus, in the present work, the main goal was to investigate whether metabolic data obtained by this combination of functional and metabolic flux analysis approaches might complement the current clinical classification of breast cancer.Altogether, our data clearly displayed different metabophenotypes that interestingly correlated well with the clinical classifications. According to a systemic perspective, this study selected several inhibitors as key targets. Thus, the main goal was to generate a metabolic profile that could be used to search for different phenotypes and evaluate whether the potential metabophenotypes might be associated with the current breast cancer classifications. The profiling was designed to select principal metabolic pathways that support cellular function in terms of both energy and biosynthetic demands as targets. Accordingly, the metabolic dependence on glycolytic flux and/or mitochondrial oxidative metabolism via TCA activity was analyzed based on the cellular responses to small molecules that inhibit key enzymatic targets for sustaining the activity of the proposed pathways. In particular, BKM120 and phenformin were used to impact glycolysis while AOA and BAM15 were used to short circuit TCA activity and mitochondrial oxidative metabolism. These drugs have been extensively used for metabolic profiling. BKM120 is an inhibitor of PI3K activity, which in turn produces an inhibitory effect on the PI3K-Akt axis that ultimately impacts glycolysis by decreasing its flux ,57,62. I13C-pyruvate pulse experiments (Specifically, the cell lines MCF7 and ZR751, which are representative of the hormone receptor-positive breast cancer cell subtype, showed stronger glycolytic dependence . Therefoeriments . Labelederiments .On the other hand, the cell lines MDA-MB-231 and MDA-MB-468, which are representative of the triple-negative subtype, displayed a different metabophenotype featuring their main reliance on TCA activity and mitochondrial oxidative metabolism. This phenotype was clearly supported by the striking sensitivity to AOA and BAM15 treatments , whereas2 [Notwithstanding the reliability of these findings, several interesting questions remain open. In particular, the resistance of MCF7 and ZR751 cells to AOA treatments is intriguing. However, our data tentatively indicate that these cell lines might recycle cytosolic NAD+ to sustain glycolysis, even if the aspartate-malate shuttle was collapsed by AOA transaminase inhibition. Our data indicated high PC activity, which was clearly deduced from the presence of m+3 citrate and m+3 aspartate . Notably2 . Of note2 .The SKBR3 cell line, which is representative of the HER2+ breast cancer subtype, displayed an intriguing intermediate metabolic phenotype between the other two cell lines already discussed above. Overall, as illustrated by our results (summarized in Nevertheless, further studies should be performed to gain more detailed information to clarify whether SKBR3, as a representative cell line of the HER2-positive breast cancer subtype, represents another independent metabophenotype or is just a special type within one of the already well-described metabophenotypes.Confirming the scope of the proposed research, this work showed that breast cancer cell subtypes present different metabophenotypes. Importantly, these metabophenotypes are clearly correlated with the current clinical classification. These results pave the way for further studies to gain a detailed understanding of many particular metabolic routes and key targets that might be beneficial to sustain the already discovered metabophenotypes. Thus, in the future, important metabolic parameters could be crucial for potential diagnostic, prognostic and therapeutic developments.


CS-M08sh exhibited the highest K concentration (14.50 g kg−1), an increase of 29% and 49% over TM-1 and AM UA48, respectively. Other CS lines had higher Mg, P, and S than the controls. A similar trend was found at the MS location. This research demonstrated that chromosome substitution resulted in higher seed macronutrients in some CS lines, and these CS lines with a higher content of macronutrients can be used as a genetic tool towards the identification of desired seed nutrition traits. Also, the CS lines with higher desired macronutrients can be used as parents to breed for improved nutritional quality in Upland cotton, Gossypium hirsutum L., through improvement by the interspecific introgression of desired seed nutrient traits such as Ca, K, P, S, and N. The positive and significant (p ≤ 0.0001) correlation of P with Ca, P with Mg, S with P, and S with N will aid in understanding the relationships between nutrients to improve the fertilizer management program and maintain higher cottonseed nutrient content.Nutrients, including macronutrients such as Ca, P, K, and Mg, are essential for crop production and seed quality, and for human and animal nutrition and health. Macronutrient deficiencies in soil lead to poor crop nutritional qualities and a low level of macronutrients in cottonseed meal-based products, leading to malnutrition. Therefore, the discovery of novel germplasm with a high level of macronutrients or significant variability in the macronutrient content of crop seeds is critical. To our knowledge, there is no information available on the effects of chromosome or chromosome arm substitution on cottonseed macronutrient content. The objective of this study was to evaluate the effects of chromosome or chromosome arm substitution on the variability and content of the cottonseed macronutrients Ca, K, Mg, N, P, and S in chromosome substitution lines (CS). Nine chromosome substitution lines were grown in two-field experiments at two locations in 2013 in South Carolina, USA, and in 2014 in Mississippi, USA. The controls used were TM-1, the recurrent parent of the CS line, and the cultivar AM UA48. The results showed major variability in macronutrients among CS lines and between CS lines and controls. For example, in South Carolina, the mean values showed that five CS lines  had higher Ca level in seed than controls. Ca levels in these CS lines varied from 1.88 to 2.63 g kg Macronutrients are involved in different physiological and metabolic processes. For example, K, Zn, and P are involved in CO enzymes ,2; P is  enzymes ,4; Mg, S enzymes  due to t enzymes ,6. Nutri enzymes ,7,8. Mac enzymes . In addi enzymes . The sur enzymes .Medicago truncatula [Nutrient accumulation in seeds is controlled by several processes, including nutrient uptake, translocation, redistribution, and accumulation ,12. Inheuncatula ; limiteduncatula . While Quncatula ,21, and uncatula ,23, it iuncatula .Molecular and genetic research on cottonseed macronutrients is needed. Chromosome substitution can be used to infuse new genetic variation into a crop species, some of which could significantly affect seed macronutrient contents. Since the genetics of cottonseed traits associated with chromosome substitution germplasm lines is scarce , the objp < 0.0001) influenced by location, line, and their interactions (p < 0.0001) influenced by line, and location × line interactions. The effects of line, location, and their interactions on some macronutrients such as Mg, P, C, N, and S indicated the importance of the environmental factors of each location and the genetic makeup of each line on the accumulation of these nutrients in the cottonseed.ANOVA analysis showed that Mg, P, C, N, and S were significantly , a significant increase over TM-1 (−1. Line CS-T04 had the highest concentration of Mg (2.85 g kg−1). Lines CS-M04 and CS-T04 had higher P concentrations than the controls and the rest of the CS lines, with CS-M04 being the highest (5.38 g kg−1). Both CS-M08sh and the control AMUA48 exhibited the highest concentration of C, but four CS lines  were higher than TM-1. Seven CS lines were higher in S than the controls, with CS-T04 being the highest. Four  out of nine lines had higher N than the controls, with CS-T02 having the highest concentration (47.79 g kg−1). Lines CS-B02, CS-B04, CS-B08sh, CS-M02, CS-T08sh, CS-T04, and CS-T02 had higher S than the controls, with CS-T04 being the highest (5.39 g kg−1).In South Carolina, the mean values showed that five CS lines  had higher Ca levels in seed than both controls . Ca levever TM-1 . Lines CIn MS, the concentration of Ca was higher in CS-M08sh, CS-T02, and CS-T04, with CS-M08sh being the highest . PotassiTwo CS lines (CS-T02 and CS-T04) showed higher Ca in both locations compared to the controls. CS-T04 had higher Mg concentrations in both locations. Three CS lines  showed superiority over TM-1, but only CS-T02 and CS-T04 showed superiority over both controls. Cultivar AM UA48 was the best performer for C concentration in both locations. CS-T02 exhibited a higher N concentration than the controls. Four CS lines  exhibited a higher concentration of S than the controls.p ≤ 0.0001) correlation in both locations; for example, P positively correlated with Ca, P positively correlated with Mg, S positively correlated with P, and S positively correlated with N . A similar observation was noticed for lines CS-B02, CS-B04, CS-B08sh, CS-M02, CS-M04, CS-M08sh, CS-T02, and CS-T04 for Mg concentration; lines CS-M04 and CS-T04 for P concentration; and lines CS-B08sh, CS-T02, CS-T04, and CS-T08sh for N concentration. Other CS lines showed negative responses for some macronutrients; for example, CS-M02 and CS-M04 showed reduced N compared to the controls.Since cotton chromosome substitution lines are near-isogenic to the recurrent parent TM-1 with one chromosome or chromosome arm introgressed from sly used ,29,30,31ronments . In thisronments ,33. In aIn MS, similar responses for macronutrients in CS lines were shown when compared to the controls, exhibiting positive, negative, or non-responsive effects of chromosome or chromosome arm substitution as in the SC location. The higher concentration of some nutrients in some CS lines reinforced our explanation that the accumulation of seed nutrients in CS lines were due to introgressed chromosomes as these CS lines are near-isogenic and the only differences between these CS lines and the parent or control are the chromosomes or chromosome arms under study. For example, Ca concentration was high in lines CS-M08sh, CS-T02, and CS-T04 compared with the controls. A similar observation was recorded for K in CS-M02 compared to TM-1; CS-T04 and CS-T08sh for Mg compared with the controls; lines CS-M04, CS-M08sh, CS-T02, and CS-T04 for P compared with the controls; lines CS-B08sh, CS-M08sh, and CS-T02 for N compared with the controls; and lines CS-B02, CS-B04, CS-B08sh, CS-M04, CS-M08sh, CS-T02, CS-T08sh for S content. The negative response of nutrients in some CS lines was shown in, for example, CS-B02, CS-B04, and CS-M04 for C. It must be noted here that some seed nutrients showed different location responses in some CS lines, reflecting the significant effects of environmental conditions at each location. Previous research on other seed traits also showed different responses, depending on the environmental factors of each location, including soil type, temperature, drought, and other environmental conditions . For exaAmong the macronutrients evaluated in the CS lines, some showed a positive response at one location, while others showed a negative response at the same location, and others showed a positive response in both locations. Previous research conducted on other seed traits found similar observations. For example, ref.  found hoA similar observation was noted at the Mississippi location, with different responses of the CS lines due to location effects. These observations demonstrated that chromosome substitution resulted in a higher concentration of macronutrients in some CS lines, with no change or reduced concentrations in others. The positive or negative response of these macronutrients was due to both location and CS line genotype, as explained by others above ,34. The p ≤ 0.0001) positive correlation between P and Ca, P and Mg, S and P, and S and N in both locations may suggest the stability of these traits across locations, and these traits may help growers for fertilizer management and advance our knowledge of nutrient uptake and translocation. The wide distribution, especially in Ca, K, N, and S, and significant variability of nutrients among CS lines and between CS lines and the controls signified the potential use of CS lines with high levels of these nutrients for future breeding programs to increase the nutritional quality of cottonseed. The right skewed, left skewed, or peak trends, shown by the nutrient distribution between CS lines, showed complex interactions of these nutrients in these lines.The positive correlation of P with Ca and Mg; N with Ca and K; S with Ca, Mg, P, and N in SC; the positive correlation of P with Ca and Mg; N with Mg; the negative correlation of N with P; and the negative correlation of S with P and positive correlation with N in MS indicated that the correlation between some nutrients can be influenced by complex genetic and environmental factors . The conG. tomentosum and G. mustelinum is possible. This is the first report on the effects of chromosome or chromosome segment substitution on macronutrients in cottonseed. We were able to discover specific chromosomal associations with cottonseed macronutrient contents gene(s) and developed some novel germplasm with the gene(s) of improved macronutrient contents from interspecific crosses using the wild species.This research demonstrated that the improvement of cottonseed nutritional quality using chromosomes or chromosome segments from the wild species G. barbadense L. (CS-B), G. tomentosum Nutt.Ex Seem (CS-T), and G. mustelinum Watt (CS-M), respectively, into Upland cotton , the recurrent parent of the CS lines, and AM UA48, Reg. No. CV−129, . The nine CS lines were: CS-B02, CS-B04, CS-B08sh, CS-M02, CS-M04, CS-M08sh, CS-T02, CS-T04, CS-T08sh; TM-1 and AM UA48 were used as controls. The developed lines are described in detail elsewhere [262829Nine cotton chromosome substitution lines were used and represented by the substitution of chromosome two, four, and the short arm of chromosome eight (8sh) from three tetraploid species of ewhere 26282930,403 in a microwave digestion system. The concentrations of nutrients were determined by inductively coupled plasma spectrometry (ICP) (Thermo Jarrell-Ash Model 61E ICP and Thermo Jarrell-Ash Autosampler 300) [The macronutrients Ca, K, Mg, P, C, N, and S were analyzed in the ground, dried whole seed samples. Collected seeds from each CS line and control were ground with a Laboratory Mill 3600 , and analyzed by digesting 0.5 g of dried ground seed in HNOler 300) . The nutler 300) ,42. Seedv/v HCl. Then, 5 mL of 5 M HCl and 5 mL of ammonium molybdate–ammonium metavanadate was used. The concentration of P was determined by a Beckman Coulter DU 800 spectrophotometer as previously described by others [The concentration of P in cottonseed was determined by the yellow phosphor vanado–molybdate complex method , and as y others ,42. The G. barbadense, G. tomentosum, and G. mustelinum, because an individual CS line is considered to be near-isogenic in a background of TM-1. Correlation analyses were conducted in SAS [Cotton lines were grown in a randomized complete block design with four replications within each location. Proc Mixed   was condd in SAS  using PrThe current research demonstrated the possibility of increasing major macronutrient  levels in cotton using wild species germplasm introgression. In this research, we used a comparative method  to detec


Compounds reduced cell colony formation of both cell lines and inhibited the growth and viability of IGR39 cell spheroids more efficiently compared to triple-negative breast cancer spheroids.Heterocyclic compounds are one of the main groups of organic compounds possessing wide range of applications in various areas of science and their derivatives are present in many bioactive structures. They display a wide variety of biological activities. Recently, more and more attention has been focused to such heterocyclic compounds as azoles. In this work, we have synthesized a series of new imidazole derivatives incorporating a benzenesulfonamide moiety in their structure, which then were evaluated for their cytotoxicity against human triple-negative breast cancer MDA-MB-231 and human malignant melanoma IGR39 cell lines by MTT assay. Benzenesulfonamide-bearing imidazole derivatives containing 4-chloro and 3,4-dichlorosubstituents in benzene ring, and 2-ethylthio and 3-ethyl groups in imidazole ring have been determined as the most active compounds. Half-maximal effective concentration (EC After huge breakthrough in pharmaceutical chemistry in early twentieth century, treating more and more previously incurable or difficult-to-treat diseases has become common practice in doctor’s offices nowadays. Research and synthesis of various functionalized heterocycle compounds have contributed significantly to this progress. Heterocycles are one of the largest classical divisions in organic chemistry and they play important role in modern drug design due to their wide range of biological properties . A lot oTherefore, based on recent studies, benzenesulfonamide-bearing imidazole derivatives were synthesized in this work. Two cancer cell lines, namely TNBC cell line MDA-MB-231 and human melanoma cell line IGR39 were selected to test cytotoxic activity and compound effect on cell colony-forming ability. Both cancer types are considered very aggressive and have a low survival prognosis. Resistance to drugs occurs quite often in the case of melanoma treatment with targeted therapeutics, due to BRAF mutations . More th2) was prepared in the reaction of amine 1 with 1-chloropropanone in small amount of water at reflux  was treated with various α-haloketones in water/1,4-dioxane solution to obtain compounds 4–9, which were later used as precursors for synthesis of corresponding imidazoles 10–15 in similar reaction conditions as were in product 3 synthesis (10–15 (or the rest in this work) can be purified without using any organic solvents. For purification, these imidazole derivatives can be dissolved into 5% sodium hydroxide aqueous solution instead. Then, after filtration, the acidification of the obtained solution with glacial acetic acid will provide purer forms of the desired products without any significant loss to the purified amount of it. We also wanted to modify some of the pharmacokinetic properties of 1H-imidazole derivatives 10–15 to see, how it would affect the cancer cells. We decided to perform S-alkylation of 1H-imidazole 11 to reach higher solubility in organic solvents and lipophilicity. As a result, in the first attempt, imidazole 11 was treated with ethyl iodide in DMF overnight to obtain compound 16. In a 1H NMR spectrum for 16, the triplet assigned to the protons in the CH3 group at 1.30 ppm and quartet assigned to the protons in the CH2 group at 3.15 ppm have proved the presence of ethyl moiety in the molecule. Elemental analysis data of 16 confirmed that imidazole 16 did not form hydroiodide salt. Moreover, to increase reaction rate, we used triethylamine as a base catalyst, which shortened the reaction time to 3 h.The 3-((2-oxopropyl)amino)benzenesulfonamide (t reflux . The strtomerism . Howeversolution . Moreoveynthesis  25]. We. We2) wa4–9 were dissolved in glacial acetic acid and treated with urea to afford oxoimidazoles 17–22  was used in reactions with α-haloketones in water/1,4-dioxane mixture to obtain precursors 25–30 for further synthesis of imidazoles 31–36 , that reduced cell viability below 50%, have been identified between non-hydroxylated compounds, especially with imidazolone fragment. 4-Hydroxybenzenesulfonamide derivatives with thioimidazole ring (compounds 31–36) were practically inactive against both tested cell lines. It is worthy to notice, that the most active compounds in separate groups contained either 4-chloro  or 3,4-dichloro  substituents. The least active compounds were those containing cyano group in phenyl ring (the only active compound was 8). Two compounds (23 and 16) contain 2-ethylthio and 3-ethyl substituents in imidazole ring, which could contribute to their high anticancer activity, and the importance of this fragment in this position could be of great interest in the future research.Tested imidazole derivatives showed different activity against human triple-negative breast cancer and human melanoma cell lines at 100 µM concentration . Almost 8, 16, 19, 23, 25, and 28, which reduced cell viability of at least one cell line below 10%, and they were then tested more thoroughly both against human cancer cell lines and normal cells lines (fibroblasts (HF) and endothelial cells (EC)). The effective concentrations that reduce cell viability by 50%  have been established  [Compound s series B. Moreov >50 µM) . In gene >50 µM) , and for >50 µM) . However >50 µM)  and ther >50 µM) . Meanwhi >50 µM) .Doxorubicin, which is considered as a chemotherapeutic agent against triple-negative breast cancer, reduces MDA-MB-231 cell viability below 50% at 5 μM concentration , and str8, 16, 19, 23, 25, and 28 were tested for their effect on IGR39 and MDA-MB-231 cell colony formation at 50% of their established EC50 concentrations.Both types of cancer are characterized as recurrent, thus we decided to test the compound effect on single cell ability to survive and proliferate using clonogenic assay. The most active agents 23, which was the most cytotoxic in MTT assay, showed comparable activity with compounds 8 and 16 against both cell lines. In the presence of compound 23, MDA-MB-231 cell colony area dropped down to 27.1 ± 6.6%, compared to the control (p < 0.05)  . All thrAs previously mentioned, the strategy of combinational therapy is being extensively studied and investigated whether this approach could reduce the resistance of cancer to available therapy. It has been established MERTK inhibitor UNC2025 reduces colony formation of BRAF mutant and BRAF wild-type cell lines at 300–500 nM concentration . Combina50 value could reduce MDA-MB-231 cell colony formation up to 40% [Many studies have been done on MDA-MB-231 cell colony formation in the presence of drugs used for triple-negative breast cancer treatment, novel compounds, or their combinations. Franco et al.  establisp to 40% . Combinap to 40% . In summ8, 16, 19, 23, 25, and 28 on melanoma IGR39 and triple-negative breast cancer MDA-MB-231 cell spheroid growth . Melting points were determined on a MEL-TEMP  melting point apparatus and are uncorrected. IR spectra  were recorded on a Perkin–Elmer Spectrum BX FT-IR spectrometer  using KBr pellets. The 1H and 13C-NMR spectra were recorded in DMSO-d6 on a Bruker Avance III  spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) calibrated from TMS (0 ppm) as an internal standard for 1H NMR, and DMSO-d6 (39.43 ppm) for 13C NMR. Elemental analyses  were performed on an Elemental Analyzer CE-440 . Compounds 2-23 and 25-42 synthesis methods, identification data and NMR spectra can be found in Reagents were purchased from Sigma-Aldrich . The reaction course and purity of the synthesized compounds were monitored by TLC using aluminum plates pre-coated with silica gel 60 F2. They were used until the passage of 20.The human melanoma IGR39 and the human triple-negative breast cancer MDA-MB-231 cell lines, as well as the human endothelial cells (EC) CRL-1730 were obtained from the American Type Culture Collection . Human foreskin fibroblasts (HF) CRL-4001 were originally obtained from ATCC and kindly provided by Prof. Helder Santos . IGR39, MDA-MB-231, EC, and HF were cultured in Dulbecco’s Modified Eagle’s GlutaMAX medium ). Medium was supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin (Gibco), and 10% fetal bovine serum (Gibco). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO3 cells/well). After 24 h, the cells were treated with 100 μM of various concentrations of tested compounds. Only medium without cells was used as a positive control, and the medium with 0.5% DMSO (Sigma-Aldrich Co.) served as a negative control. After 72 h, the medium from cells was discarded and the fresh medium containing 0.5 mg/mL of MTT solution (Sigma-Aldrich Co.) was added. Cell were incubated for the next 4 h. Then the liquid was aspirated, and the colored formazan product was dissolved in 100 μL DMSO (Sigma-Aldrich Co.). The absorbance was measured at 570 and 630 nm using a multi-detection microplate reader. Compound effect on cell viability was calculated using a formula:Cell viability was tested using 3--2,5-diphenyltetrazolium bromide  assay, as described elsewhere . BrieflyA—mean of absorbance of tested compound,A0—mean of absorbance of blank ,NCA—mean of absorbance of negative control .50 of the most active compounds, the same MTT procedure has been applied, only the compound serial dilutions from 50 µM to 1.56 µM have been made in a medium and added to the cells in triplicate repeats. EC50 value that represent the concentration of a compound causing 50% reduction of cancer cell metabolic activity has been calculated using Hill equation.For establishing EC2 cells/well) and grown in DMEM GlutaMAX supplemented with 10% FBS  and 1% antibiotics  at 37 °C in a humidified atmosphere containing 5% CO2. After 24 h, the fresh medium containing tested compound 8, 16, 19, 23, 25, and 28 at a concentration representing 50% of calculated EC50 values were added to cells, followed by incubation at 37 °C in a humidified atmosphere containing 5% CO2 for the next 10 days (for IGR39 cell line) and 12 days (for MDA-MB-231 cell line), replenishing the media with the compounds once a week. Cells treated with medium containing 0.5% DMSO served as a negative control. After incubation, the colonies were stained with 0.1% Crystal Violet (Sigma-Aldrich Co.) solution. To start with, the media from cells was removed and the cells were washed once with sterile PBS. Then, the cells were fixed in a 4% formaldehyde (Thermo Scientific) solution and washed with PBS two times to remove the fixative, and stained with Crystal Violet solution for 20 min. After the stain had been removed, the remaining stain residues were washed three times sterile water. Lastly, the plates were dried overnight, and imaged with SYNGENE G:BOX gel doc system , using GeneSys software version 1.5.5.0, followed by quantification Gene tools software version 4.3.8.Colony formation assay was applied to evaluate the inhibitory effect of the most active compounds on cell ability to survive, and proliferate by forming colonies, as described elsewhere . Briefly2. Then, cells were trypsinized, centrifuged, and seeded into ultra-low attachment 96-well plate in a volume of 100 µL . The plate was placed on a magnetic drive  and incubated for 2 days at 37 °C in a humidified atmosphere containing 5% CO2. Then the fresh medium containing tested compound concentration representing 100% of calculated EC50 values was added to the wells. The spheroids were captured every two days using the Olympus IX73 inverted microscope (OLYMPUS CORPORATION). The quantitative analysis of compound anticancer activity in spheroids was performed using ImageJ  and Microsoft Office Excel 2016 software.Magnetic 3D Bioprinting method was used to form spheroids, as described elsewhere . Brieflyp < 0.05.All biological experiments were repeated at least three times, calculating the mean and standard deviation. The data was processes using Microsoft Office Excel 2016 software . Statistical analysis was performed by using Student’s t-test. The level of significance was set as In this study, the chemical transformations of benzenesulfonamides were carried out and a series of 1-substituted imidazoles with aliphatic, aromatic fragments were synthesized and evaluated for their anticancer activities in 2D and 3D models in vitro.In the MTT assay, compounds were more effective against human triple-negative breast cancer MDA-MB-231 cell line, compared to malignant melanoma IGR39 cell line. Benzenesulfonamide-bearing imidazole derivatives containing 4-chloro and 3,4-dichlorosubstituents in benzene ring, and 2-ethylthio and 3-ethyl groups in imidazole ring have been determined as the most active compounds. These compounds reduced cell colony formation of both cell lines. Tested derivatives inhibited the growth and viability of IGR39 cell spheroids more efficiently compared to triple-negative breast cancer spheroids.The obtained results suggest that benzenesulfonamide-bearing imidazole could be a suitable core for the anticancer compounds against such aggressive and invasive tumour types as malignant melanoma and triple-negative breast cancer.