MLL-rearranged leukemia. Serendipitous observations during high-throughput drug screens indicated that the use of DOT1L inhibitors might be expandable to multiple myeloma (MM). Through pharmacologic and genetic experiments, we could validate that DOT1L is essential for growth and viability of a subset of MM cell lines, in line with a recent report from another team. In vivo activity against established MM xenografts was observed with a novel DOT1L inhibitor. In order to understand the molecular mechanism of the dependency in MM, we examined gene expression changes upon DOT1L inhibition in sensitive and insensitive cell lines and discovered that genes belonging to the endoplasmic reticulum (ER) stress pathway and protein synthesis machinery were specifically suppressed in sensitive cells. Whole-genome CRISPR screens in the presence or absence of a DOT1L inhibitor revealed that concomitant targeting of the H3K4me3 methyltransferase SETD1B increases the effect of DOT1L inhibition. Our results provide a strong basis for further investigating DOT1L and SETD1B as targets in MM.The histone 3 lysine 79 (H3K79) methyltransferase (HMT) DOT1L is known to play a critical role for growth and survival of  MM is an aggressive hematologic cancer characterized by the monoclonal expansion of plasma cells secreting high amounts of immunoglobulins. Despite recent progress in therapies, MM still remains an incurable disease and emergence of drug resistance is unfortunately a common feature –4. New tDue to the high production of immunoglobulins, the protein production machinery in the ER of MM cells is under stress, which is mitigated by a set of pathways known as the unfolded protein response (UPR) in order to ensure survival –10. The in vitro and inhibits growth of established MM xenografts in mice. Our findings confirm a recent study in which DOT1L was suggested as a target in MM [MLL-rearranged leukemia, where fusion proteins of MLL with a variety of partners recruit DOT1L to a set of genes (e. g. HOXA9) that are critical to maintain leukemic cells in an undifferentiated state [Through pharmacologic and genetic approaches, we found that inhibition of DOT1L, a H3K79 methyltransferase, profoundly reduces viability of a subset of MM cell lines et in MM . Selectiet in MM , 15 haveet in MM  has evened state , 16–21. ed state .MLL-rearranged leukemia [DOT1L in 6 MM cell lines in the context of a whole-genome pooled CRISPR screen [Previous studies reported that epigenetic factors are involved in the biology of MM . During leukemia , 16, we leukemia , we obseleukemia  in a dosleukemia  , and continuous intravenous infusion [in vivo MM mouse xenograft model. NOD-SCID mice bearing established MM1-S tumors were treated with 75 mg/kg Compound 11 s. c. once or twice daily. Significant tumor growth inhibition was observed with the twice-daily schedule, while a once-daily schedule only led to a moderate effect on tumor growth . Interestingly, genes involved in the ER stress pathway and protein translation, including ATF4, ASNS, ERN1 (IRE1α), DDIT3, HERPUD1, and MYC were among these genes . This relatively long treatment duration was chosen, because DOT1L inhibitors are known to act slowly , potentise genes  and 2C. se genes . H3K79mese genes .ATF4  could explain the observed changes using ISMARA . This coATF4  nor ISMAATF4  showed aATF4 .P-value ≤ 10-10, average difference between groups at least 4-fold), we noted a large number of differences: 1557 transcripts for a total of 1089 genes showed higher expression in sensitive cell lines, 637 different transcripts for a total of 271 genes showed lower expression  were not among the genes with differential baseline expression. We also did not observe consistent differences that would distinguish sensitive from insensitive cell lines in terms of basal levels or DOT1L inhibitor-induced changes of the key ER stress regulators IREα  or phospho-PERK   .To quantify the antibody production in MM cell lines, we measured secreted light chains by ELISA assays. No significant difference between sensitive and insensitive cell lines was observed in absence of DOT1L inhibitor , and theSETD1B in the DOT1L inhibitor arm relative to the control arm was the most pronounced sensitizing effect  identification of sensitizers (i. e. genes whose inactivation further enhances the effect on cell viability) could have practical therapeutic value and (2) identification of rescuers (i. e. genes whose inactivation restores viability in presence of DOT1L inhibitor) could yield mechanistic insights into the DOT1L dependency of some MM cell lines. Four sensitive, one intermediate, and one insensitive cell line were screened. Following transduction with the sgRNA libraries and selection, cells were treated with either DMSO or the DOT1L inhibitor Compound 11 . 14 daysg effect . Among tSETD1B is a member of the MLL H3K4me3 methyltransferase family [SETD1B sgRNA construct that led to SETD1B protein loss upon induction  occurred in the combination group, overlapping substantially with each of the two other experimental conditions  [SETD1B targeting. Furthermore, protein synthesis in RPMI8226 cells was reduced in a combinatorial manner as well  –35, were as well . ProteinSETD1B KO) or a combination of both. Genome-wide H3K79me2 and H3K4me3 profiles were then acquired by ChIP-seq  and KMS-27 Cas9 (insensitive) cells containing inducible sgSETD1B were treated with either DMSO, 1 μM SGC0946, dox  [When we conducted genome-wide CRISPR screens in the presence or absence of a DOT1L inhibitor in MM cell lines, the H3K4me3 methyltransferase SETD1B emerged as a robust sensitizer. We could validate that targeting lude MYC  and the , PRDM1) . In summMLL-rearranged leukemia, DOT1L is thought to maintain expression of leukemia stem cell genes through recruitment via MLL fusion proteins to these gene loci, leading to higher H3K79me2 levels. To investigate whether DOT1L exerted such a locus-specific effects on its target genes or on ATF4 target genes in MM, we conducted ChIP-seq experiments. These studies revealed no distinct H3K79me2 profile at genes that are modulated upon DOT1L inhibition. Moreover, reduction of H3K79me2 at those modulated genes was comparable in sensitive and insensitive cells, although mRNA expression was not modulated in the latter. Thus, we did not find distinguishing behavior of MM cells on the chromatin level. Remarkably, SETD1B knockout led to very few differential H3K4me3 peaks in both sensitive and insensitive cells (data not shown) despite SETD1B being a H3K4 methyltransferase. This could well be a consequence of redundancy among H3K4 methyltransferases, although it raises the question what mediates the observed sensitization and gene expression modulation.In SETD1B enhances phenotypic and transcriptional effects of DOT1L inhibition in sensitive MM cell lines, which may be an additional therapeutic angle. To our knowledge, no selective SETD1B inhibitors have been described yet, and our data questions whether its methyltransferase activity is critical in the MM context. Additional work is needed to address this question and guide drug discovery.In summary, our discovery indicates a novel opportunity for DOT1L inhibitors in cancer treatment. Concomitant targeting of For additional information, see Supplementary Materials and Methods.A whole-genome CRISPR screen was performed in 6 MM cell lines , in which each gene was targeted by 10 sgRNAs. Experimental procedures were detailed previously , 42. FolThe drop-out value for each sgRNA was calculated using the Bioconductor R package EdgeR . The plaTo identify statistically significant sensitizers and rescuers between the treatment and DMSO groups of cell lines at day 14, for each sample a gene-summarized value was first calculated as the median count of sgRNAs targeting it. The edgeR package was then used to fit negative binomial generalized log-linear model to the summarized count data and to conduct gene-wise statistical tests for the contrast ~Treatment-DMSO.Details can be found in the Supplementary Material and Methods. Briefly, cells were crosslinked with 1% formaldehyde and lysed in SDS buffer. DNA was fragmented by sonication. ChIPs for H3K79me2 and H3K4me3 were then performed and eluted DNA fragments were subjected to NGS.For detection of newly synthetized proteins, Click-iT reagents and buffers were used . MM cells were treated during 7 days either with DMSO, 1 μM of SGC0946 or 1 μM ribociclib. As a control, cells were treated with 20 μM cycloheximide for 8 h. For analysis of newly synthetized proteins, cells were then washed and grown in methionine-free media for 30 min containing Click-iT HPG (50 μM). After incubation, cells were washed with PBS and then fixed with 3.7% formaldehyde, washed with 3% BSA and then 0.5% Triton-X-100 was added to the cells. For the detection of Click-iT HPG, Click-iT reaction cocktail containing the AlexaFluor 488 azide. The analysis of nascent protein synthesis was then performed by flow-activated cell sorter (FACS) .


Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us.These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile. Leukemia is the most common malignancy and the second most frequent general cause of childhood death. It is classified as acute lymphoid of the B- or T-lineage being the most prevalent type in children, and as acute myeloid. Although tremendous improvements have been made in the treatment of leukemia in the past few years, there is still a large proportion of patients who do not benefit from the available therapy. The overall survival of pediatric acute lymhoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients treated with the current chemotherapy regimens is above 80 and 70%, respectively , 2.The standardized treatment protocols consist of the same repertoire of cytostatic drugs which have been used for the past decades. They differ in the drug dosage, the time of administration and in the drug combinations. State-of-the-art genomic techniques have enabled the identification of new genetic alterations, which could be targeted by novel compounds; however, genetic-based approaches have not fully revealed the direct cause of inter-individual differences in the sensitivity to cytostatic drugs –6. ThereWe turned our attention to cellular metabolism which represents a limiting process in cell proliferation and survival. Recent data have indicated that a number of cytostatic drugs trigger metabolic reprogramming, which impairs their effects and, in the long run, could cause resistance. Our group previously described that leukemia cells treated with L-asparaginase (ASNase) reprogrammed their metabolism and increased fatty acid oxidation (FAO) together with autophagy in order to compensate for asparagine and glutamine depletion . PharmacOur aim was to investigate how the basal metabolic profile of leukemic cells interferes with the effectiveness of the compounds currently used for treatment. We focused on cytostatic drugs used in the treatment of childhood leukemia with an emphasis on ASNase. ASNase represents a crucial drug used in the treatment of ALL; it is also incorporated in the front-line treatment of adult leukemia , 10. Unf2). The cultured cells were split every 2 to 3 days and maintained in exponential growth phase.Human B-cell precursor leukemia cell lines , T-cell leukemia cell lines , AML cell lines  and cell lines derived from the blast crisis of chronic myeloid leukemia (CML), which manifest as AML  and as ALL (BV-173), were used . All samples were obtained with the informed consent of the children’s parents or guardians. The study no. 201528848A was approved by the Ethical Committee of the University Hospital Motol, Prague, Czech Republic. Healthy controls were isolated from buffy coats  using Ficoll-Paque PLUS . To enrich samples for B-lymphocytes, buffy coat was pre-treated with RosetteSep™ Human B Cell Enrichment Cocktail  prior to Ficoll-Paque PLUS.The isolated blasts were maintained in RPMI-1640 medium with GlutaMAX™ supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 μg/mL). For the MTS assay, insulin-transferrin-sodium selenite supplement was added to the culture media .3H] palmitic acid  in the presence or absence of etomoxir . Medium was collected to analyze the amount of released 3H2O that was formed during the cellular oxidation of [3H]-palmitate -palmitate.We used 19 human leukemia cell lines of different origin , 4 T-ALL and 7 of myeloid origin ); in all of them, we determined glycolytic function, mitochondrial respiration and FAO  revealed that glycolysis and mitochondrial respiration, but not FAO, clustered leukemia cells separately according to the hematopoietic lineage from which they were derived Fig. a, b, S1.Next, we tested which cellular processes could participate in a given metabolic state. The relative activity of major signalling pathways influencing basal metabolism was determined in 15 leukemia cell lines of different origin  and Daunorubicin (DNR). The last 2 drugs are included in the treatment of a broader spectrum of cancers. First, sensitivity to ASNase did not correlate with sensitivity to VCR and DNR, which demonstrated that the tested leukemia cell lines are not generally sensitive or resistant to cytostatic drugs. However, due to their intrinsic properties they respond independently to drugs with different mechanisms of action  and their sensitivity to cytostatic drugs. For this purpose, we used Canonical Correlation analysis (CCA), which explores the relationship between 2 multivariate sets of variables. In our case, set 1 included all metabolic parameters, and set 2 consisted of the lineage origin and IC50s  that effectively decreased the level of ATP, but at the same time did not completely inhibit cell proliferation. Thus, we counted live cells after 48 and 72 h of treatment with different concentrations of Oligomycin A (20 nM–2 μM). The results showed that 20 nM Oligomycin A inhibited the proliferation of leukemia cells to the same level as higher concentrations . Leukemia cells were pre-treated with 20 nM Oligomycin A for 1 h and then co-treated with different concentrations of ASNase or left untreated for 48 or 72 h. Then, live cells were counted using flow cytometry. The results showed that Oligomycin A pre-treatment increased the resistance of leukemia cells to ASNase compared to cells treated with ASNase alone (adjusted (bonferroni) p-value< 10Basal mitochondrial membrane potential (MMP) was measured as TMRE fluorescence in 10 human leukemia cell lines of different origin . Additionally, mitochondrial content was determined as the amount of mtDNA by real-time PCR. TMRE is an MMP-dependent dye and its fluorescence intensity is in proportion to both MMP and mitochondrial mass. TMRE fluorescence alone, or TMRE fluorescence normalized to mtDNA amount, clustered leukemia cell lines regardless of their origin (data not shown). On the other hand, in the tested leukemia cell lines, TMRE fluorescence significantly correlated  with sensitivity to ASNase  and 2 healthy controls ). We also determined the sensitivity of primary leukemia cells to ASNase by MTS assay Table . InteresThe aim of this study was to elucidate if metabolic predisposition of leukemia cells (both cell lines and primary cells) influences their response to therapy. We wondered whether there is a basal metabolic profile that would predict which leukemia patients are sensitive or resistant to a given treatment. Since our previous work showed that ASNase affects the bioenergetics of leukemic cells, we were particularly interested in the link between the sensitivity to ASNase and the basal metabolic status of the cells as assessed before treatment. In addition to ASNase, we also tested the sensitivity to VCR and DNR, cytostatic drugs with a different mechanism of action, which are used in the treatment of a wide variety of cancers. We first examined the metabolic activity, including glycolysis, mitochondrial respiration and FAO in 19 cell lines. We performed HCA in order to cluster cell lines with a similar metabolic profile. Based on both glycolytic function and mitochondrial respiration, the cell lines were very similarly gathered into clusters. On the other hand, FAO clustered the cell lines differently. The first cluster, consisting of exclusively lymphoid leukemia cell lines, associated the cell lines with a lower glycolytic function and a lower mitochondrial respiration. The second cluster, grouped the cell lines with a higher glycolytic function and a higher mitochondrial respiration and contained all myeloid leukemia cell lines as well as some T-ALL cell lines. Next, aiming at the confirmation of this phenomenon in the primary samples, we assessed glycolytic function and mitochondrial respiration in leukemia cells isolated from ALL and AML patients and performed HCA. The first cluster which was divided according to glycolytic function consisted mostly of samples from ALL patients and the second cluster combined both, AML patients, ALL patients and healthy controls. After subsequent examination of the samples for their susceptibility to cytostatics, we found that ALLs from the high glycolytic cluster were less sensitive to ASNase treatment than their counterparts in the low glycolytic cluster. Moreover, a similar association was observed also in the tested cell lines. T-ALL lines from the high glycolytic cluster (and high respiration) were resistant to ASNase. Interestingly however, some AML patients were sensitive to ASNase to the same extent as sensitive ALL patients. This can be explained by the FAB classification according to which these AML patients fall into the M1, M4 or M5 subgroups. These morphological subgroups were shown to be more sensitive to ASNase treatment by in vitro testing . This coIn cell lines, IC50 of ASNase did not correlate with IC50 of VCR or DNR and also, the sensitivity to these cytostatics was not associated with any specific metabolic phenotype demonstrating that their effect was less influenced by the basal metabolic status of leukemia cells.We have previously described specific signaling pathways that regulate bioenergetic processes after ASNase treatment . We wereSince we found an association between the metabolic predisposition and the consecutive response to ASNase treatment, we next asked which metabolic parameter correlates with IC50 of ASNase and therefore with the potential treatment efficacy. Statistical analysis based on CCA revealed that higher ATP-linked respiration, higher OCR/ECAR and lower basal respiration significantly correlated with increased sensitivity to ASNase. To functionally confirm this relationship we used Oligomycin A, a specific ATP synthase inhibitor. After its administration, we treated the cells with ASNase and examined the changes in cell survival and growth. We confirmed that cells with lower ATP synthase activity, i.e. lower ATP-linked respiration, are more resistant to ASNase. The sensitivity to VCR and DNR did not change, which corresponded with the results of the CCA. Reduced ATP synthase activity, in relation to a resistant phenotype, has been described in colorectal carcinoma cells and 5-fluorouracil . Our assSince Oligomycin A treatment leads to increased MMP , we examEven though ASNase is a crucial component in the treatment of ALL, its precise administration is still being tested in some specific ALL prognostic-risk groups . MoreoveOur study provides novel findings on the role of cellular metabolism in the treatment response of leukemia patients. We have shown that metabolic phenotype in ALL cells can predict response to ASNase treatment in which less sensitive patients display high glycolytic action when compared to more sensitive patients with low glycolytic action. We also found correlation between ATP-linked respiration and basal MMP with the sensitivity to ASNase. These data support the role of cancer metabolism and open new potential ways in the diagnostic and treatment management of resistant leukemia patients.Additional file 1: Supplementary Table S1. Genetic characterization of leukemia cell lines.Additional file 2: Supplementary Table S2. List of primary antibodies.Additional file 3: Supplementary Figure S1. Absolute values of metabolic parameters measured in 19 leukemia cell lines. (A–D) Parameters calculated from glycolytic function. (E–H) Parameters calculated from mitochondrial function. (I) OCR/ECAR ratio calculated after glucose injection during the Glycolysis stress test measurement. (J) FAO rate. AML is presented as black graphs, B-ALL as white and T-ALL as gray graphs. (K) Levels of signaling proteins in the leukemia cell lines were measured by immunoblotting. β-actin was used as a loading control. Relative quantification  was calculated in ImageJ. * p < 0.05, ** p < 0.01, *** p < 0.001. The immunoblot is a representative result of three independent experiments.Additional file 4: Supplementary Figure S2. Effect of OXPHOS inhibitors on the growth and mitochondrial respiration of leukemia cell lines. (A) The effect of Oligomycin A  on the growth and on the course of mitochondrial respiration of NALM-6 cells. (B) The effect of Antimycin A  on the growth and the course of mitochondrial respiration of NALM-6 cells. Cells were counted 48 and 72 h after the treatment. Cell Mito Stress Test was performed after 24 h of treatment. Measurements were done in three biological replicates and the data are presented as mean ± SD.Additional file 5: Supplementary Figure S3. Functional study on the correlation between ETC complex III activity and sensitivity to ASNase. Effect of Antimycin A (10 ng/ml) on the sensitivity of leukemia cell lines  to ASNase. Cells were pretreated with Antimycin A for 1 h or left untreated and then co-treated with ASNase for 48 h. Absolute cell counts were obtained from three independent experiments; data were normalized to untreated controls and are presented as mean ± SD. Measurements were done in three biological replicates and the data are presented as mean ± SD.Additional file 6: Supplementary Figure S4. Cluster analysis of patient samples according mitochondrial respiration. Hierarchical cluster analysis of primary leukemia cells and healthy control samples based on parameters calculated from mitochondrial function. Type of leukemia and IC50 ASNase [IU/ml] are indicated for each patient. For more information, see Table


The latter showed an improvement in IC50 values of ca. three orders of magnitude for the lung and bone cancer cell lines as well as fibroblasts in comparison with ALN. The development of OSILs with high cytotoxicity effect towards the tested cancer cell types, and containing an anti-resorbing molecule such as ALN may represent a promising strategy for the development of new pharmacological tools to be used in those pathological conditions.Herein the quantitative synthesis of eight new mono- and dianionic Organic Salts and Ionic Liquids (OSILs) from alendronic acid (ALN) is reported by following two distinct sustainable and straightforward methodologies, according to the type of cation. The prepared ALN-OSILs were characterized by spectroscopic techniques and their solubility in water and biological fluids was determined. An evaluation of the toxicity towards human healthy cells and also human breast, lung and bone (osteosarcoma) cell lines was performed. Globally, it was observed that the monoanionic OSILs showed lower toxicity than the corresponding dianionic structures to all cell types. The highest cytotoxic effect was observed in OSILs containing a [C Alendronic acid is an aminobisphosphonate derivative that has shown efficacy in postmenopausal osteoporosis, malignant hypercalcemia and Paget’s disease . AlendroBisphosphonates bind at the bone mineral surface, where they potently inhibit osteoclast-mediated bone resorption and subsequently embed in the bone, being released only during subsequent resorption . In contAll bisphosphonates rapidly reduce bone resorption, which leads to decreased bone formation because resorption and formation are coupled. Within three to six months, equilibrium is reached at a lower rate of bone turnover .It is described that alendronate in rats exhibited 200 to 1000 times more potency than etidronate and approximately 100 times more in comparison with clodronate or tiludronate . It is rThe introduction of specific functional groups in the BPs structure can lead to modifications in their physicochemical, biological, therapeutic and toxicological properties. In recent literature studies, alendronic acid is reported as an FDA drug already approved for the prevention and treatment of osteoporosis in men and women, either postmenopausal or glucocorticoid-induced . HoweverThe combination of Active Pharmaceutical Ingredients (APIs) with biocompatible organic counter-ions has rendered over the last decade an attractive class of materials entitled API–Organic Salts and Ionic Liquids (API-OSILs) ,8,9,10. Thus, considering the pharmaceutical importance of bisphosphonate drugs as antiresorptive and potential antitumoral agents, it is of paramount importance that novel formulations of such drugs, which render higher bioavailability and lower systemic toxicity than the latter are developed. Hence, in this paper, we report our latest results on our research line regarding bisphosphonate-based OSILs. In particular, we describe the synthesis of eight new Ionic Liquids and Organic Salts from alendronic acid (ALN–OSILs) as mono- and dianion by combination with one or two units of biocompatible cations, respectively. The desired compounds were prepared in quantitative yields by two distinct sustainable and straightforward methodologies, according to the type of cation. All prepared ALN–OSILs were characterized by spectroscopic techniques and their solubility in water and biological fluids was determined. Finally, evaluation of the toxicity towards human healthy cells and lung, breast and bone (osteosarcoma) cell lines was performed.H-imidazol-3-ium [C2OHMIM]) cations relied on the known information about their biocompatibility [The selection of protonated organic superbases non-5-enium [DBNH]) and quaternary ammonium (cholinium [Ch] and 1-(2-hydroxyethyl)-3-methyl-1tibility ,23. Desptibility ,20,22.1–4 consisted of the addition of the diluted superbases to a dispersion of ALN (pathway A). These particular compounds, termed Superionic Liquids of APIs, were prepared according to a previous methodology reported by us for zoledronic acid [The preparation of compounds nic acid  as well nic acid . While b2OHMIM] cations yielded four more ALN-based OSILs (compounds 5–8) by following another methodology already described by our group  and  cation, where the mono- and dianionic are, respectively, a paste and a foam. This inversion of the trend is probably related to specific interactions of ALN with the electron-rich imidazolium ring  and [DNBH][ALN], to which no glass transition nor crystallization (Tc) temperatures were observed in the experimental conditions. However, [TMGH][ALN] showed different behavior in comparison with the remaining ALN–OSILs in the first cycle, in particular a cold crystallization (Tcc) peak at 107.1 °C  and on different cancer cell lines, namely T47D (breast), A549 (lung) and MG63 (osteosarcoma). The results obtained with the starting materials of the synthesis, as well as the prepared ALN–OSILs upon incubation for 24 h are presented in The cytotoxicity of the prepared ALN–OSILs was determined on human cells, by means of IC50 values determined at 72 h of incubation were higher than those obtained with a culture period of 24 h. The IC50 values obtained for paclitaxel in the tumor cell lines were the lowest ones, which is in line with its well-known cytotoxic potential. However, it showed no selectivity between healthy and cancer cells.Globally, it was observed that the IC2OHMIM] and [Ch] appeared to be less deleterious than alendronic acid. Comparatively, OSILs containing [TMGH]2, [C2OHMIM] and [C2OHMIM]2 seemed to be more cytotoxic to breast cancer T47D cells. In the case of lung cancer A549 cell line, high cytotoxic activity was observed in cell cultures supplemented with the OSILs containing [TMGH]2, [C2OHMIM] and [Ch]. Finally, osteosarcoma MG63 cell line seemed to be particularly sensitive to the OSILs containing [TMGH]2, [C2OHMIM] and [C2OHMIM]2.In the case of the ALN–OSILs, the obtained data clearly shows that the monoanionic OSILs elicited lower cytotoxicity than the corresponding dianionic versions for all cell types. Regarding gingival fibroblast cell cultures, the OSILs containing one unit of [DBNH], [ALN] was found to be particularly active against lung cancer and osteosarcoma cell lines while retaining very low toxicity towards healthy cells. These enhanced biological properties, in addition to the absence of polymorphism in this monoanionic compound, suggest that this room temperature ionic liquid could be a very promising alendronic acid formulation.Overall, [C2O  a methanolic solution of 1 or 2 molar equivalents of organic superbase (15 mg/mL) was added dropwise at room temperature under magnetic stirring. After reacting for 1 h, the solvent was evaporated and the desired product was dried under vacuo for 24 h.To a dispersion of alendronic acid  in MeOH/H2O under magnetic stirring at room temperature. After 1 h, the solvent of the clear solution was evaporated, and the desired product was dried under vacuo for 24 h.The halide salts of the selected ammonium and methylimidazolium cations were dissolved in methanol and passed slowly through an anion-exchange column A-26(OH) . The freshly formed methanolic solutions of the corresponding hydroxide salts  were consequently added dropwise to alendronic acid  dispersed in HToxicity studies are described as supporting information.


Enhancer of zeste homolog 2 (EZH2) is considered an important driver of tumor development and progression by its histone modifying capabilities. Inhibition of EZH2 activity is thought to be a potent treatment option for eligible cancer patients with an aberrant EZH2 expression profile, thus the indirect EZH2 inhibitor 3-Deazaneplanocin A (DZNep) is currently under evaluation for its clinical utility. Although DZNep blocks proliferation and induces apoptosis in different tumor types including lymphomas, acquired resistance to DZNep may limit its clinical application.To investigate possible mechanisms of acquired DZNep resistance in B-cell lymphomas, we generated a DZNep-resistant clone from a previously DZNep-sensitive B-cell lymphoma cell line by long-term treatment with increasing concentrations of DZNep (ranging from 200 to 2000 nM) and compared the molecular profiles of resistant and wild-type clones. This comparison was done using molecular techniques such as flow cytometry, copy number variation assay (OncoScan and TaqMan assays), fluorescence in situ hybridization, Western blot, immunohistochemistry and metabolomics analysis.AHCY) gene in the resistant clone. AHCY is a direct target of DZNep and is critically involved in the biological methylation process, where it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine. The amplification of the AHCY gene is paralleled by strong overexpression of AHCY at both the transcriptional and protein level, and persists upon culturing the resistant clone in a DZNep-free medium.Whole exome sequencing did not indicate the acquisition of biologically meaningful single nucleotide variants. Analysis of copy number alterations, however, demonstrated among other acquired imbalances an amplification (about 30 times) of the S-adenosyl-L-homocysteine hydrolase (This study reveals one possible molecular mechanism how B-cell lymphomas can acquire resistance to DZNep, and proposes AHCY as a potential biomarker for investigation during the administration of EZH2-targeted therapy with DZNep. The development of drug resistance to cancer chemotherapeutics remains a major concern in most treatment regimens. Several epigenetic-based therapies are under investigation or being employed for treatment of patients with lymphomas of B-cell origin. This is due to the important role that epigenetic alterations play in promoting tumor development and progression via downregulation of tumor suppressor genes . These eEZH2 gain-of-function mutations and overexpression are considered important drivers of oncogenesis because of their role in silencing tumor suppressor genes regulating apoptosis, cell cycle regulation, proliferation, migration and differentiation [Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is involved in cellular differentiation and development in both health and disease. EZH2 promotes transcriptional repression by catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3) - a repressive histone mark. In lymphoma and other malignancies, ntiation –14. Due ntiation –20. The ntiation , 18, 21.DZNep directly inhibits the enzyme S-adenosyl-L-homocysteine hydrolase (AHCY) that catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine (SAH) to L-homocysteine and adenosine. The direct inhibition of AHCY by DZNep leads to the build-up of the substrate SAH, which in turn causes a negative feedback inhibition of methyltransferases such as EZH2 . Proper AHCY as a potential biomarker that could be of predictive relevance for therapeutic inhibition of EZH2 using DZNep.In this study, we investigated the underlying molecular mechanism of resistance of a B-cell lymphoma model to DZNep using a DZNep-resistant clone generated from a DZNep-sensitive cell line. We identified DZNep  was dissolved in sterile water following the manufacturer’s recommendation as previously described .2.The sporadic Burkitt lymphoma cell line BLUE-1  Germany) was cultured in RPMI 1640  medium enriched with 20% fetal calf serum . Cell lines were tested and confirmed mycoplasma negative with the MycoAlert Mycoplasma Detection kit . All cell lines were incubated at 37 °C at 5% COGeneration of a DZNep resistant clone was achieved by splitting the BLUE-1 culture into a control group and a treatment group  and propidium iodide . Doubling time was determined by seeding the cells in 6-well plates at a cell density of 2 × 10^5 cells/ml. The number of vital cells were counted after 24 h, 48 h and 72 h by flow cytometry. The doubling time during the exponential growth phase was subsequently calculated using the formula DT = Tln2/ln(Xe/Xb) (https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_Guide.ashx), where DT represents the doubling time (in hours), T symbolizes the incubation time (in hours), Xe indicates the cell number at the end of the incubation time, and Xb is the cell number at the beginning of the incubation time.The BD Accuri C6 flow cytometer  was used for the measurement of apoptotic cells and determination of the doubling time of cells. Measurement of apoptosis was performed after staining 3 × 10Cell lysis and Western blot were carried out as described previously . Twelve B2M) and succinate dehydrogenase (SDHA) genes were used as endogenous controls and the ΔΔCt method [RNA was isolated using the RNeasy Midi Kit  adhering to the manufacturer’s recommendations and reverse transcribed to cDNA using TaqMan Reverse Transcription reagents  on a T3 thermocycler . Real-time RT-PCR was carried out using TaqMan Gene Expression Assays and TaqMan Gene Expression Master Mix  following the manufacturer’s protocol on the StepOnePlus Real-Time PCR System . Beta-2 microglobulin  following the manufacturer’s instructions. Cluster Generation was done with the aid of TruSeq PE Cluster Kit v4  and the resulting templates sequenced on an Illumina HiSeq2000 sequencer (at least 150 million reads with a sequencing depth of greater than 160x) using the Illumina HiSeq SBS 250 cycle kit v4.Genomic DNA was isolated from the cell lines using QIAamp DNA Mini Kit , following the manufacturer’s instructions. WES was performed on genomic DNA at the Berlin Institute of Health core facility Genomics, Berlin, Germany. Sequencing libraries were prepared with the SureSelectData analysis was performed using BWA-MEM  to map eAHCY gene on chromosome 20. The assay covers intron 7 and exon 8 on the reference genome GRCh37 and was performed according to the manufacturer’s recommendations. Data analysis was done using the CopyCaller software version 2.1 .CNV analysis was performed on genomic DNA from the respective BLUE-1 cell lines, controls and patient samples by applying the TaqMan copy number assay (assay ID: Hs02422126_cn) to the OncoScan CNV assay Kit was also used to perform copy number analysis according to standard protocols (Affymetrix) . The ChrB-cell clonality studies were performed to determine the immunoglobulin heavy chain (IGH) rearrangements for BLUE-1, BLUE-1 K10 and BLUE-1R10 using a multiplex PCR method developed within the EuroClonality/BIOMED-2 collaborative study, BMH4-CT98–3936  for all AHCY gene-specific probes (product name: AHCY-20-OR) and green-labeled chromosome 20-control (centromeric) probes (product name: CHR20–10-GR) . DakoCytomation Hybridizer  was used for FISH probe hybridization, while nuclear counterstaining was done with the aid of Dako fluorescence mounting medium containing DAPI . Visualization and analysis (of at least 50 intact nuclei) were performed with the Zeiss Axio Imager Z1  and the Isis imaging software version 5.3.1 .Immunohistochemistry (IHC) was performed using sections of formalin-fixed paraffin-embedded (FFPE) cell line blocks as described . The priProfiling of short tandem repeats (STR) for authentication of the cell lines BLUE-1, BLUE-1 K10 and BLUE-1R10 using the StemElite kit (Promega) was performed as previously described . Convent.idat files) using the ‘conumee’ R package in Bioconductor [The intermediates of methionine and polyamine metabolism in BLUE-1, BLUE-1 K12 and BLUE-1R12 cell extracts were measured by liquid chromatography-tandem mass spectrometry following an established protocol . Genome-onductor , 40.p values less than 0.05 were considered significant.Statistical analysis was done using the GraphPad Prism 5 software . Statistical significance was evaluated using the Mann-Whitney U test (two-tailed) for pairwise comparisons, and one-way ANOVA with the Tukey post-hoc test for group comparisons. We generated a DZNep-resistant clone by subjecting the DZNep-sensitive Burkitt lymphoma cell line BLUE-1  to progrTo determine the identity of the generated clone, we explored the STR profile and determined the clonality of BLUE-1R10. The results were then compared with those of its corresponding control BLUE-1 K10 and the parent cell line BLUE-1. The STR profile analysis for the three cell lines when compared with the DSMZ STR profiling database revealed an STR profile of a BLUE-1 cell line, confirming their authenticity. Furthermore, the IGH chain gene rearrangement patterns for the three cell lines were identical  and increasing thereafter  in the level of both adenine and adenosine in comparison to its control BLUE-1 K12 , adenine and adenosine in BLUE-1 and the control cell line BLUE-1 K12. The resistant clone (BLUE-1R12), however, displayed a significant increase , there was an almost two-fold increase in the AHCY copy number. This copy number gain doubled following continuous cultivation of the clone in a DZNep-free medium (from BLUE-1R10 to BLUE-1R11 and BLUE-1R12). This implies that the AHCY copy number gain, once initiated, does not require DZNep pressure to persist. Although this copy number event may reflect a sort of genomic compensation in the resistant clone due to prolonged AHCY inhibition, it is unlikely that this genomic aberration would decrease to baseline levels upon prolonged withdrawal of DZNep pressure.Since AHCY is a direct target of DZNep-mediated EZH2 inhibition, we focused on validating the identified AHCY amplification was more pronounced in BLUE-1R12 in comparison to BLUE-1R10 further affirms the persistence of AHCY copy number gain. The chromosome 20 polysomy observed in the cells of each cell line may reflect the level of genomic instability usually occurring in cancer cells [Moreover, the FISH data which revealed that the er cells , 53. Theer cells , 54. AltThe results of our metabolomics studies which revealed an analogous distribution for SAH, adenine and adenosine in both BLUE-1 and BLUE-1 K12 signifies that following long-term culture of BLUE-1, there is no peculiar alteration of the balance exerted by these intermediates within the cell. The increase in adenine and adenosine levels of the resistant clone (BLUE-1R12) when compared to BLUE-1 K12 is in line with the increased expression of AHCY, which catalyzes the hydrolysis of SAH to adenosine and L-homocysteine.AHCY gene among others. However, little is known about the role of AHCY copy number gain in driving B-cell lymphomas. For this reason, we analyzed the AHCY copy number of 12 primary lymphoma samples using the TaqMan CNV assay. The absence of AHCY copy number gain in the patient samples analyzed implies that it is unlikely that AHCY copy number gains play a driving role in B-cell lymphoma pathogenesis. In addition, the absence of AHCY copy number alterations from in silico studies signifies that alterations in AHCY are rare in primary B-cell lymphoma however, in solid tumors, the frequency of AHCY amplification can be as high as 20% [Previous works have linked copy number gains on chromosome 20q with the pathogenesis of tumors such as colon cancer , colorech as 20% , 42.AHCY is one possible mechanism of acquired resistance to DZNep-mediated apoptosis and propose AHCY as a potential biomarker to stratify patients during the use of DZNep. Although AHCY alterations are rare in primary B-cell lymphomas, it may still be important to screen for modifications of this gene in patients prior to the initiation of EZH2 based therapy with DZNep. These findings might be valuable in predicting patients, who will benefit from EZH2 inhibition using DZNep once it progresses into clinical studies.Acquired drug resistance poses a great challenge in cancer therapy. It is important to recognize mechanisms of resistance for novel drugs about to enter clinical trials so that one can monitor for early signs of development of drug resistance. DZNep is a promising epigenetic drug that is in the pre-clinical phase of clinical approval, but has shown promising effects for selected cancer patients. We show that copy number gain of Additional file 1: Figure S1. Confirmation of the identical B-cell clonality of BLUE-1K10 and BLUE-1R10.Additional file 2: Figure S2. Karyotypes of the cell lines BLUE-1, BLUE-1R10 and BLUE-1K10.Additional file 3: Figure S3. OncoScan Copy number analysis in the of BLUE-1 cell lines.Additional file 4: Figure S4. Analysis of AHCY copy number in primary lymphoma samples.Additional file 5: Table S1. Comparative copy number analysis in the BLUE-1 cell lines as determined by OncoScan assay.Additional file 6: Figure S5. Full-length blot for Western blot image presented in Fig. Additional file 7: Figure S6. Full-length blot for Western blot image presented in Fig.


Vine pruning residues are by-products of the wine industry that have not received much attention in the past, in spite of being rich in bioactive compounds. In this study, we aimed to test whether an ohmic extract of vine pruning residue (VPE) has anti-colorectal cancer (CRC) properties, and whether responses differ according with cell’s mutation profile. VPE decreased human CRC cell proliferation, accompanied by DNA effects and cell cycle modulation. VPE also increased cell sensitivity to the chemotherapeutic drug 5-FU. Our results suggest that tumors harboring BRAF mutations may be more responsive to VPE than KRAS mutated tumors. These effects of the extract were not completely reproduced by the most abundant constituents tested individually at the concentrations present in the effective dose of VPE. Globally, our results indicate that VPE, a polyphenol enriched extract produced by ohmic heating of vine pruning residue, has anti-colorectal cancer potential, including sensitizing to a chemotherapeutical drug, and its use in functional foods or nutraceuticals could be exploited in personalized anti colorectal cancer dietary strategies. Valorization of this lignocellulosic residue should encourage bio-waste recycling, adding value to this agricultural by-product and promoting the sustainable use of natural resources. Environmental concerns have prompted the desire to reduce the impact of the agro-food industry, and transform today’s linear economy into a more efficient circular model. In line with this, the scientific community has been devoting major efforts to the search for bio-waste recycling methodologies that promote sustainable growth, based on the efficient use of natural resources ,2.Grapes are a major agricultural product, and according to the 2019 Statistical Report on World Vitiviniculture , 77.8 miMany grapevine by-products, such as grape pomace, seeds, stems, and vine leaves, have been extensively characterized in terms of chemical composition and biological properties ,6, and hVine pruning residues constitute valuable biomass that can be used in a biorefinery scheme, considering their high content in antioxidant compounds (phenolic compounds derived from lignin processing) ,12,13, aRecently, we have reported on ohmic heating, an environmental friendly polyphenol extraction technique, that allowed an increase in phenolic compound extraction from vine pruning residue, relative to extraction at room temperature . In addiResveratrol and quercetin  have been found in many grapevine by-products, together with other compounds . Among tThe anti-colon cancer (anti-CRC) potential of polyphenol enriched ohmic vine pruning extract was tested using two human CRC cell lines, HCT116 and RKO, with different molecular signatures representative of the most common mutation types of CRC, in order to characterize possible molecular targets and mechanisms of action. Moreover, the major individual constituents present in the extract were tested alone, in an attempt to identify possible active principles. To identify possible effects on sensitivity to the chemotherapeutical drug 5-fluorouracil (5-FU), combination treatments were also performed.Cell culture media (RPMI-1640 and MEM) fetal bovine serum (FBS), antibiotic antimycotic solution, thiazolyl blue tetrazolium bromide (MTT), apigenin (AP), quercetin (Q), ellagic acid (EA), hesperidin (H), etoposide (ET), ribonuclease A (RNase A), propidium iodide (PI), Hoechst 33342, 5-fuorouracil (5-FU), and all reagents not otherwise specified, were purchased from Sigma-Aldrich . SYBR Gold nucleic acid gel stain was acquired from Invitrogen . Vine pruning was harvested in the Portuguese region of Minho , and samples were dried and crushed as described by Jesus and collaborators 2019) [19 [20]. v/v), with electrical conductivity adjusted to 2.3 mS/cm with addition of NaCl (thus guaranteeing a homogeneous current flow), 80 °C, for 60 min under agitation (840.0 V/cm). The room temperature extract (RT) was prepared by placing the vine pruning inside the reactor and maintaining under constant agitation by a magnetic stirrer. At the end of each treatment, the extracts obtained were immediately cooled in an ice bath, and then filtered with a cellulose filter, with a pore size of 0.45 μm. The ethanol was evaporated in an orbital shaker at 40 °C and 150 rpm, and the extracts lyophilized and stored in amber flasks and frozen until use. The phenolic composition of the extracts was determined by Jesus and collaborators (2020) . Th. Th16]. In viability assays, AP and Q were the most promising compounds in HCT116 cells, and H in the case of RKO cells, because they seemed to produce similar effects to those of VPE on cell viability. Quercetin, however, in addition to being present in both RT and VPE extracts and just 2 times more concentrated in VPE relative to RT , seemed None of the effects of the individual compounds seemed to be due to DNA damage induction, as demonstrated by the comet assay results. In the comet assay, the percentage of DNA in comet tails (% tail intensity) reflects the presence of DNA beaks. Our data also show the expected DNA damage induction by the positive control etoposide (a known genotoxic agent)  and, to DNA damage is known to induce cell cycle arrest and/or cell death by apoptosis, and further experiments were performed in order to address effects on these parameters.Data from cell cycle modulation analyses reflected the observed genotoxicity of ET and VPE. Individual compounds were without effect on cell cycle regulation in both cell lines, which is also in agreement with the absence of DNA damage observed by the comet assay .In the comparison between VPE and RT, VPE showed, therefore, to be much more efficient than RT against both colon cancer cell lines, possibly due to its much higher phenolic content, although no individual major constituent could be identified as the active principle. This means that the interesting anti colon cancer effect of VPE is due to the complex mixture of compounds present, and not to any its main constituents individually, although some of the individual compounds also showed significant decrease in cell viability.Due to 5-FU’s significance in the treatment of CRC, combination assays with VPE and two of the most effective individual constituents AP and H with the chemotherapeutic drug were performed, and effects on proliferation and cell death studied in both cell lines. 5-FU alone induced both decreased cell viability and increased cell death in both cell lines, as expected. Interestingly, the combination 5-FU + VPE significantly enhanced 5-FU’s effects on both parameters, which indicates an increased cell responsiveness to the pharmaceutical drug induced by VPE. With regard to the individual compounds, in co-treatment with 5FU, AP and H had similar effects to VPE in RKO cells decreasing viability to a similar extent as VPE. However, those similarities were not present when induction of cell death was considered and effects of individual compounds were also absent in HCT116 cells; VPE showed, therefore, to be more effective in potentiating the response to 5-FU than any of its main flavonoids used individually. This indicates that, although a good source of individual bioactive flavonoids, in particular of AP, VPE as a whole would be more effective in anti-colon cancer dietary strategies, used, for example, in functionalized foods, than its individual compounds.The combination treatment 5-FU+VPE showed that the effects in RKO were more pronounced than those in HCT116, which is in agreement with the previous responses to VPE alone. Due to the different mutation profiles of the cells used, and the fact that BRAF mutated cells (RKO) responded better to VPE than KRAS mutated cells (HCT116), also in combination treatments with 5-FU, our results suggest that patients with colorectal tumors harboring BRAF mutations would benefit most from combination treatments VPE+5-FU, compared with patients with tumors harboring KRAS mutations. Although this is a significant outcome of our study, future work is required to confirm this trend and clarification of the molecular mechanisms involved. In order to confirm the molecular profile that would benefit the most from VPE and VPE+5-FU, further experiments should test more cell lines with either BRAF or KRAS mutations, and quantify expression levels of phosphorylated ERK  and AKT phosphorylation. Since the PI3K/AKT signaling pathway is not expected to be constitutively activated in BRAF mutated cells, but only in KRAS mutated cells, determination of VPE treatment effects on p-AKT levels would also be relevant to establish extract’s effects on the MAPKinase and/or the PI3K/AKT pathways. Furthermore, in spite of the fact that our data suggest an enhancement of the effect of the drug 5-FU by simultaneous treatment with VPE, the establishment of a synergy between VPE and 5-FU will require further experiments.Taken together, our results show that ohmic heating extraction of vine pruning residue produces a polyphenol enriched extract (VPE) with anti-colorectal cancer potential, including the ability to sensitize cells to 5-FU, a chemotherapeutic drug. The use of VPE in functional foods or nutraceuticals could be exploited in personalized anti colorectal cancer dietary strategies, because higher sensitization to the drug 5-FU is achieved in BRAF mutated tumors, relative to KRAS mutated tumors. Valorization of this lignocellulosic residue of the wine industry should encourage bio-waste recycling, adding value to this agricultural by-product and promoting the sustainable use of natural resources.


The volume of the spacer and RSP decreased at rates of 4.6 and 0.44% per week, respectively, whereas the target dose coverage was maintained until the end of treatment. The PGA spacer was considered effective for pediatric cancer patients undergoing chemo-proton therapy.The clinical utility of a recently developed bioabsorbable polyglycolic acid (PGA) spacer has not yet been established in pediatric patients; therefore, we aimed to investigate its utility during chemo-proton therapy for pediatric cancer. Proton depth–dose curves were obtained in a water phantom with or without the spacer. Computed tomography (CT) scans were performed for the PGA spacer immersed in saline for 2 weeks to measure CT numbers and estimate the relative stopping power (RSP) for the proton beams. The spacer was placed in a patient with sacral Ewing sarcoma receiving 55.8 Gy [relative biological effectiveness (RBE)] in 31 fractions and was evaluated using CT scans performed every other week. In addition, the images were used to quantitatively evaluate changes in volume and RSP of the spacer and dose distributions in normal tissues. The spacer immersed in saline had a CT number of 91 ± 7 (mean ± standard deviation) Hounsfield units, and the corresponding RSP was predicted to be 1.07 ± 0.01. The measured RSP agreed with the predicted one. The volumes of the large bowel and rectum receiving ≥45 Gy(RBE) (V Recent advances in multidisciplinary treatment have improved the survival of pediatric cancer patients, but in exchange, they face the problem of living with late complications . AlthougPrevious studies reported that placement of a spacer GORE-TEX®  could effectively displace normal tissues from irradiation fields and provide a dosimetric advantage and long-term clinical benefits throughout the treatment duration . HoweverRecently, a bioabsorbable spacer was developed . This spThe purpose of this study was to physically and clinically investigate the effectiveness of a PGA spacer implanted in a pediatric patient. Two key considerations were: (i) changes in dosimetric parameters on treatment plans and (ii) degradation of the spacer compared with adult patients. Degradation was evaluated through volume and proton stopping power relative to that in water (relative stopping power [RSP]).We evaluated tissue equivalence of the PGA spacer for proton irradiation using computed tomography (CT) scans and proton range measurements. A CT image is expressed as a spatial distribution of ‘CT number’, which corresponds to the X-ray linear attenuation coefficient in medium. CT number can be converted into RSP with a heuristic conversion method . The spaWe prepared a 20 × 10 × 1 cm (thick) PGA spacer . To simulate the spacer placed in a body, the spacer was soaked in distilled water containing 0.9% NaCl at ~26°C and kneaded enough by hand to remove air. The spacer was CT-scanned  with 2-mm slice thickness at 120-kV voltage, and the images were analyzed with open-source ImageJ software  . A largeProton RSP was determined by calculating the ratio of water equivalent length to physical length of the spacer. The water equivalent length was derived from the shift in the percent depth dose (PDD) curves with and without the spacer. In a water phantom, a parallel plate chamber  served as a scanning chamber. Measurements were made using passively scattered proton beams delivered from the synchrotron PROBEAT-III  at the Nagoya Proton Therapy Center , 21. TheCT images and dose distribution in an 11-year-old male patient with Ewing sarcoma at the sacrum were evaluated. The patient received multiagent chemotherapy for tumor size reduction followed by spacer implantation by surgery. A 20 × 10 × 0.5 (thickness) cm PGA spacer was shaped to an appropriate size before implantation. It was folded into a two-layer sandwich and fixed together with an adhesion barrier Seprafilm®  in the retroperitoneal space between the tumor and the intestinal tract. It is encouraged that the spacer be used together with the adhesion barrier to avoid induction of the ileus . There wDmax) to the rectum, large bowel, small bowel and bladder ≤55.8 Gy(RBE). The dose covering 98% of a planning target volume (PTV) was planned to be at least 95% of the prescribed dose (D98% > 95%). A single field uniform dose treatment plan of three fields using a spot scanning system was used to deliver a dose of 55.8 Gy(RBE) in 31 fractions to the patient. Proton therapy was performed from the fourth to tenth week after spacer placement.Following recovery from surgery, the patient received chemotherapy with ifosfamide and etoposide. The patient underwent CT scans with 2-mm slice thickness for treatment planning of proton therapy. Our method for proton therapy planning was described previously , 23. TheIn order to assess the improvement in dose distribution in the OAR due to the PGA spacer, CT images with 5-mm slice thickness before spacer placement were fused with the treatment planning CT images with bone matching on MIM Maestro using a fusion optimization tool. Then, dummy plans were created and delivered doses on the dummy plans were calculated by overlaying the dose distribution of the treatment plan on the CT images. The impact of the PGA spacer on target coverage and OAR dose was evaluated by comparing dose–volume histograms (DVHs) on the dummy plan and treatment plan.The patient received verification CT scans at 2-week intervals to check anatomical changes during fractionated therapy. These CT scans provided geometric shapes and RSP of the spacer in the patient’s body. Because the double-layered spacer was expanded by gas produced in the degradation process, we excluded the parts of produced gas when drawing the contours of the spacer manually. The thickness of the spacer could not be measured with high precision because it was inserted along the shape of organs. By using VQA and MIM Maestro, doses at critical organs were checked by applying the beam configurations of the first treatment plan to the anatomy of the verification CT scan. The DVH of the verification plans were compared with the first plan.3. 3.Water absorption of the PGA spacer was measured after it was immersed in saline for 2 weeks. The CT number of the spacer before it was immersed in saline was −730 ± 20 Hounsfield units  and the CT number of the saline was 18 ± 5 HU. The physical density of the spacer before immersion in saline was 0.22 ± 0.01 g/cmThe water equivalent shift of the reference Bragg peak position was observed by placing the spacer in the field of the beams. The water equivalent length and physical length of the spacer were measured to be 10.8 ± 0.2 and 10.3 ± 0.1 mm, respectively. The spacer therefore had a proton RSP of 1.06 ± 0.02.98% in the PTV was >95%. On the other hand, the implantation of the PGA spacer resulted in dose reduction in the rectum and large bowel. The Dmax at the rectum was 56.7 Gy(RBE) before the implantation of the PGA spacer and 46.7 Gy(RBE) after the implantation. Large differences in the volume receiving ≥45 Gy(RBE) (V45Gy) in the rectum were found as well: 48.5% before the implantation and 0.01% afterwards. The Dmax at the large bowel was 56.9 Gy(RBE) before spacer implantation and 34.2 Gy(RBE) after. The V45Gy at the large bowel was 7.2% before and 0% after spacer implantation.In our phantom study, the mean CT number of the spacer immersed in saline for 2 weeks was 91 HU, and the density of the spacer was almost uniform. This indicted that all parts of the spacer contained sufficient water. The measured RSP of the spacer was in good agreement with the predicted RSP based on CT scan data when the spacer was placed in saline for a sufficient time. These results demonstrated the tissue equivalence of the PGA spacer in wet conditions and suggested that CT number would provide a reliable RSP of the PGA spacer placed in a patient’s body. In our case, the PGA spacer offered a significant dose reduction in the rectum and large bowel, so the patient surely benefited from the use of the spacer.It is assumed that an increase in the RSP of the PGA spacer resulted from water absorption. The RSPs of the spacers in dry and wet conditions were calculated from their material compositions using a method from Ref.  in orderThe sustained effectiveness of a PGA spacer in pediatric patients has been a concern. We did not measure the CT–RSP relationship in a PGA spacer during the degradation process in this study; therefore it is not clear whether the CT number of the degraded spacer provides a reliable RSP. However, we concluded that the tissue equivalence of the degraded spacer might have been ensured because the majority of the spacer is composed of water and PGA molecules are released outward. That is why we calculated dose distributions with CT numbers of the degraded spacer in a patient’s body. Our hypothesis was that the degradation velocity of the PGA spacer implanted in a pediatric patient might be faster than that in adult patients because a pediatric patient body generally tends to induce electrolyte and acid–base imbalance during cancer therapy. CT images revealed regression of the spacer volume probably due to a hydrolysis reaction of the materials in the patient. It was assumed that the reaction generated carbon dioxide gas, which expanded the spacer to form an internal cavity. The morphological change in the spacer may have an influence on the position and volume of OAR, which can also happen when a double-layered spacer is implanted in other patients. We adopted the double-layered structure of a 0.5-cm thick spacer because it was too difficult to trim a 1-cm thick spacer. For subsequent cases, the 1-cm thick spacer has been used to avoid the morphological change. In our clinical case, the spacer showed a volume reduction rate similar to that reported for adults, which was not concordant with our hypothesis. This implied that a PGA spacer placed in a pediatric patient exerted a beneficial effect as seen in adult patients when the patient’s condition is managed well during treatment.In our case, the RSP reduction rate (0.44% per week) was one order of magnitude lower than the volume reduction rate (4.6% per week). Thus, the RSP change during proton therapy was considered to be of limited significance. Dosimetric analysis showed that the PGA spacer allowed for protecting of OAR throughout the treatment period and suggested that this approach should be effective in pediatric cancer treatment as well as in adults. However, the volume and RSP of the PGA spacer decreases as hydrolysis proceeds, and it becomes difficult to stop proton beams when the treatment is protracted. Its effect has a notable impact on radiotherapy, which requires a longer period especially in pediatric cancer treatment for bone and soft tissue. Future work should therefore include regular CT scans designed to evaluate dose deposition in OAR. In addition, it might be necessary to modify and re-optimize the dose distribution during the course of the patient’s treatment.It should be noted that this study evaluated the benefit of a PGA spacer applied to only one pediatric patient. So further clinical cases are needed to validate the effectiveness of the PGA spacer. A phase I trial (UMIN000039288) is currently ongoing in order to evaluate the safety and efficacy of the spacer in pediatric patients, and the results are awaited.A bioabsorbable spacer is a promising tool for sparing normal tissues in proton therapy. Our data demonstrate that a spacer implanted into a pediatric patient served the same function as in adult patients. Pediatric patients with bone and soft tissue tumors are often treated with radiation over a longer period than adults, so regular CT scans should be carried out to check the dose distribution in future clinical cases.Supplementary_Fig1_rraa087Click here for additional data file.


Ribonucleic acids (RNAs) are generally linear chains of nucleotides which function in many cellular processes, best known in protein biosynthesis. In the last decade, circular RNAs have been discovered which are circularized after their synthesis and differ in important features from linear RNAs. These circular RNAs have meanwhile been implicated in important cellular processes in health and disease. Here, we present a comprehensive database, circ2GO, compiling and analyzing circular RNAs found in lung cancer cell lines providing the data in tables as well as visualizing it in transcript maps and in heatmaps. Importantly, we also provide easy-to-use online tools to find circular forms of genes associated with specific molecular functions, biological processes or cellular components or predict their targeted microRNAs. This resource will enable researchers to rapidly identify circular RNAs relevant for their specific research question.Circular RNAs (circRNAs) play critical roles in a broad spectrum of physiological and pathological processes, including cancer. Here, we provide a comprehensive database—circ2GO—systematically linking circRNAs to the functions and processes of their linear counterparts. circ2GO contains 148,811 circular human RNAs originating from 12,251 genes, which we derived from deep transcriptomics after rRNA depletion in a panel of 60 lung cancer and non-transformed cell lines. The broad circRNA expression dataset is mapped to all isoforms of the respective gene. The data are visualized in transcript maps and in heatmaps, to intuitively display a comprehensive portrait for the abundance of circRNAs across transcripts and cell lines. By integrating gene ontology (GO) information for all genes in our dataset, circ2GO builds a connection between circRNAs and their host genes’ biological functions and molecular mechanisms. Additionally, circ2GO offers target predictions for circRNA—microRNA (miRNA) pairs for 25,166 highly abundant circRNAs from 6578 genes and 897 high-confidence human miRNAs. Visualization, user-friendliness, intuitive and advanced forward and reverse search options, batch processing and download options make circ2GO a comprehensive source for circRNA information to build hypotheses on their function, processes, and miRNA targets. The covalently closed continuous loop makes circRNAs resistant against degradation by exonucleases, and hence they have a longer half-life than their linear counterparts . GrowingAt the molecular level, circRNAs can affect the cell homeostasis, e.g., by interacting with RNA binding proteins (RBPs) , or by fAt the cellular level, circRNAs are important regulators in many cellular processes, such as cell signaling , embryonFor the accurate and transcriptome-wide identification of circRNAs, deep RNA sequencing (RNA-seq) approaches which comprehensively cover the circRNA spectrum need to be employed. Since circRNAs are not poly-adenylated (poly-A), they are often strongly depleted from transcriptome sequences based on poly-A enrichment. In contrast, preparing sequencing libraries with rRNA depletion retains circRNAs for RNA-seq in the next step. Hence, we sequenced rRNA-depleted RNAs from 60 lung cell lines (57 lung cancer cell lines and 3 non-transformed lung cell lines) in replicates generating 3.8 billion reads in total, including 2.8 million backsplicing reads quantifying 148,811 circRNAs derived from 12,251 genes .Here, we created the online database circ2GO to provide easy access to this large dataset. The integration of a broad spectrum of important orthogonal data and unique search and prediction options will foster and enhance circRNA research by providing hypotheses for pathways and miRNA targets linked to a circRNA. Mapping circRNAs to the genes in relation to all splice isoforms provides an important overview on how circularization can impact gene function and will also raise awareness for the many different circRNAs that can be derived from the same gene as well as their connection to different linear isoforms.The circRNA dataset was obtained by the sequencing of rRNA-depleted RNAs from 60 lung cell lines  with a total of 175 replicates. A total of 148,811 circular RNAs were detected from 12,251 genes. Each entry in the database contains a circRNA name, position, transcript of origin, expression level, gene symbol, GO annotations, and miRNAs with predicted binding sites within the circRNA. The design of the circ2GO website is intuitive and user-friendly. Generally, users can search for, obtain, and visualize information for individual circRNAs, or all circRNAs derived from a specific gene. They can search for all circRNAs derived from genes which are linked to a specified molecular function, biological process, or cellular component (GO). Lastly, they can also search for all circRNAs harboring a binding site for a specified miRNA, or vice versa. These comprehensive search options, visualization features for transcript maps and expression heatmaps, batch analyses, and download options present valuable information to the user .Uniquely, circ2GO includes the transcript map as a visualization module that depicts the position and abundance of all circRNAs derived from one gene and its relation to all known transcripts. The circRNA transcript map panel allows users to gain more detailed information on the circRNA position in relation to all transcripts of a queried gene, as well as their absolute abundance a. The maThe circRNA heatmap depicts the circRNA read counts in each cell line. The expression profile for the gene of interest can be viewed either as a heatmap (multiple circRNAs included) a, or as Gene Ontology (GO) is an important bioinformatics project that aims to uniformly define the representation of gene characteristics and gene products in all species. The main uses of GO are retrieving functional profiles of gene sets by performing enrichment analyses, as well as GO term annotation of individual genes in the categories of molecular function, biological process, and cellular component. All GO terms are listed for the gene of interest in the transcript map section.As a unique feature, circ2GO offers a reverse search, i.e., the option to search for all circRNAs derived from genes involved in a specific molecular function, biological process, or cellular component via the “GO Search” module in circ2GO. Users can find circRNAs by GO terms and download the data with circRNA expression profiles for further functional exploration of circRNAs.The basic search option can be applied for a GO ID, a complete GO term, or a part of it a. The adMicroRNAs (miRNAs) are small, single-stranded and highly-conserved non-coding RNA molecules, which can bind to target mRNAs and silence their protein expression by mRNA destabilization or translational inhibition ,33. Circhttp://www.mirbase.org) [Hence, we included a prediction of miRNA binding sites within circRNAs into circ2GO. For this circRNA–miRNA dataset, 25,166 highly abundant circRNAs from 6578 genes were filtered with the threshold of at least 2 reads  in any cell line. 897 high-confidence human miRNAs were downloaded from miRBase (ase.org) . The prease.org)  and miRaase.org) .The circRNA–miRNA search tab allows the search either for all circRNAs targeting a specified miRNA, miRNA family or miRNA seed region sequence, or for all miRNAs with binding sites in a specified circRNA, or in all circRNAs of a specified gene . ApproxiThis batch download option enables users to easily transfer data for further individual analysis. The users can download the circRNA dataset completely or partially, by selecting cell line names, genes, or miRNAs by multi-line text input with cell line, circRNA, miRNA, gene symbol or miRNA sequence. Approximate string matching is supported. The circRNA dataset is formatted into two different levels: (1) the gene level which contains an aggregation of all circRNAs of one gene; (2) the backsplice level which contains all individual circRNAs separately. The circRNA–miRNA database is also available for download.In three ways, the presented data have been selectively validated: (1) The existence of selected circRNAs identified by RNA-seq has been experimentally validated by RT-PCR and RT-qPCR in our previous study . (2) We Tophat2 [-a 6 -m 2 -g 1 -p 16. Unmapped reads were extracted as a new bam file and were then mapped again to the reference genome with the TopHat-Fusion module (included in TopHat2). CIRCexplorer2 [The circRNA dataset contained within circ2GO was derived from RNA-seq data. Libraries for RNA sequencing were prepared by depleting ribosomal RNA. Raw reads were mapped using Tophat2  with parxplorer2  was usedThe GO annotations were downloaded from the Ensembl BioMart . The GO DESeq2 [n = 175). A total of 25,166 highly abundant circRNAs from 6578 genes were filtered with the threshold of at least 2 reads  in any cell line. With the bed file based on the circRNA coordinates and strand, getfasta was used to obtain sequences for the circRNA exons. All exons within the span of the circRNA splice sites were included. Pieces of exonic sequences were concatenated sequentially to generate a complete circRNA sequence. A total of 897 high-confidence human miRNAs were downloaded from miRBase [DESeq2  was util miRBase . miRNA–c miRBase  and Targ miRBase , respectShiny application was built with RStudio [https://github.com/airbox11/circ2GO.circ2GO was implemented using HTML and in R language (v3.6.0)  with shi RStudio . Part ofhttps://circ2GO.dkfz.de.The circ2GO database is freely and without registration available at The functions of circRNAs are gaining considerable interest across many areas of life sciences and have become a key focus in cancer research. To date, thousands of circRNAs have been detected in various species and tissues. While several functions have been proposed for these circRNAs, our understanding of their precise biological roles and significance is still limited for the vast majority of circRNAs. With circ2GO, we present a comprehensive database for human circRNAs, including their expression in a broad cell line panel, their associated GO terms regarding molecular functions, biological processes, and cellular components, as well as comprised miRNA binding sites predicted by two independent algorithms. Visualizations in transcript maps and heatmaps, advanced forward and reverse search options, batch search and download options, combined with its intuitive and easy use will make circ2GO a valuable tool for circRNA research.We imagine that the most widespread applications of circ2GO will be: (1) the comparison of all circRNAs for a given gene or transcript, including their expression levels; (2) the search for cell lines with a particularly high or low expression of a specific circRNA; (3) the search for GO terms associated with a circRNA by virtue of its linear counterpart to form hypotheses about its potential impact on the functions of pathways to be experimentally tested; (4) the search for all circRNAs derived from genes involved in a specific molecular function, biological process, or cellular component of interest; (5) the search for abundant circRNAs harboring binding sites for a particular miRNA of interest, and their prioritization based on their expression and number of binding sites; (6) the search for all binding sites of high confidence within a specific circRNA of interest to form hypotheses about its potential function as ceRNA (competing endogenous RNA) to be experimentally tested.While the expression data provided in circ2GO are limited to lung-derived cell lines, other functions of circ2GO are not restricted to lung cancer or cancer research in general, but can be applied to other areas of human life sciences. The “GO Search” tool and the “microRNA search” tool can also be applied if the same circRNA has been identified in any other context. While the expression patterns are derived from a broad panel of human lung cell lines, the sequencing depth as well as the rRNA depletion (instead of polyA-enrichment) of our underlying transcriptomic study gives a comprehensive picture of the landscape of human circRNAs. For comparison, circ2GO includes 148,811 distinct circRNAs, while human studies stored in one of the leading circRNA databases, circBase, add up to only 92,375 circRNAs. Hence, circ2GO provides a map of human circRNAs with a large depth. Moreover, the transcript map visualization, the “GO Search” options, and the “microRNA Search” options are fully separate of the underlying expression dataset, and can therefore be universally applied, independent of the user’s research area for this large set of circRNAs.


Discovering that metals are essential for the structure and function of biomolecules has given a completely new perspective on the role of metal ions in living organisms. Nowadays, the design and synthesis of new metal-based compounds, as well as metal ion binding components, for the treatment of human diseases is one of the main aims of bioinorganic chemistry. One of the areas in vanadium-based compound research is their potential anticancer activity. In this review, we summarize recent molecular and cellular mechanisms in the cytotoxic activity of many different synthetic vanadium complexes as well as inorganic salts. Such mechanisms shall include DNA binding, oxidative stress, cell cycle regulation and programed cell death. We focus mainly on cellular studies involving many type of cancer cell lines trying to highlight some new significant advances. The discovery that metals are essential for the structure and function of biomolecules has given a completely new perspective on the role of metals in living organisms . It has cis-[Pt(NH3)2Cl2] (cisplatin) [In the year 1965, Barnett Rosenberg serendipitously discovered the Pt(II) coordination compound, splatin) , one of splatin) . Furthersplatin) . Both cosplatin) ,9. Unforsplatin) ,11. Howesplatin) .Nowadays, the design and synthesis of new metal-based compounds, as well as metal ion binding components, for the treatment of human diseases is one of the main aims of bioinorganic chemistry . Metal-b2VO4− [This new approach to the design and synthesis of new metal-based molecules has not excluded vanadium, which is the 18th most abundant element in our planet’s crust and the 2nd most common element in sea water, in regard to transition metal concentration (between 30 and 35 nM), where it exists mainly in the form of H2VO4− . It is n2VO4− . Importa2VO4− . In the 2VO4− .One of the areas of vanadium research is its potential anticancer activity. Recently, reviews describing its mechanism of anticancer activity have been published ,20,21,22In classical chemotherapy, anticancer compounds directly target DNA, causing lesions and ultimately triggering cell death. This is in accordance with the cisplatin paradigm in which one of the major therapeutic pathways of the platinum-based complex is based on interaction with DNA to generate inter- and intra-strand crosslinks. This leads to transcription inhibition, disruption of the DNA repair system and ultimately to apoptosis . Nowaday1) to the salmon sperm DNA, accompanied with a partial insertion of the ligand between the base stacks of the DNA. These experimental results have been confirmed by the results of molecular docking [1) exhibited cytotoxicity against breast, liver and colon cancer cell lines [2 and 3) (containing trifluoropentanedione and trifluoro-1-phenylbutanedione) has shown that investigated complexes preferred minor groove binding with DNA [4) exhibited stronger DNA binding than cisplatin [5 and 6) exhibited cytotoxic activity against human cervical carcinoma cells by direct interactions with DNA, thus increasing the level of arrest cells in stage G2/M. The DNA interaction ability has been determined also for the phenantroline vanadium complex (7–10) with simultaneous cytotoxic activity against human ovarian and breast carcinoma cells [11–13) interacted with calf-thymus DNA (CT-DNA) through a non-classical intercalative mode and they could efficiently cleavage plasmid pBR322 DNA upon exposure to ultraviolet light. Additionally, all investigated complexes exhibited anti-proliferative activity against many human tumor cell lines [14–17), which could bind with CT-DNA and which cleaved supercoiled plasmid DNA in the presence of H2O2, and also exhibited cytotoxicity against a cervical cancer cell line by inducing apoptosis [18) [19 and 20) [21–25) complexes [Furthermore, in the case of vanadium, many studies on its interaction with DNA have been performed. Mohamadi et al.  have use docking . Additioll lines . Anotherwith DNA . Interesisplatin . Importaisplatin  have demma cells . Furtherma cells  has showll lines . A similpoptosis . The DNA and 20)  or VO(IIomplexes .26 and 27) for its PDT action, which showed dual activity: light-activated VO2+-DNA crosslink formation and singlet oxygen (1O2) induced mitochondria-targeted PDT. Interestingly, the BODIPY-based vanadium complex (27) exhibited remarkable photocytotoxicity against cervical and breast cancer cell lines via apoptotic pathway in visible light (400–700 nm) compared with low dark toxicity [28 and 29), and this effect was observed upon irradiation with visible light [30–33) had DNA binding propensity and it was shown that these interacted with CT-DNA through minor groove binding mode; however, the complex with isonicotinoylhydrazone of 2-hydroxy acetophenone (32) showed the highest photo-induced DNA cleavage activity [An interesting approach to anticancer therapy provides photodynamic therapy (PDT) which is based on selectively damaging the photo-exposed cancer cells, leaving the unexposed healthy cells unaffected . Kumar etoxicity . In othele light . Additioactivity .34) inhibited relaxation activity of human topoisomerase IB in a dose-dependent manner. However, the inhibition was incomplete, suggesting that the inhibitory effect of the vanadium compound is reversible [Importantly, many indirect mechanisms that affect DNA structure and stability have been determined. Topoisomerases are enzymes that control the topological state of DNA through the re-joining or breaking of DNA strands . There aversible .The structure and activity of DNA-binding vanadium compounds are summarized in Oxidative stress is a complex issue . This co3 (36) and vanadium(IV) sulfate oxideVOSO4 (37) significantly increased the ROS level in human lung cancer cells. However, the higher ROS level was induced by complexes containing vanadium(+IV) in the coordination center [38) inhibited growth of lung carcinoma cells by activating the axis of Rac1-NADPH oxidase leading to oxidative stress [39) triggered apoptosis in human colorectal carcinoma cells through mitochondrial outer membrane permeabilization, possibly by altering cellular redox status [40) and sodium metavanadate (36) increased ROS generation; however, they did not induce a sustained increase of ROS generation, but the level of ROS reached a plateau instead [41 and 42) [43 and 45) by use of the same type of cancer cells. The complex with phenanthroline (45) showed the highest cytotoxic activity which correlated with the strongest increase of ROS [45–47) also induced the ROS generation in a human pancreatic cancer cell line [48 and 49) showed cytotoxicity against a human liver cancer cell line by decreasing the mitochondrial membrane potential and increasing the ROS levels [Oxidative stress as a mechanism of the cytotoxic activity of vanadium-based compounds is well documented. In one of the most recent studies, vanadium salts, sodium metavanadate NaVOn center . These rn center . For pere stress . The vanx status . In the  instead . Additio instead . Oxidati and 42) . Leon et and 42)  have inve of ROS . Interesell line . In anotS levels .2-modified) quartz surface has been prepared. The obtained results showed that the VO2-modified quartz surface (releasing ions from surface) interrupted the mitochondrial electron transport chain and then elevated the intracellular ROS levels in the cholangiocarcinoma cells [An interesting study has been performed by Li et al.  in whichma cells .35, 40, 50) should reduce their toxicities in human normal cells without affecting their antitumor activities in cancer cells (selective cytotoxicity). This is consistent with results for an oxidovanadium(IV) complex with 3-quinic acid (51), which exhibited antioxidant activity as well as selective cytotoxicity against a human breast cancer cell line without increase of the ROS level [52). The new complex showed cytotoxic activity against breast cancer cell lines without generating reactive oxygen species in the cells and producing damage of DNA. Moreover, the complex did not affect the normal proliferation of the breast epithelial mammal cells [53) showed moderate cytotoxicity against lung and cervix cancer cell lines with simultaneous slight increments of ROS levels and decrease of the GSH/GSSG ratio [54) caused a concentration-dependent inhibition of cell human osteosarcoma cells and ROS generation. The alterations in the GSH/GSSG ratio were proposed as the main mechanisms [55) also showed ROS-dependent cytotoxicity against a human lung cancer cell line [An interesting and intriguing study performed by Wang et al.  suggestsOS level . The synOS level ,58, appeOS level  have conal cells . These iSG ratio . AdditioSG ratio . In anotchanisms . Besidesell line .56) has been proposed as a promising chemoprotectant against oxidative stress and nephrotoxicity induced by cisplatin [56) significantly attenuated cyclophosphamide-induced oxidative stress in the liver as evident from levels of reactive oxygen species, nitric oxide and lipid peroxidation [Interestingly, an L-cysteine-based oxidovanadium(IV) complex  exhibit photocytotoxic activity against breast and cervical cancer cells through ROS generation [In case of photodynamic therapy (PDT) (described in the DNA subsection), induction of oxidative stress has been documented. Ferrocenyl-terpyridine oxidovanadium(IV) complexes (neration . Moreoveneration . The str37) caused molecular oxygen-dependent DNA strand breaks as well as molecular oxygen dependent 2′-deoxyguanosine (dG) hydroxylation to form 8-hydroxyl-2′-deoxyguanosine (8-OHdG) [4 (37) with dG in argon did not generate any significant amount of 8-OHdG [The free radical generation may be associated with DNA damage. It has been determined that vanadium(IV) ((8-OHdG) . Moreovef 8-OHdG . For somf 8-OHdG ,37,47,481, S, G2 and M phases [1 and G2 are “gaps” during which time cells prepare for the next phase [2 arrest following DNA damage [The cell cycle is the sequence of stages through which a cell passes between one cell division and the next. This process includes four stages: GM phases . In S phxt phase . The pasxt phase . Cyclin/xt phase . If aberxt phase . During xt phase . The sysxt phase . MoreoveA damage ,71. The A damage . DeregulA damage .36) caused G2/M cell cycle arrest in prostate cancer cells, which is evidenced by the increase in the level of phosphorylated cdc2(cdk1) at its inactive Tyr-15 site. Importantly, the results revealed that ROS-mediated degradation of cdc25C is responsible for vanadate-induced G2/M cell cycle arrest [61) induced G2/M phase cell cycle arrest in a human thyroid carcinoma cell line [61) decreased the expression of cyclin D1 and increased the expression of p21 protein in papillary thyroid carcinoma-derived cells; however, the cell cycle profile was similar to the untreated cells [62) arrested the cell cycle in G2/M phase whereas pirydone-based vanadium complex (63–65) arrested it in the G0/G1 phase [62 and 63) induced dephosphorylation of the Retinoblastoma protein (Rb) and together had a pronounced increase of cyclin-dependent kinase inhibitor p21 protein expression [66) induced cycle arrest in the G2/M phase with simultaneous triggering of the p53/p21 pathway in pancreatic cancer cell lines [67 and 68) [69–72) arrested the cell cycle in the S and G2/M phases in hepatocellular carcinoma cell lines [73 and 74) caused a G0/G1 phase cell cycle arrest in the same type of cancer cell lines [0/G1 phase cell cycle arrest, induced by organic vanadium complexes (75–78), was shown in human neuroblastoma cells [61) [79 and 80) arrested the cell cycle at the S phase in human colon cancer and ovarian carcinoma cell lines [The impact of vanadium-based compounds on cell cycle progression was determined and described earlier . More ree arrest . Furtherell line . Interesed cells . In otheG1 phase . Furtherpression . These sll lines . Inducti and 68) . An oxidll lines . Contrarll lines . Additioma cells . Cell cylls [61) . Additioll lines . All abo2/M cell cycle arrest in pancreatic cancer cells. Additionally, cell cycle disruption with simultaneous ROS generation was shown in lung carcinoma [Some studies, described in the previous subsection, have found a connection between DNA binding and cell cycle arrest ,28,31,40arcinoma  and ostearcinoma .Apoptosis is programed cell death with distinct genetic and biochemical pathways that play a critical role in development and homeostasis in normal tissues . Apoptos3VO4) (61) induced apoptosis in the oral squamous cell carcinoma cell line [61) induced typical features of apoptosis including DNA fragmentation, loss of mitochondrial membrane potential and activation of caspase-3 in thyroid cancer cells harboring RET/PTC1  [Induction of apoptotic cell death by vanadium-based compounds is well established. Sodium orthovanadate (Naell line  as well ell line . Moreovegements) .81) induced apoptosis in gastric cancer lines via the mediation of the intrinsic apoptotic pathway  [82) upregulated the expressions of p53 and caspase 3 and 9 with simultaneous downregulation of Akt, mTOR and VEGF expressions in human breast cancer cell lines [82) induced apoptosis in a breast cancer animal model [83) triggered apoptosis by activation of both extracellular (through caspase 8) and intracellular (through caspase 9) apoptosis-inducing pathways leading to activation of downstream caspase 3 in the human T-leukemic cells [84) induced apoptosis in liver hepatocellular carcinoma cells using the p53-p21 pathway-dependent way [67 and 68), was also determined in cervical cancer cells [85) induced upregulation of proteins such as caspase 3, caspase 6, caspase 7, caspase 10, caspase 11, Bcl-x and DAPK, as well as downregulation of ones such as PKB/AKT and DIABLO [86 and 87) showed remarkable apoptotic photocytotoxicity in visible light and specific localization to endoplasmic reticulum (ER) in ovarian and breast cancer cell lines [In the case of some vanadium-based complexes, it has been exhibited that vanadium complex (ase-3/9) . Moreovell lines . Additioal model . On the ic cells . A vanaddent way . A p53-der cells . A compld DIABLO . Moreoved DIABLO . An intell lines .3 (36) and VOSO4 (37), exhibited apoptotic-inducing cytotoxicity against non-small lung cell carcinoma cells with simultaneous increase of ROS level [62) and the vanadium complex with pyridinonate (63) induced apoptosis through generation of ROS in malignant melanoma cells [39) actuated apoptosis through mitochondrial outer membrane permeabilization in human colorectal carcinoma cells; however, this was in a caspase-independent manner, possibly by altering cellular redox status and inflicting DNA damage [Induction of oxidative stress may lead to apoptotic cell death . VanadiuOS level . Pisano ma cells . InteresA damage .88 and 89) induced cell death in colon cancer cells via the activation of RIPK3 and necroptosis pathway [45, 47) also trigger the necroptosis pathway in a human pancreatic ductal adenocarcinoma cell line [66) induced autophagic process in pancreatic cancer cells with simultaneous increase in the RAGE protein level [90) breast cancer cells [91) exhibited strong apoptotic activity against hepatocellular carcinoma cell lines under normoxic conditions. However, this was completely lost under hypoxic conditions [Evading programed cell death is one of the hallmarks of cancer . Therefo pathway . Furtherell line . In anotin level . Autophaer cells . An intenditions . This wanditions . Autophanditions . StructuSummary of the described molecular and cellular mechanisms of vanadium compounds are illustrated in Many other mechanisms in the anticancer activity of vanadium compounds have been determined which are described in other reviews.Kioseoglou et al.  describeIrving and et al.  discusse4VO3 was exhibited to prevent T cell activation by downregulating the expression of proinflammatory cytokines including IL-2, IL-6, TNF-α, and IFN-γ [Interestingly, both pro- and anti-inflammatory effects have been documented for V-containing compounds . The immnd IFN-γ . Triggernd IFN-γ .Furthermore, in addition to potential anticancer activity, vanadium compounds exhibit a well-established antidiabetic activity . VanadatThe studies described in this review suggest that the molecular and cellular mechanisms of vanadium compounds depend on many factors, including the oxidation state of the vanadium cation, organic ligands, spatial structure and also the type of cancer cell lines. That is why we can observe so many, sometimes mutually exclusive, mechanisms of cytotoxicity.2+ ion hinders oxidation of the metal ion, V(IV) to V(V) [The literature review revealed that the chemical form of vanadium-based complexes  influences their physicochemical properties and thus their biological properties. For instance, the presence of a strong binding ligand in the coordination sphere of the VO to V(V) . FurtherAlthough there is some evidence that the structure and physicochemical properties of vanadium complexes impact their biological activity, the correlation of the chemical form of vanadium-based complexes versus their mechanism of action still remains to be elucidated. Moreover, the differences in physicochemical and biological properties of the compounds may stem from very different experimental  conditions. The results of chemical studies on physicochemical properties of the compounds should be assessed very carefully as the experimental conditions leading to these results are generally very different from biological conditions. Furthermore, there are still very few in vivo studies and an almost complete lack of innovative approaches based on targeted therapies. In view of this, further biological research, focusing on a more in-depth analysis of cytotoxic activity using the most modern techniques, is required.In conclusion, the above considerations underline the anticancer potential of vanadium-based compounds. Through modification of the chemical form of vanadium-based complexes, we can influence affinity for DNA, oxidative stress or the type of cell death induced by vanadium-based compounds in cancer cells. On the other hand, due to many factors, it is difficult to precisely define structure–activity relationships.