We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS) expression, nitric oxide (NO) production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.Endothelial cells (ECs) express fibroblast growth factor (FGF) receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs) were used to generate  Normal vascular function is critically dependent on physiological endothelial cell (EC) responses to altered tissue nutrient demand and an appropriate stress response. The endothelium generates many factors that regulate vascular tone, adhesion of circulating blood cells, smooth muscle proliferation, and inflammation; as such, EC dysfunction has emerged as a relevant risk marker for cardiovascular events in the general population, particularly older individuals , patientin vivo α-Klotho delivery resulted in reversal of endothelial dysfunction, reduction of the elevated blood pressure, and prevention of arterial wall hypertrophy and perivascular fibrosis [Both FGF-23-deficient and α-Klotho deficient animal models exhibit similar phenotypes as patients with impaired renal function, e.g., accelerated aging with the development of extensive arteriosclerosis and vascular calcification , 11. A nfibrosis . Interesfibrosis . Furtherfibrosis . ImportaIn this study, we present data that show differential expression of functional full-length α-Klotho protein in human ECs. In addition, the study provides evidence that this full-length α-Klotho protein is required for EC response to FGF-23.This study conforms to the principles outlined in the Declaration of Helsinki. Human tissues were obtained with local ethical approval and informed written consent from three sources: surgical specimens from the human tissue bank at University Hospitals Coventry and Warwickshire NHS Trust, UK , artery and kidney tissue from Warwick Medical School, UK , and brain tissue was kindly donated by the New York Brain Bank (NYBB) at Columbia University.n) corresponds to the total repeat experiments performed collectively by using all 2 or 3 cell sources. Cells were treated with EC medium  containing 5% FBS and 0.5 mM β-glycerolphosphate for HAECs, and CSC medium  containing 5% FBS for HBMECs. FGF-23  was used for cell treatments. Passages of cells used in this study were less than or equal to eight. HBMECs were assessed regularly to confirm their central nervous system properties. The cells were stained with antibodies for Von Willebrand factor to confirm EC origin. These cultures were analyzed routinely for astrocyte contamination by staining with anti-glial fibrillary acidic protein. Cell lysates of human pulmonary microvascular endothelial cells  and human cardiac microvascular endothelial cells  for Western blot were commercially obtained .Three different age-matched sources of human aortic endothelial cells (HAECs)  and two different age-matched sources of human brain microvascular endothelial cells (HBMECs)  were obtained. The number  and rabbit polyclonal isotype antibody (Cat No. ab27478) at 1:100 to 1:500 were used. Sections were stained with Vectastain Universal Elite ABC (Avidin and Biotinylated horseradish peroxidase macromolecular Complex solution) kit  before counterstaining with hematoxylin.in vitro assay kit . Briefly, cells were seeded until 50% confluent in complete medium. FGF-23 treatment was then performed in DMEM containing 0.5% FBS. XTT stock solution equal to 20% of the culture medium volume was added to media. Media containing XTT was then transferred to a 96-well plate and the absorbance was measured at 450 nm. Background absorbance readings at 690 nm were subtracted from readings taken at 450 nm.Cell proliferation was assessed using XTT NO levels were assessed with a Colorimetric Nitric Oxide assay kit  according to the manufacturer’s instructions. The unstable nature of NO means its rapid oxidative degradation to nitrite and nitrate. Subsequently the nitrate was reduced to nitrite with vanadium (III) chloride and then the total nitrite was quantified by Griess reaction. The tested samples were fresh cell lysate after cell treatment experiments. Cells were washed with cold PBS, resuspended in ice-cold Assay Buffer, and homogenized quickly on ice. Then the samples were centrifuged for 5 minutes at 4°C at top speed to remove any insoluble material. The collected supernatant underwent a deproteinization step before being used in the assay. After these reactions, the absorbance was measured with a test wavelength at 540 nm and a reference wavelength at 630 nm. NO production for each sample was normalized by protein concentration.Western blot technique was used for protein analysis of cell lysates as previously described . Brieflyα-Klotho small interfering RNA (siRNA) was purchased from Invitrogen . Cells were seeded until 60% confluent and placed in opti-MEM I Reduced Serum Medium . Transfection was achieved using Lipofectamine reagent (Cat No. 15338–100) and PLUS reagent (Cat No. 11514–015) from Invitrogen , at the manufacturer’s recommended concentrations. For all experiments, 1 μM α-Klotho siRNA was used over a transfection time of 24 hours followed by EC medium and the other cell treatments. Validation of siRNA efficacy experiments was performed .5’- ACT CCC CCA GTC AGG TGG CGG TA-3’ and reverse, 5’- TGG GCC CGG GAA ACC ATT GCT GTC-3’ [Total RNA was isolated from cell lysates using an RNeasy kit  following the manufacturer's protocol. Reverse transcription of total RNA (100–200 ng) was carried out using Superscript 3 reverse transcriptase  with random hexamers . The generated cDNA was used as the template for PCR of α-Klotho using BIOTAQ DNA polymerase . The primers for α-Klotho were: forward, T GTC-3’ , 17. PCRpost-hoc analysis was performed to compare data among experimental groups if there were more than 2 groups.Data were shown as mean ± SD. The 2-tailed Student’s test was used to compare data between two experimental groups. ANOVA with Bonferroni in vitro only when cells were treated with FGF-23 concentrations > 50 ng/ml. FGF-23 had no effects on the proliferation of HBMECs .We have previously shown that exposure of human aortic SMCs to high phosphate renders them FGF-23 resistant due to loss of full-length α-Klotho expression . When HAOur group and others previously reported that FGF-23-Klotho signaling stimulates proliferation in vascular SMCs  and renain vitro did not respond to FGF-23 due to the absence of full-length α-Klotho. We therefore investigated if circulating α-Klotho made HBMECs responsive to FGF-23. After adding soluble α-Klotho, HBMECs remained resistant to FGF-23 and no increased cellular proliferation or expression of eNOS was seen  , 7, HBMEwas seen .In this study we showed for the first time full-length α-Klotho protein expression in the ECs of elastic arteries, medium sized arteries, and in the microcirculation of numerous human tissues, and described differential endothelial α-Klotho protein expression along the human vascular tree. ECs express functionally active α-Klotho protein in most vascular beds; however, no α-Klotho protein was found in the microcirculation of brain or brain tissue derived ECs. We reveal that FGF-23 mediated eNOS protein expression, NO production, and cell proliferation in ECs with the expression of membrane α-Klotho protein. On the other hand, there was a lack of functional response of brain derived ECs to FGF-23, regardless of the presence of the endothelial FGF receptors or soluble α-Klotho. Our results suggest that FGF-23-mediated endothelial functions require the presence of full-length membrane α-Klotho protein.et al. showed that EC function depends on α-Klotho using a mouse α-Klotho knockout model [et al. utilized coronary artery ECs to highlight that in states of α-Klotho deficiency, FGF-23 mediated NO production is attenuated, leading to an increase of reactive oxygen species (ROS) [Several previous studies support our postulations that full-length membrane α-Klotho is a necessity in modulating EC response to FGF-23. Previously, Saito ut model . Furtherut model , 21. Likes (ROS) . Two reces (ROS) .2+ channel and restoring endothelial Ca2+ handling. Another cell surface receptor system modulated by hormonal α-Klotho could also be essential for brain microvascular ECs integrity and physiological function. A α-Klotho overexpressing mouse model experienced extended life span that may in part be due to α-Klotho binding to a cell-surface receptors, resulting in repressed intracellular signals of insulin and insulin-like growth factor 1 pathways [Given the importance of endogenous α-Klotho, it is interesting that our data displayed its absence in ECs of human brain microvessels. Traditionally, ECs of human brain microvessels (the major component of the blood-brain barrier (BBB)) could be distinguished from other tissue ECs due to several unique characteristics, with the most important being the highly resistant tight junctions that provide high transendothelial electrical resistance and retard paracellular flux , 26. We pathways .in vivo α-Klotho gene delivery [et al. showed the importance of cellular α-Klotho, utilizing HUVECs and human fibroblasts to investigate the role of α-Klotho in cellular inflammatory response to cell senescence. They showed that intracellular, not circulating, α-Klotho was essential for suppressing senescence associated inflammation [ECs are very well placed to respond to humoral α-Klotho protein, particularly on the cell surface exposed to plasma. It is therefore interesting to note that soluble α-Klotho was able to mitigate phosphate and FGF-23 mediated endothelium dependent vascular contractility through increased NO production . Endothedelivery . Liu et ammation . These sPrevious clinical studies have shown that elevated circulatory FGF-23 is associated with endothelial dysfunction in CKD patients . Similaret al., using human coronary artery ECs, also showed that NO production increases after FGF-23 treatment and this was dependent on the presence of α-Klotho [As stated recently, Richter α-Klotho ; howeverin vivo study setup in the future would be needed to validate this postulation.In addition, our results also showed a dose-dependent response of HAECs to FGF-23, raising an important consideration for future studies into clinical application. The dose of FGF-23 required to stimulate proliferation was much higher than the dose to increase eNOS expression and NO production in HAECs. Mechanisms of vascular injury such as age-related vascular dysfunction usually begin with a lower expression or inactivation of eNOS followed by decreased production of NO . Therefoin vitro cellular experiments; our findings would need validation by additional in vivo studies. We recognize that the primary HBMECs monolayer culture system may be limited by HBMEC transformation and loss of specific BBB features. While single-cell type culture systems are indispensable to studying the role of individual cell-types, our data will need to be interpreted cautiously against these limitations. It is important to highlight that immunostaining of tissue provided the same observation as the in vitro work and both primary endothelial cell cultures were grown in comparable cell culture conditions. Future work using a co-culture systems or organ culture may in part address these concerns and help confirm our data. Furthermore, in this study, we have utilized brain tissue from the cerebellum and hippocampus regions to investigate the expression of α-Klotho in brain microvessels. As previously demonstrated by our group [Our study has some limitations. First, these mechanistic studies only used ur group , α-Klothur group , 35, we In summary, our results have shown that human ECs differentially express functional full-length α-Klotho protein. And that it is this full-length α-Klotho protein that modulates EC response to FGF-23. We speculate that intracellular, cell surface and cleaved α-Klotho is likely to play different roles for endothelial and vascular function. Future studies will have to investigate its potential role in health and diseases.S1 Fig(TIF)Click here for additional data file.


Thalidomide and its derivatives, lenalidomide and pomalidomide , have significantly changed the treatment landscape of multiple myeloma, and the recent discovery of cereblon (CRBN) as their direct biological target has led to a deeper understanding of their complex mechanism of action. In an effort to comprehend the precise mechanisms behind the development of IMiD resistance and examine whether it is potentially reversible, we established lenalidomide‐resistant (‐LR) and pomalidomide‐resistant (‐PR) human myeloma cell lines from two IMiD‐sensitive cell lines, OPM2 and NCI‐H929, by continuous culture in the presence of lenalidomide or pomalidomide for 4–6 months, until acquirement of stable resistance. By assessing genome‐wide DNA methylation and chromatin accessibility in these cell lines, we found that acquired IMiD resistance is associated with an increase in genome‐wide DNA methylation and an even greater reduction in chromatin accessibility. Transcriptome analysis confirmed that resistant cell lines are mainly characterized by a reduction in gene expression, identifying SMAD3 as a commonly downregulated gene in IMiD‐resistant cell lines. Moreover, we show that these changes are potentially reversible, as combination of 5‐azacytidine and EPZ‐6438 not only restored the observed accessibility changes and the expression of SMAD3, but also resensitized the resistant cells to both lenalidomide and pomalidomide. Interestingly, the resensitization process was independent of CRBN. Our data suggest that simultaneous inhibition of DNA methyl transferases and EZH2 leads to an extensive epigenetic reprogramming which allows myeloma cells to (re)gain sensitivity to IMiDs. CRBNcereblonDNMT1DNA methyl transferase 1dscDNAdouble‐stranded cDNAMMmultiple myelomaMS‐MCAmethylation‐specific melting curve analysisNoEno enzymeSAMS‐adenosylmethionine1et al., The introduction of novel agents for the treatment of multiple myeloma (MM), mainly proteasome inhibitors and immunomodulatory agents (IMiDs), has significantly improved the survival rates of the patients, and both classes of drugs stand as the main treatment options for MM  together with CUL4A, DDB1, and RBX1, where it serves as a substrate receptor  as the direct target of thalidomide, suggesting a model that explained the drug's teratogenic effects , or nucleosome occupancy can potentially regulate the expression of CRBN in these cell lines. By performing Acce22.12 in a humidified atmosphere and under sterile conditions. Cell density and viability were determined using the cell counter NC‐250 . For the development of IMiD‐resistant cell lines, we treated OPM2 and NCI‐H929 continuously with increasing doses of either lenalidomide  or pomalidomide  for 4–6 months, until cell viability and proliferation were not affected, as previously described  whenever the cells consecutively exhibited viability above 90%  in the presence of the drug. The cells were not treated with any compounds for a minimum of 7 days before all the experiments performed in this study.The human myeloma cell lines JJN3, OPM2, RPMI‐8226, U266, KMS12‐BM, and NCI‐H929 were purchased from DSMZ  and were grown using the recommended cell culture medium by DSMZ, at 37 °C with 5% COOther chemotherapeutic agents used in the cell culture in this study were 5‐azacytidine  and EPZ‐6438 (MedChem Express).2.25 and 4 × 105 cells·mL−1) in 96‐well plates and were left untreated (control) or treated with serial doses of lenalidomide and pomalidomide. Following incubation for 3 days, 100 μL of XTT Labeling Reagent was added in each well, and after a further incubation of 2–4 h, absorbance was read at 450 and 650 nm (reference wavelength). Generation of dose–response curves and calculation of IC50 values for lenalidomide and pomalidomide for each cell line were performed using graphpad prism 6 software .Cell proliferation was determined using the XTT Cell Proliferation Kit II . Briefly, cells were seeded in different concentrations , according to the manufacturers' protocol. All samples were analyzed in a FACS Calibur (BD Biosciences). All experiments were performed in a triplicate and repeated at least once.2.3A total of 89 bone marrow samples were obtained from patients with MM diagnosed with MM between 2007 and 2009, according to the IMWG criteria, at the Department of Hematology, Rigshospitalet, Denmark. From these samples, 48 were obtained at diagnosis and 41 at relapse. For all the samples, the CD138+ plasma cells were isolated from the mononuclear cell population from each bone marrow sample with the use of a RoboSep . All included patients provided written consent in accordance with the Helsinki Declaration. The Danish National Ethical Committee approved the conduction of this study.2.4Total DNA and RNA were isolated from each cell line using All Prep DNA/RNA/miRNA Universal kit  according to the manufacturers' protocol. The quantity (260 nm) and quality (260/280 and 260/230 ratios) of total DNA and RNA were measured by spectrophotometry on a NanoDrop‐1000 spectrophotometer .2.5CRBN was performed by MS‐MCA as described, using 1 μg of bisulfite‐converted DNA as a template  and unmethylated genomic DNA (Roche) were also bisulfite‐converted and used as positive (methylated) and negative (unmethylated) control, respectively.The melting peaks were calculated using the 2.6Ct values transformed into a linear‐scale expression quantities, and raw Ct values into the NormFinder , many of which have been shown to be stably expressed in cancer  and were therefore used for normalization of all qPCR data in this study. Relative gene expression was calculated by using the comparative threshold method  according to the manufacturers' instructions. Subsequently, the samples were diluted 1 : 10 in DNase/RNase‐free water and qPCR was performed in duplicate in a 96‐well format using 5 μL of cDNA and SYBR Green I Master Mix (Roche) to a total volume of 20 μL. The PCR amplification was carried out with the following PCR cycling conditions: one cycle of 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s, and one cycle of 72 °C for 10 min, on a LightCycler® 480 instrument II (Roche Diagnostics). All the qPCR primers used in this study are shown in Table 2.7Whole‐cell lysates were isolated from all cell lines using lysis buffer (RIPA buffer) with the addition of a combination of protease inhibitors . Protein concentration was determined by generating a 6‐point standard curve using Bradford assay in duplicate. Equal amounts of protein (50 μg) were then loaded to SDS/PAGE gels, followed by transfer to a polyvinylidene difluoride membrane. After blocking with 5% BSA in TBS‐T, membranes were incubated with the relevant primary antibody overnight at 4 °C, washed, and incubated for 1 h with a HRP‐conjugated anti‐rabbit secondary antibody . Proteins were detected using chemical luminescence . The following primary antibodies were used in this study: rabbit polyclonal CRBN antibody , rabbit polyclonal IKZF1 antibody , and secondary anti‐rabbit polyclonal H3 antibody (Abcam).2.86 cells·mL−1. The cells were attached to individual slides by centrifugation of approximately 200 μL of cell suspension at 600 r.p.m. for 2–4 min. Following a standard procedure of fixing, washing, and blocking the attached cells, the slides were stained for CRBN using the HPA045910 antibody from Sigma, at a 1 : 1000 dilution. The stained slides were finally microscopically evaluated and scored independently by two hematopathologists, with a cutoff of 30% used for CRBN positivity.Cells were harvested and washed with PBS to a concentration of 4–5 × 102.9et al., g for 5 min, and then resuspended in 60 μL PBS. For nuclei isolation, 1 mL of lysis buffer  was added, and the cells were centrifuged at approximately 700 g for 5 min at 4 °C after an incubation of approximately 10 min on ice. The supernatant was removed and the nuclear pellets were resuspended in 1 mL wash buffer  and centrifuged again at 3000 r.p.m. for 5 min at 4 °C. The supernatant was removed and the following was added to each tube: 76.75 μL 1× NEB buffer 2, 7.5 μL 10× NEB buffer 2, 45 μL 1 mol·L−1 sucrose, 5 μL 32 mmol·L−1 S‐adenosylmethionine (SAM), and 15 μL 4 U·μL−1 M.SssI (or H2O for NoE tube). The reaction mixtures were flicked to mix and then incubated at 37 °C for 7.5 min. An additional 5 μL of SAM was added and the samples were incubated for further 10 min. Prewarmed (37 °C) 300 μL Stop Solution  and 3 μL Proteinase K (20 mg·mL−1) were added to each tube, and each reaction mixture was incubated at 55 °C for 16 h. The DNA was then purified by phenol/chloroform extraction and ethanol precipitation and finally redissolved in 21 μL nuclease‐free water for the subsequent analyses. One microgram of DNA was bisulfite‐converted using the Zymo EZ DNA Methylation Kit, and subsequent quality control of M.SssI treatment was performed as previously described , diluted 1 : 2, and then qPCR was performed in duplicate, normalizing to a biological duplicate of total input.For ChIP, we harvested 1 × 102.11et al., N = 340 327), cross‐reactive probes (N = 42 558) , and probes with a high detection P‐value (above 0.01) were filtered before further analyses, leaving a total of 522 554 probes.The Infinium HumanMethylationEPIC BeadChip array , containing a total of 866 836 probes, was used to analyze genome‐wide DNA methylation patterns. Data import and analysis, normalization, background and dye bias correction, and calculation of β values were performed using the R package minfi , probesAccessibility for each cell line (Acc) was defined as the difference between the corrected β‐values (range 0–1) of the M.SssI‐treated sample and of the NoE sample . After removing negative values of Acc for each cell line, Acc and NoE values of every resistant cell line were then subtracted from their respective values in their sensitive counterpart, resulting in delta‐methylation (ΔMeth) and delta‐accessibility (ΔAcc) values. ΔMeth and ΔAcc values  were used in the subsequent analyses as values for changes in DNA methylation and accessibility upon acquired IMiD resistance, using a cutoff of Δβ value ± 0.20 for changes at each locus.2.12A total of 1 μg of RNA was used for RNA‐seq. We sequenced ten cell lines , all in technical duplicate, leaving a total of 20 samples. The sample quality control, library preparation and quality control, sequencing, and data quality control were performed by BGI. Briefly, library preparation was performed using oligo‐dT beads for enrichment with mRNA containing poly‐A tails. RNA was then fragmented and reverse‐transcribed to double‐stranded cDNA (dscDNA) using random hexamer primers. Each library was then sequenced at a depth of 20M clean reads using SE50 sequencing on the BGISEQ500 platform. The dscDNA was then end‐repaired and ligated to the bubble adapter with protruding T of 3′ end. During PCR amplification step, the fragments were separated into single strands, amplified, and cyclized to form ‘DNA nanoballs’, which were finally sequenced at a depth of 20M clean reads.2.13et al., P‐value using the Benjamini–Hochberg method (P < 0.05).RNA‐seq data were aligned to the human reference genome (GRCh37/hg19) using Tophat, as part of the cufflinks pipeline. Aligned reads were then assembled into transcripts and quantified using the cufflinks pipeline . Comparison of mean values across different subgroups was made by ANOVA , with the analyses performed in graphpad prism 6. When multiple comparisons were made, the P‐values were corrected with the Bonferroni method. The level for significance was set at 0.05 with the following annotation: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.All the statistical analyses and calculations for Acce33.150 of both drugs for each cell line. Using the two most IMiD‐sensitive cell lines, namely OPM2 and NCI‐H929, we were able to generate lenalidomide‐ and pomalidomide‐resistant cell lines  by continuously culturing the cells in the presence of IMiDs for a period of 4–6 months  of CRBN, we observed that in both OPM2‐ and NCI‐H929 IMiD‐resistant cells, that area maintained enrichment of histone marks associated with active transcription  and was depleted of H3K27me3 . Even more interestingly, none of the probes showed significant decreases in chromatin accessibility, with all ΔAcc values for all probes being higher than the cutoff of −0.20  but probably through other cis or trans regulatory mechanisms.Furthermore, using Acce3.4SssIble, we were able to compare the combined chromatin accessibility and methylation changes between IMiD‐sensitive cell lines for a total of 522 554 probes across the genome. Interestingly, we found that acquired resistance for both lenalidomide and pomalidomide in OPM2 was primarily associated with a global decrease in chromatin accessibility and to a much lesser extent with DNA methylation changes  and EPZ‐6438, with panobinostat and EPZ‐6438 as the second most effective , an HDAC inhibitor (panobinostat), and an EZH2 inhibitor EPZ‐6438), tested both as monotherapy and in different combinations. All IMiD‐resistant cell lines were treated with different doses and combinations of these epidrugs for 48 h and then exposed to either no further treatment (control) or 10 μive Fig. B. Furthe8, testediDs Fig. C. Even miDs Fig. D. Overal3.6SMAD3 as a commonly downregulated gene across all four IMiD‐resistant cell lines with subsequent upregulation and normalization of expression after epigenetic resensitization  and EPZ‐6438 (doses ranging from 1 to 10 μm) in combination significantly increased the sensitivity of cells to both lenalidomide and pomalidomide in seven of eight cell lines , which means that it does not offer information about the entire genome. However, it is still a high‐throughput, but at the same time cost‐effective and bioinformatically manageable method, covering the most important regulatory genomic areas, such as gene promoters and enhancers.Using Acceet al., Chromatin accessibility has consistently been shown to affect gene expression . Despite the impressive In another interesting finding, we were able to inverse chromatin accessibility with a combination of 5‐azacytidine and EPZ‐6438 and restore sensitivity to both lenalidomide and pomalidomide in all four cell lines with acquired IMiD resistance. This combination was shown to almost entirely reverse the chromatin accessibility to the initial state and restore sensitivity, and interestingly, it was shown to be effective in inducing apoptosis independently of CRBN expression. This is, to our knowledge, the first report on combined DNA methyl transferase 1 (DNMT1) and EZH2 inhibition and its effects on epigenetic reprogramming and drug resistance.et al., et al., et al., The justification for this combination is extensively supported by previous data. In a study from our group, we showed that decreased chromatin accessibility in malignant cells (with a concomitant decrease in gene expression) was only partly associated with increased DNA methylation on the same probes, suggesting that the probes without DNA methylation most possibly obtain the repressive H3K27me3 histone mark  signaling pathway , bypassing CRBN through epigenetic reprogramming might overcome the effect of these mutations. However, the precise mechanism through which this combination can effectively restore IMiD sensitivity has to be further inquired, as a better understanding of the complexity of the CRBN pathway and IMiD interactions may unravel novel biological targets for therapy.In conclusion, our study is the first to ever show that acquired IMiD resistance is mainly an epigenetic event that is potentially reversible through a combination of two epigenetic compounds, 5‐azacytidine and EPZ‐6438. These drugs have been shown to have relatively low levels of toxicity, thus making them very good candidates for a prospective phase I study to examine their potential as ‘IMiD resensitizers’, which may improve the outcome of treatment of MM patients with drug‐resistant myeloma clones and a potentially high‐risk disease.KD designed this study, collected and analyzed the data, and wrote the manuscript. ASH, JC, LSK, FA, and NEUH contributed with data collection for this study. HFMP and LS contributed with data analysis for this study. COC, PG, GL, and KG were senior supervisors of this study and contributed to the design of this study, data interpretation, and preparation of the manuscript.Fig. S1.  Dose‐response curves for lenalidomide and pomalidomide in (A) OPM2 and (B) NCI‐H929, as well as their resistant counterparts.  Methylation specific melting curves for the CRBN promoter of CD138+ plasma cells from (C) 48 patients with newly diagnosed MM and (D) 41 patients with relapsed MM. The blue curves represent the unmethylated (left), methylated (right) and 50% methylated  controls, while the green curves, which all fall under the unmethylated control, represent the DNA samples from the patients. The promoter of CRBN was unmethylated in all the patients analyzed in this study.Fig. S2. (A) Proportional genomic distribution (promoters vs non.promoter areas) of all the probes with accessibility or DNA methylation changes across all IMiD‐resistant cell lines. A bit less than half of the probes showing significant changes in either accessibility or DNA methylation in the resistant cell lines map to promoter areas of known genes. (B) Apoptosis response of OPM2‐PR to either no treatment or 10 μm of lenalidomide or pomalidomide for 72 h, followed by a 48 h pretreatment with different epigenetic drugs. The most effective combination in restoring the apoptotic effect of IMiDs to the resistant OPM2‐PR cells was 5‐Azacytidine and EPZ‐6438. (C) Apoptotic response of H929‐PR without any pretreatment (black bars), with pretreatment only with 0.5 μm of 5‐Aza (green bars), with EPZ‐6438 (blue bars) and with both (red bars). The combination of 5‐Aza and EPZ‐6438 is effective in resensitizing the H929‐IMiD‐resistant cells in a similar manner to OPM2‐LR and OPM2‐PR. (D) Kernel density scatter plot of the accessibility changes (x axis) and DNA methylation changes (y axis) in OPM2‐PR treated with 5‐Aza and EPZ‐6438 for 48 h, compared to the paternal OPM2. The cluster of probes exhibiting decreased accessibility observed in OPM2‐PR  is significantly decreased, with more probes showing increased accessibility and decreased methylation.Fig. S3.  Volcano plots of differentially expressed genes for OPM2‐PR (A) and H929‐PR (B) compared to their paternal cell lines. The dots in red represent the differentially expressed genes with an absolute value of log2 fold change above 1 and an adjusted P‐value (Benjamini–Hochberg method) below 0.05. There is a slight predominance of downregulated genes in both cases, supporting the data from AcceSssIble. (C) Venn diagram of all the Illumina probes mapping to known genes showing either decreased accessibility or increased DNA methylation in OPM2‐PR and all the downregulated genes found in RNA‐seq of OPM2‐PR, showing only little/some overlap between epigenetic deregulation and gene expression. (D) Expression pattern of SMAD3 in OPM2 (IMiD sensitive), OPM2‐LR and OPM2‐PR (IMiD resistant) and the epigenetically resensitized OPM2‐LR and OPM2‐PR, shown in normalized counts for gene length (fpkm: Fragments Per Kilobase of transcript per Million mapped reads). SMAD3 follows the same expression pattern in the H929 sensitive, resistant and resensitized cell lines.Fig. S4. Apoptosis measurements in five primary IMiD‐resistant or partly resistant cell lines: KMS12‐BM, MOLP2, EJM, MOLP8 and U266.Click here for additional data file.


Results from flow cytometry showed that luteolin could induce apoptosis and caspase-3 activation and induce cell cycle arrest at G2/M phase in a dose- and time-dependent manner in EC1 and KYSE450 cells. JC-1 test results showed that membrane potential of mitochondria after luteolin treatment was down-regulated and this was an indicator for intrinsic apoptosis. Western Blot results showed the expression of cell cycle regulatory protein p21 and p53 increased and three apoptosis related proteins that participate in mitochondrial apoptotic pathway, namely, Bim, CYT-c and cPARP, also increased in luteolin treated cells compared with control groups. We further confirmed that luteolin could significantly inhibit the growth of ESCC tumors in xenograft mouse models and no evidence of systemic toxicity was observed. Our results suggest that luteolin can induce cell apoptosis and cell cycle arrest in G2/M phase through mitochondrial pathway in EC1 and KYSE450 cell lines and proper utilization of luteolin might be a practical approach in ESCC chemotherapy.In current study, we investigated the anti-tumor effect of luteolin in human ESCC cell lines  Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, and it is characterized by high mortality and striking geographic variation throughout the world with the highest incidence rate in east Asia , 2. ESCCet al reported that luteolin can induce G2/M arrest in both KYSE510 ESCC and OE33 EAC cell lines [et al reported that luteolin can induce G0/G1 cell cycle arrest in Eca109 human ESCC cell line in vitro [in vitro and in vivo.Luteolin is a kind of natural plant flavonoid, which can be derived from a variety of vegetables, fruits, and medicinal herbs in glycosidic form . Like otll lines , 18. Wanin vitro . And thein vitro and try to explore the underlying mechanisms. Moreover, we investigated the anticancer potential of luteolin in vivo in ESCC xenograft mouse models.Thus, we designed current study to focus on the cell cycle arrest and cell apoptotic effect of luteolin on esophageal cancer cells p < 0.05). Considering the degree of differentiation and cell origins, we chose EC1 and KYSE450 cell lines in further experiments. The half maximal inhibitory concentration (IC50) fell in 20 and 60 μM range in these cell lines. We chose 20 and 40 μM as experimental concentrations in further experiments to avoid severe cytotoxic side effect. Plate colony formation assay showed that different concentrations of luteolin could reduce the number of EC1 and KYSE450 cell colonies compared with control groups. Colony-forming efficacies of EC1 and KYSE450 cells were compromised with the increase of concentration of luteolin. Both colony numbers (p < 0.05) and in colony sizes decreased . These results indicated that luteolin could induce cell apoptosis via activating caspase-3.The effect of luteolin on cell apoptosis was further investigated by flow cytometry. The apoptotic rates at 72 h after different treatments are shown in Figure p < 0.05, Figure JC-1 test results show that with luteolin concentration increased, mitochondrial membrane potential decreased  and luteolin treated group. The results showed that growth of EC1-EGFP tumors was significantly inhibited by administration of luteolin Figure . Tumor sin vitro, including cell lines from hepatocellular carcinoma, cholangiocarcinoma, leukemia, thyroid cancer, breast cancer, prostate cancer, melanoma, lung cancer, oral squamous cancer, stomach cancer, gastric cancer, colon cancer, as well as ESCC and EAC [Chemotherapy may be used to stall the development of ESCC and extend survival of ESCC patients , 25. Cyt and EAC , 26–37.in vitro and compromise their colony-forming efficacies. This is consistent with findings from Zhang's and Wang's research groups [50 ranging from around 35 to 73 μM [50 of luteolin falling in the range of 20 to 60 μM. IC50 varies in different cancer cell lines. This indicates that different cancer cell lines might have different sensitivity to luteolin induced cell cycle arrest. Considering results from CCK-8 test and the degree of differentiation and origins of these four ESCC cell lines, EC1 and KYSE450 cells were chosen for further experiments. Results from flow cytometry revealed that EC1 and KYSE450 cells were arrested in G2/M phase after being treated with luteolin. Several studies have reported that luteolin can promote apoptosis but differentially induce cell cycle arrest in different types of cancer cell lines, either G1/S or G2/M arrest [et al and Wang et al, it is postulated that luteolin can also differentially induce cell cycle arrest in different ESCC cell lines [In current study, we further confirmed the anti-proliferation effect of luteolin in EC1 and KYSE450 ESCC cells. Results from CCK-8 assay showed that luteolin could suppress the growth of four ESCC cell lines in a dose-dependent manner h groups –19. In tto 73 μM , 19. In M arrest , 38–40. ll lines , 19.CIP1/ WAF1 is a kind of CDK inhibitors (CKIs), which can regulate the activities of CDKs [CIP1/ WAF1 upregulation [CIP1/ WAF1 and p53, and this can further induce apoptosis and inhibit cell proliferation in EC1 and KYSE450 cell lines.In eukaryotes, two Cyclins (Cyclin A2 and Cyclin B1) and cyclin-dependent kinase 1 (CDK1) play central role in G2/M transition. P21 of CDKs . In cellgulation , 42–49. L and BimS protein expression accompanied with CYT-C overexpression and cPARP cleavage. Based on these findings, we propose that luteolin can induce apoptosis in EC1 and KYSE450 cells through the mitochondrial pathway.There are two main pathways of apoptosis in mammals, intrinsic pathway, also called mitochondrial pathway, and extrinsic pathway. Mitochondrial pathway is the main target in developing anti-tumor drugs , 51. P53in vivo in xenograft mouse models for non-small cell lung cancer, squamous cell carcinoma of head and neck, breast cancer [Furthermore, we constructed ESCC xenograft mouse models by subcutaneous injection of EC1-EGFP cells in nude mice. The results showed that tumor sizes in luteolin treated groups were significantly smaller than that in control groups. No significant cytotoxic side effects were observed since the body weight in both groups showed no difference. This is consistent with findings from other groups that luteolin can significantly inhibit cancer growth t cancer , 52–54.in vivo study has also demonstrated that luteolin can significantly inhibit the growth of tumor in ESCC xenograft mouse models. This has further elucidated the mechanisms of luteolin in ESCC treatment and prevention, and indicates that proper utilization of luteolin might be a practical approach in ESCC chemotherapy.In conclusion, current study has demonstrated that luteolin can induce cell cycle arrest in G2/M phase and apoptosis through mitochondrial pathway in EC1 and KYSE450 cell lines, and All experiments in present study were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The animal experiments were approved by the Animal Care and Use Committee at Zhengzhou University. The number of animals used was minimized, and all necessary precautions were taken to mitigate pain or suffering.2 at 37°C. DMSO was used as drug delivery vehicle in all the in vitro experiments, and the final concentration of DMSO in negative control was 0.1%. Cells were checked routinely and trypsinized until they reached 80-90% confluency. Luteolin was purchased from Enzo Life Sciences  and dissolved in DMSO solution prior to use.The human Esophageal squamous carcinoma cell lines  were purchased from Shanghai cell bank (China Academy of Science) and cultured in DMEM medium (KYSE450/KYSE30), RPMI 1640 (EC1/EC9706)  which were supplemented with 10% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin  respectively under a humidified atmosphere of 5% CO3 cells were seeded in 100μl per well DMEM/RPMI 1640 in a 96-well plate. 100 μl luteolin at concentrations of 0, 10, 20, 40, 80 μM was added to the medium, and the cells were cultured for different time lengths (48 h and 72 h). Subsequently, 10 μl of the CCK-8 solution was added to each well and incubated at 37°C for another 2 h. The absorbance at 450 nm was measured on a spectrophotometric plate reader. Each group was repeated in three different wells.The cell viability was examined via cell counting kit-8  according to the manufacturer's instruction . BrieflyThe ability of cells to form macroscopic colonies was determined by a plate colony formation assay . Single The influence of luteolin on apoptosis and cell cycle distribution were detected with Annexin V-FITC-/PI apoptosis kit  and cell cycle kit  separately according to the manufacturer's instructions. KYSE450 and EC1 cells were exposed to luteolin at concentrations of 0, 20, and 40 μM for 24 h or 72h. Then, cells were trypsinized, collected by centrifugation at 2000 rpm for 5 min and washed with PBS. Then corresponding regents and solution were added and incubated according to manufacturer's instructions respectively. At the end of incubation, cell apoptosis distribution was analyzed on a flow cytometer , and cell cycle data was analyzed with ModFit software .Caspase-3 activity and mitochondrial membrane potential (ΔΨm) analysis were isolated using CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit  and mitochondrial membrane potential detection kit (JC-1)  according to the manufacturer's instructions respectively. Luteolin at concentrations of 20 and 40 μM were used as treatment for 72h. DMSO solution was used as control.After being treated with luteolin at concentrations of 0, 10, 20, 40 μM for 72h, whole cell proteins were extracted with RIPA lysis buffer and protein concentrations were determined by the standard BCA method . Then whole cell proteins (50 μg) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes . Membranes were blocked in TBS containing 0.1% Tween-20 and 5% dry milk, and probed with primary antibody overnight at 4°C. Primary antibody directly against Bim, cPARP, CYT-C, p21, p53 and GAPDH  were used at a dilution of 1:1000, followed by incubating with HRP-conjugated secondary antibodies for 2 h at room temperature. Signals were visualized using BeyoECL Plus  and captured using an Image Quant LAS-4000 . All the experiments were repeated three times. The protein quantification of the Western blot results was normalized to GAPDH and then compared to the control group.in vivo. They were cultured in pathogen-free sterile conditions with continuous access to sterile food and water. For tumorigenicity assays, 5×106/100 μl EC1-EGFP cells were subcutaneously injected into the upper portion of the right hind limb of BALB/c Nude mice to construct ESCC xenograft models. After tumor grows, the xenograft mouse models were randomly divided into two even groups, control and treatment (50mg/kg luteolin) groups. Mice in the control group were injected with HPBCD and mice in the treatment group were injected with luteolin (50mg/kg) to enterocoelia as intervention. Tumors were observed by FluorVivo  and size was measured using calipers every 3 days. Cervical dislocation was conducted after mice being euthanized by Isoflurane  inhalation 18 days after luteolin intervention. At the end of this experiment, tumors were harvested and weighed. The condition of mice was observed every day and the mice were weighed twice a week.Fourteen six-week-old female BALB/C-nu mice with green fluorescent protein marker of human esophageal cancer (EC1-E-GFP) were used in this study to test the therapeutic effects of luteolin All statistical analyses were conducted by using the SPSS 19.0 statistical software . Results are expressed as means ± SD. Two-tailed independent-samples Student's t-test was used to determine the difference between two mean values, and two-sided p values less than 0.05 were considered statistically significant.


Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.The online version of this article (10.1186/s12943-017-0745-1) contains supplementary material, which is available to authorized users. Despite advances in our understanding of the etiology and pathogenesis of cancer, the failure rate of novel therapies remains high. Current drug discovery efforts have predominantly utilized standard cancer cell lines due to their ease of proliferation and amenability to numerous preclinical applications. While these models have yielded key insights into mechanisms of malignant transformation and drug response, recent data suggests that the selective pressure of cell culture leads to a divergence from the tumors from which they were derived . Therefo- have emerged as important tools for preclinical and translational research. PDX models have several advantages over standard cell line xenografts including preservation of gene expression patterns, continuance of tissue histology and maintenance of chromosomal architecture. Despite their benefits, PDX models are limited by several factors including slow growth, variable engraftment rate and lack of sustained growth in vitro. While PDX cells may be grown in 2D and 3D cultures for short periods of time, most PDX cell lines cease proliferating and undergo senescence within several passages [Patient-derived xenografts (PDX) - in which patient tumor and stromal tissue is implanted directly into immunocompromised animals and maintained in vivo passages .Recently, Conditional Reprogramming (CR) technology has been successfully used to achieve sustained expansion of human normal and tumor epithelial cells. This method is based on co-culture of epithelial cells with growth-arrested mouse 3 T3-J2 fibroblast feeders in the presence of a Rho kinase inhibitor . ImportaThe PDX tumors used in this study were originally established through subcutaneous implantation of patient tumors into immune-deficient NSG mice and consecutively passaged in vivo. Cryopreserved PDX tumors were enzymatically disassociated to form a single cell suspension and co-cultured with irradiated 3 T3-J2 feeder cells in Conditional Reprogramming Media (CRM). The number of viable cells recovered following dissociation varied greatly, owing to variations in total starting biomass and viability of the sample prior to processing  as well as to JQ1 (IC50 = 57.1 nM) . Conversely, the KRAS (G12C) NSCLC LG0567F model is moderately sensitive to MEK inhibition by selumetinib  [50 > 1 μM). Similarly, the KRAS (G12R) NSCLC HLXF-056 model is exquisitely sensitive to taxotere in vivo (TGI > 100%) but sensitivity is lost in vitro.We next sought to determine if the CR-PDX lines could be used for in vitro drug sensitivity screens and if so, whether the cells maintained sensitivity to targeted inhibitors in vitro as observed by the parental PDX tumor in vivo. CR-PDX cells were removed from CR conditions and treated with targeted agents with known in vivo response at concentrations ranging from 0.01-10 μM. Staurosporine was used as a positive control and viability was assessed after 3 to 5 days Fig. . In in v IC50 = 5.1 nM  and mice bearing CR-LG0567F tumors were treated with selumetinib . CR-HLXF-036LN maintained identical growth kinetics and drug sensitivity to parental tumors. Following treatment with savolitinib, both parental and CR-HLXF-036LN exhibited complete tumor growth inhibition and intratumoral inhibition of MET phosphorylation  technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. This method offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods provided here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.Additional file 1: Table S1.PDX Model Characteristics. (DOCX 12 kb)Additional file 2: Figure S1.Figure S2. CR-PDX cells are amenable to in vitro chemosensitivity screening. CR-PDX cells were removed from CR conditions and treated with targeted agents at concentrations ranging from 0.01-10 μM. Staurosporine was used as a positive control and viability was assessed after 3 to 5 days (a) CR-OV0857F, (b) CR-LG0567F (c) CR-HLXF-036LN, (d) CR-HLXF-056. Figure S3. CR-PDX cells are amenable to gene knockdown studies using siRNA. CR-OV0857F cells were reverse transfected with indicated siRNAs for 120 h. Knockdown efficiency was confirmed by qPCR and the effect of knockdown on viability was assessed at the end of study. (PPTX 3628 kb)SNP Fingerprinting of parental and conditionally reprogrammed samples reveals homology across all sample pairs. Sample phylogeny shows clear concordance with key SNPs, confirming sample identities. Phylogenic clustering was used to identify matched parental and conditionally reprogrammed (CR) samples.


Nitroxides are stable free radicals that contain a nitroxyl group with an unpaired electron. In this paper, we present the properties and application of nitroxides as antioxidants and anticancer drugs. The mostly used nitroxides in biology and medicine are a group of heterocyclic nitroxide derivatives of piperidine, pyrroline and pyrrolidine. The antioxidant action of nitroxides is associated with their redox cycle. Nitroxides, unlike other antioxidants, are characterized by a catalytic mechanism of action associated with a single electron oxidation and reduction reaction. In biological conditions, they mimic superoxide dismutase (SOD), modulate hemoprotein’s catalase-like activity, scavenge reactive free radicals, inhibit the Fenton and Haber-Weiss reactions and suppress the oxidation of biological materials . The use of nitroxides as antioxidants against oxidative stress induced by anticancer drugs has also been investigated. The application of nitroxides and their derivatives as anticancer drugs is discussed in the contexts of breast, hepatic, lung, ovarian, lymphatic and thyroid cancers under in vivo and in vitro experiments. In this article, we focus on new natural spin-labelled derivatives such as camptothecin, rotenone, combretastatin, podophyllotoxin and others. The applications of nitroxides in the aging process, cardiovascular disease and pathological conditions were also discussed. Th. Th13) i14a, 14b) exhibited high antiproliferative activities against four tested tumour cell lines  [Eighteen spin-labelled analogues of camptothecin were tested against MDA-MB-231 cells. Two compounds (ctively) .15). Its derivatives are present in Derris elliptica, Lonchocarpus utilis and other Legumes (16) was also tested in A-549, DU-145, Kb and Kbvin cancer cell lines.Another compound of plant origin is rotenone flavonoid (Rot)  known as a glycolysis disrupting compound. When nitroxide and 2-DG were administered individually, no effect on the normal and cancer cell viability was noted, whereas 2 μM Mito-CP11 and 1 mM 2-DG (24 h incubation) caused 50% reduction in HepG2 viability without no influence on normal hepatocytes. An incubation period of 6 h for the cells with a combination of drugs (concentrations as above) disrupted the energy production processes resulting in 60% reduction of ATP content in the cancerous cells. The cytotoxicity of the combination of Mito-CP11 and 2-DG was confirmed by the induction of apoptosis, increase in caspases 3 and 7 activity, and altered expression of anti- and proapoptotic proteins. The results showed a synergistic effect of Mito-CP11 and 2-DG [The studies performed on HepG2 cell line showed that Mito-CP therapy . On the and 2-DG .17) . L-NNP reduced cell viability and increased ROS production and lipid peroxidation accompanied by a reduction in mitochondrial potential and GSH level in hepatoma cells . L-NNP resulted in a higher level of proapoptotic Bax and lower content of antiapoptotic Bcl-2 proteins. The cytotoxic properties of this compound were also confirmed in some in vivo studies performed on mice. Hepatic cancer bearing mice administered with high doses of L-NNP  lived longer, had a higher body mass and smaller volume of tumours compared with untreated and 5-fluorouracil treated animals (20 mg/kg of body mass). Additionally, a high dose of nitroxide resulted in a 126-fold higher level of ROS in the tumour tissue than in an untreated control. Simultaneously, it did not alter ROS level in the normal liver and kidney tissues . A high efficiency of L-NNP was explained by a significant increase in the oxidative stress in cancer cells, which they were not able to overcome  due to the low activity of antioxidative enzymes. Thus, nitroxide has proapoptotic properties and induces oxidative stress selectively in cancer cells [The anticancer activity of nitroxides towards HepG2 liver cancer cell line was also demonstrated by Guo et al. (2012)  while in17) . It was er cells .18) (An adamantyl nitroxide derivative (18)  revealed19)  , is a nat et al. . This coO-substituted carbonic ether moiety, showed anticancer activities against four tumour cell lines, viz. MDA-MB-231, MCF-7, human leukaemia cell line (K562) and human lung cancer cells (A549 cells) with IC50 values in the range of 1 to 180 nM [cis and trans. The cis form exhibits better tubulin binding properties than the trans form.Combretastatin A-4 derivative, which contains a 3′-o 180 nM . This co20 a–d), while the second group included spin-labelled combretastatin and a piperidine derivative with different substituents (21) [Two series of spin-labelled combretastatin derivatives were synthesised and their cytotoxic activities were tested using four tumour cell lines . The first group of spin-labelled combretastatin derivatives contained piperidine, pyrroline and pyrrolidine residues (nts (21) .Spin-labelled combretastatin from first group compounds exhibited significantly less potent cytotoxicity against HepG-2 cell lines than the starting substance 3-amino-deoxycombretastatin A-4. While the nitroxides of the second group exhibited greater antitumor activity than etoposide, a clinically used anticancer drug, used as a reference compound.22) nitroxide therapy (The studies of Wu and colleagues (2006)  revealed therapy . This co14) have recently been reported and their antitumor activity in human non-small cell line (A549) has been established. Of the eighteen tested compounds, fourteen were characterised by a low IC50  [About 20 novel spin-labelled camptothecin derivatives (.090 μM) .15, 16) was determined against A-549 cell line. All spin-labelled derivatives exhibited a cytotoxic activity against tested cancer cell lines, with IC50 values ranging from 0.471 to 0.738 mg/mL. However, spin-labelled rotenones showed significantly less toxicity than paclitaxel used as a reference compound [The toxicity of spin-labelled rotenone derivatives (compound .23) were used as a cancer drug against A549 cell line (23d and 23f) displayed anticancer activity at IC50 values of 2.76 and 2.38 μM, respectively, against lung cancer cells. The compound (23f) revealed approximately seven-fold more cytotoxicity against A-549 cell line in comparison to 5-FU [Fluorouracyl is widely used for the treatment of many tumors such as colon and rectal cancer, breast cancer, gastrointestinal cancers , head and neck cancer, basal cell cancer of the skin and actinic keratosis (squamous cell), neuroendocrine tumors, thymic cancers, cervical cancer, bladder cancer and hepatobiliary cancers. 5-FU was also used as a combination chemotherapy for lung cancer ,72,73. Sell line . Two com to 5-FU .11 [50 0.38 and 0.89 μM for abovementioned cell lines, respectively (for 48 h) and triggered arrest of cell cycle in G0/G1 phase. The efficacy of nitroxide was compared with vandetanib—a drug commonly used in thyroid cancer therapy. Both compounds reduced cell viability at a similar level, but only Mito-CP11 induced PARP fragmentation and reduced expression of RET proto-oncogene. Therefore, both compounds act in different ways and the only nitroxide could induce apoptosis. These results were confirmed using some in vivo studies on mice, bearing induced medullary thyroid carcinoma. Additionally, nitroxide was reported to have an approximately equal level of anticancer activity of vandetanib, but Mito-CP11 reduced body mass of mice at a lower extent. Therefore, Mito-CP11 seems to be potent and less toxic than commonly used drugs [The studies on TT and MZ-CRC-1 medullary thyroid carcinoma cell lines indicated that these cells are prone to Mito-CP11 . It was ed drugs .19) is an anticancer drug used in treating patients with advanced anaplastic thyroid cancer. This compound possesses varying ability to cause vascular disruption in tumours and inhibition of tubulin polymerisation prevents cancer cells from producing microtubules.Combretastatin A4  revealed the highest cytotoxicity against HepG-2, BGC-832 and HeLa cancer cell lines tested in comparison to 3-amino-deoxycombrethastin A-4 inhibitor of tubulin polymerisation and cytotoxic compounds.Additionally, the spin-labelled analogues (12), which at 10 μM concentration (24 h) led to 80% reduction in the viability of A2780 human epithelial ovarian cancer cell line and slightly reduced the viability of normal human smooth muscle cells. It also increased oxidative stress and activity of caspase-3 accompanied by a drop of phosphorylation of STAT3 in cancerous cells. This compound acted differently in normal cells, being much less toxic and acting as antioxidant [Selvendiran et al.  investigioxidant .50 was 3 fold lower). This difference could be explained by the differential activity of Tempol in normal and cancerous cells, where it acted as antioxidant and pro-oxidant, respectively. In fact, it increased oxidative stress and upregulated p21WAF1/CIP1 in HL-60 cells. Additionally, Tempol triggered cell cycle arrest in G1 phase. The authors proved that p53 protein (which is not expressed in HL-60 cells) is not obligatory for cells to undergo oxidative stress-induced apoptosis and also the levels of Bax and Bcl-2 did not change significantly after Tempol treatment. Possibly, p21WAF1/CIP1 induced the p53-independent pathway of apoptosis [−/− mice, being especially prone to lymphoma development [−/− mice lived for 30 weeks, while the animals administered with 10 mg of nitroxide per gram of food lived two-times longer. Tempol lowered oxidative stress in mice thymocytes, from both Atm−/− and wild-type. In the first type of mice, nitroxide prevented further decrease in the mitochondrial potential and lowered the level of oxidative stress marker haemo-oxygenase-1. The authors proposed a chemopreventive action affected by the antioxidant properties of nitroxide. They also observed that Tempol caused a notably slower gain of body mass and slower proliferation rate of thymocytes in Atm−/− mice. In combination with the slower proliferation of splenocytes under in vitro conditions, nitroxides also influence the signalling pathways connected with cell proliferation [−/− mice were also used as a model in other studies, devoted to CTMIO (26) nitroxide. In this case, a stronger chemoprevention in the Atm−/− mice was reported [Gariboldi et al.  investigpoptosis , the autpoptosis . Nitroxielopment . This tyferation . Atm−/− reported .The oxidative mechanism of action of Tempo nitroxide was observed in mouse lymphoma cell line (L5178Y). This nitroxide induced a time- and concentration-dependent intracellular production of ROS and depletion of glutathione. It also increased caspase-3 and 7 activities, decreased expression of anti-apoptotic proteins including Bcl-2, Bcl-xL and McL-1 and consequently led to apoptosis .27). It was observed to be cytotoxic towards Yoshida’s sarcoma, both in vivo in rats and in vitro in mice cell lines of the neoplastic phenotype. It was a suppressor of tumorigenesis and inducer of apoptosis in cancerous cells while being nontoxic to the normal cells [Another nitroxide that might find application in chemotherapy is Tempicol-3  and increase TNF-α expression in the brain tissue when administrated in rats with doxorubicin (DOX), paclitaxel (PXL) and docetaxel. On the other hand, Pirolin decreased DOX-induced oxidative damage and protected DNA against DOX- and PTX-induced damage. PL reduced PARP-1 cleavage and nNOS expression evoked by DOX and DTX. We found nitroxides as the new cytoprotectants of the central nervous system against DOX-, DTX- and PTX-induced oxidative stress .Both Mito-CP and Mito-CP-Ac exhibited similar dose- and time-dependent antiproliferative effects when tested on human pancreatic (MiaPaCa-2 and Panc-1), human epidermoid carcinoma (A431) and bladder (253J) cancer cells .The studies conducted on the human melanoma cell lines  showed that Mito-CP effectively induced oxidative stress and in consequence depolarization of the mitochondrial membrane and apoptosis. Additionally, these tests exhibited a greater efficacy of nitroxide in comparison to a proto-oncogene BRAF inhibitor, Vemurafenib (PLX4032) .It was shown that the redox nanoparticles (RNPN) including curcumin and Tempol induced strong apoptosis in the prostate cancer (PC-3) cells compared to free curcumin. While, the intravenous injection of RNPN to old nude mice (BALB/C) suppressed tumour growth in vivo, which was due to the increased bio-availability and significant ROS scavenging at the tumour sites .15, 16) were used against DU-145, Kb and Kbvin cell lines. Rotenone revealed anticancer activity by the induction of apoptosis via inhibiting the microtubule assembly [50 0.075 and 0.092 μg/mL, respectively) [The previously mentioned spin-labelled rotenone derivatives (assembly . The spictively) .21a–c) exhibited significant cytotoxicity against human leukemia cell line K562, gastric cancer cell line SGC-7901 and HeLa cancer cells and were more active than etoposide, an antineoplastic drug [The spin-labelled derivatives of combretastatin A-4 (tic drug .21a–c) revealed more cytotoxicity against two cell lines, human gastric cancer (BGC-823) and HeLa cancer cell lines than 3-amino-deoxycombrethastin A-4, which is an inhibitor of tubulin polymerisation and a cytotoxic compound.Nitroxides  revealed lower cytotoxicity against all cell lines than 3-amino-deoxycombretastatin A-4. On the other hand nitroxides (21a–c) exhibited the greatest cytotoxicity against all cancer cell lines than the first group. The mechanistic analysis showed that this compound (21a) can effectively inhibit tubulin polymerisation to prevent mitosis in cancer cells, leading to cell cycle arrest and apoptotic cell death better than the etoposide reference drug [Some spin-labelled combretastatin derivatives used against HepG-2 cell lines were also tested against K562, SG7901 and HeLa cell lines. Compounds (nce drug .Some spin-labelled fluorouracil derivatives were tested as anticancer compounds against prostate cancer cell line (DU-145) as well as parental Kb and multidrug-resistant Kbvin cell lines. However, these studies revealed little lower anticancer activity of spin-labelled compounds than fluorouracil .14a) and (14b) showed the highest cytotoxicity against the Kbvin cell line IC50 .The previously described camptothecin and its spin-labelled analogues were also tested on Kb and Kbvin cell lines. Spin-labelled derivatives  exhibited antitumor activity against human nasopharyngeal carcinoma Kb, lung cancer AS49 and stomach carcinoma SGC-7901 cells, mouse leukaemia L12 10 and P388 cells. Nitroxides (29a–c) displayed comparable or superior activity to etoposide for the inhibition of these cell lines. The cell line p388 showed the highest sensitivity for the used compounds [The spin-labelled podophyllotoxin derivatives (ompounds .30 and 31), exhibited better cytotoxic activity against all cell lines in comparison to etoposide used as a reference anticancer drug. Nitroxides (30) showed a higher cytotoxic activity against Kbvin cells line than etoposide [Spin-labelled podophyllotoxin derivatives exhibited better antitumor activity than podophyllotoxin. Three spin-labelled podophyllotoxins analogues were tested against four human cancer cell lines . Both compounds, (toposide .33 and 34). These compounds displayed a cytotoxic activity against the tested cancer cell lines ; however, the IC50 value was higher in comparison to etoposide. On the other hand, nitroxide after 33 h showed a higher cytotoxic activity than etoposide (IC50 6.30 to > 10 μM) against Kbvin cell line.Using the same cell lines, Yang and colleagues (2014) , used anAnother spin-labelled ester derivative of podophyllotoxin was used against neoplastic cell lines . Most of these compounds showed cytotoxicity against all cancer cell lines, except for SPCA-1 .35) was tested against leukaemia and osteosarcoma cell lines. This compound can arrest cycle in the S phase and induce apoptosis by the activation of caspases-3, 8 and 9. In a DNA fragmentation experiment, it also caused DNA laddering pattern, further confirming that it can induce apoptosis. The release of cytochrome-c and the increase of proapoptotic proteins Bax and Bak also indicated apoptosis through mitochondrial pathway [Another spin labelled derivative of podophyllotoxin ( pathway .Cis-platin (36) is one of the most commonly used chemotherapeutic agents , which led to cell cycle arrest and induced apoptosis in HeLa human cervical carcinoma and H1299 human lung carcinoma cell lines [37), induce cancer resistance against a drug at a 2.5-fold slower rate than cis-platin. When used in combination for the treatment of leukaemia, cis-platin and Nx-Pt I  showed a synergistic effect. After 60 days of the treatment, 100% of mice were cured, while the drugs administered alone showed no effect as almost all mice were dead. In case of nitroxide-platin complex, the slower development of resistance and mentioned synergism suggested that anticancer mechanism of the complex is different from that of cis-platin alone. These complexes possess antioxidative properties that alleviate side effects, such as nephro- or neurotoxicity, which results in the higher viability of animals and efficacy of therapy [c agents . It has ll lines . Some in therapy .Doxorubicin (DOX) belongs to the anthracycline antibiotics group that is commonly used in anticancer therapy of many tumours. However, it is also toxic towards normal cells, especially those of heart, liver and kidneys. This adverse action is related to the conversion of DOX to semichinone radical, which reacts with DNA and/or oxygen resulting in the generation of the superoxide anion radical. The latter, after dismutation to hydrogen peroxide takes part in the Fenton reaction, which yields hydroxyl radical. It induces oxidative damage to lipids, proteins and DNA of many cells, especially those with a low antioxidant pool such as cardiomyocytes. The application of nitroxides may alleviate side effects of DOX, protect normal cells from oxidative stress and enhance the cytotoxic activity of DOX against cancer cells ,31,92.39), in reducing the toxicity induced by DOX in normal cells. These derivatives differed in substituent at position 4 of the piperidine ring. The experiments carried out on B14 immortalised hamster fibroblast cell line revealed that the most effective compound in alleviating the side effects of DOX was Tempace. Similarly, to Tempo and Tempol, it increased the viability of cells unlike Tempamine, which at higher concentrations (500–2000 μM) acted as a pro-oxidant and enhanced the toxic effect of DOX. The effect of the compounds on lipid peroxidation induced by DOX was also evaluated. Again, Tempamine was the only nitroxide that slightly increased damage to lipids, while the three other nitroxides had protective properties. EPR studies revealed that the bioreduction of nitroxides may be significant for their protective activity. The lowest rate of reduction was noted for Tempace, which was also the most efficient antioxidant to protect from DOX-induced toxicity. The least efficient Tempamine was reduced much faster. It has been confirmed that the type of substituent at position 4 of the piperidine ring is crucial for the activity of nitroxides [Czepas et al. (2008)  evaluatetroxides . Antioxitroxides .N in alleviating DOX-related side effects. This molecule consists of an ethylene glycol copolymer and aminomethylstyrene (PEG-b-PMNT) (40) with attached nitroxide. These macromolecules are pH-sensitive and preferentially accumulate at inflamed sites, for instance, the cancer environment. The microenvironment of a cancer site is characterised by a lowered pH due to hypoxic conditions and enhanced glycolysis (Warburg effect). Under such conditions, polymer releases Tempo that interacts with cancerous cells. ROS generated by DOX lead to activation of NF-κB that promotes cancer progression and drug resistance. The antioxidant effect of Tempol reduces the level of DOX-derived ROS and prevents the development of resistance towards drugs in cancer cells. The authors showed that in comparison with free Tempol, RNPN circulated longer in the blood of mice, accumulated at cancer site at a high level and significantly reduced the activity of NF-κB in the nucleus of colon-26 cancer cells. The administration of RNPN resulted in a significant drop of ROS and TNF-α levels when compared to free Tempol and the untreated controls. The subsequent administration of DOX few days after RNPN resulted in an approximately 6-fold reduction in tumour volume in mice, while DOX alone yielded much less evident effect. The influence of RNPN on DOX-induced toxicity towards normal cell was also evaluated. The administration of RNPN (unlike Tempol) significantly reduced the activity of biomarkers of cellular damage CPK and LDH and reduced the level of ROS and lipid peroxidation in cardiomyocytes of mice. In summary, RNPN reduced the level of inflammatory cytokines, production of ROS and cardiotoxicity, while simultaneously enhanced the anticancer activity of DOX. It was more potent than free Tempol, which is administered intravenously and transported through the whole body, does not act as precise as RNPN when specifically released in the microenvironment of a tumour [Yoshitomi et al.  investiga tumour .42). Dexrazoxane (41), which is used in DOX therapy to prevent oxidative stress and alleviate chemotherapy-related side effects, was used as the reference. The analysis of mitochondrial fractions of heart and cancer cells of rats revealed that Mito-T-4 accumulated strongly in mitochondria of cardiomyocytes than cancer cells. While DOX and Mito-T-4 combined therapy led to a significant reduction in the body mass of the rats (comparing to control and single DOX), it alleviated some cardiotoxicity parameters such as plasma glucose level and changed lymphocyte and granulocyte counts. Dexrazoxane combined with DOX, on the other hand, alleviated the reduced levels of plasma albumin, alanine-1-transferase, bilirubin, glucose and lipids. In order to assess cardiotoxicity, the damage to cardiomyocytes was also evaluated. The high extent of damage induced by DOX was lowered by the co-administration of Mito-T-4, but it was slightly less efficient than dexrazoxane. The relevant difference between Mito-T-4 and dexrazoxane was that only nitroxide enhanced DOX toxicity against cancer cells. Both alleviators inhibited apoptosis and the induced autophagic protective pathway, thus protecting the heart of rats and reducing the oxidative damage to DNA induced by DOX in this organ. Therefore, Mito-T-4 might be used in DOX therapy to increase its anticancer activity and protect the heart from its adverse effects [Dickey and colleagues (2013)  employed effects .50 range of 24 μM to ≥300 μM. In contrast nitroxide derivatives, viz. TroH, TroS and α-TOS, could not inhibit the proliferation of any cancer cells [Recently twelve new spin-labelled conjugates of known antioxidants trolox (TroH), trolox succinate (TroS), α-tocopheryl succinate (α-TOS) have been reported and compared with their unlabelled counterparts. TroS inhibited the growth of human cancer cells: myeloma, mammary adenocarcinoma, hepatocarcinoma, T cells leukaemia, histiocytic lymphoma and T-cellular leucosis in an ICer cells .One of the theories of aging is Harman’s theory of free radicals ,95,96. IThe ROS can be generated from exogenous sources like air and water pollution, ionizing and ultraviolet radiations, tobacco smoke, cooking , drugs , transition metals , pesticides, organic solvents hypoxia (ischemia and reperfusion), hyperoxia, acute exercise, etc. ,103,104.However, in homeostasis, there is a counterbalance between ROS generation and its elimination. The disruption of equilibrium between ROS formation and its removal leads to oxidative stress and associated diseases. The disorder may be due to overproduction of reactive oxygen species or degradation of the antioxidant system or both. Moreover, each cell also has a suitable repair system to repair defective proteins, lipids, and DNA. However, the scope of these systems is limited and may be damaged by ROS. As age increases, the activity of antioxidant and repair systems decreases as the release of reactive oxygen species increases.Aging is a result of increased intracellular oxidative stress and chronic inflammation, which leads to the damage of nucleic acids, proteins and lipids . A highe2−•), precursor of other oxygen reactive species. The dismutation of O2−• by SOD leads to the generation of a very toxic agent to the cell, namely, hydrogen peroxide. However, hydrogen peroxide is rapidly eliminated by glutathione and thioredoxin in the mitochondrial matrix and other enzymes such as catalase and glutathione peroxidase [According to the mitochondrial theory of aging mitochondria act as the primary sources as well as the primary targets of ROS. The oxidative stress in mitochondria plays an important role in aging and health status of many vital organs . A decreroxidase ,113,114.The high levels of ROS are detrimental to cells, leading to the onset of aging and many acute and chronic aging-related diseases such as hypertension, atherosclerosis, coronary heart disease, cancer, metabolic syndrome, diabetes mellitus, chronic pulmonary disease, cataracts, age-related macular degeneration, myocardial infarction, cerebrovascular disease, sepsis, rheumatoid arthritis, and neurodegenerative disease (Alzheimer’s and Parkinson’s diseases) ,115.However, a moderate level of oxidative stress may induce and modulate an adaptive cellular response, which can be beneficial for redox signaling and cell survival . The antNitroxides may also be used as antioxidants to delay aging especially for those having accelerated oxidative stress due to overweight, obesity leading to diabetes mellitus, inflammation, cardiovascular disease, and others.Our previous paper showed that nitroxide protects biological materials from the effects of ionizing radiation. Nitroxide protects the healthy cells but not the cancerous ones from ionizing radiation. The differential effect of nitroxides in both cells correlates with the oxidative state in the tumor tissue as opposed to the reducing status of normal cells . NitroxiTempol and its diamagnetic derivatives (hydroxylamines) are widely used in in vivo and in vitro studies. The application of Tempol as a protective agent against oxidative stress in biomedical studies has extensively been described by Wilcox . Tempol In the fat-fed models of metabolic syndrome, Tempol was found to prevent left ventricular hypertrophy and heart failure . Tempol Caloric restriction is known to prolong life . It was Tempol showed a profound effect on body weight, atherosclerosis, hyperlipidemia, and inflammation in established models of obesity-associated hyperlipidemia and atherosclerosis .In older mice, plasma lipid peroxidation and C-reactive protein were significantly increased as compared to younger ones, which were counteracted by an oral dose of Tempol.The Tempol therapy, which leads to a reduction in mortality, is based on the alleviation of chronic inflammation and improvement of the immune system function .Tempol mitigated renal damage in animal models of hypertension associated with obesity ,134. TemThe administration of Tempol significantly reduced the progressive sclerotic and proliferative glomerular changes in hypertensive rats and prevented the occurrence of hypertension, increased urinary excretion of protein, and lipid peroxidation ,136. In It was reported that Tempol can remove ROS and attenuate the inflammation and tissue damage associated with carrageenan-induced pleurisy in rats .Nitroxides can be used as a preventative or pharmaceutical drug against age-related macular degeneration (AMD) and cardiovascular disease.43), ester of cyclopropanecarboxylic acid (44), and Tempol-H hydroxylamine (OT-551) (45) were used as preventive and pharmaceutical drugs in age-related macular degeneration (AMD) [Tempol and its derivatives, e.g., Tempol-hydroxylamine (Tempol-H) (on (AMD)  , and omega-3-polyunsaturated fatty acids .The early etiology of AMD involves the degradation of retinal pigment epithelial (RPE) cells, which are sensitive to oxidative stress . Tempol-I was shown in young adult smokers that Tempol effectively reversed the damage of impaired endothelial function. This effect was caused by the inhibition of oxidative stress and increased bioavailability of nitric oxide ,143.Recently, Tempol has been shown to relieve and prevent paclitaxel-induced neuropathic pain in rats by reducing the levels of inflammatory cytokines and free radicals in dorsal root ganglia .Nitroxides are the promising candidates of cancer therapy. Several studies have confirmed their efficacy against breast, liver, lung, thyroid, ovarian and lymphatic cancers. Their anticancer effects have been established by reducing the viability of tumor cell lines, slowing down ATP production, damage to electron transport chain, mitochondrial mitosis, induction of apoptosis (by regulating levels of pro- and anti-apoptotic proteins), cell cycle arrest in G1 phase, strong RFT production and the induction of oxidative stress in cancer cells and tumor tissue. The effect of nitroxides is highly precise and selective; meanwhile, in normal cells, they exhibit completely different activity. Generally, they act as protectants and antioxidants slightly affecting the viability of healthy cells. They protect against weight loss and in some cases effectively prevent the onset of cancer and prolong the life of the experimental animals.11 with fluvastatin in breast cancer and with 2-DG in liver cancer. Nitroxides were also found to be highly effective against thyroid cancer. It has also been shown that nitroxides may be more effective than commonly used antineoplastic drugs such as 5-fluorouracil, vandetanib and many others. Nitroxides decreased the toxicity in Chinese hamster cells (line B14), mouse cardiomyocytes and SHR rats, being treated with doxorubicin. Their protective effect was associated with a decrease in oxidative stress and the consequent inhibition of oxidation of proteins and lipids, reduction in cardiac damage and inhibition of apoptosis in this organ. On the other hand, nitroxides when combined with a drug intensify the oxidative stress in the malignant cancer cells.Several reports have documented the synergistic effects of nitroxide with anticancer drugs, for example, Mito-CPcis-platin derivatives, exhibited in vitro toxicity against several tumour cells and when these complexes were co-administered with cis-platinum under in vivo experiments, a high therapeutic value and significantly slower development of drug resistance were observed.Nitroxides, ligated to Thus, it can be said that spin-labelled analogues of natural antineoplastic compounds could be a better choice as an antitumour agent than their precursors. Anti-inflammatory, angiogenic and antioxidant properties of nitroxides give the opportunity to use them as effective drugs and antioxidants to protect the cells and tissues from oxidative stress.


V600E mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. Methods: Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma (A673) and atypical synovial sarcoma (SW982), were treated with vemurafenib and the effects on cell growth, apoptosis, cell cycle progression and cell signaling were determined. Results: Vemurafenib induced a strong cytostatic effect in SA-4 cells, mainly due to cell cycle arrest, whereas only moderate levels of apoptosis were observed. However, a high dose was required compared to BRAFV600E mutated melanoma cells, and removal of vemurafenib demonstrated that the continuous presence of drug was required for sustained growth inhibition. A limited growth inhibition was observed in the other three cell lines. Protein analyses demonstrated reduced phosphorylation of ERK during treatment with vemurafenib in all the four sarcoma cell lines confirming that the MAPK pathway is active in these cell lines, and that the pathway can be inhibited by vemurafenib, but also that these cells can proliferate despite this. Conclusions: These findings indicate that vemurafenib alone would not be an efficient therapy against BRAFV600E mutated sarcomas. However, further investigations of combination with other drugs are warranted.The BRAF Sarcoma is a heterogeneous group of rare tumors, generally arising from mesenchymal cells and accounting for approximately 1% of all human cancers and 10–20% of childhood solid cancers , and theV600E) [Mutated BRAF are described in several cancers and can be targeted by selective inhibitors, such as vemurafenib . The RAFV600E) . This amV600E) ,11.V600E mutation accounts for more than 90% of all reported BRAF mutations [V600E mutation is present in 50% of melanomas and has also been identified in subsets of many other cancer types, including colorectal cancer (10–15%), papillary thyroid carcinoma (30–50%) and non-small cell lung cancer (1.3%) [V600E mutation has also been reported in GIST, several cases of clear cell sarcoma, and liposarcoma [V600E mutation has been described in sarcoma cell lines of different subtypes, including SW872, SA-4, A673 and SW982 [The BRAFutations . The BRAr (1.3%) ,15,16,17osarcoma ,19,20,21nd SW982 ,21,22.V600E, and resulted in a response rate of >50% and approximately 7 months of median progression-free survival for melanoma patients [V600E mutation; however, vemurafenib as monotherapy was unsuccessful due to resistance, which occurred as a consequence of a rapid, EGFR-induced reactivation of the MAPK pathway, observed already at 24–48 h after treatment [V600E mutation was successfully treated with vemurafenib and displayed tumor regression after 8 weeks of treatment [V600E inhibitor [V600E mutated sarcomas, by determining its efficacy in vitro in four BRAFV600E mutated sarcoma cell lines.Vemurafenib was initially approved by the Food and Drug Administration (FDA) in 2011 as a therapy for melanoma patients with mutated BRAFpatients ,23. In creatment ,24. Imporeatment . Vemurafreatment . FurtherV600E, namely SA-4 and SW872, classified as liposarcomas, as well as Ewing sarcoma cell line A673 and an atypical synovial sarcoma cell line SW982 lacking the SYT-SSX translocation. The WM9 melanoma cell line, known to harbor a hemizygous BRAFV600E mutation, was included as a positive control, whereas the LPS510 liposarcoma cell line was included as a negative control line for all BRAFV600E mutated sarcoma cell lines, as it is wild-type for BRAF according to our DNA sequencing data. We evaluated the expression of wild-type and mutated BRAF mRNA by next-generation sequencing and found similar levels of wild-type and mutated BRAF transcripts in both SA-4 and SW872 cell lines, whereas LPS510 only expressed wild-type BRAF  profiling to ensure correct identity of the cell stock and confirmed negative for mycoplasma infection using the VenorGeM Mycoplasma detection kit (Minerva Biolabs) for PCR.SA-4 (CRL-7938), SW872 (HTB-92), A673 (CRL-1598) and SW982 (HTB-93) cells were used as adherent human cancer cell model systems and were all obtained from ATCC. SA-4 is derived from a liposarcoma, whereas SW872 is derived from an undifferentiated liposarcoma. The A673 cell line represents Ewing sarcoma  and SW98XT Human All Exon v5 protocol and Illumina sequencing by synthesis technology on a HiSeq 4000 instrument (2 × 150 bp). Preprocessing, mapping of the reads and calling of variants was performed as previously described [Three of the sarcoma cell lines  were exome-sequenced at the Oslo University Hospital Genomics Core Facility using Agilent SureSelectescribed . RNA-seqN-morpholino) propanesulfonic acid (MOPS) running buffer  and transferred to PVDF membrane . Membranes were blocked with TBS-T containing 5% bovine serum albumin (pERK) or 10% non-fat dry milk for the other antibodies and incubated over night while shaking. The antibodies used were as follows: pan-BRAF (1:200) , BRAFV600E (1:1000) , phosphorylated p44/42 MAP kinase (pERK) (1:1000) , p44/42 MAP kinase (ERK) (1:1000)  and anti-α-Tubulin (1:2000) . Anti-Mouse (1:5000)  or anti-Rabbit (1:5000)  was used as secondary antibodies. Following incubation with secondary antibody, the membrane was incubated in a development solution  . The emitted signal was detected using the digital developer G:BOX (Syngene) Cambridge, UK, and images captured were analyzed using the GeneSnap software.Cells were resuspended in Lysis buffer  and protease inhibitors (#04693124001), both from Roche, and subsequently boiled at 95 °C. Cell lysates were separated on a 4–12% PAGE gel  with 3-  was dissolved in dimethyl sulfoxide (DMSO) to a 10 mM stock solution. The dual BRAF/MEK inhibitor RO5126766  was dissolved in DMSO to a 1 mM stock solution. When used in experiments, drugs were further diluted in complete growth media. Cells treated with DMSO at a concentration corresponding to the highest drug concentration were included as controls in all experiments and referred to as vehicle.n = 3) and presented relative to control.1000–2000 cells were plated in 96-well plates one day before drug treatment and subsequently treated with vemurafenib (0.31–5 µM) or vehicle only. Cell growth was assessed by MTS  assay 72–96 h post treatment , following the protocol recommended by the manufacturer. The absorbance was measured at 450 nm. The data was shown as mean of three biological experiments  at excitation/emission = 352/461 nm. FLOWJO v7.6.5 software and Watson settings were used to analyze the data. Data are presented as mean of three biological experiments.10,000 cells were plated in 6-well plates, and on the subsequent day treated with the indicated concentrations of vemurafenib or vehicle only for 48 h. Cells were trypsinized, washed with PBS and fixed and permeabilized with 96% ethanol and stored at −20 °C at least 2 h. Cells were washed with PBS twice and stained with 2 µg/mL of the DNA-binding dye, Hoechst 33258 . Approximately 1 × 101000–2000 cells were plated in 96-well plates and on the subsequent day, treated with vemurafenib at the indicated concentrations or vehicle only. The Cellplayer reagent that reports caspase 3/7 activity  was also included in a final concentration of 2.5 µM. The cells were monitored by live cell imaging (IncuCyte Zoom from Essen BioScience), where phase-contrast and green fluorescent images (488 nm) were recorded and used to estimate the confluence and the number of apoptotic cells, respectively. Data is presented as number of caspase-3/7 active cells per well and as means of three technical replicates. At 72 h, data is also shown as relative to cell density, normalized to control.1000–2000 cells were plated in 96-well plates one day before drug treatment, and subsequently treated with vemurafenib  or vehicle only. Replacement of media containing drug or control was performed twice a week for the entire duration of the experiment (approximately 240 h). To determine if continuous presence of drugs is necessary to achieve cell growth inhibition, drug containing media was exchanged with regular growth media after approximately 240 h. Cells were monitored by time-lapse microscopy,  with phase-contrast images taken every third hour for the duration of the treatment, and cell growth estimated based on cell density. Data is presented as mean of three technical replicates.t-test was used to compare the read-out of drug-treated with control-treated cells. p < 0.05 were considered statistically significant.Data presented is obtained from at least three independent experiments, unless otherwise stated. Two-tailed paired Student’s V600E sarcoma; however others could benefit from vemurafenib in combination with additional therapies and further work is required to explore this.We conclude that vemurafenib can have therapeutic potential for selected cases of BRAF


Raf Kinase Inhibitory Protein (RKIP) expression has been profiled for a number of unique tissue cancers. However, certain tissues have not been explored, and oral and oropharyngeal cancers stand out as high priority targets, given their relatively high incidence, high morbidity rate, and in many cases, preventable nature. The purpose of this study was to examine changes in RKIP expression and phosphorylation in tissues resected from oral cancer patients, and compare to results generated from immortalized cell lines raised from primary oral cancer tissues, including oral squamous cell carcinoma line 4 (SCC4) and human squamous cell carcinoma line 3 (HSC3). Out of 4 human samples collected from male and female patients across various ages with variable risk factors, we observed an across the board reduction in RKIP expression. Two human samples demonstrated a significant increase in phosphorylated RKIP when normalized to total RKIP, however all 4 were increased when normalized to total cellular protein. The immortalized oral cancer cell culture HSC3 revealed significant increases in phosphorylated RKIP with no change in total RKIP expression, while line SCC4 demonstrated an increase in both total and phosphorylated RKIP. Results presented here indicate that oral cancers behave similarly to other cancers in terms of changes in RKIP expression and phosphorylation, although immortalized cell line expression profiles significantly differ from human tissue biopsies. Oral squamous cell carcinoma is the most common cancer in the orofacial cavity, accounting for approximately 90% of all cancers of the mouth . Te AmerRKIP is a scaffolding protein capable of binding to and inhibiting Raf kinase . Raf kinPrevious studies identify RKIP as an important protein determinant in many types of cancers, including prostate, melanoma, colorectal, liver, and breast -10. IndeOral squamous cell carcinoma line 4 (SCC4) and human squamous cell carcinoma line 3 (HSC3) cells were grown as previously described . Cell cuAll procedures utilizing animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and were conducted in accordance with policies for the ethical treatment of animals established by the National Institute for Health. Male Sprague-Dawley rats 175 – 200g in weight  was used for trigeminal ganglia (TG) dissection, as described previously . 25 μg aliquots were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride  membranes for Western blotting, using antibodies specific to phosphorylated RKIP , total RKIP (SantaCruz Biotechnology) and β-actin (SantaCruz Biotechnology). Appropriate secondary antibodies  were utilized with enhanced chemiluminescence  to visualize protein immunoreactivity. Samples were independently analyzed 3-4 times; images are representative of all trials. Pixel densities of scanned Western blots were quantified using NIH image 1.62, with samples normalized as indicated in figures.Cells and tissues were homogenized by 20 strokes in a Potter-Elvejm homogenizer on ice in homogenization buffer  and human squamous cell carcinoma line 3 (HSC3). Cell lysates were generated of these cells under naïve, low serum conditions, to more closely represent physiological signifcance. Furthermore, we compared the protein expression profiles of these cancer cell lines to that of a primary cell model, trigeminal ganglia (TG) neurons. The inclusion of other immortalized cell lines for control purposes would have confounded result interpretation given that most cell lines are derived from cancer tissues, and that any immortalized cell line would represent an abnormal transcriptional environment. In , WesternWe next performed similar analysis on tissue biopsies of oral cancers collected from patients. In this analysis, we compared changes in RKIP expression and phosphorylation between cancerous biopsies and healthy tissue control samples taken from the same patient. Therefore, for each patient, normal tissue (N) was compared to cancerous tissue (C). In , WesternPatient data were examined to determine whether patient history and/or tumor characteristics could account for sample-to-sample differences observed in . Table  outlinesRKIP transcript and protein expression has been characterized in numerous cancer types, except for oral cancers. Since oral squamous cell transformation rate is relatively high compared to other cancers, changes to mitogenic signaling modulators such as RKIP are highly significant to understanding the pathogenesis of the disease. Here, we performed proteomic analysis of RKIP phosphorylation and expression from two well-studied immortalized oral cancer cell lines and multiple squamous cell carcinoma biopsies. Our results indicate that RKIP expression is reduced in cancerous tissue biopsies, similar to what is found in other cancer types ,11-14. FTe role of RKIP in cancer and tumor progression was first identified in prostate cancer cells in 2003 . Here, iRKIP phosphorylation primarily serves to redirect the protein away from its inhibition of Raf Kinase and into a scaffolding role with other signaling proteins, such as G-protein receptor kinase 2 ,24. It iFrom results presented here, proteomic analysis of oral cancers could serve as an important screening tool to predict metastatic potential and assist with determining optimal treatment for the patient.