The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression.A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy.Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines.The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands. Cancer cells, similarly to any other cell and living system, respond to mild changes of external temperature by activating homeostatic biological mechanisms, in an attempt to sustain a tolerant intracellular environment and prevent death. Temperatures above 41°C are toxic both to the tumor vasculature and to the cancer cells themselves, and are used to heat tumors (Oncothermic Hyperthermia) aiming to suppress their growth, achieve regression or to sensitize them to radiotherapy and chemotherapy .Nevertheless, temperatures below 41°C, at the so called fever-range hyperthermia, have also a direct effect on cancer cell and tissue biology, sensitizing tumors to radiotherapy and chemotherapy. Whole body hyperthermia between 38–40°C has been used in the treatment of wide-spread metastatic tumors in combination with chemotherapy , 6. ProtIn addition, mild hyperthermia as a complementary therapy to cancer immune-therapy has been presented by several preclinical and clinical studies, by improving antitumor immune responses. The hyperthermia- induced improved immune response includes HSPs generation, antigen presenting cells activation and lymphocytes trafficking changes . FurtherThe Heat-Shock Proteins (HSPs) are sharply up-regulated under hyperthermic conditions, as their higher affinity to accumulating unfolded proteins releases the HSF-1 transcription factor bound to HSPs which enters the nuclei to initiate HSP gene transcription . HSPs prIn any case, cancer cell response to hyperthermia may depend on both temperature levels and exposure times, additional ambient conditions and, certainly, on the cell type under investigation. In the current study, we examined the effect of fever-range hyperthermia on a wide range of cancer cells, providing evidence that its effect on viability and proliferation is cell-dependent. Hypothermia, a far less studied condition in cancer cell systems, was also investigated. The effect of hyper- and hypothermia on HSP90 expression was further examined. Finally, hyperthermic chemosensitization was examined in two glioblastoma cell lines (T98G and U87MG) and two lung cancer cell lines (A549 and H1299). T98G and A549, the only thermo-tolerant cell lines were examined in comparison with two representative, thermo-sensitive cell lines of the same tumor type, U87MG and H1299, respectively, with the appropriate chemotherapy drugs for each type of cancer, together with mild hyperthermia, in order to examine a possible sensitisation or resistance.A549 , H1299 , MCF7 , U87MG , DU145 , PC3  and MRC5  cell lines were cultured using DMEM basal medium  and T98G  cell line was grown in MEM basal medium . Both basal culture mediums were supplemented with 10% FBS , 100 units/ml Penicillin and 100μg/ml Streptomycin  and 2mM L-Glutamine . Cells were maintained at standard conditions, 37°C, 5% CO2 in humidified atmosphere and were used upon reaching 70–90% confluency.The ability of cell proliferation under different incubation temperatures was tested by a proliferation assay. Cells were seeded in 96-well plates at a density of 1000 cells/well, the plates were incubated at 37°C for 3h to facilitate adherence and normal growth and then were placed at 34°C, 37°C and 40°C, respectively, for three consecutive days. Proliferations assays for all the examined cell lines and growth temperatures were performed simultaneously. Cell proliferation measurements were performed in a 24h interval using the AlamarBlue Cell Viability Reagent  measured at 540nm excitation and 590nm emission wavelengths, in a FLUOstar Omega microplate reader (BMG LABTECH). The AlamarBlue assay  is a reliable method for cell viability . This asGlioblastoma cell lines (U87MG and T98G) were cultured under standard conditions, as previously mentioned. These cell lines were similarly incubated at 34°C and 40°C for 72h. Cells were lysed using a sucrose lysis buffer supplemented with a protease and phosphatase inhibitor cocktail . Protein concentrations were measured based at the BCA protein assay kit . Specific rabbit polyclonal primary antibodies were used for western blot analysis, anti-HSP90  and anti-Caspase9 .Whole fraction samples were separated on discontinuous SDS gels using 10% separating and 5% stacking gels. Forty micrograms of the cell extracts were loaded on the gel. Immunoblotting was performed utilizing PVDF-PSQ membranes (Millipore Corp.). Following a blocking step with 5% non-fat dry milk in 150 mM NaCl, 10 mM Tris, pH 7.5 containing 0.1% (v/v) Tween 20 (TBS-T) at room temperature (RT) for 2h, the membranes were hybridized overnight at 4°C with the primary antibodies. The membranes were then hybridized for 2h at 37°C with the secondary antibody, goat polyclonal to rabbit IgG (H+L)-HRP  and finally developed in Amersham ECL Western blotting detection reagents and analysis system  utilizing Chemidoc MP Imaging System .In chemosensitization experiments, 1000 cells/well were plated in a 96-well plate in the appropriate culture medium. Cells were incubated with clinically established drugs; lung cancer cell lines: A549 and H1299 with 100 μΜ of Cisplatin and glioblastoma cell lines: T98G and U87MG with 500 μΜ of temozolomide. The cells, simultaneously with the drug treatment, were incubated for 24h at 37ºC and 40ºC, respectively, while cells’ viability was assessed after 24h incubation period by AlamarBlue assay and the cells survival percentages (%) were calculated. Statistical analysis by the 2way ANOVA test  and graph presentation has been performed using the GraphPad Prism Version 5.01 statistical package . The experiments were performed three times in order to confirm the significance of the results.For immunofluorescence staining, cells were grown on No. 1.5 glass coverslips, fixed in 3.7% paraformaldehyde/PBS pH 7.4 for 20 min at 37°C and then permeabilized in PBS/0.1% v/v Triton X-100 pH 7.4 for 5 min at room temperature. In addition, cells were blocked in PBS/5% w/v BSA pH 7.4 for 20 min and stained with various primary antibodies: anti-ki67 mouse monoclonal  anti-Caspase9 rabbit polyclonal , anti-HSP90 rabbit polyclonal , for 1 h at RT. Cells were washed in PBS pH 7.4, incubated with appropriate CF 488 and 564 secondary antibodies at RT and DNA was counterstained with Hoechst 33342 . After final washes coverslips were mounted in homemade Mowiol mounting medium. Imaging was performed on a customized Andor Revolution Spinning Disk Confocal System built around a stand  with a 60x lens and a digital camera (Andor Ixon+885) . Image acquisition was performed in Andor IQ 2 software. Optical sections were recorded every 0.3 µm. All confocal microscopy images presented in this work are 2D maximum intensity projections of z-stack images .Image intensity analysis for the obtained data sets has been performed using ImageJ 1.47v  software. Image processing macros have been custom developed in order to quantify the levels of the examined proteins (% Fluorescence Intensity) for Caspase 9 and HSP90 in the area of interest.Image analysis and quantification of ki67 expression in the nucleus have been performed using custom developed macros in ImageJ software 1.47v . A population of n cells (n≥ 20) has been analyzed, for ki67 quantification. The cells were categorized in three classes, according to their pixel units, representing ki67 expression levels. Class I was the lower class including cells with 1000–5000 pixels in the green channel (ki67 imaging) and Class III was the higher class including cells with more than 7501 pixels, while Class II included 5001–7500 values, respectively.The two-dimensional (2D) average projection of z-stack images were quantified using a standard size square area where integrated intensity values have been measured. Statistical analysis by the 2way ANOVA test  and graph presentation has been performed using the GraphPad Prism Version 5.01a statistical package .Following a 3-day incubation period under fever range hyperthermia, changes of cell proliferation/viability were strongly dependent upon each cell line . The norHypothermia at 34°C, on the other hand, had a rather homogeneous effect, resulting in reduction of both the normal MRC5 fibroblasts and of all cancer cell lines viability . This reGiven that the cell proliferation/viability assessed by the AlamarBlue assay is a result of combined proliferation and death events, we further examined the cell proliferation using the ki67 proliferation index. After 3 days of cell incubation, hyperthermia resulted in an increased fraction of cells in class III, inT98G and A549 (1.3 and 1.4 fold respectively) cell lines compared to normothermia. This dropped by 1.1 to more than 45 fold in the rest of cell lines . CharactHypothermia resulted in decrease of cell accumulation in the class III group by 1.2 up to more than 45 fold in all cell lines with the exception of DU145 prostate cancer cell line, where this was increased by 1.6 fold.Confocal immunofluorescence images of Caspase 9 expression in cell lines following exposure to hyperthermia and hypothermia are presented in Hyperthermia sharply induced Caspase 9 in the U87MG thermo-sensitive cell line, which also exhibited the most profound reduction of cell viability in Alamarblue experiments. Of interest, both T98G and A549 thermo-tolerant cell lines, Caspase 9 levels were reduced by hyperthermia, suggesting an apoptosis suppressing effect of hyperthermia in these cell lines. The most thermo-sensitive of all, MRC5 cell line, however, did not show increased Caspase 9 levels suggesting death by independent of Caspase 9 pathways.Hypothermia induced Caspase 9 in U87MG, DU145 and MCF7 cells, in agreement with the reduced viability, demonstrated in Alamarblue experiments. In the rest of cell lines, Caspase 9 expression remained stable or it was reduced in the case of T98G cells, suggesting that if apoptosis pathways are activated by hypothermia these are Caspase 9 independent.Western blot analysis performed in the thermo-tolerant T98G and the thermo-sensitive U87MG glioblastoma cell lines are presented in Representative confocal microscopy and immunofluorescence images of the effect of hyperthermia and of hypothermia on HSP90 on cell lines are shown in Hyperthermia strongly increased the HSP90 levels in the T98G thermo-tolerant cell line, whilst a sharp drop was recorded in the thermo-sensitive PC3 and U87MG cell lines. On the other hand, the HSP90 levels were clearly reduced in all cell lines examined under hypothermia. Western blot analysis performed in the thermo-tolerant T98G and the thermo-sensitive U87MG glioblastoma cell lines are shown in Exposure of the thermo-tolerant A549 and the thermo-sensitive H1299 lung cancer cell lines to cisplatin, the key drug used in the clinical practice for the treatment of lung cancer, showed that fever range hyperthermia strongly sensitized H1299 to the drug but its effect on the A549 cell line was minimal .Exposure of the thermo-tolerant T98G and the U87MG thermo-sensitive glioblastoma cell lines to temozolomide, the only approved drug for the treatment of human glioblastoma, showed that hyperthermia at 40 ºC strongly sensitized the U87MG cell line to the drug, whilst no sensitizing effect was noted for the T98G one .et al also reported a stimulatory effect of mild hyperthermia at 38°C on U87MG cell line that was sharply reversed at 40°C [The effect of fever-range hyperthermia on normal and cancer cell biology and its eventual role and influence in cell sensitivity to chemotherapy and radiotherapy remains poorly understood, requiring further investigation. Mild hyperthermia has been reported to have an inhibitory effect on cell proliferation. In a previous study, however, Morrisey  at 40°C . The con at 40°C , but a det al [On the other hand, the therapeutic role of hypothermia should not be underestimated and should be thoroughly examined in animal models, as about half of the examined cell lines demonstrated a 3–7 fold reduction of viability at 34°C. The current knowledge on its effect on cancer cell is limited. Hypothermia at 28°C seems to protect preferentially normal fibroblasts compared to cancer cells against 5-fluorouracil . In our et al , provideWe further examined whether the death effect induced by mild temperature changes in several cell lines is Caspase-9-mediated. The aspartic acid specific protease Caspase-9 is involved in the mitochondrial death pathway. Release of cytochrome c from mitochondria activates apaf-1 (apoptosome), which in its turn cleaves the pro-enzyme of Caspase-9 into its most active form. Nevertheless, cleavage is not essential for Caspase-9 apoptotic activity . In our vivo to be tumoristatic and to have a synergistic effect with chemotherapy and other target therapies [HSP90, on the other hand, is a heavy member of the HSP family, with an important role in tumor growth, being also linked with poor prognosis in breast cancer and other malignancies . The HSPherapies . The finHyperthermia has been also studied in combination with chemotherapy drugs. It has been previously reported that simultaneous hyperthermia and cisplatin induced cell death in T leukemic cells by different molecular mechanisms, in that way the enhanced cisplatin-induced cytotoxicity by hyperthermia can be explained . SimilarIt is concluded that cancer cells respond differentially to mild temperature changes, whether these are towards mild hypothermia or fever-range hyperthermia. Thermo-tolerant and thermo-sensitive cells lines have been identified to fever range hyperthermia, which encourages research to identify suitable methods for clinical grouping of human tumors according to their thermal predilection. Such characterization would allow clinical trials with non-toxic localized or total body hyperthermia in patients predicted to be sensitive to such therapies. Of interest, mild hypothermia had a suppressing effect on cell proliferation in all cells examined, suggesting that hypothermia would suppress tumor replication and metabolism in most human tumors, further supporting the radio-sensitization hypothesis through increased oxygenation by reducing oxygen and metabolic demands.


Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. Comparative analysis of cell lines in combination with system-wide profiling techniques may disclose cross cell-type commonalities and variations of biological processes. This knowledge can be used for understanding cancer metabolism, identifying anticancer drugs, in vitro evaluation of predicted drug targets and outlining mechanisms of action of therapeutic agents2Human cancer cell lines are widely used model systems for studying cellular mechanisms underlying cancer by analyzing their perturbation-response patterns in simplified experimental conditionsAdvances in omics technology have enabled simultaneous measurement of molecular components interacting within complex interconnected networks and hereby provided insights into cellular functions and phenotypic states of the cells. However, analyzing genome-wide data and in particular gaining new biological knowledge, is a nontrivial effort56891012Preserved proliferative signaling and avoidance of growth suppressors have been identified as hallmarks of cancer1719in silico toxicity by employing GEMs for 83 human healthy cell-types. Next, we experimentally tested the effect of an L-carnitine analogue, one of the potential antimetabolite that was predicted to be effective on all cell lines. Significant difference in proliferation of cultured cell lines in presence or absence of selected analogue, confirmed our computational prediction. Finally, we presented high or moderate protein staining level of the CPT1A and CPT1B, which were targeted by the L-carnitine analogue in cell lines using the antibodies generated in the Human Protein Atlas (HPA).In this study, we first followed a systematic approach and analyzed the global mRNA expression pattern of the 20,314 protein coding genes in eleven human cancer cell linesnot detected. Based on this cut-off the number of protein coding genes expressed ranged from 11,000 to 12,500 across the eleven cell lines. Approximately 43% of the expressed genes for each cell line were found in the category of high and medium RNA expression levels and the average transcript abundance score in a log2 scale ranged from 4.90 for the epidermoid carcinoma cell line (A-431) to 5.30 for the metastatic breast adenocarcinoma cell line (MCF-7) (RNA-Seq have been employed to identify the tissue-specific expression of genes across the major human tissues and combined with the antibody based profiling of the same tissues23242526 (MCF-7) . The tra (MCF-7) . In tota (MCF-7) .http://www.metabolicatlas.org/) in the Systems Biology Mark-up Language format.A generic genome-scale metabolic model of cancer has been developed to capture the common metabolic functions between cancer typesThe resulting CL-GEMs contained 5,297 to 5,584 reactions associated with 2,193 to 2,328 genes and involving 4,209 to 4,432 metabolites . In totaWe used CORE metabolite profiles of cancer cell linesCancer cells reprogram their metabolic network to support cell proliferation by synthesizing all the cellular components and the required Gibbs free energy29We also checked the number of the flux carrying reactions in each model  and founNext, using the transcriptome expression profile of genes included into the CL-GEMs, we investigated the global shifts between the cell lines. Kruskal-Wallis one-way analysis of varianceThus, comparing GEMs through constituent parameters  and also using mRNA expression pattern disclosed a considerable difference between metabolic models for the eleven cell lines. This indicated the necessity of developing fine-tuned CL-GEMs to capture the metabolic alterations that are specific to each cancer cell line.We analyzed the differences in mRNA expression pattern of the individual pathways across the eleven cell lines. We calculated the fraction of cell lines in which each metabolic subsystem had an average high transcript expression (H) and low transcript expression (L), and transferred the results into the (H − L) and (H + L) coordinates to observe the behavior of subsystems across cell lines . We founIn contrast, lipoic acid metabolism and glucocorticoid biosynthesis revealed a relatively high fraction of L across all cell lines, which followed by retinol metabolism and androgen metabolism. Lipoic acid has been shown to reduce the proliferation/viability and to increase apoptosis of tumor cells3436373940We next studied the inter-cell expressional variation of subsystems to explore the heterogeneous behavior of metabolic pathways across the different cell lines. Bile acid recycling, fatty acid desaturation, ascorbate and aldarate metabolism, formation and hydrolysis of cholesterol esters, and vitamin D metabolism showed higher heterogeneity among all subsystems in HMR2 . In CACOin silico toxicity test using the previously published GEMs for 83 different cell types in the human body. We checked if the use of the antimetabolites would potentially disrupt the metabolic tasks in energy production.We used reconstructed CL-GEMs to predict the potential antigrowth factors through analyzing the robustness of metabolic networks against the induced perturbations. The metabolite-centric approach based on the concept of antimetabolites was employed to introduce the growth inhibiting perturbations. By testing the essentiality of metabolites present in the HMR2 on growth of cancer cell lines, we predicted 138 antimetabolites that can kill the growth on any of eleven studied cell lines and 106 of them were found to be effective in all cell lines. However, using these antimetabolites for the treatment of the cancer may have toxic effects to healthy cell-types in the human body. Therefore, we performed We finally predicted 85 potential antimetabolites that can inhibit growth of any cell lines studied here, while not being toxic to healthy cells . We founFurthermore, we categorized 85 predicted antimetabolites based on their relevant subsystems in HMR2 and calculated the average mRNA expression level of the subsystems across cell lines . It was de novo biosynthesis43Following the prediction of L-carnitine as an antimetabolite and considering the connection of the L-carnitine shuttle to the mitochondrial β-oxidation, the analogue of L-carnitine were proposed as a potential antigrowth factor targeting the growth in all eleven cell lines . L-carniβ-Oxidation was found to have high average mRNA expression levels in all eleven cell lines. This pathway consists of recurrent series of reactions that result in cyclical shortening of fatty acids and production of acetyl CoA, NADH and FADH. Generated NADH and FADH are used for ATP production through electron transport chain, while acetyl CoA enters the citric acid cycle (TCA)444542434447The use of L-carnitine analog inhibits the carnitine palmitoyltransferase (CPT) 1 and 2 enzymes that catalyze the L-carnitine and fatty acyl CoAs. We used perhexiline malate salt (perhexiline) that inhibits CPT1 and partly CPT2 to mimic the effect of the potential L-carnitine antimetabolite. To confirm our prediction about the use of L-carnitine analogue as potential antigrowth factor for all cell lines, we tested the effect of Perhexiline on the proliferation of prostate carcinoma cell line (PC-3) and A-431. Perhexiline was used to imitate the L-carnitine mechanism of effect since mitochondrial enzyme CPT1, as a member of carnitine acyltransferases family of enzymes, is responsible for translocation of conjugated L-carnitine and long chain FAs from cytosol to the mitochondria . We treaWe also analyzed protein expression level of CPT1 and CPT2 using the antibodies generated in the HPA projectRecent advances in understanding cancer cell biology and continuous development of high throughput analytical methods have revealed that cancer is remarkably complex and heterogeneous. Increased understanding of this genomic heterogeneity between different tumors may open a new window towards the development and optimization of therapeutic methods for specific cancer types. GEMs provide a mechanistic description of relationships between genes and metabolic functions, and they can therefore be used for the interpretation of large experimental datasets. In this study we reconstructed CL-GEMs for eleven human cancer cell lines based on RNA expression data and used the generated models to understand the heterogeneity of metabolic networks in cancer cell lines. Our analysis revealed notable heterogeneity across CL-GEMs, where each model had an average of 353 different genes (heterogeneity degree of 0.78), 272 different metabolites (heterogeneity degree of 0.69) and 517 different reactions (heterogeneity degree of 0.74) in comparison to other models. Comparing CL-GEMs with GEMs for 83 healthy cell-types, we found a slight tendency towards the uniformity of metabolic networks in CL-GEMs and the heterogeneity of models preserved to a large extent. This finding indicates inadequacy of reconstructing a generic cancer model to capture metabolic alterations in transformed cells.We analyzed the RNA expression pattern of individual pathways to identify the transcriptome based distribution of subsystems across CL-GEMs. We found that genes involved in fatty acid biosynthesis and cholesterol biosynthesis had highest average mRNA expression level in most of the cell lines, which shows the importance of biosynthesis pathways in transformed cells. On the other hand, lipoic acid metabolism and glucocorticoid biosynthesis revealed lowest average expression level among subsystems consistent with previously published studies indicating their positive correlation to reduced cell proliferation/viability and increased apoptosis of tumor cellsin silico toxicity test based on previously generated GEMs for 83 healthy human cell types, proposed as potential antimetabolites. Analogues of L-carnitine were proposed as potential antimetabolites, due to their key role in L-carnitine shuttle pathway, and the contribution of L-carnitine shuttle to the mitochondrial β-oxidation and consequently to the fatty acid synthesis. Two cell lines with different phenotypic origins, PC-3 and A-431 were selected for in vitro testing of our prediction using Perhexiline to mimic the effect of L-carnitine analogue. Both cell lines revealed significant reduction in cells viability in presence of Perhexiline, validating the relevancy of predicted target. The effect of Perhexiline on viability of Hep-G2 has previously been verified by ref. Metabolite essentiality, defined as metabolites necessary for cell growth and survivor, has previously been usedPlasticity is one of the key factors to maintain fitness of transformed cells and β-oxidation of fatty acids may prepare some of this plasticity by supplying metabolic intermediates for cell proliferation, suppressing pro-apoptotic subsystems, eradicating potentially cytotoxic lipids, and empowering the production of NADPH and ATP when required51525354Despite promising performance of GEMs in identification of biomarkers and drug targets for cancer, there are cautions and limitations regarding this approach. Unlike bacteria, reconstructing and testing the functionality of GEMs is more complex when it comes to cells and tissues. Constructing new generation of unified GEMs that integrate metabolism with other cellular processes, especially signaling and regulation, remains as a nontrivial challenge. Different assumptions to estimate the connection between transcription, translation and flux rate challenges the accuracy and compatibility of models generated by incorporating omics data into GEMs.In conclusion, we used CL-GEMs to investigate the differences in the metabolism of cell lines and explore potential antigrowth factors, which may impede the growth of cancer cells, and experimentally verified the inhibitory effect of the L-carnitine analogue, one of the predicted antimetabolite. The study of cancer metabolism using GEMs has revealed new therapeutic opportunities, as well as more profound understanding of metabolic reprogramming of cancer cells. Over the past decades we learned that targeting single pathways or enzymes is not enough to cure cancer and agents proposed here as antigrowth factors may be combined with other therapeutic agents and methods to get desired results. The approach presented here may also be extended to study the combinatory effect of potential and standard therapeutic agents to find more effective cancer specific therapies, and may present new possibilities towards personalized medicine.The tINIT algorithmhijd is the heterogeneity degree of model i based on parameter j, jIv). In this formulation, n is the number of models , m is the number of the constituent parameters , and jIv is a unique union of corresponding parameter across all models. For example, I.v1 represent a union vector of genes present at least in one of the models.A new measure of heterogeneity was used in our study to capture the divergence between metabolic networks based on their constituent parameters: genes, reactions and metabolites. For each model, heterogeneity degree formulated as:To compare the CL-GEMs based on RNA expression levels, we built a vector of expected transcriptome expression values of genes linked to metabolic subsystems for each model, and merged these vectors into n × m unified ratio matrix. Here, n is number of subsystems and m is the number of the models. We used this matrix to perform multi-comparison analysis employing Kruskal-Wallis non-parametric test with significance level of 0.01. Finally, we performed pair wise analysis of models using post hoc Fisher's LSD test with confidence level of 0.05.To identify the heterogeneous expression of metabolic subsystems across the cell lines, we calculated the ratios of high and low RNA expression level for genes associated to reactions inside the CL-GEMs in logarithmic scale. For each pathway, we calculated the average ratio of high expression level and low expression level and integrated the results into two different n × m ratio matrices, high matrix (HM) and low matrix (LM), respectively. Here, n is number of pathways and m is the number of models. We used a subsystem-specific metrics, HM-LM and HM + LM, to compare the expression pattern of pathways across the cell lines.Inhibition of essential metabolites in cancer cell lines can also disrupt the metabolism of healthy cells, similar to chemotherapeutic drugs. To avoid this side effect, we used reconstructed GEMs for 83 healthy cell types and investigated the response of these metabolic models against inhibition of essential metabolites. We excluded the essential metabolites that disrupted energy metabolism and redox balance in healthy cells. Our approach was based on the central role of energy producing tasks for all cell types, and also high likelihood of neoplastic shifts in energy metabolism of transformed cells.ueous One Solution Cell Proliferation Assay (MTS) (Promega) on PC-3 and A-431 according to instructions from the manufacturer . In brief, the two cell lines were treated with 2, 4, 8 and 20 μM of Perhexiline Malate salt  for 24 and 48 hours respectively. Perhexiline was dissolved in DMSO, and corresponding concentrations of DMSO in medium were used as controls. In addition, controls consisting of cells growing in only cell culture medium were included. For all concentrations and corresponding DMSO-controls eight replicates were analysed, and for the controls consisting of cells in only medium 16 replicates were analysed. In the follow up experiment the two cell lines were treated with 4 and 6 μM respectively of Perhexiline Malate salt  for 48 hours. This time 16 replicates were used for all conditions and controls.A proliferation assay was performed using the colorimetric CellTiter 96® AQin-vitro experiments. S.S. validated the functionality of the models. J.N. and M.U. conceived the project. P.G., A.M. and J.N. wrote the paper and all authors were involved in editing the paper.P.G. reconstructed the models and P.G. and A.M. performed the analysis of the data. A.A., C.K. and M.U. performed the Supplementary MaterialSupplementary Dataset 1-10


Despite immune suppression, GVHD is highly incident even after allo-HSCT using human leukocyte antigen (HLA)-matched donors. Therefore, alternative and more effective therapies are needed to prevent or control GVHD while preserving the beneficial graft-versus-cancer (GVC) effects against residual disease. Among novel therapeutics for GVHD, oncolytic viruses such as myxoma virus (MYXV) are receiving increased attention due to their dual role in controlling GVHD while preserving or augmenting GVC. This review focuses on the molecular basis of GVHD, as well as state-of-the-art advances in developing novel therapies to prevent or control GVHD while minimizing impact on GVC. Recent literature regarding conventional and the emerging therapies are summarized, with special emphasis on virotherapy to prevent GVHD. Recent advances using preclinical models with oncolytic viruses such as MYXV to ameliorate the deleterious consequences of GVHD, while maintaining or improving the anti-cancer benefits of GVC will be reviewed.Allogeneic hematopoietic stem cell transplantation  has a curative potential for many hematologic malignancies and blood diseases. However, the success of allo-HSCT is limited by graft- Donor T cells can be very toxic to the recipient, even with close human leukocyte antigen (HLA) matching of the donor and patient. These donor T cells have the potential to attack and damage multiple organs and tissues of the allo-transplanted recipient, resulting in high risk for morbidity and mortality [Allogeneic hematopoietic stem cell transplantation  involves the transfer of multipotent hematopoietic stem cells from a healthy donor to a genetically non-identical recipient . Allo-HSer (GVC) , which ier (GVC) . Howeverortality .ex vivo agent to control GVHD and also augment the anti-cancer effects mediated by GVC [Despite the progress over the past several years regarding prophylaxis to better control GVHD, the need remains for more effective methods that guarantee not only the control and management of GVHD but also the maintenance, or even improvement, of GVC. Recently, virotherapeutics with oncolytic viruses or genetically modified viruses, have demonstrated potential to promote and improve the GVC effect. For example, myxoma virus (MYXV), a rabbit-specific virus that can mediate oncolysis in a wide spectrum of human cancers ,9,10,11,d by GVC ,13.The pathophysiology of GVHD will be reviewed, focusing on the successes and failures of the associated research and the potential impact of GVHD on allo-HSCT in clinical practice. We also review the current, and most promising emerging therapies to prevent or control GVHD and to elaborate on the strengths and limitations of these strategies. Furthermore, the status of novel therapies to prevent, and control GVHD while preserving GVC will be summarized. Emphasis will be placed on the virotherapeutic strategy with MYXV, which appear to possess the dual consequences of controlling GVHD while at the same time augmenting the anti-cancer effects of GVC.Allo-HSCT is the only curative therapy for many chemotherapy-refractory or relapsed hematologic malignancies . The genSeveral stem cell sources have been used for allo-HSCT. For instance, bone marrow (BM), a rich source of hematopoietic stem and progenitor cells (HSPCs), was the sole source of donor HSPCs until the 1990s . In the mid-1990s, donor peripheral blood (PB) that had been mobilized with the cytokines granulocyte colony-stimulating factor (G-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) was increasingly used as a source of allo-HSCT ; howeverversus-cancer (GVC) and is one of the major therapeutic reasons for allo-HSCT in the treatment of hematological malignancies [Prior to allo-HSCT, patients receive conditioning regimens with chemotherapy and/or radiation in order to (a) kill residual cancer cells in the patient, and (b) inactivate the host immune system so that the allograft will not be rejected. Conditioning is followed by the intravenous infusion of donor HSPCs that seed donor-derived hematopoiesis . In addignancies . Unfortugnancies  and resugnancies ,27. The Another obstacle of allo-HSCT is delayed immune reconstitution, which is exacerbated by the prolonged post-transplant immune suppression required to reduce the risk of GVHD. In this time of immunocompromise, recipients are at very high risk of developing opportunistic diseases like fungal infections, cytomegalovirus (CMV) reactivation, and post-transplant lymphoproliferative lymphomas. It should also be noted that during active GVHD, the patient is also at very high risk of opportunistic infections due to a lack of immunocompetency against microbial infections. Although the prevention and treatment of GVHD induce additional immunocompromise, GVHD itself is an immunopathological condition.In order to increase the number of patients eligible for allo-transplantation, a balance between ameliorating GVHD and yet preserving GVC is required, as well as the implementation of parameters such as donor selection, stem cell source, conditioning, immune reconstitution, and immunosuppressive regimens . These aBarnes and colleagues initially reported GVHD as a “secondary disease” developed in mice as a result of lethal irradiation and treatment with foreign bone marrow . Later, + and CD8+, are the major effectors of GVHD and cause tissue damage by a variety of pathways including: the Fas-Fas ligand (FasL) and perforin-granzyme pathways [The development of GVHD occurs in five steps, which are schematically represented in pathways , TNF-recpathways ,46,47,48pathways .Although APCs and T cells are commonly associated with the development of GVHD, recently, several studies have indicated that non-T cells like neutrophils can also be effectors of GVHD ,50,51.In vivo studies using myeloperoxidase imaging showed that neutrophil infiltration (Ly6G+) of mouse ileum after allo-HSCT was dependent of the microbial flora [Cybb  in neutrophils disabled ROS production and led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. In humans, severity of intestinal GVHD correlates with the levels of neutrophils in GVHD lesions [Neutrophils promote T cell activation by cleaving chemokines and producing reactive oxygen species (ROS) ,53. Thusal flora . Neutrop lesions . The stu lesions .Prevention of neutrophil granulocyte infiltration and degranulation can minimize GVHD. In this setting, Giroux and co-workers showed that SMAD3 had a role in preventing neutrophils infiltration through the suppression of T helper 1 (Th1) skewing of donor CD4+ T cells.SMAD3 is a receptor that regulates transforming growth factor-β (TGF-β) signals, . TGF-β set al., demonstrated that the presence of SMAD3 was necessary to decrease the generation of monocytes, neutrophils, as well as the production of ROS by neutrophils. In contrast to wild type (WT), SMAD3- knockout (KO) grafts induced lethal GVHD in major histocompatibility complex-identical recipients. The severity of GVHD was associated with abundant leukocyte infiltration, mainly T cells (CD4+) and neutrophils (CD11b+Gr-1+) into the intestine of recipients [The studies conducted by Giroux cipients .et al., demonstrated that increased transplant-related mortality (TRM) was correlated with the expression in situ of apoptosis mediators such as TNF-α and Fas in the gastrointestinal (GI) tract during digestive GVHD, as well as increased cellular infiltration of neutrophils in target organs after allo-HSCT [In 2004, a clinical study conducted in humans by Socié llo-HSCT . TherefoThe clinical presentation of GVHD is heterogeneous, involving the skin, mucosa, GI track, liver and lungs . Based oThe incidence of aGVHD varies with incidence of grade II-IV GVHD at 40% in matched related donor (MRD) transplant to 50% matched unrelated donor (MUD) transplant . Acute GHLA disparity can be at the level of major or minor histocompatibility antigens, which are variable among individuals . The incPre-transplant conditioning regimens trigger the innate immune system with the concomitant induction of aGVHD. Chemo- and radio-therapeutic conditioning regimens performed prior the infusion of BMT donor cells induce the release of pathogen-associated molecular patterns (PAMPs), such as the endotoxin lipopolysacchraride (LPS). The interaction of LPS with toll-like receptors (TLRs) like TLR-4 initiates cellular signaling pathways that activate cytokine secretion , such asIn aGVHD, the immune response is mounted when APCs sense endogenous molecules that are activated after the conditioning regimen-mediated tissue damage, like damage-associated molecular patterns (DAMPs), which present the MHC or minor histocompatibility antigen disparate protein, and provide secondary co-stimulatory signals via CD28, ICOS, CD40, CD30, 4-IBB and OXO40 molecules, and tertiary (cytokine) signals for activation of mediators of aGVHD such as different subsets of the allo-reactive T cells (mediators of aGVHD). The pivotal role of T cells in aGVHD is supported by the complete abrogation of GVHD following depletion of T cells from the allo-graft, an approach that remains the most effective in preventing aGVHD . HoweverIn vitro and in vivo studies have demonstrated that stimulation of APCs with ATP leads to increase expression of CD80 and CD86 and induce a cascade of pro-inflammatory events such as the phosphorylation of signal transducer and activator of the transcription 1 (STAT1), production of IFN-γ and donor T cell expansion, and the reduction of regulatory T cell numbers. Therefore, ATP neutralization, blockage or genetic deficiency of P2X7R during the development of GVHD can improve survival [As mentioned before, conditioning can induce tissue injury and cell death. Dying cells release adenosine 5′-triphosphate (ATP), which is not only a danger signal that triggers innate immune response pathways, but also is the main endogenous ligand of the P2X7 receptor (P2X7R). Activation of P2X7R is a critical step in the pathogenesis of GVHD . In vitrsurvival .Furthermore, the interaction between ATP and P2X7R promotes assembly and activation of the Nod-like receptor protein 3 (Nlrp3) inflammasome, an intracellular multiprotein complex that controls the activation of inflammatory caspase-1 in response to exogenous and endogenous stress or danger signals . After cIn vivo studies have demonstrated that inhibition of co-stimulatory molecules reduces acute GVHD [Co-stimulation signals are required to achieve T cell activation, proliferation, differentiation and survival . In vivoute GVHD ,77,78,79ute GVHD .As mentioned above, alloreactive donor T cells are the critical mediators of aGVHD. Alloreactive donor T cells consist of several subsets with different stimuli responsiveness, activation thresholds, and effector functions . The comcGVHD affects 30%–80% of patients receiving allo-HSCT  with a 5Classically, Th2 responses have been involved in the pathophysiology of cGVHD. However, we now recognize that the biology of cGVHD is much more complex and thus other factors such as thymic dysfunction, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF), B cells and autoantibodies, and Th1/Th2/Th17 cytokines, as well as regulatory T cells (Tregs), need to be taken into consideration .The treatment of cGVHD is based on its severity. For instance, mild cGVHD is treated with topical immunosuppressive agents or systemic steroids. Moderate to severe cGVHD requires treatment with systemic immunosuppression including prednisone with continued administration of calcineurin inhibitor for steroid sparing . A majorDespite the progress in understanding the pathophysiology of GVHD, the limited success of established therapies for prevention and treatment of GVHD remains unsatisfactory. The ideal clinical scenario for allo-HSCT would be prevention of GVHD while retaining GVC. However, it has not been easy to de-segregate GVHD from GVC, since the main effectors for both GVHD and GVC are T lymphocytes that share many common pathways for these two outcomes. T lymphocytes contained in the donor graft have crucial roles in the pathogenesis of GVHD; therefore, prophylaxis of GVHD has been focused on the depletion, tolerization or functional incapacitation of donor T cells . The strVarious methods have been tested to manipulate donor T cells to retain therapeutic effector functions against residual tumor cells in the patient without causing GVHD. In this section, current strategies that are applied in the clinic for the prevention and treatment of GVHD, are reviewed. In addition, emerging strategies and their potential to improve patient outcomes are discussed.A myelosuppressive regimen is administered prior to allo-HSCT to deplete stem cells from the host, thus making more available engraftment space for the donor graft, and to reduce graft rejection mediated by host immune cells . HoweverThis section summarizes some of the most relevant drugs and therapeutic strategies used for the inhibition or depletion of allo-reactive T cells.MTX inhibits dividing allo-reactive T-cells  resultinCalcineurin inhibitors such as cyclosporine A (CYA) , and tacCorticosteroids at high doses are the first-line treatment for established GVHD ; howeverCyclophosphamide has been used post-transplant since the 1980s for the prevention of aGVHD, which occurs via inhibiting T cell division and is used after myeloablative conditioning in related and unrelated allo-transplants .MMF has a potent cytostatic effect on T and B lymphocytes . MMF hasSirolimus (rapamycin) forms a complex with the mammalian target of rapamycin (mTOR), inhibiting biochemical pathways with the concomitant reduction of DNA transcription/translation of protein synthesis, cell cycle progression, which ultimately result in T cell immunosuppression . Sirolimex vivo negative selection of T cells, ex vivo positive selection of CD34+ stem/progenitor cells, and use of antibodies against T cells in vivo.T cell depletion prophylaxis for GVHD was undertaken in the 1980s, and 1990s. The three main depletion strategies were + stem/progenitor cells, but also NK cells and dendritic cells [In vivo administration of the polyclonal anti-thymocyte globulin (ATG) antibodies, pre- and peri-allo-HSCT, simultaneously targeted donor and host T cells resulting in the control of GVHD and avoided graft rejection. However, one of the challenges was that the ATG antibodies also target B cells, NK cells and APCs, without reducing the incidence of cGVHD [In the 1970s, GVHD was lethal in recipients of HLA-mismatched bone marrow . In the ic cells . Depletiic cells , was anoic cells .Polyclonof cGVHD .+, CD6+ and CD8+ have been implemented. However, the success of this strategy remains limited [MAbs like T10B9, which targets the T-cell receptor, in combination with CYA have been used to minimize graft rejection; however no improvement in long-term survival has been observed. On the contrary disease relapse, and the risk of opportunistic infections were increased . Alemtuz limited ,130,131.+ with immune-magnetic beads is an effective method of depleting allo-reactive donor T cells prior to transplant, and results in significant reduction of aGVHD and cGVHD [CD34nd cGVHD ,133. Hownd cGVHD ,134.There is no specific Food and Drug Administration (FDA)-approved treatment for established aGVHD or cGVHD. Currently, the first-line therapy for GVHD is with corticosteroids like prednisone . CorticoAdjuvant topical steroids to reduce the steroid toxicities have been shown to have some success. In a study evaluating oral beclomethasone, this steroid improved outcomes in patients with GI aGVHD . In anotA number of approaches to reduce the symptoms of GVHD that include the use corticosteroids in combination with other agents have been tested. These other agents have included horse rabbit anti-thymocyte globulin ATG; daclizumab, a monoclonal antibody that targets CD25 (IL-2Rα) present on activated T lymphocytes, antibodies against TNF , pentostatin, MMF, and sirolimus. Unfortunately, these pharmacological agents administered with corticosteroids failed to improve response rates . and areA second line of treatment of GVHD includes the use of anti-metabolites , extracorporeal photopheresis or light therapy, which improve GVHD outcomes in skin, liver, and mouth tissue. Photopheresis is complicated and requires up to six months to see improvement of the GVHD symptoms.For cGVHD treatment, prolonged administration of corticosteroids in combination with calcineurin inhibitors such as cyclosporine or tacrolimus has been used as a typical standard therapy. However this combination therapy has not improved patient outcomes .Although some of the above mentioned therapies have been used a second-line therapy in the treatment of SR-GVHD, none have been adopted as a standard salvage therapy for either SR-aGVHD or SR-cGVHD. Pre-clinical evidence has indicated that ruxolitinib, an inhibitor of the Janus kinase (JAK) 1 and 2, has potent anti-inflammatory properties .et al., showed that inhibition of the JAK1/2 signaling with ruxolitinib resulted in reduced proliferation of effector T cells, suppression of production of proinflammatory cytokines in response to mouse allo-antigen, and increase in Foxp3+ regulatory T cells [Since activated JAKs are required for T-effector cell responses in different inflammatory diseases, their blockade could reduce GVHD . Using a T cells . Therefo T cells .Bortezomib, a proteasome inhibitor, was approved by the FDA for treating patients with multiple myeloma and mantle cell lymphoma . AlthougB cells contribute to immune responses through the production of antibodies, inflammatory cytokines, antigen recognition and other immunoregulatory functions. A growing body of research indicates that in addition to T cells, B cells play an important role in the immune pathology and tissue damage characteristic of GVHD . Thus, tIn vitro and in vivo preclinical studies and phase I/II clinical trials have demonstrated that the retroviral-mediated transfer of the suicide gene HSV-TK into donor T cells prior to infusion allows for the efficient control of donor T cell allo-reactivity [The cellular suicide gene approach is a promising tool to manipulate donor T cell effector functions in order to augment the GVC effects, promote immune reconstitution, and to prevent or control GVHD . The celactivity . Importaactivity . Currentactivity .There are some limitations of the HSV-TK approach. In immunocompromised patients, TK may lead to undesired elimination of transduced cell populations as a result of the immunogenicity of this viral protein. In addition GCV is a drug used to treat cytomegalovirus (CMV) reactivation, which commonly affects immunocompromized patients. Administration of GCV in these patients to treat CMV induces undesired TK-cell killing .A different suicide gene strategy has been explored using inducible human caspase 9 transgene (iC9), which is a hybrid protein consisting of a human FK 506-binding protein (FKBP12) linked to a modified human caspase 9 lacking the caspase recruitment domain (CARD). The iC9 transgene can be dimerized and activated by administration of a bio-inert small molecule drug AP1903. A phase I/II clinical trial suggests that patients had immediate and sustained protection from major pathogens in the absence of aGVHD or cGVHD .+CD25+Foxp3+. Tregs constitute 5%–10% of peripheral CD4+ T cells, and play a pivotal role in the induction of immunologic tolerance and maintenance of immune homeostasis [in vitro expansion of Tregs through T cell receptor (TCR)-mediated activation with IL-2 [in vitro conversion of conventional T cells (Tcon) into Tregs in the presence of TGF-β [Naturally occurring Tregs are immunophenotypically defined by CD4eostasis ,156. Addeostasis ,157,158,eostasis ,156,159.eostasis ,160 reprof TGF-β , or via of TGF-β . Neverthof TGF-β .+CD25− T cells. Using a leukemic model, they demonstrated that the infusion of engineered Tregs in combination with donor bone marrow and splenocytes not only prevented recipients from lethal GVHD, but also the GVC effect was maximally preserved [In 2010, Cao and co-workers  reportedreserved . AlthougCo-stimulatory molecules provide the second signal that fully activates alloreactive T cells. The co-stimulatory pathways are grouped in two major families: the immunoglobulin (Ig) superfamily and the TNF/TNF receptor (TNFR) family. Since co-stimulatory molecules promote GVHD, different therapeutic approaches have been aimed to target and block such molecules. Herein, we briefly describe some of the best-known co-stimulatory molecules and their action of blockade with pharmacological agents to abort GVHD.+ T cells increases T cell survival proliferation and cytotoxic T lymphocyte (CTL) activities. The use of anti-CD137L mAb inhibits aGVHD, while exacerbating cGVHD. The anti-CD137L mAb inhibits the activity of donor T cells in the following GVHD mouse models: DBA/2 (donor)→F1 (host); and C57BL/6 (donor)→F1 (host) [CD137 is a member of the TNF receptor family that functions as a co-stimulatory molecule for T cells. CD137 ligation on CD81 (host) .CD28 is constitutively expressed on naive T cells. CD28 has two ligands, B7-1 and B7-2, which are expressed on APCs; and the activation of T cells induces the up-regulation of these ligands. B7-1 and B7-2 also have high-affinity for the inhibitory receptor named as CTLA-4, whose expression is induced on activated T cells . ReductiICOS is a member of the CD28 family that is expressed on activated T cells and memory T cells. It is constitutively expressed on B cells, macrophages, and dendritic cells and is upregulated on APCs and some non-lymphoid tissues by TNF or LPS . BlockadPD-1 is a member of the B7:CD28 superfamily and is involved in keeping the cellular immune system in check. PD-1 is expressed on activated CD4, CD8 T cells, as well as NK cells, B cells, and macrophages. PD-1 has two ligands, PDL-1 and PDL-2 that are expressed on APCs after cellular activation. Some studies have shown that blockade of the PD-1/PD-L1 pathway accelerates GVHD-induced lethality . In acco+ T cells. CD40 signals activate DCs and activated DCs promote the activity of CD8+ T cells and CD4+ T cells. Studies using a DBA/2 (donor)→F1 (recipient) aGVHD or cGVHD model demonstrated that blockade of the CD40 pathway inhibits aGVHD and cGVHD [+ T-cell proliferation [+ T cell-mediated GVHD [+ T cell-mediated GVHD [CD40L pathway is involved in the GVHD lethality. CD40 is expressed on DCs and B cells, and CD40L is expressed on activated CD4nd cGVHD . Studiesnd cGVHD  by promoferation . Apart fted GVHD , HVEM isted GVHD .Recently, intracellular molecular targets in T cells have been recognized as being critical for regulating alloreactivity. Some of these intracellular targets include epigenetic regulation in donor T cells, notch signals in donor T cells, mitochondrial ATPase, the proteasome, protein kinases, microRNAs and the JAK-STAT pathways.in vivo via a direct effects on donor Tregs [Epigenetic regulation in all cells is modulated via histone acetylation and methylation along with DNA methylation . Histoneor Tregs .In vivo, studies have demonstrated that Notch regulates allo-reactive T cells that mediate GVHD. In mouse allo-HSCT models, inhibition of Notch in donor-derived T cells resulted in the reduction of severity and mortality of GVHD [Signaling mediated by Notch activation is important in cellular processes such as differentiation, survival and homeostasis . In the  of GVHD ,184.1F0 adenosine triphosphate synthase (F1F0-ATPase), which induces selective apoptosis of alloreactive T cells, and reverses GVHD in BMT models without affecting hematopoietic engraftment or the immune reconstitution [During GVHD T cells proliferate in response to allo-antigens, and this process demands increased levels of ATP as a source of energy. Studies have revealed that proliferation of T cells increased aerobic glycolysis and oxidative phosphorylation. Alloreactive T cells also display increased superoxide production and decreased amounts of anti-oxidants. Bz423 is an inhibitor of the mitochondrial Ftitution .Proteasome inhibition has been shown to induce apoptosis of allo-reactive T cells . StudiesIn vivo studies have demonstrated that both PKCα, and PKCθ contribute to GVHD. Pharmacological inhibition of PKCα and PKCθ with R524 prevented GVHD and preserved GVC responses. R524 hampers T cell proliferation in vivo and in vitro, and reduces the expression of chemokine receptors and the production of chemokines, preventing the development of GVHD [Protein kinase Cθ (PKCθ) maintains the immunological synapse between T effector (Teff) cells and ligated-APCs, and is a key regulator of T-cell receptor (TCR) signaling. PKCθ promotes T-cell allo-reactivity and GVHD induction ,189. Stu of GVHD .In vivo studies using mouse GVHD models showed that microRNA-155 (miR-155) was up-regulated during activation of T cells after allo-HSCT [MicroRNAs are small single-stranded noncoding RNAs, which influence molecular pathways that control the development and function of innate and adaptive immune responses . In vivollo-HSCT . Lethal llo-HSCT . Likewisllo-HSCT .+ T cells [+ T cells increased nTregs post-BMT and prevent GVHD lethality [Regulation of donor T cells with specific signal transducer and activator of transcription (STAT) family proteins has shed light into the role of these proteins in GVHD regulation. Activation of STAT1 is an early event in GVHD and correlates with early cytokine storm . Experim T cells . Transfeethality . Murine ethality . Therefoethality . OverallLeporipoxvirus genus of the Poxviridae family, MYXV causes the lethal disease called myxomatosis in European rabbits (Oryctolagus cuniculus) [ex vivo purging agent to functionally eliminate specific malignant cell populations from human hematopoietic stem cell transplants [Oncolytic viruses are promising new agents for targeting and killing cancer cells ,200. Amoniculus) ,202. Intniculus) ,203,204.niculus) ,206. Secniculus) . Thus, tniculus) . The abiniculus) . In anotniculus) ,210,211.niculus) ,213,214,niculus) ,213. Thensplants ,212,213.ex vivo MYXV treatment) [+ T cells into the liver, intestines, skin, lung, kidney and spleen, consistent with the classic pathology of allo-HSCT GVHD. On the other hand, mice injected with human BM or PBMCs that had been pretreated ex vivo with MYXV for 1 h prior to transplant survived until the time of sacrifice without evidence for the disease [+ T lymphocytes. In addition, ex vivo MYXV treatment of human HCT samples did not impair normal human hematopoietic stem and progenitor cell (HSPC) engraftment in the immunocompromised NSG recipients and yet selectively inhibited expansion of transferred donor CD3+ T cells in the transplant recipient tissues such as spleen, liver and lungs [ex vivo pre-treatment of the human HCT specimens with MYXV attenuated the subsequent induction of GVHD. Importantly, the ex vivo MYXV treatment did not compromise the ability of the donor human HCT to mediate GVC as assessed by the elimination of pre-seeded human U266 MM in the majority of the recipient NSG mice.Recently, we reported that immunodeficient NSG mice xeno-transplanted with MYXV-pretreated human HSPC samples, such as BM or PBMCs, showed prolonged survival times compared to mock-treated transplant controls  . In this disease . This stnd lungs . These rIn the same study, human-to-human haplo-mismatched mixed lymphocyte reactions (MLRs) were also tested to investigate whether MYXV could prevent the expansion of allo-reactive lymphocytes in the context of human antigen allo-stimulation. The results indicated that MYXV prevented responder cell proliferation in the MLRs, suggesting that MYXV prevents the expansion of alloreactive lymphocytes from primary human BM samples when stimulated with HLA-mismatched human donor leukocyte samples .ex vivo MYXV treatment for just 1 h prior to transplant affects the post-transplant activities of primary human CD3+ T lymphocytes found in HCT samples derived from either BM or PBMCs. Second, this ex vivo treatment of human HCT samples with MYXV delayed or eliminated the development of lethal GVHD in xeno-transplanted NSG mice, while sparing the GVC effects.To summarize, two striking results were derived from this work . First, ex vivo virotherapy with MYXV prevented GVHD caused by human T cells in the allo-HSCT xeno-transplant model [in vitro studies performed with MYXV revealed that MYXV prevented GVHD by impairing the subsequent functionality of human T lymphocytes from the donor transplant [+ T cells, the activation of these virus-bound T cells is required to launch productive infection with MYXV. Importantly, the activation of MYXV-adsorbed human T cells caused the inhibition or suppression of T cell proliferation, as well as the downregulation of effector molecules such as IL-2, IL-2Rα and IFN-γ. These results help explain the mechanism by which ex vivo MYXV virotherapy prevents GVHD in the xenogeneic model [in vitro studies suggest a dual role of ex vivo MYXV virotherapy to prevent GVHD, but yet also augment the GVC effects of donor human lymphocytes [Although nt model , the mecansplant . This stic model . Furtherphocytes .ex vivo MYXV virotherapy of human BM or PBMCs prevented GVHD in a xeno-transplanted model and yet poorly infected resting human T cells ex vivo, one intriguing question that needed to be addressed was how MYXV selectively prevented GVHD while still permitting GVC against pre-seeded human myeloma. Thus, by exploiting a xeno-transplant model that assessed both GVC and GVHD, we demonstrated that MYXV prevented GVHD did not impair either the GVC effects of the human HSPC transplant graft or the engraftment of the normal stem cells [ex vivo pre-treatment of human BM with MYXV, followed by infusion in immuno-compromised mice with pre-seeded human myeloma, was a more effective anti-cancer strategy than just the direct injection of free MYXV virions, supporting the conclusion that the intravenous injection of free virus did not reach the pre-seeded disease in the BM as efficiently as when delivered via transplant leukocytes loaded ex vivo with virus [Adoptive transfer of mature T cells contained in the allo-HSCT improves patient prognosis through the highly beneficial anti-tumor effects of GVC, which relies on donor T cells becoming engaged and activated in clearance of minimal residual disease . This GVem cells . Importath virus . Very reth virus   by which MYXV mediates these dual effects will be critical to translate this new technology to the clinic.Despite significant progress in allo-HSCT, GVHD remains a major clinical complication of this therapy. Many therapeutic strategies have emerged in the last two decades with the aims of preventing or controlling GVHD and improving patient outcomes after allo-HSCT. Unfortunately, most strategies also reduce GVC against residual disease remaining in the patient and have only shown incremental benefits in mitigating GVHD. Development of safer strategies to prevent and treat GVHD, while still preserving or even augmenting GVC, will particularly help higher-risk transplant populations. Populations at higher risk for GVHD include those who are older or lack a suitably matched donor, including racial and ethnic minorities.


Triple-negative breast cancer (TNBC) is associated with a higher incidence of recurrence and distant metastasis and a poor prognosis, whereas effective treatment strategies remain to be established. Finding an effective treatment for TNBC has become imperative. We examined the effect of the combination of S-1 (or 5-FU in an in vitro study) and eribulin in TNBC cell lines. The in vitro effect of the combination was examined in four TNBC cell lines  using a combination index and isobolograms. In addition, we assessed the effect of the combination in an MDA-MB-231 tumor xenograft model. A synergistic effect was observed in three TNBC cell lines , and in an in vivo study, the combination of S-1 and eribulin resulted in significantly higher antitumor effects compared with S-1 or eribulin alone. 5-FU induced epithelial-mesenchymal transition (EMT) change in the TNCB cell line, as supported by the decreased expression of epithelial marker and the increased expression of mesenchymal markers. Meanwhile, TGF-beta induced EMT changes in a TNBC cell line and decreased the sensitivity to 5-FU. This result suggests that 5-FU-induced EMT changes reduce the sensitivity to 5-FU. In contrast, eribulin induced a mesenchymal-epithelial transition (MET) in a TNBC cell line. The EMT phenotype induced by 5-FU was also canceled by eribulin. We demonstrate that the combination of S-1 (5-FU) and eribulin exerts a synergistic effect for TNBC cell lines through MET-induction by eribulin. Therefore, this combination therapy may be a potential treatment option for TNBC. HER-2). TNBC accounts for between 9% and 16% of all breast cancers and is associated with a poor prognosis  is not sensitive to conventional chemotherapies. TNBC is a clinical phenotype characterized by a lack of overexpression or gene amplification of hormone receptors (estrogen receptor [ER] and progesterone receptor [PR]) and human epidermal growth factor receptor-2  is an oral fluoropyrimidine derivative composed of 1-(2-tetrahydrofuryl)-5-fluorouracil , combined with two modulators of 5-FU activity, 5-chloro-2, 4-dihydroxypyrimidine (gimeracil) and potassium oxonate (oteracil)  is a non-taxane, microtubule dynamics inhibitor with a novel mechanism of the action, which induces irreversible mitotic block at the G2-M phase and apoptosis  and eribulin.Because a monotherapy of S-1 or eribulin has shown promising activity in TNBC and eribulin have a manageable toxicity and a modest incidence of neuropathy, which appears to be lower than with other microtubule inhibitor agents, the combination of S-1 and eribulin is a hopeful combination regimen for breast cancer including TNBC. Phase I trial of S-1 and eribulin combination therapy for advanced or recurrent breast cancer pretreated by anthracycline and taxane is being executed in Kinki University Hospital. However, no preclinical studies of this combination have been performed. In this study, we investigated the combination effect of S-1 (5-FU) and eribulin on TNBC cell lines both 2 at 37°C, passaged every 3–4 days.This study was performed using four TNBC cell lines  and two non-TNBC cell lines . These cells were obtained from ATCC  or CLS  and were maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and cultured under a humidified atmosphere of 5% COin vitro study and eribulin was provided by Eisai Co. These agents were dissolved in dimethyl sulfoxide (DMSO).S-1 was prepared by mixing tegafur, gimeracil, and oteracil potassium  at a molar ratio of 1:0.4:1 in 0.5% HPMC. 5-FU was obtained from Sigma-Aldrich for the Cell growth was assessed using a standard 3- 2, 5-diphenyltetrazolium bromide (MTT) assay and a previously described method 2 are the concentrations required for single agents to achieve a% drug effect  and (D)1 and (D)2 are the concentrations of 5-FU and eribulin used in combination to achieve the same effect. In a normalized isobologram, the diagonal line represents the additive effect. Experimental data points, represented by dots located below, on, or above the line, indicate synergism, additively, and antagonism, respectively. The CI equation determines the additive effect of drug combinations, with synergism being defined as greater than the expected additive effect and antagonism being defined as less than the expected additive effect. Thus, CI values <1, 1, and >1 indicate synergism, additively and antagonism, respectively.where (Da)Total RNA was converted to cDNA using a GeneAmp® RNA-PCR kit . The cDNAs were used for PCR analysis using oligonucleotide primers specific for E-cadherin (forward 5’-TTAAACTCCTGGCCTCAAGCAATC-3’ and reverse 5’-TCCTATCTTGGGCAAAGCAACTG-3’) N-cadherin (forward 5’-CGAATGGATGAAAAGACCCATCC-3’ and reverse 5’-GGAGCCACTGCCTTCATCGTCAA-3’) vimentin (forward 5’-TGAGTACCGGAGACAGGTGCAG-3’ and reverse 5’-TAGCAGCTTCAACGGCAAAGTTC-3’) and Snail2 (forward 5’-ATGCATATTCGGACCCACACATTAC-3’ and reverse 5’-AGATTTGACCTGTCTGCAAATGCTC-3’). The PCR was performed using Thermal Cycler Dice  under the following conditions: 95°C for 5 min, 50 cycles of 95°C for 5 s, and 60°C for 10 s. The primers were purchased from Sigma-Aldrich or TaKaRa and were used with SYBR® Premix Ex Taq (TaKaRa). GAPDH was used as an internal control to normalize and compare each sample.Immunoblot analysis was performed as described previously  in PBS containing 50% Matrigel were injected subcutaneously into each mouse. Thirty days following the inoculation of the cells, the mice were divided randomly into 4 groups , and treatment was initiated. S-1 was administered orally on days 1 to 16, and eribulin dissolved in saline containing 2.5% DMSO was injected intravenously on days 1, 5, 9, and 13. The doses of S-1 and eribulin were 8.3 mg/kg and 0.1 mg/kg, respectively. The tumor volumes were estimated using the formula [(width)2 × length]/2 (mm3). To evaluate the anti-tumor effects of the S-1, eribulin, and a combination of S-1 and eribulin, the tumor sizes and body weights were measured twice per week. Antitumor effects were expressed as % T/C (treated versus control), dividing the tumor volumes from the treatment groups by those of the control groups and multiplying by 100.Female BALB/c-nu/nu mice  were used for the Formalin-fixed tumors were paraffin-embedded. 4 μm sections were used for HE staining, and IHC using antibodies against E-cadherin (1:100) and to vimentin (1:100). The methods used in this section have been described previously  to calculate the SD and to test for statistically significant differences between the samples using a Student's 50 values of 5-FU and eribulin for the cell lines fell within the ranges of 2.3-13.0 μM and 0.4-4.3 nM, respectively. There was no difference in cellular sensitivity between the TNBC cell lines and the non-TNBC cell line, except for the MX-1 cells  . These results showed the EMT changes in MDA-MB-231 cells reduced cellular sensitivity to 5-FU.We analyzed the mechanisms of the synergistic interaction between 5-FU and eribulin. Several reports have demonstrated that established 5-FU-resistant cell lines exhibit EMT changes , we observed that MDA-MB-231 cells displayed EMT-like changes in cell morphology, including the elongation of the cell shape and cell scattering, which are characteristic features of cells undergoing EMT  Figure a. In agrin vivo, we examined the antitumor activity of this combination in an MDA-MB-231 tumor xenograft model. Mice bearing MDA-MB-231 tumors were prepared and treated with S-1 , eribulin , or S-1 plus eribulin. The mean tumor volumes (mm3) on day 36 following the initial treatment of the 4 groups  were 1900 ± 473, 1055 ± 197, 900 ± 235, and 199 ± 26, respectively . S-1 or eribulin alone inhibited the tumor growth , and a combination of S-1 and eribulin inhibited the tumor growth of MDA-MB-231 xenografts more intensively (T/C = 10.5%). Body weight loss related to treatment with S-1 or eribulin was not observed in any of the groups  or additive (1/4) effects against TNBC cell lines in vitro and in vivo experiments. The action of eribulin on MET may improve the 5-FU resistance, resulting in produced a synergistic effect. Because of evidence of an association between EMT change and resistance to several other anti-cancer agents, these actions of eribulin provide a rationale for the combination of these agents. Moreover, a previous report demonstrated that a portion of TNBC tumors among clinical samples exhibit EMT changes and these subsets are significantly associated with a high histological grade  and eribulin exerts a synergistic anti-tumor effect against TNBC cell lines


We have also reported that CD14+ cells isolated from healthy donors are converted to the immunosuppressive CD14+HLA-DRlo/neg phenotype when cocultured with tumor cell lines or primary tumor cultures. Therefore, one potential mechanism of CD14+HLA-DRlo/neg generation in cancer patients is the recruitment of CD14+ cells to the tumor microenvironment. In this study, we tested the ability of multiple human lung cancer cell lines to recruit CD14+ cells, convert normal donor CD14+ cells to CD14+HLA-DRlo/neg, and the impact on tumor survival under stress conditions in the presence of CD14+HLA-DRlo/neg cells. Monocyte recruitment by cell line supernatants was studied using freshly isolated CD14+ cells and measured in real time with live cell imaging. Monocytes from 10/10 unique healthy donors migrated towards H23 supernatant in a dose dependent fashion. Supernatants from other human lung cancer cell lines including H889, H1155, H1581, and H1975 also recruited healthy donor monocytes 6 to 9 fold greater than culture media alone. The CD14+ phenotype resulting from interaction with lung cancer cell lines was studied in a 2 day coculture system. Coculture of H1581 with monocytes from 27 of 29 unique healthy donors resulted in a downregulation of monocyte cell surface expression of HLA-DR by 48.7% ± 22.1%, a phenotype known to mediate both local and systemic immune suppression. C14+ HLA-DR downregulation after coculture was observed with H23  and H889 . Importantly, CD14+ cells promoted lung tumor cell line survival under stress conditions. After 6 days of low serum growth conditions, the survival and proliferation of H23 and H1581 was approximately 2 fold greater when cocultured with CD14+ cells than without monocytes. H889 continuously exposed to cisplatin in culture had significantly improved survival when co-cultured with monocytes for 2 days prior to cisplatin exposure. In conclusion, we have demonstrated multiple lung cancer cell lines recruit and transformation monocytes to the HLA-DRlo/neg phenotype. These data suggest monocyte cancer cross-talk results in improved tumor survival when exposed to the likely in vivo conditions of a nutrient poor environment or chemotherapy independent of local immune suppression and angiogenesis.We have previously shown that a higher burden of CD14+HLA-DR


Our results indicate that pathway activation profiling is a promising strategy for evaluating the safety and potency of iPSC lines in translational medicine applications.Many attempts have been made to evaluate the safety and potency of human induced pluripotent stem cells (iPSCs) for clinical applications using transcriptome data, but results so far have been ambiguous or even contradictory. Here, we characterized stem cells at the pathway level, rather than at the gene level as has been the focus of previous work. We meta-analyzed publically-available gene expression data sets and evaluated signaling and metabolic pathway activation profiles for 20 human embryonic stem cell (ESC) lines, 12 human iPSC lines, five embryonic body lines, and six fibroblast cell lines. We demonstrated the close resemblance of iPSCs with ESCs at the pathway level, and provided examples of how pathway activity can be applied to identify iPSC line abnormalities or to predict  IPSC lines are powerful tools which can be used to study human embryonic development [The discovery of human induced pluripotent stem cells (iPSCs) in 2006 completely revolutionized the field of stem cell biology, and these cells are expected to play a key role in the development of many regenerative medicine therapies. IPSC lines can be obtained by reprogramming somatic cells using ectopic expression of transcription factors, such as OCT4, SOX2, KLF4, and C-MYC or alternative reprogramming cocktails , 2. Simielopment , as modeelopment , 6. The elopment .These applications require the selection and characterization of iPSC lines that reliably, efficiently, and stably differentiate into disease-relevant cell types. However, despite the fact that iPSCs theoretically have the full potential of embryonic stem (ES) cells, there are several studies showing evidence that induction of iPSCs can generate cell lines with genetic or epigenetic variability and abnormalities that could affect iPSC differentiation potential and clinical safety –15. ListThese findings raised the question of the variability of iPSC lines and whether iPSCs and ESCs are fully equivalent. To address these concerns, many recent attempts have been made to evaluate the safety and potency of iPSCs using genome-wide readouts of cell function, such as transcriptomes and methylomes, however, the results are often ambiguous and sometimes contradictory , 17. Forin vitro differentiation capacity of these ESC and iPSC lines [To address the issue of variability and safety in iPSCs, Bock et al. previously established a reference set of DNA methylation maps and gene expression profiles for 20 human ESC lines, 12 human iPSC lines, five embryonic body lines, and six fibroblast cell lines, and further provided data on the SC lines . While tSC lines , which cSC lines , 20. PatSC lines . In thatSC lines , 22, andSC lines . Using aSC lines .vs. ESCs from the GSE25970 data set and the resemblance of iPSC with ESC lines at the PAS level in comparison with EB and fibroblast lines. Additionally, we provided examples of how iPSC line abnormalities can be identified using our bioinformatics algorithm, and how pathway activation correlated with the known iPSC differentiation potential. We showed that the PAS algorithm can provide a powerful bioinformatics tool for quality control of iPSC lines prior to clinical application.Here, we defined the typical pathway profiles of iPSCs http://www.ncbi.nlm.nih.gov/geo/) and EBI ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) databases were searched for the most representative data set to include 10 or more microarray profiled ESC and iPSC lines and any iPSC differentiation assay linked to the profiled iPSC lines. One dataset, which met these requirements, was GSE25970 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25970) which consists of 20 human ESC lines, 12 human iPSC lines, six fibroblast cell lines, and five embryoid body (EB) cell lines, and includes in vitro differentiation data for each iPSC line. Next, we used these data to calculate PAS scores for 271 pathways, including more than 3500 genes, in iPSC and ESC lines. The raw PAS data, including p- and q-values, for all 271 pathways in 20 ESC and 12 iPSC lines derived from the GSE25970 dataset are presented  for ESC and iPSC lines shown on Figure NCBI Gene Expression Omnibus (GEO)  algorithm , 26, whiin vitro data. Our results demonstrate that pathway activation, as measured with Regeneration Intelligence, is a promising strategy for iPSC quality control.We analyzed existing stem cell transcriptome data (GSE25970) to compare iPSCs, ESCs, and fibroblasts at the pathway level. We found that iPSCs and ESCs show a similar pattern of pathway activity that is distinct from the pathway activity of fibroblast and EB cell lines. We defined a new quality score for iPSCs based on PAS, and demonstrated how it can be applied to identify iPSC abnormalities using in vitro differentiation data were combined into a valuable data set consisting of genome-wide reference maps of gene expression and DNA methylation in a collection of pluripotent cell lines, including 20 human ESC lines, 12 human iPSC lines, six fibroblast, and five EB cell lines, which can be used to analyze the transcriptional properties of these ESC and iPSC lines in comparison with founder lines. By using the genome-wide reference maps of gene expression and DNA methylation in ESC, iPSC, EB, and fibroblast of the GSE25970 data set combined with our algorithm, we were able to demonstrate close resemblance of iPSCs with ESCs at the PAS level in comparison with fibroblast and EB cell lines, which formed separate clusters in a PAS heat map. Furthermore, we built a new PAS-based quality score that was able to identify impaired iPSC lines. Several iPSC lines that we predicted as having aberrant pathway signaling also showed a poor in vitro neural differentiation pattern. However, it is worth pointing out that one iPSC line displaying a lower than average PAS quality score, had an increased differentiation capacity, indicating that above as well as below average differentiation capacity may be detected by PAS quality score evaluation. Thus, these results clearly demonstrated that our algorithm shows promise for assessing iPSC differentiation capacity in silico.Hu et al.  demonstrvs. fibroblast characteristics, and that the cAMP pathway was also among the top 20 least variable pathways in ESC and iPSC lines, thus, defining a key role of this pathway in stem cell differentiation. One of the major advantages of the signaling pathway analysis bioinformatics algorithms , are their unique ability to quantify perturbations for signaling pathways relative to control samples on a mathematical basis [LASSO model coefficients identified nine signaling pathways as strong predictors of iPSC versus fibroblast characteristics. Interestingly, whole genome analysis of gene expression during dopaminergic differentiation of human ESC and iPSC lines revealed that among the distinct pathways which are activated during neuronal differentiation, the cAMP signaling pathway was found to play a significant role in the differentiation of dopaminergic neurons. The role of cAMP signaling was confirmed by small molecule perturbation experiments , indicatal basis , 24. It al basis , 26 we whttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25970). Data set GSE25970 contains the information on 43 cell lines profiled using Affymetrix HT Human Genome U133A arrays: 20 human ESC lines, 12 human iPSC lines, six fibroblast cell lines, and five EB cell lines [The most representative data set available that included 10 or more microarray profiled ESC and iPSC lines and any iPSC differentiation assay linked to the profiled iPSC lines, was chosen from the NCBI GEO and ArrayExpress databases  for each of the 271 pathways analyzed, lgorithm , optimiza value of beyond tolerance interval flag, which equals 0 or 1; and ARR (activator/repressor role) serves as a discrete value which equals to the following numbers: −1, −0.5, 0, 0.5 or 1 depending on the topological role of the particular gene/protein in the signaling pathway p, respectively. Results for the 271 pathways PAS that were obtained for each sample of ES and iPS cell lines can be found in supplement materials (Here, CNRn is the ratio of the expression level of a gene n in the case sample and in the control; BTIFn is aterials .In order to obtain an iPSC line differentiation assessment we established a reference PAS value corridor using ESC line PAS as a gold standard. The ESC PAS quality score was set as average of PAS values across all ESC lines ± SD of the same PAS across all ESC lines for each pathway. For each of the 12 iPSC lines we calculated the number of signaling pathways with PAS values inside of the previously described ESC reference corridor. We used the PAS-based quality score as a metric to identify iPSC outliers that may be functionally impaired.


Cannabinoids, originally derived from Cannabis sativa, as well as their endogenous and synthetic counterparts, were shown to induce apoptosis of glioma cells in vitro and tumour regression in vivo via their specific receptors, cannabinoid receptors CB1 and/or CB2. CB2 are abnormally expressed in human gliomas and glioma cell lines. Most of the analysed gliomas expressed significant levels of CB2 receptor and the extent of CB2 expression in the tumour specimens was related to tumour malignancy. A synthetic cannabinoid, WIN 55,212-2, down-regulated the Akt and Erk signalling pathways in C6 glioma cells that resulted in reduction of phosphorylated Bad levels, mitochondrial depolarization and activation of caspase cascade leading to apoptosis. We examined whether synthetic cannabinoids with different receptor specificity: WIN55,212-2 (a non-selective CB1/CB2 agonist) and JWH133 (a CB2-selective agonist) affect survival of four human glioma cell lines and three primary human glioma cell lines. WIN-55,212-2 decreased cell viability in all examined cell lines and induced cell death. Susceptibility of the cells to JWH133 treatment correlated with the CB2 expression. Cannabinoids triggered a decrease of mitochondrial membrane potential, cleavage of caspase-9 and effector caspases. Induction of cell death by cannabinoid treatment led to the generation of a pro-apoptotic sphingolipid ceramide and disruption of signalling pathways crucial for regulation of proliferation and survival. Increased ceramide levels induced ER-stress and autophagy in drug-treated glioblastoma cells. We conclude that cannabinoids are efficient inhibitors of human glioma cells growth, once the cells express specific type of cannabinoid receptor.


Stephania delavayi Diels. against breast carcinoma cell lines MDA-MB-231 and MCF-7. FK-3000 suppressed CDC25B phosphorylation directly and indirectly via p38 MAPK phosphorylation. CDC25B dephosphorylation decreased levels of cyclin B and phospho-CDC-2, and ultimately induced cell cycle arrest at the G2/M phase. The p38 MAPK inhibitor, SB 239063 blocked FK-3000-induced p38 MAPK phosphorylation and nuclear accumulation, but did not completely rescue cell death. Conclusively FK-3000 exerts its antiproliferative effect through two pathways: i) G2/M cell cycle arrest via downregulation of cyclin B and phospho-CDC2 by p38 MAPK phosphorylation and CDC25B dephosphorylation, and ii) p38 MAPK-independent induction of apoptosis.We evaluated the cytostatic effect of 6,7-di-O-acetylsinococuline (FK-3000) isolated from  In 2014, the American Cancer Society reported that cancer was the second leading cause of death in the USA, estimating that >1,665,540 new cancer cases would be diagnosed that year and 585,720 cancer deaths would occur in the USA. To develop effective treatments, scientists have isolated anticancer agents from natural materials and identified a number of promising drug candidates , which was isolated from activity  and exhi000, whicMammalian cell division is controlled by cyclins and cyclin-dependent kinases (CDKs), which form various cyclin-CDK heterodimeric complexes (protein kinase holoenzymes) that regulate different phases of the cell cycle. Positive regulators of CDK function are upregulated in most cancer cells, whereas the expression of negative regulators are downregulated. Accordingly, cyclin D1, CDK4, cyclin E, cyclin A, and Wee1 are upregulated in the Long-Evans Cinnamon rat model of hepatocellular carcinoma ; CDK4 pl1/S cell cycle transition, and CDC25B and CDC25C control mitosis , and CDC2  of 6.5 μM against MCF-7 cells and 1.2 μM against MDA-MB-231 cells, and causes G1/S and G2/M phase arrest  arrests cells in the Grylation  and blocansition ,21.2/M phase arrest in human breast carcinoma cell lines MDA-MB231 and MCF-7.In the present study, we investigated the antiproliferative mechanisms of FK-3000 by examining its effect on cell cycle regulatory proteins. Our results show that FK-3000 decreased levels of phosphorylated CDC25B (phospho-CDC25B) but neither CDC25A nor CDC25C, and induced GS. delavayi Diels. was separated by chromatography on a Sephadex LH-20 column . Fraction 3 (700 mg) was further purified by C18 high performance liquid chromatography   or negative [e.g., INK4 family  p38 MAPK-independent induction of apoptosis (We demonstrated that FK-3000 exerts an antiproliferative effect through two pathways: i) Gpoptosis . Althoug