The protein profile of a 92-biomarker panel was analyzed on cells treated with 0 or 0.1 µM doxorubicin during 6 or 72 h, under normoxia or hypoxia. Hypoxia decreased viability of HepG2 and SNU499. HepG2 was least and SNU449 most tolerant to doxorubicin treatment. Cytotoxicity of doxorubicin increased over time in HepG2 and Huh7. The combination of doxorubicin + hypoxia affected the cells differently. Normalized protein expression was lower for HepG2 than Huh7 and SNU449. Hierarchical clustering separated HepG2 from Huh7 and SNU449. These three commonly used cell lines have critically different responses to chemotherapy and hypoxia, which was reflected in their different protein expression profile. These different responses suggest that tumors can respond differently to the combination of local chemotherapy and embolization.Hepatocellular carcinoma is often treated with a combination of doxorubicin and embolization, exposing it to high concentrations and hypoxia. Separation of the possible synergistic effect of this combination in vivo is difficult. Here, treatment with doxorubicin, under hypoxia or normoxia in different liver cancer cell lines, was evaluated. Liver cancer cells HepG2, Huh7, and SNU449 were exposed to doxorubicin, hypoxia, or doxorubicin + hypoxia with different duration. Treatment response was evaluated with cell viability, apoptosis, oxidative stress, and summarized with IC Intermediate hepatocellular carcinoma (HCC) is treated with image-guided transcatheter tumor therapy by locally infusing a chemotherapeutic agent in a drug delivery system, so called transarterial chemoembolization (TACE) ,2,3,4. TTumor hypoxia is a key hallmark of all solid tumors, including HCC. An inevitable consequence of chemoembolization therapy is the de novo formation of hypoxic regions within the tumor . HypoxicHuman cancer-derived cell lines are the most widely used in vitro models to study cancer mechanistically and to validate the efficacy of cancer treatments . One of In this study, the objective was to investigate the tolerance to DOX and/or hypoxic treatment of three commonly used liver cancer cell lines ,26,27. H2 was confirmed by measurement of HIF1α and PDK1 after CoCl2 treatment confirmed presence of hypoxia  of HepG2 cells to DOX decreased 1600-fold from 6 to 72 h of exposure in normoxic conditions . These cell lines were selected based on previously published data on their response to DOX and their morphological features.50 values for HepG2 and SNU449. Huh7 and HepG2 are epithelial cell lines, characterized by a high expression of E-cadherin and low expression of Vimentin [50 values of DOX from different studies vary vastly. For example, after 48 h DOX exposure on HepG2 cells IC50 values varied from 0.038 to 1.9 µM [50 values for DOX in human carcinoma cell lines decrease with exposure time [50 is reported for HepG2 cells when increasing exposure time from 6 to 48 h, and similar effects (30-fold decrease) are observed in a ovarian cancer cell line when increasing exposure time from 2 to 12 h. [50 decreased between 60- and 500-fold, depending on cell line and increase in exposure time. This suggests that our cell lines were highly responsive to DOX. Interestingly, the SNU449 cell line showed higher tolerance at 72 h than after 48 h, having possibly built up resistance to DOX treatment. Several mechanisms have been described to contribute to the acquired resistance to DOX [The observed tolerance to DOX was significantly different between the cell lines, where HepG2 cells exhibited the lowest and SNU449 cells the highest tolerance. For example, at 72 h exposure to DOX there was a 580-fold difference between ICVimentin . In contVimentin ,31. The Vimentin ,31. In sVimentin . HoweverVimentin ,34 up toVimentin ,35. Alsoo 1.9 µM ,37. Thise to DOX . Firstlye to DOX . Unfortue to DOX ,41. Thise to DOX . In our e to DOX , which ce to DOX . An in vThe difference in viability of cells after treatment with DOX is also reflected in their different expression of the apoptotic marker Annexin V. Under normoxia, Annexin-V increased in the Huh7 and SNU449 cells treated with 0.1 and 1 µM DOX for 24 h. This is in line with our viability data, which show a decrease of 3% in cell viability of Huh7 cells after 24 h of exposure to 0.1 µM and 29% after 24 h of exposure to 1 µM DOX in normoxic conditions. In contrast, the SNU449 cells had no increased levels of Annexin V, after treatment with DOX in hypoxic conditions. This is in line with our viability data, which show that SNU449 cells experience no decrease in cell viability at 0.1 µM and 1 µM DOX. Under hypoxia, Huh7 cells showed a significant increase of Annexin V, after 24 h exposure to 0.1 µM and 1 µM DOX. This partially corresponds to our viability data, which show no decrease in cell viability of Huh7 cells after 24 h of exposure to 0.1 µM, but a 15% after 24 h of exposure to 1 µM DOX in hypoxic conditions. Remarkably, HepG2 cells showed no change in the apoptotic marker Annexin V, suggesting other mechanism may be responsible for the decreased viability. Despite the widespread clinical use of DOX, its antiproliferative and death-inducing signal cascades are not yet fully understood. Studies have suggested that decreased viability seen after DOX treatment is a result of apoptosis, necrosis, cell cycle arrest, and senescence ,46,47, w2 resulted in varying responses of the three cell lines. HepG2 and SNU449 cell viability declined when exposed to hypoxia. Huh7 had unaffected or slightly increased cell viability in hypoxic conditions, suggesting that Huh7 has a higher tolerance to hypoxia. It is important to note that HIF1α activity varies between different cancer cell lines under the same level of hypoxia [2-induced hypoxia, but recovered to normal levels at 72 h. This suggests that these cells adapted to the hypoxic conditions over time. In contrast, Huh7 has a higher tolerance to hypoxia, but also lower baseline levels of stabilized HIF1α and PDK1 in normoxic conditions. In agreement to our findings, a study on six human breast cancer cell lines found high basal levels of VEGF A and low HIF-1α and HIF-2α induction was correlated with improved survival under hypoxia [2, and then activates transcription of both VEGF A and CAIX [Inducing hypoxia with CoCl hypoxia . The difand CAIX . In our and CAIX ,50 was dTolerance of cancer cell lines to chemotherapeutic agents can increase or decrease in hypoxic conditions. Increased tolerance to chemotherapeutics under hypoxic conditions are observed in human embryonal carcinoma testicular germ cell tumor cell lines , HCC cel2 does not create an actual oxygen deprivation, but mimics hypoxia by binding to prolyl hydroxylase containing domain proteins that activate HIF-1α, there is still an abundance of reactive oxygen species, which might have contributed to the DOX cytotoxicity [2. A low exposure of 0.1 µM DOX induced oxidative stress in all cell lines after 24 h, which is in line with previous reports [2-induced hypoxia was combined with DOX treatment. This suggests that oxidative stress did not contribute to DOX cytotoxicity in the SNU499 cell line. Elevation of intracellular mitochondrial ROS levels in tumors is known to activate PKD1 and NF-κB, leading to upregulation antiapoptotic proteins such as A20 and cIAPs [It has been hypothesized that cells that proliferate less in hypoxia will be more tolerant to chemotherapeutic agents, as the chemotherapeutic agents act on dividing cells . DOX hastoxicity . In linetoxicity , we foun reports . A highend cIAPs . Therefond cIAPs .2 [2. In our study, we found different levels of HIF1α and PDK1 in the three cell lines under the same oxygen conditions, which could explain differences in their adaptation to hypoxia and their sensitivity to DOX. In addition, there were differences in generation of ROS between the different cell lines under normoxia and hypoxia, before and after DOX treatment, which would further contribute to the cytotoxic effect of both hypoxia and DOX.DOX is known to upregulate and activate HIF-1α, and HIF-1α targeting strategies have been suggested to enhance the effect of DOX treatment . Possibl2 . It is t2 is a hypoxic mimetic that functions by stabilizing HIF-1α. It has been shown that the difference in oxygen levels during hypoxia could affect cell behavior, where induction of apoptosis was observed when oxygen levels decreased below 0.5%, but not at oxygen levels of 1–3% [Another possible explanation of the different responses between cell lines, both in historical data and our results, could be the difference in how hypoxia was created. Chemical hypoxia with CoCl of 1–3% . Interes of 1–3% , which iA final important finding in our study was the difference in oncogenic protein profile between cell lines. Previous studies have shown a high similarity in gene expression between Huh7 and HepG2 cell lines and HCC tumor tissue, based upon rank-based gene expression ,27. The 2, Sigma-Aldrich, Darmstadt, Germany) stock solutions were prepared by dissolving CoCl2 in sterile water (25 mM) and filtering through a syringe filter (0.22 µm).Doxorubicin hydrochloride (DOX HCl) was purchased from Toronto Research Chemicals, Canada. DOX stock solutions (100 mM) were prepared by dissolving the DOX in DMSO . RIPA buffer, resazurin sodium salt and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich . Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute medium (RPMI), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco. Cobalt (iii) chloride hexahydrate (CoClFBS) and 1% antibiotic–antimycotic solution . Cells were cultured at 37 °C with 5% CO2 under standard cell culturing conditions  before making the 0, 0.01–1000 µM DOX concentration range in CCMFBS + CoCl2  + DOX was added to each well. Plates were again incubated in a humidified incubator with 5% CO2 at 37°C for the desired exposure time , with and without chemically induced hypoxia  or hypoxic conditions  for 6, 24, and 72 h. HIF1α and PDK1 expression was measured using Hypoxic Response Human In-Cell ELISA Panel  according to manufacturer’s guidelines. Plates were scanned using an Odyssey imager . Expression values were obtained by subtracting background values from wells with negative controls for each condition and normalized to the corresponding Janus Green fluorescence values for each well.Cells were seeded into 113851) in a microplate format and by flow cytometry. DCFDA is fluorogenic dye that measures ROS activity within the cell. After diffusion in to the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into DCF, a highly fluorescent compound with maximum excitation and emission spectra of 495 nm and 529 nm respectively. Cells were seeded into 96-well plates with clear bottom and black sides at a density of 2.4 × 104 cells/well for the microplate assay, and into 24-well plates at 1.2 × 105 cells/well for flow cytometry and left to adhere overnight. On the next day, cells were stained with 25 µM DCFDA for 45 min at 37 °C, according to manufacturer’s guidelines. Following this step, cells were exposed to 0, 0.1 µM and 1 µM DOX in normoxic (CCMFBS) or hypoxic conditions . After 6 h of treatment, fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths using a Fluostar Omega plate reader and BD FACSCallibur. Results of the microplate assay are shown as fold change fluorescence. Results of the flow cytometry measurements are shown as percentage of mean DCF signal intensity of control conditions.Oxidative stress was measured using DCFDA—Cellular Reactive Oxygen Species (ROS) Detection Assay Kit  or hypoxic conditions  for 24 h, following which apoptosis was assessed by flow cytometry using eBioscience™ Annexin V Apoptosis Detection Kit FITC (88-8005-72) according to manufacturer’s guidelines. Briefly, cells were washed with 1× Binding Buffer and incubated with a 1/20 dilution of fluorochrome-conjugated Annexin V for 10 min, after which fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths using BD FACSCallibur. Results are shown as percentage of mean Annexin V signal intensity of normoxic untreated condition.Cells were seeded into 24-well plates at 1.2 × 106-well plates at 2 × 105 per well and left to adhere overnight. DOX stock solution was diluted to 0.1 µM in CCMFBS (+ CoCl2). Eight different conditions were tested per cell line: normoxia or chemically induced hypoxia with exposure to 0 or 0.1 µM DOX during 6 or 72 h, with three replicates per experimental condition. Medium and cell debris were removed and remaining cells washed with PBS. Cells were detached with 100 µL ice-cold RIPA containing protease inhibitors (Sigma Aldrich), and cells were scraped off and aspired :50% inhibitory concentration (IC50) of DOX. DOX IC50 values were determined per cell line, for each exposure time, for both normoxic and hypoxic conditions. Differences between DOX IC50 with and without hypoxia per cell line was tested statistically with a 2-way ANOVA and Tukey’s multiple comparisons test (significant when adjusted p < 0.05) in GraphPad Prism. The effect of hypoxia was assessed by taking the ratio of cell viability under hypoxic and normoxic conditions. The effect of hypoxia on cell viability was tested statistically with a one-sample t-test (difference from 1) in GraphPad Prism.The blank wells were empty in the plate while control wells contained cells in medium without any additional DOX treatment. Cell viability data vs. DOX concentrations were plotted in GraphPad Prism  and fitted with the “inhibitor vs. response—variable slope analysis” to determine the Protein expression data from all different treatments on the three cell lines C was obt2, Huh7, and SNU449) have critically different responses to chemotherapy and hypoxia. This was also reflected in their oncogenic protein profile and their response to hypoxia and oxidative stress. A synergic effect of hypoxia and treatment with DOX was only observed in HepG2 cells, while Huh7 and SNU449 might have developed escape mechanisms from both treatment and hypoxia. These escape mechanisms are of uttermost importance when studying chemotherapeutic agents. Adaptation of tumor cells to hypoxia is believed to be the main driver for selection of more invasive and therapy-resistant cancer phenotypes [In this study, we present our findings that three established and commonly used liver cancer cell lines (HepGenotypes . Our resenotypes .


Correction to: BMC Cancer (2018) 18:325https://doi.org/10.1186/s12885-018-4261-5Following publication of the original article , the autAdditional file 1:Table S1. Sequences of the qPCR primers used in this study. Table S2. Cell numbers per well used in this study for cell proliferation, cell cycle and immunofluorescent assays. Table S3. KPNA7 silencing efficiencies of the cell lines used in Fig. 1 24 h after siRNA treatment. (DOCX 17 kb)


The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use. Cyclin dependent kinase CDK7), along with cyclin H and the accessory protein MAT1, forms the CDK-activating kinase (CAK), which functions to activate CDKs by T-loop phosphorylation . CAK is , along wCell cycle deregulation is a common feature of cancer and a number of cell cycle CDK inhibitors have been approved as cancer therapeutics, most prominently CDK4/6 inhibitors, exemplified by palbociclib, for the treatment of advanced oestrogen receptor positive (ER+) breast cancer . By contThe emergence of resistance to cancer treatment remains a major problem in cancer therapy. Cancer cells can adapt and become resistant to drugs in a multitude of ways, including, but not limited to, mutations that disrupt drug-target engagement, activating mutations in drug targets and downstream pathways, changes in gene expression levels of targets, altered drug metabolism and alterations in cell death pathways . In addiWhilst CDK7 inhibitors (CDK7i) represent a promising therapeutic approach, development of resistance in the clinic is possible, therefore identifying potential resistance mechanisms preemptively is of great importance. We have developed models of acquired resistance to both ICEC0942 and THZ1 in the breast cancer cell line MCF7, identifying upregulation of ABC-transporters as mediators of resistance. These findings may be informative for future patient selection strategies and combination treatment regimes.50 became growth-arrested after 1–2 weeks, so could not be continually passaged. Cells treated with 800 nM ICEC0942 remained growth arrested until resistance developed after 4 months, eventually giving rise to the ICEC0942-resistant line (MCF7-942R).To investigate mechanisms of acquired resistance to the CDK7i ICEC0942 and THZ1, we generated MCF7-derived drug resistant cell lines through prolonged culturing in the presence of ICEC0942 or THZ1. THZ1 resistance was achieved by initially exposing the cells to 50 nM THZ1, approximately the concentration required for 50% growth inhibition. This was increased gradually up to a concentration of 250 nM, with passaging once a week, until the cells became stably THZ1-resistant after 3 months. A different strategy was used to achieve ICEC0942 resistance, as even cells exposed to drug concentrations well below ICR showed a 29-fold resistance to ICEC0942  showed a 13-fold resistance to THZ1 .THZ1 inhibits CDK7 by covalently targeting a cysteine residue (C312), which lies outside the ATP-binding domain , whereasR cells, ABCG2 expression was increased 140-fold, relative to MCF7 cells , ABCG2 (HEK293ABCG2) or empty vector (HEK293)  and ABCG2 (P < 0.0001) and THZ2 response  [R) [R    and a miR) [R) , 29. qRT [R Fig.  and flow [R Fig. . NCI-ADRµM) Fig. , whereasely Fig. .Fig. 6ChR was confirmed to have high expression and pump activity of ABCG2, by qRT-PCR  as frozen stocks. The NCI Adriamycin-resistant cell line (NCI-ADRR) was provided by Dr. Ernesto Yagüe, and OVCAR8 by Dr. Paula Cunnea, both from Imperial College London . The MCF7 mitoxantrone-resistant cell line (MCF7-MXR) was provided by Dr. E Schneider from the University of Maryland . HEK293 cells, stably transfected with either empty pcDNA 3.1 vector (HEK293), pcDNA3.1 containing ABCB1 (HEK293ABCB1) or ABCG2 (HEK293ABCG2) have been described [R, which were maintained in phenol-red free RPMI, supplemented with 10% FCS and 1 nM estradiol. Cell lines were cultured up to a maximum of 20 passages beyond initial thawing. Cell cultures were routinely tested for Mycoplasma infection by assay of culture supernatants, and found to be negative, using the MycoAlert Mycoplasma Detection Kit .MCF7 cells were obtained from the ATCC (escribed . All celR) was established by growing MCF7 cells in the continued presence of ICEC0942 at 800 nM for 4 months, splitting when cells neared confluence. The MCF7 THZ1-resistant cell line (MCF7-THZ1R) was established by initially growing MCF7 with THZ1 at 50 nM. Cells were split when they neared confluence and the THZ1 concentration then raised by 25 nM increments, up to a maximum of 250 nM, over a 3-month period. The independent MCF7 ICEC0942-resistant cell line (ICR-MCF7-942R) was established by growing MCF7 cells in the continued presence of ICEC0942 at 400 nM for 3 months, splitting when cells neared confluence. After drug resistance was established, the cell lines were maintained in culture with 800 nM ICEC0942 (MCF7-942R), 400 nM ICEC0942 (ICR-MCF7-942R), or 250 nM THZ1 (MCF7-THZ1R), with medium and drug changes every 3–4 days.The MCF7 ICEC0942-resistant cell line (MCF7-942ICEC0942 synthesis has been described  and was 50) were calculated from log dose-response curves in GraphPad prism v7.0. All dose-response curves are presented with error bars for standard errors of the mean (SEM) of six technical replicates. A minimum of three independent experiments were conducted and the average fold resistance of the drug resistant cell lines, MCF7-942R and MCF7-THZ1R, relative to the parental cell line, MCF7, was determined thus; fold resistance = GI50 resistant cell line/GI50 parental cell line [The sulphorhodamine B (SRB) assay was used to assess drug sensitivity, as previously described . Drug coell line . For ABCWhole cell lysates were prepared in RIPA buffer (Sigma-Aldrich), supplemented with protease and phosphatase inhibitors , and immunoblotting was carried out as previously described . AntibodR and NCI-ADRR. Cells were trypsinised and resuspended in phenol red-free DMEM at a concentration of 1 × 106 cells per ml. Twenty microliters of the EFFLUXX-ID Green Detection Reagent (in DMSO) was diluted in 1 ml DMEM (phenol red-free). Cells (2 × 106) were incubated with 50 µl of the diluted EFFLUXX-ID Green Detection Reagent and the fluorescence intensity of each sample was measured for 10,000 events every 5 min for 30 min, on a BD LSR II Flow Cytometer . The same procedure was used to assess the pump efflux activity of ABCG2 in, MCF7, MCF7-THZ1R and MCF7-MXR, except Hoechst 33342 [The EFLUXX-ID Green Multidrug Resistance Assay Kit  was used to assess the pump efflux activity of ABCB1 in MCF7, MCF7-942st 33342  was usedR cells was carried out by reverse transfection, using Lipofectamine RNAiMAX transfection reagent (Thermo Scientific), with ABCG2-specific siRNA  or non-targeting siRNA, (Dharmacon ON-TARGETplus Nontargeting Pool) in either six well plates , or 96 well plates , at a final concentration of 25 nM. ABCB1 knockdown in MCF7-942R cells was carried out in the same way but with ABCB1-specific siRNA (Dharmacon SMARTpool ON-TARGETplus) or non-targeting siRNA (Dharmacon ON-TARGETplus Nontargeting Pool). RNA preparation, qRT-PCR, protein preparation, immunoblotting and drug sensitivity assays were then carried out as described above. Primers used in sequencing and qRT-PCR are listed in Supplementary Table Knockdown of ABCG2 in MCF7-THZ1t-test. The Pearson correlation coefficient was used for gene expression analyses. To assess whether ABC-transport inhibitors significantly alter the GI50 of CDK7i, in CDK7 inhibitor-resistant cell lines, the extra sum-of-squares F test was used.Statistical analyses were carried out in Graphpad prism v7.0. Pairwise comparisons were performed using the Student Supplementary Methods.Supplementary Figure Legends.Supplementary Figures.


Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines  with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances. Histone deacetylases (HDACs) remove acetyl groups from histone or non-histone proteins and regulate gene expression as components of multiprotein repressor complexes ,2,3,4. IOur group studies HDAC function and the therapeutic potential of HDAC inhibitors in urothelial carcinoma (UC), the most common type of urinary bladder cancer. In accord with observations in many other cancers, we have previously observed that experimental downregulation or pharmacological inhibition of class I HDACs, in particular of HDAC1 and HDAC2 combined, strongly impairs proliferation and survival of UC cells . The staIn a previous investigation of HDAC4 expression in UC cell lines and published microarray data sets, we found variable levels of HDAC4 protein and mRNA, respectively, in UC cell lines and tissues . OverexpIn accord with the data from the large TCGA study, we observed that HDAC5 was consistently lower expressed in UC cell lines than in normal urothelial cells. Taken together, these findings suggest a possible tumor-suppressive function of HDAC5 in UC. We therefore characterized in detail the effects of stable expression of HDAC5 on four urothelial carcinoma cell lines with different phenotypes, which reflect the heterogeneity of the disease. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were further followed up by comprehensive proteomics and transcriptomics analyses. While HDAC5 has previously been identified as a factor in fibrosis and cancer progression, this is, to our knowledge, the first report demonstrating that HDAC5 on its own may induce EMT in certain circumstances. In accord with our previous report, overexpression of HDAC4, in comparison, had only weak effects on UC cells.To determine the endogenous protein levels of class II HDACs, HDAC4, HDAC5, and HDAC7, protein lysates from ten different UC cell lines (UCCs) and two non-cancerous controls, namely the benign urothelial cell line HBLAK and the primary urothelial culture UP245, were subjected to immunoblotting. Expression of HDAC4 and HDAC7 was variable among the cell lines. For instance, SW1710 and 5637 cells had stronger HDAC4 expression compared to the low levels in HT1376, UM-UC-3, 253J, J82, 639V and HBLAK. The expression of HDAC4 was nearly undetectable in RT112, VM-Cub-1, BFTC905, and UP245 cells. HDAC7 expression was also variable, being clearly stronger in 5637, UM-UC-3, 253J, as well as the controls HBLAK and UP245, compared to other UCCs. Notably, HDAC7 regularly appeared as a double band, which could correspond to several isoforms known to be generated by alternative splicing. In contrast to HDAC4 and HDAC7, HDAC5 levels in UCCs were uniformly low and often below the detection limit. The highest expression was observed in the normal UP245 culture A.Due to its low expression, we were primarily interested in the effects of HDAC5 overexpression in UCCs. Therefore, lentiviral-based vectors were used to introduce HDAC5 into four UCCs; paired control cell lines were generated using vector only. The parental four cell lines used, RT112, VM-Cub-1, SW1710 and UM-UC-3, differ in their phenotypes with decreasing epithelial and increasing mesenchymal morphology in that order. Stable overexpression of HDAC5 was achieved in all four cell lines B.We next measured cell proliferation by MTT assay at different time points over 96 h. Until 72 h (48 h in the case of UM-UC-3), HDAC5 and vector-only transduced cell lines grew quite similarly, but at 96 h proliferation of HDAC5-expressing RT112, SW1710 and UM-UC-3 cells was clearly diminished  in comparison to their respective vector-only cells . InteresThe decrease in proliferative ability over time conferred by HDAC5 was also reflected in clone formation assays. The ability to form clones following seeding at low density in tissue culture plates was strongly diminished in HDAC5-transduced RT112, SW1710 as well as UM-UC-3 cells, and to a lesser extent in VM-Cub-1, compared to their respective vector-only controls . Upon seAmong UCCs, almost exclusively, cell lines with a more mesenchymal morphology form colonies in soft agar. Accordingly, the morphology of HDAC5-transduced VM-Cub-1 cells changed towards a more mesenchymal morphology and the cells grew in a more dispersed pattern rather than as tight colonies a. We therefore investigated markers of epithelial-mesenchymal transition by immunoblotting. Indeed, in VM-Cub-1 HDAC5-transduced cells, the amounts of the epithelial markers Cytokeratin 5 and E-Cadherin were diminished compared to the control, and the expression of the mesenchymal marker Vimentin was increased to a similar level as in SW1710 and UM-UC-3 cells b. In theSince a more mesenchymal phenotype is often associated with increased migratory ability, we compared HDAC5-transduced to vector-only transduced UCCs in cell migration assays. A clear increase in migration was seen for HDAC5-expressing VM-Cub-1 cells over the entire duration of the experiment, whereas no significant difference in migration velocity was observed among vector-only and HDAC5-transduced SW1710 cells. RT112 and UM-UC-3 cells appeared to migrate slightly faster at earlier time points, but the differences were not statistically significant c. To characterize the overall changes in the proteome of the UCCs following HDAC5 overexpression, we performed high-throughput proteomics analysis by mass spectrometry. In accord with the morphological changes, by far the most profound alteration of the proteome was observed in VM-Cub1 cells where more than half of the quantified proteins (1747 proteins) were significantly differently abundant in HDAC5-transduced versus vector-only cells. In comparison, a moderate number of significant changes were observed in SW1710 (145 proteins), and UM-UC-3 cells (154 proteins), and comparatively few in RT112 (33 proteins) . HierarcFinally, we searched for pathways that might underlie the different reaction of VM-Cub-1 to HDAC5 overexpression compared to the other cell lines. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis identified metabolic processes, especially “amino sugar and nucleotide sugar metabolism”, as well as “proteasome” and “ribosome” as consistently enriched in VM-Cub-1 compared to the other three UC cell lines . The chap ≤ 0.05 and at least two-fold differences. As in the proteome analysis, more moderate numbers of genes were differentially expressed in SW1710 and UM-UC-3, namely 1,265 and 733 genes, respectively. Likewise, the lowest number of changes was observed in RT112, with 261 genes differentially expressed  to an aggressive, undifferentiated and mesenchymal cell line (UM-UC-3). The most interesting case was however the VM-Cub-1 cell line, which is considered as representative of the basal subtype and moderately differentiated. This cell line was not significantly impeded in its growth, but underwent a dramatic phenotypic change with obvious EMT and acquisition of the ability for anchorage-independent growth. The different response to HDAC5 between VM-Cub-1 cells and the other cell lines was not only visible morphologically, but was also reflected in the proteome and transcriptome of this cell line. Compared to the other cell lines, changes in VM-Cub-1 were more numerous and more pronounced. It is not clear why VM-Cub-1 reacts differently from the other UCCs to HDAC5. Evidently, SW1710 and particularly UM-UC-3 already possess mesenchymal morphology in the absence of HDAC5. RT112, on the other hand, is more strictly epithelial and grows in tighter clusters than VM-Cub-1. Indeed, we have observed in a previous study that VM-Cub-1 cells but not 5637, another strictly epithelial cell line, underwent EMT in response to overexpression of the lncRNA HOTAIR . InteresIt is indeed intriguing that expression of HDAC5 on its own is capable of inducing EMT in certain circumstances, and notably, appears to be more effective than even TGFβ, which can promote enhanced migration and invasion in some UCCs . While tObviously, overexpression of any protein in a cell line has the potential to cause unspecific effects irrelevant to its normal biological function. This is also a concern in our study. However, the comparison between the effects of HDAC4, which were modest at most, and those of HDAC5, which were prominent, indicates a high degree of specificity and suggests that the effects observed with HDAC5 overexpression reflect biologically relevant actions.In conclusion, according to global gene expression analyses, the class IIA HDAC4 and HDAC5 are both lower expressed in urothelial carcinoma than in normal bladder tissue. In UC cell lines, however, HDAC5, but not HDAC4 is consistently weakly expressed. Accordingly, lentiviral transduction of HDAC5, but not HDAC4 had significant effects on cellular properties. Supporting a tumor-suppressive function of HDAC5 in UC, its overexpression diminished growth in three out of four cell lines. However, induction of EMT in a fourth cell line hints at a potential of HDAC5 to promote a more aggressive phenotype in individual cases. Strikingly, we found HDAC5 overexpression on its own can be capable of inducing EMT. Our findings call for further analyses of the relation between HDAC5 on the one hand, and EMT and fibrosis, on the other hand, in cancer and other diseases.Finally, the overall aim of our project is defining an optimal HDAC target profile for HDAC inhibitors for the treatment of UC . In this2. As a benign urothelial control, we used the HBLAK cell line  VM-Cub1, RT112, SW1710, and UM-UC-3 were provided by Dr. M. A. Knowles , Dr. J. Fogh  and Dr. B. Grossmann  or by the DSMZ . They were cultured in DMEM GlutaMAX-I  supplemented with 10% fetal calf serum  at 37 °C in a humidified atmosphere of 5% COll line  and TNFα ) were used at 5 ng/mL and 20 ng/mL, respectively.The plasmid pcDNA3.1+HDAC5-FLAG was a kind gift from Eric Verdin (Addgene plasmid #13822) . BrieflyColony forming potency of vector and HDAC5 expressing UCCs was assessed by Giemsa staining. One thousand cells (in triplicates) were seeded in 6 well plates and cultured for 8 d or 10 d, for RT112, until macroscopically visible colonies had developed. Then, colonies were washed with PBS, fixed in methanol and stained with Giemsa .Soft agar assays were performed by a standard method  in DMEM Cell viability as an indicator of proliferation was determined by MTT -2,5-diphenyltetrazolium bromide, Sigma-Aldrich) assays as described . One tho4 cells/well) were seeded around Ibidi inserts in 50 µl medium on either side. The next day, inserts were detached to form a gap between the two sections. Cells were once gently washed with media and left to migrate for up to 24 h. Images were taken at the indicated time points and cell migration was measured as the distance between opposite cell fronts and plotted. Measurements were performed in triplicates. Vector-only and HDAC5-transduced UCCs  and MACSQuantify software as previously described .Immunoblot analysis of whole cell extracts was performed as described . Cells wCells were harvested with Qiazol  and lysates were stored at −80 °C. Total RNA was then isolated by the Qiagen RNeasy Mini Kit . RNA quality was checked by spectrophotometry. w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate, 30 mM Tris-HCl, pH 8.0) and prepared for mass spectrometric analysis, as described elsewhere [To study the effect of HDAC5 overexpression on selected cell lines, quadruplicates from individual culture dishes were prepared from RT112, VM-Cub-1, SW1710, and UM-UC-3 cells expressing HDAC5 as well as corresponding and HBLAK vector-only cells. Cells were harvested and protein lysates prepared in an aqueous urea containing buffer  was used with standard parameters. Spectra were matched against sequence data from the Homo sapiens proteome . Further search parameters and parameters for peptide and protein acceptance and quantification were essentially as described previously .Further analysis of quantitative data was carried out with Perseus  within the R environment . Here, only proteins showing at least two different peptides were considered and proteins showing at least three valid quantitative values in at least one group in the respective comparison. For the identification of HDAC5 affected proteins and differences between the cell lines, a two way ANOVA followed by a Benjamini Hochberg correction and by Tukey’s honest significance tests was carried out on log2 label-free quantification intensities after missing values were filled in with random values from a normal distribution . Additionally, Student’s t-test were calculated for pairwise comparisons of HDAC5-transduced and vector-only cells and cutoffs were determined by the significance analysis of microarrays method . Differences of the mean values of log2 label-free quantification intensities were further used for categorical annotation enrichment analyses.Total RNA samples used for transcriptome analyses were quantified  and quality measured by capillary electrophoresis using the Fragment Analyzer and the Total RNA Standard Sensitivity Assay . All samples in this study showed high quality RNA Quality Numbers . The library preparation was performed according to the manufacturer’s protocol using the ‘TruSeq Stranded mRNA Library Prep Kit’ from Illumina®. Briefly, 250 ng total RNA were used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation and library amplification. Bead purified libraries were normalized and finally sequenced on the HiSeq 3000/4000 system  with a read setup of 1x150 bp. The bcl2fastq tool was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing. Data analyses on fastq files were conducted with CLC Genomics Workbench . The reads of all probes were adapter trimmed (Illumina TruSeq) and quality trimmed . Mapping was done against the Homo sapiens (hg38)  genome sequence. After grouping of samples  according to their respective experimental condition, multi-group comparisons were made and statistically determined using the Empirical Analysis of DGE . The Resulting P values were corrected for multiple testing by FDR and Bonferroni-correction. A P value of ≤0.05 was considered significant.RNA-Seq data were further analyzed with the Ingenuity Pathway analysis software  using the expression data with p(FDR) ≤ 0.05 and fold-change ≥ 1.7. The number of gene hits with this filter were 606 (RT112), 8000 (VM-Cub-1), 2419 (SW1710), and 1527 (UM-UC-3) in HDAC5-expressing cells compared to their vectors.p < 0.05 was considered as statistically significant.Data are represented as the mean with standard deviation (SD). Statistically significant differences between two groups were analyzed using the unpaired Student’s t-test with GraphPad. A minimum p value of


Cell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws.To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification.Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method.OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping.The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs. Using immunosurgery (2), mouse ESCs (mESCs) were first derived in 1981 . Later, other groups made unsuccessful attempts to generate human ESCs (hESCs) (5), followed by generation of ESCs in primates , and eventually, in 1998, hESCs were derived (8). This coincided with the advent of their putative counterpart, human embryonic germ cells (hEGCs) (9). Typical hESC morphology is an initial characteristic that helps in the derivation process. The morphology of hESCs is very similar to pluripotent embryonal carcinoma cells (ECCs), with a high ratio of nuclei to cytoplasm, pale nuclei, and prominent nucleoli (10). Four major factors play a critical role in the hESC derivation process. (a) The source and stage of pre-implantation embryos from cleavage (11) to blastocyst (8). Recently, it was shown that embryos obtained following in vitro embryoid body (EB) formation or by in vivo teratocarcinoma formation, to investigate further differentiation potential (21). The chromosome content of the cell line is another issue that can be evaluated by G-binding or the CGH-array method (22). One of the challenges in banking any cell type is the method of freezing used. The use of an ideal method for cryopreservation can improve the survival rate and proliferative capacity of post-thawed hESCs (23). Studies have demonstrated that fewer than 5% of hESCs survived an equilibrium slow-freezing procedure using 10% dimethylsulphoxide (DMSO) in fetal calf serum; in contrast, high viability among hESCs was reported when using a vitrification procedure for the cell lines using an “open pulled-straw” method with a small volume of cells (13). Vitrification is a state-of-the-art method used for the freezing of a small number of cells, including gametes and embryos, and is used for the cryopreservation of hESCs using an open pulled-straw method (13). Vitrification is also a good choice of method to use shortly after the derivation of hESCs that are in urgent need of cell line preservation (23).Following their initial derivation, hESCs must be cryopreserved and expanded for further characterization of specific gene and marker expression to assess their undifferentiated status (13). In addition, their capacity to differentiate into the three germ layers  and germ cells, to demonstrate their pluripotency, should be evaluated, either by Here, we report the vitrification of new outgrowths to save newly derived hESC lines (Yazd1-3) using Cryotech and Cryowin tools. Whole, zona-free blastocysts were cultured on an MEF feeder layer in microdrop culture. The purpose of this study was first to derive and characterize new hESC lines and then using Cryotech and Cryowin tools for their vitrification .Chemicals were purchased from Sigma Aldrich . Culture media and supplements were purchased from Invitrogen and Gibco (UK), unless otherwise stated.The vitrified donated embryos (n = 10) were warmed as described elsewhere (24) and cultured in a microdrop system with G series medium  plus 5% human serum albumin (Vitrolife) till getting to the blastocyst stage. The fresh donated embryos were cultured in the same culture medium for in vitro blastocyst development.et al., unpublished data). MEFs and YhFFs#8 were mitotically inactivated using Mitomycin-C (by adding 2 mg Mitomycin-C to 200 ml DMEM/10% FBS) for 2 and 4 hrs, respectively. After washing three times with Dulbecco's phosphate buffered saline (D-PBS), cells were detached using 1 x trypsin/EDTA, centrifuged at 1000 rpm for 5 minutes, and then re-plated in 50 µl drops onto dishes  at a concentration of 3,500 cells per 50 µl and covered with mineral oil . The following day the microdrop medium of feeders was replaced with KOSR/HES medium  and left for 2 hrs for conditioning prior to use. Each dish was plated with seven microdrops of feeder layer.MEFs were derived from Naval Medical Research Institute (NMRI) mouse embryos according to ethical guidelines relating to animals and cultured as described elsewhere (25). Briefly, 13 days after the appearance of the vaginal plug, fetuses were recovered from the uterus and their heads, spinal cords, and livers were removed. Following enzymatic and mechanical treatment, the resulting cell suspension was transferred to a T25 tissue culture flask containing Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), and antibiotics, then incubated at 37°C in 5% COTo obtain intact zona-free blastocysts, and based on the grade of each individual blastocyst, embryos were either cultured to fully hatched blastocyst stage or were treated with pronase  to lyse the zona pellucida. The intact zona-free blastocysts were plated onto the microdrops of MEF feeders. Yazd1 cells were initially generated using VitroHES culture medium (ready-to-use from Vitrolife), Yazd2 cells were derived and expanded in KOSR/HES medium, while for Yazd3 cells were, the blastocysts were initially cultured in DMEM/10% FBS overnight, and the following day the medium was exchanged for KOSR/HES medium. Small modifications to the culture conditions for the derivation of each individual Yazd hESC line (Yazd1-3) are shown in Table I.All three cell lines were derived using intact embryo co-culture on a microdrop of MEF feeders. All lines were later expanded on both MEFs  and Yazd HFFs (YhFF#8). Yazd1 cells were generated from a fresh embryo, which was cultured to the blastocyst stage (grade 3CB) in VitroHES medium. Yazd2 cells were derived from a frozen-warmed embryo, which was allowed to develop to the early cavity stage (grade 1) on KOSR/HES medium. Yazd3 cells were generated from an embryo which lost its zona during the freezing and thawing process and was allowed to develop to the blastocyst stage (grade 2). All cell lines were expanded using KOSR/HES medium. Yazd1 and Yazd2 hESC lines contained normal karyotypes ; however, the Yazd3 karyotype was initially a triploid 69, XXY.To establish the Yazd1-3 hESC lines following initial hESC outgrowth formation, the cells were mechanically passaged using a mouth pipette and pulled Pasteur pipettes. To detach the colonies, they were first treated with collagenase type IV (1 mg/ml) at 37°C in 5% CO-3 hESC lines, according to the scale of the culture. Colonies within the microdrops for master cell-banking were cryopreserved using Cryotech and Cryowin tools  for the vitrification method (13). For larger scale cultures, such as central well dishes or tissue culture flasks , at later passages colonies were frozen using a slow-freezing solution consisting of 10% DMSO in FBS. In summary, for vitrification, small pieces  of hESC colonies were scraped off and placed into a small drop of KOSR/HES medium. Then, colonies were transferred into homemade vitrification solutions 1 and 2 (VS1 and VS2) containing ethylene glycol and DMSO, first at 10%, then at 20%, respectively, in basal medium . Small colonies were drawn onto the Cryotech and Cryowin tools, plunged into liquid nitrogen, and then transferred to long cryovials, which were sealed with a cap located in the cryocane and placed in a liquid nitrogen storage container. For thawing, a Cryotech or Cryowin was taken from the liquid nitrogen container tank and then placed into a drop of KOSR/HES medium. The cells were washed five times by transferring them through more drops before being placed onto microdrops containing feeders in KOSR/HES medium and incubated at 37°C at 5% COTwo different methods were applied for freezing the Yazd1The identification of pluripotency-associated cell markers was carried out using IF localization, as previously described (26). In summary, Yazd1-3 hESC colonies were washed with PBS containing 1% FBS before being fixed in cooled methanol  and differentiated colonies in the monolayer culture system (after 7 days) were collected. The resulting pellets were suspended in 1 mL TRI reagent (Sigma) and total RNA was extracted according to the standard protocol supplied by the manufacturer. Total RNA was treated with DNase I  to remove genomic DNA. First-strand cDNA synthesis was performed using the Superscript II reverse transcriptase system . PCR was performed using the prepared cDNA and primers for different genes (Table III) by Platinum Blue PCR Super Mix  in triplicate. PCR cycling conditions were as follows: 5min at 95°C for initial denaturation, followed by 40 cycles of 45 sec at 95°C, 45 sec at 59°C-65°C (see Table III), and 45 sec at 72°C.To investigate the chromosomal content of Yazd1-3 hESC lines, the karyotype of ten cells in metaphase of each Yazd cell line was determined by a standard G-banding procedure. Briefly, cells were cultured in flasks without feeder for 5 to 6 days to form mesenchymal-like cells (27). Following treatment with colchicine (10 µg/ml), harvested Yazd hESC-derived cells were stained using a standard G-banding technique. G-bandings were analyzed under light microscopy  using applied spectral imaging software.In vitro differentiation of Yazd1-3 hESC lines was performed as previously described (13), using EB formation and gene expression profile assessment after 4, 7, and 14 days for mesoderm, endoderm, ectoderm, and germ-cell development. The same was done for spontaneously differentiation in the monolayer culture with DMEM/10% FBS (after 7 days).Sixteen fresh (n = 6) and frozen (n = 10) supernumerary embryos were donated for research by infertile couples after they had provided full, written, informed consent according to the Shahid Sadoughi University of Medical Sciences Ethical Committee (ethics committee reference number: IR.SSU.REC.1394.104). For research purposes, all embryos were coded to ensure patient anonymity.in vitro, the zona pellucida was lysed as described above, and plated onto a microdrop of MEFs  in KOSR/HES medium. The first outgrowths after two and three days were as shown in Figure 3B and C, respectively. As indicated in Figure 3C, a population of cells were overgrown, resembling putative hEGC clusters (28), which were distinguished from these areas after several passages of hESC colonies . To derive Yazd3 cells, a frozen embryo donated for stem cell research was thawed. During thawing the zona pellucida was destroyed and the zona-free embryo was allowed to develop to blastocyst stage (grade 2) in vitro, plated onto a microdrop of MEFs , and then incubated overnight in DMEM/10% FBS. On the following day attachment of the embryo onto the feeder layer and the medium was replaced with KOSR/HES. The first outgrowths after two and five days were as shown in Figure 4B and C, respectively. Following passaging, as previously described (13) and explained above, for Yazd2 cells a compact area  was distinguished from other parts of the colony from which, in later passages, typical hESC colonies were generated . Summary information of modifications to the techniques used for derivation of Yazd1-3 hESC lines is shown in Table I.In the current study, sixteen fresh (n = 6) and frozen (n = 10) donated surplus human embryos were used to generate Yazd1-3 hESC lines using a microdrop culture system (13)  and MEFs as feeder layer cells. The pre-implantation human embryos  were used as described above following pronase treatment (for Yazd1 and Yazd2) in a 50 μl microdrop co-culture system . The Yazd1 cell line was produced from a fresh 3CB full blastocyst donated for stem cell research , which was plated onto a microdrop of MEFs . After two days, the outgrowth of cells was observed . Following eight passages, colonies with a typical hESC morphology were obtained . The Yazd1 cell line was derived and initially expanded using VitroHES (Vitrolife) culture medium. For the generation of the Yazd2 cell line, a frozen-warmed embryo, which had been donated for stem cell research was developed to the early blastocyst stage (grade 1) in vitro (13).All Yazd cell lines (1-3) were initially derived and expanded on MEFs . Later, YhFFs#8  and Matrigel  were applied to maintain undifferentiated Yazd hESCs in culture. Half of the KOSR/HES medium was replaced with fresh medium each day. Non-adherent petri-dishes with EB medium were used to produce EBs  to induce spontaneous differentiation NANOG, SOX2, and OCT4/POU5F1  determined by RT-PCR and contained normal 46, XX karyotypes . Yazd2 hESCs (passage number 8+38+10) displayed expression of pluripotency-associated markers such as SSEA4 , TRA-1-60 , and TRA-2-49  and genes such as NANOG, SOX2, and OCT4/POU5F1 , as assessed by IF and RT-PCR, respectively. The initial karyotype of Yazd2 hESCs was normal, 46, XY, as revealed by a G-binding procedure . The undifferentiated Yazd3 hESC colonies (passage number 68) were characterized by SSEA4 , TRA-1-60 , and TRA-2-49  using IF, and NANOG, SOX2, and OCT4/POU5F1 using RT-PCR . Yazd3 was an abnormal hESC line with a karyotype of 69, XXY in its early passages .Yazd1 hESCs (passage number 7+5+10) showed positive expression of pluripotency associated markers, SSEA4 , TRA-1-60 , and TRA-2-49 , as assessed using an IF technique. Undifferentiated Yazd1 cells expressed the pluripotency genes in vitro using different EB formations  and monolayer cultures , with assessment of the expression of specific genes for the three germ layers and germ cells. Differentiated cells expressed specific genes of the three embryonic germ layers  and germ cells (VASA and DAZL).The pluripotent differentiation capacity of the first three Yazd hESC lines (Yazd1-3) was proven This study reports the derivation of Yazd1-3 hESC lines, initially using an MEF feeder layer in a microdrop culture system, and the banking of these cell lines using Cryotech and Cryowin tools for vitrification, which has started since 2008. Later Yazd hESCs were expanded on YhFF#8 while establishment and characterization.NANOG, SOX2, and OCT4/POU5F1. Furthermore, the differentiation potential of the cell lines in EB formation and monolayer culture was determined using RT-PCR for the three embryonic germ layers and germ-cell-specific genes.Following the generation of Shef lines using the microdrop culture system, we have applied this method for the derivation of new Yazd hESC lines (13). In recent studies, the efficiency of hESC derivation was reported to be more than 50% (29). In this study, however, three new hESC lines (Yazd1-3) were generated from sixteen fresh and frozen embryos, representing a success rate of 18.75%. This difference could be due either to the source of the embryos or the culture conditions, which in our case involved conventional KOSR/HES medium with MEFs as a feeder layer. The success rate for hESC generation from fresh embryos in this study was about 16.66%, whereas it was 20% for hESC lines derived from frozen embryos. As far as we know, this is the first report to describe the vitrification of hESCs using Cryotech and Cryowin tools; however, no comparisons were made between the efficiency of this method with the conventional open pulled straws method. This new method is applicable for the preservation of early outgrowths in early passages of pluripotent stem cells, and in our experience: hESCs. The new Yazd1-3 hESC lines were characterized by specific pluripotency markers, such as SSEA-4, TRA-1-60, and TRA-2-49, and pluripotency genes, including The use of a microdrop culture system was first reported in 1994 for the culturing of bovine ESC (30). In an open culture system, secreted growth factors may be lost or diluted, whereas it has been demonstrated that a combination of a co-culture and a microdrop system had the highest rates of blastocyst formation and hatching in comparison with an open co-culture system (31). Wakayama and colleagues (32) derived mESC lines using an efficient micro-scale method. Single blastomeres were co-cultured with MEFs in individual wells of 96-well plates. Following that, Aflatoonian and co-workers generated hESC lines using a microdrop culture system (13).In assisted reproductive techniques (ART), a laser is commonly used to assist with different processes, such as assisted hatching (33) and blastomere and trophectoderm (TE) biopsies (34). In addition, laser-assisted technology has been used for ICM biopsies in mice  and humans . In the present study, no ICM isolation methods, such as immunosurgery and laser-assisted biopsy, were used. The Yazd1-2 hESC lines were derived from blastocyst and early cavity embryos respectively, which were extruded from the zona pellucida by pronase treatment. It should be noted; however, that pronase is derived from bacteria and is therefore not suitable for the derivation of clinical grade hESCs in the future (14). The Yazd3 hESC line was derived from a zona-free embryo following the thawing process. While the ICM is known to be the usual source for hESC derivation, the development of hESCs from single blastomeres of cleavage- and morula-stage embryos  has also been reported, but these methods have not been used routinely.To avoid animal contamination following the co-culture of hESCs with MEFs, xeno-free derivation of hESC lines can be achieved by using human feeders including HFFs (18), HFGFs (13), human endometrial-derived fibroblasts (19), hESC-derived feeder (EDF), and human placental fibroblast feeders (43). In this regard, hESCs derived fibroblast-like cells conditioned medium demonstrated to support undifferentiated hESC proliferation under feeder-free conditions (44).Herein, MEFs were used for the initial derivation of Yazd1-3 hESC lines, followed by expansion of the cell lines on MEFs and YhFFs#8. Mesenchymal stem/stromal cells have been derived from Yazd hESC lines (27), although hESC-derived MSCs have yet to be used as feeders.The Yazd cell lines, which were characterized as described above and have been used during workshops (45), are available for other research groups and companies to use, according to Yazd Reproductive Sciences Institute rules and laws, as the owner of the cell lines.New hESC lines (Yazd1-3) were derived on microdrops of an MEF feeder layer, without immunosurgery, using whole blastocyst culture. Following expansion of the cell lines, YhFFs#8 were used as a xeno-free condition with KOSR/HES. This study is the first report of Cryotech and Cryowin tools being used for the vitrification of hESCs in the very early stages of derivation. Further studies are in progress to evaluate the efficiency of this method compared with other techniques and tools.The authors declare no conflicts of interest.


Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumour of the skin with growing incidence. To better understand the biology of this malignant disease, immortalized cell lines are used in research for in vitro experiments. However, a comprehensive quantitative proteome analysis of these cell lines has not been performed so far.Stable isotope labelling by amino acids in cell culture (SILAC) was applied to six MCC cell lines . Following tryptic digest of labelled proteins, peptides were analysed by mass spectrometry. Proteome patterns of MCC cell lines were compared to the proteome profile of an immortalized keratinocyte cell line (HaCaT).In total, 142 proteins were upregulated and 43 proteins were downregulated. Altered proteins included mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, several proteins of the histone family and their variants were highly abundant in MCC cell lines.The results of this study present a new protein map of MCC and provide deeper insights in the biology of MCC. Data are available via ProteomeXchange with identifier PXD008181. Merkel cell carcinoma (MCC) is a rare malignant tumour of the skin with neuroendocrine differentiation , 2 and gA genomic profiling study of patients with MCC revealed that the most frequent abnormalities are related to the TP53 gene and the cell cycle pathway. Further abnormalities were found in the PI3K/AKT/mTOR pathway and DNA repair genes . However2 in a humidified incubator. For SILAC labelling cells were grown in SILAC Media supplemented either with 13C6l-Lysine-2HCl (heavy) and l-Arginine-HCl (light) or with l-Lysine-2HCl (light) and l-Arginine-HCl (light) . Cells were cultured for at least ten cell doublings. Three biological replicates were cultured per each cell line, and these were merged into one sample upon cell lysis for further treatment. The labelling efficiency was estimated using the method described by Rappsilber et al. [The human Merkel cell carcinoma cell lines BroLi, MKL-1, MKL-2, PeTa, WaGa, and MCC13 were a kind gift of Prof. Houben . HaCaT, ®-Total Protein Extraction Kit” containing TM buffer  according to manufacturer’s manual. Protein precipitation was performed using modified Wessel–Fluegge method as described elsewhere [MCC cells were lysed using the “Chemiconlsewhere –17. Lyse® PepMap100 . Both columns were operated at 60 °C in the column compartment as described earlier [Methanol, was purchased from Merck , 98% formic acid, acetonitrile, trifluoroacetic acid were purchased from Sigma-Aldrich . HPLC grade water was prepared using an in-house Milli-Q plus device from Millipore , and trifluoroethanol was purchased from Alfa Aesar . Reverse phase separation of tryptic peptides was conducted on a nanoRSLC UltiMate3000  HPLC system consisting of an autosampler, thermal compartment and pumping module. PepMap C18 trap-column  was used for sample loading and desalting. The analytical column used for peptide separation was a 75 µm ID × 50 cm length Acclaim earlier . A totalAll raw data were converted into Mascot “mgf” files by using Bruker’s Data Analysis and these files were searched against the Swissprot database (version of November 2016) of human proteins using ProteinScape V 3.1.5 474  and Mascot V2.6 . Protein quantitation was performed using WARP-LC V1.3.136 .All samples were analysed as technical triplicates to ensure statistical sound data and avoid artefacts due to variations in ionization efficiency.Detailed information on separation gradient, the MS settings, and the data search and quantitation can be found in supplemental information . Proteins in MCC cell lines with H/L ratios of > 1.5 or < 0.5 where considered as significantly differently abundant. To put differential protein abundance into biologic context, Cytoscape  in combination with ClueGO/CluePedia (a Cytoscape plug-in) was used with default parameters except for following: Database Gene Ontology Biological Process, levels between 4 and 13 and GO Fusion set on true . p-valueThe SILAC method was used in six MCC cell lines to determine quantitative changes of proteins at the proteome level. Proteins detected in MCC cells were compared to the reference cell line HaCaT Fig. . We chosIn order to visualize the similarity and the difference between the particular cell lines, a heat map was created showing all quantified proteins. A list of proteins (overlapping and specific) for each sample is provided as Additional file Next, the protein profile of MCC cell lines was compared with the reference cell line HaCaT. Proteins present specifically in the MCC cells were determined. Then a Venn diagram was constructed. Since BroLi was the cell line with the least number of proteins and the difference between HaCaT and BroLi was small, the cell line BroLi has been omitted. Figure It is of crucial importance and of highest interest to identify and quantify biological processes involved in the biology of cancer. Term Enrichment Analysis using ClueGO showed that multiple pathways where affected by differentially represented proteins Fig. . CellulaIn total, 317 dysregulated (i.e. up- and downregulated) proteins with significance threshold of p < 0.05 were identified. Proteins altered > 1.5-fold were considered as upregulated and proteins altered < 0.5-fold were considered as downregulated. Based on these criteria, 142 proteins were identified as upregulated and 43 proteins were downregulated. The differently abundant proteins and their ratios are shown in Tables Bioinformatic analysis revealed that different cell lines have individual protein profiles. None of the dysregulated proteins was present in all tested cell lines at the same time. However, a high occurrence of histone variants was detected in all cell lines except in BroLi. In more detail, only three upregulated and two downregulated proteins were identified in the BroLi cell line. BroLi cell line is a very slowly growing cell line with a doubling time of 5 days  and thisFor the BroLi cell line, mitoferrin-1 was found to be 9.64-fold upregulated compared to HaCaT cell line. Mitoferrin-1 is a protein involved in the mitochondrial iron transport and storage . As ironIn MKL-2 cells, protein-arginine deiminase type-6 was upregulated 9.47-fold compared to the control cell line making it the most differently regulated protein for this cell line. This protein is an enzyme involved in post-translational modifications, which can have substantial effects on the structure and function of proteins. Citrullination is one such post-translational modification being catalysed by the family of protein arginine deiminase (PADs) enzymes. Five isoenzymes (PAD1-4 and 6) are known and they were identified in different types of tissue . An overFurthermore, we identified the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) to be the most upregulated protein in the PeTa cell line. The hnRNPs are a group of proteins binding to RNA and playing a role in mRNA processing . So far,In the WaGa cell line, SLX4IP (SLX4 interacting protein) was the protein showing the highest upregulation. SLX4 is a DNA repair protein and it coordinates structure-specific endonucleases  but its Finally, clathrin light chain B was the protein with the highest upregulation in the MCC13 cell line. Clathrin light chain B is a part of the clathrin protein, which is the main component of vesicles involved in intracellular transport. Recently, it was reported that clathrin light chains promote cell migration and therefore may play a role in cancer metastasis .In the PeTa cell line various histones and their variants were found to be dysregulated compared to the control cell line HaCaT. Histones are substantial components for the packaging of the DNA in the chromosomes. The smallest packaging units are nucleosomes consisting of DNA wrapped around a histone octamer. A histone octamer in turn consists of two copies of each of the core histones: H2A, H2B, H3, and H4, being the smallest units. The linker histone H1 holds the nucleosome together and is the fifth member of the histone protein family . Beside In the current study, proteins from all five major histone families with 15 different subfamily members were differently abundant in MCC cell lines compared to control samples. In particular, H2A1H and H2B1O were found to be overexpressed in MKL-1 and H2B1H and H2B2E were upregulated in MKL-2. Most of the highly abundant histone variants were found in the cell line PeTa: H2A1B, H2AJ, H1.4, H3.3, H1.5, H2B1N, and H2B2E. Furthermore, H2B1O and H2B1D were identified to be upregulated in WaGa, and H2B1C, H2B1K, H2B2F and H2AJ in MCC13. The role of histone variants in the development of carcinomas has been discussed and described in a number of publications –46. As dAnother interesting group of proteins that were identified as dyregulated in several MCC cell lines were the heat shock proteins (HSPs). It is a group of proteins that inhibit the unfolding or denaturation of cellular proteins and therefore being known as molecular chaperones whose expression is induced by stress. The major groups are classified according to their sizes and imply HSP10, HSP27, HSP40, HSP60, HSP70, and HSP90. Recent studies have shown that HSPs are highly expressed in many malignant tumours and due to their important role in cell proliferation and differentiation they are involved in carcinogenesis and metastasis , 49. In Beside HSP70, we found HSP60 and co-chaperone HSP10 to be overexpressed in the tested MCC cell lines. Actively produced by cancer cells, HSP60 exhibits a protective effect against cell stressors like chemotherapeutics. In particular, HSP60 stabilizes the anti-apoptotic protein survivin, a protein over-expressed in most human tumours, and therefore it inhibits apoptosis. Furthermore, HSP60 builds a complex with p53, which leads to the loss of the pro-apoptotic function of p53 and this process again results in inhibition of apoptosis .In conclusion, this work provides an additional insight in the biology of Merkel cell carcinoma. Multiple dysregulated proteins from various pathways were identified. The most abundant proteins were mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, the family of histone variants was frequently upregulated. In overall, each Merkel cell carcinoma cell line has its own distinct proteomic profile. This may be due to the biological heterogeneity of MCC. In this study we could demonstrate for the first time the similarities and differences between commonly used MCC cell lines.Current analysis can be significantly improved by: (a) using multidimensional separation approach for fractionation of tryptic peptides and (b) using a more sensitive mass spectrometer. We are aware of this facts and new analysis of these samples are currently being processed. However, taking into consideration that these data are the very first describing differences of putative Merkel cells we are confident that they can provide valuable help for researchers addressing this condition.Additional file 1: Additional methods information.Additional file 2: Table S1. Shows overlapping and specific proteins for each cell line.Additional file 3. Table S2. Lists all proteins that are shown in the Venn diagram (Fig.


Pancreatic cancer (PC) is the fourth most common cause of cancer death. Combination therapies with classical chemotherapeutic agents improved treatment of advanced PC at the cost of a relevant toxicity, but the 5-year survival rate remains below 5%. Consequently, new therapeutic options for this disease are urgently needed. In this study, we explored the effect of two repurposed drug candidates nelfinavir and nitroxoline, approved for non-anticancer human use, in PC cell lines. Nelfinavir and nitroxoline were tested as single agents, or in combinations with or without erlotinib, a targeted drug approved for PC treatment.The effects of the drugs on the viability of AsPC-1, Capan-2 and BxPC-3 PC cell lines were assessed by MTT. The impact of the treatments on cell cycle distribution and apoptosis was analyzed by flow cytometry. The effects of treatments on proteins relevant in cell cycle regulation and apoptosis were evaluated by western blot. Self-renewal capacity of PC cell lines after drug treatments was assessed using a clonogenic assay.When used as single agents, nelfinavir and nitroxoline decreased viability, affected cell cycle and reduced the expression of relevant cell cycle proteins. The effects on apoptosis were variable among PC cell lines. Moreover, these agents drastically impaired clonogenic activity of the three PC cell lines. Combinations of nelfinavir and nitroxoline, with or without erlotinib, resulted in dose- and cell-dependent synergistic effects on cell viability. These effects were paralleled by cell cycle alterations and more consistent apoptosis induction as compared to single agents. Treatments with drug combinations induced drastic impairment of clonogenic activity in the three cell lines.This study shows that two non-antitumor drugs, nelfinavir and nitroxoline, as single agents or in combination have antitumor effects that appear comparable, or in some case more pronounced than those of erlotinib in three PC cell lines. Our results support repurposing of these approved drugs as single agents or in combination for PC treatment.The online version of this article (10.1186/s13046-018-0904-2) contains supplementary material, which is available to authorized users. Pancreatic cancer (PC) remains the fourth most common cause of cancer death due to poor survival rate and rapid fatality after diagnosis . PC is tIdeally, combination therapies should include non-toxic drugs that act synergistically to kill tumor cells in a selective way. Several natural or synthetic compounds have been explored as single agents or in combination with standard chemotherapy in preclinical models of PC –7. CurreThere is a growing body of evidence that a number of non-anticancer drugs already approved for disparate human diseases have anticancer properties , 11. TheIn this study, we evaluated the effects of two non-anticancer drugs, nelfinavir and nitroxoline, as single agents and/or in combinations with or without erlotinib, in PC cell lines. The three drugs are already approved for human use, have a relatively low toxicity and their combination has not been tested before. Nelfinavir is a competitive inhibitor of HIV aspartyl protease used in combination with other antiretroviral drugs to treat patients with HIV infection . Chemora3--2,5-diphenyl-2H-tetrazolium bromide (MTT), crystal violet, RNAse, propidium iodide (PI), RIPA Buffer  containing 1 mM PMSF and protease inhibitor cocktail were obtained from Sigma . Mouse monoclonal anti-cyclin D3 antibody and rabbit polyclonal PARP antibody were purchased from Cell Signaling Technology, Inc. . Goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, anti-cyclin B1 antibodies were obtained from Santa Cruz Biotechnology, Inc. . Monoclonal anti-β-actin antibody was obtained from Sigma .KRAS mutations, while BxPC-3 and AsPC-1 are TP53 mutated. Cells were cultured in RPMI 1640, supplemented with 10% FBS at 37 °C, 5% CO2. Nelfinavir mesylate and erlotinib HCl (OSI-744) were obtained from Selleckchem . 8-hydroxy-5-nitroquinoline (nitroxoline) was purchased from Sigma .Human pancreatic cancer (PC) cell lines AsPC-1 and Capan-2 were purchased from Cell Lines Service . PC cell line BxPC-3 was purchased from American Type Culture Collection . The three cell lines are known to express the EGFR protein , 21. AsP3 cells/well) and were treated the following day for 48 h with erlotinib, nelfinavir or nitroxoline as single agents, or with combinations of the drugs at various concentrations as indicated. Then, the MTT solution was added to each well and incubated at 37 °C for at least 3 h, until a purple precipitate was visible. In order to dissolve formazan crystals, the culture medium was replaced with dimethyl sulfoxide . Absorbance of each well was quantified at 540 and 690 nm, using a Synergy H1 microplate reader .Cell viability was tested by MTT assay . Briefly, cells were seeded in 96-well plates . The CI was calculated by CompuSyn software, based on the Chou-Talalay equation [ICequation : a CI < 6 cells per experimental condition were harvested, fixed in 70% cold ethanol and kept at 4 °C overnight. Cells were then resuspended in 50 μg/ml PI  and 200 μg/ml RNAse  as previously described [5cells) was performed using a FACScanto II flow cytometer . Data were analyzed with FlowJo software v8.8.6  and FCS Express 5 Software .Approximately 0.5 × 10escribed . Cell cyCells were collected and lysed in RIPA buffer supplemented with protease inhibitor cocktail. Protein concentrations were determined by the BCA Protein Assay  and 30 μg were subjected to electrophoresis followed by immunoblotting. The membranes were blocked in 5% nonfat dry milk for one hour at room temperature and incubated with the appropriate primary antibodies. Then the membranes were incubated with either anti-rabbit or anti-mouse HRP-conjugated secondary antibodies. The blots were revealed by chemiluminescence using the SuperSignal West Pico Chemiluminescence Substrate  according to the manufacturer’s instructions. β-actin was used as loading control.5 events were collected. Viable cells were Annexin-Vneg, while apoptotic cells were Annexin-Vpos.To assess apoptosis, BD Pharmingen™ APC Annexin V and 7-AAD viability staining solutions  were used according to the manufacturer’s instructions. Cells were resuspended in a specific binding buffer and stained using 7-AAD for dead cell exclusion and Annexin V antibody for 30 min at 4 °C in the dark. Subsequently, the samples were washed and analyzed on a FACScanto II flow cytometer . For each sample, at least 103 cells/well) and following cell attachment they were exposed for 48 h to erlotinib, nelfinavir, nitroxoline or their combinations as indicated. Then, after medium refreshment, the plates were incubated at 37 °C with 5% CO2, until cells in the control vehicle formed colonies consisting of at least 30 cells (3–4 days). Colonies were fixed with 70% methanol and stained with 0.5% crystal violet, then rinsed with tap water, dried and counted.Clonogenic assay was performed essentially as previously described . PC cellp-value ≤0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism version 5.01 software . Comparisons of mean values were performed by an unpaired Student’s t-test. Multiple comparisons were performed by one-way ANOVA followed by Dunnett’s test. A 50 values  comparable to those obtained with erlotinib (22.8 μM). In Capan-2, nitroxoline had an IC50 value (16.9 μM) lower than those obtained with nelfinavir and erlotinib . Conversely, in BxPC-3 the IC50 of erlotinib (10.9 μM) was lower as compared to nelfinavir and nitroxoline .We analyzed by MTT the effect of erlotinib, nelfinavir and nitroxoline on the viability of three PC cell lines. The three drugs significantly affected cell viability in a dose-dependent manner, with distinct sensitivities for the three cell lines  . Combination therapies with classical chemotherapeutic agents improved treatment of advanced PC at the cost of a relevant toxicity, but the 5-year survival rate remains below 5%. Consequently, new approaches for the treatment of this lethal disease are urgently needed. In the present study, we analyzed the effect of nitroxoline and nelfinavir, two already approved non-anticancer drugs with low toxicity. These repurposed drug candidates were tested in PC cell lines as single agents, or in different combinations with or without erlotinib, a molecule already approved for PC treatment.50 values comparable, or in some case lower than those obtained with erlotinib. Conversely, in BxPC-3 the IC50 of erlotinib was lower as compared to repurposed drugs. Indeed, the IC50 of erlotinib in BxPC-3 was also lower than the IC50 observed with the same drug in AsPC-1 and Capan-2. These different effects of the drugs on the viability of the three PC cell lines were possibly related to their different genetic background. It has been previously shown that nitroxoline and nelfinavir affected cell cycle in different cancer cell lines [The effect of nitroxoline and nelfinavir on viability was analyzed in three PC cell lines AsPC-1, Capan-2 and BxPC-3 that display different genetic profiles. Interestingly, in AsPC-1 and Capan-2 these non-toxic drugs had ICll lines , 25–27. Considering that there is a substantial lack of studies combining repurposed drugs in PC and that these drug combinations might have a greater antitumor effect as compared to single agents, we determined the effect of nelfinavir, nitroxoline and erlotinib combinations on PC cells. As observed with single agents, the effects were distinct in the three PC cell lines. Overall, nelfinavir and nitroxoline in combination had a more pronounced effect on viability than single agents and this effect was more consistent at high concentrations of the drugs. Notably, the addition of erlotinib to the drug combinations in most cases did not increase the effects on cell viability. At low concentrations, only the combination of nelfinavir and nitroxoline in Capan-2 was assessed as deriving from synergistic drug interactions by CompuSyn. Conversely, at high concentrations the combinations of nelfinavir and nitroxoline, with or without erlotinib, were assessed as synergistic by CompuSyn both in Capan-2 and BxPC-3. It is interesting to note that also in AsPC-1 the combinations at high drug concentrations had enhanced effects as compared to single agents, resulting in a lower residual cell viability as compared to Capan-2 and BxPC-3 with the same drug combinations. Nevertheless, in AsPC-1 drug interactions were not assessed synergistic by CompuSyn analyses. Flow cytometry and immunoblot analyses showed that the reduced viability observed in PC cell lines with drug combinations was related to an interference with cell cycle progression and was associated to a reduction of cyclin D3 and cyclin B1 protein expression, which was more marked and homogeneous across the three cell lines at high drug concentrations. The results of flow cytometry and immunoblot analyses indicated also that apoptosis contributed to the reduced viability observed after treatment with the drug combinations. In particular, the effects of drug combinations on apoptosis across the three PC cell lines were more consistent than the effects observed with single drugs, while the addition of erlotinib to the combinations did not substantially affect apoptosis. Drug combinations had also drastic effects on clonogenic ability, indicating that these combinations had a strong impact on cell self-renewal capacity in PC cell, although it should be noted that also nitroxoline as single agent had comparable strong effects. Overall, the impact of nitroxoline as single agent on clonogenicity, as well as the corresponding effect of drug combinations was more pronounced than the effects of the same treatments on viability, as assessed by MTT.An interesting issue that is currently being investigated is how repurposed drugs exert their anticancer actions. Disparate off-target anticancer effects have been proposed and new targets are emerging for each drug. In particular, nitroxoline was shown to inhibit MetAP2, SIRT1, SIRT2 and was also shown to bind and inhibit cathepsin B, although the relevance of the latter target has been questioned because of the relatively high concentrations of the drug necessary to induce this effect . NelfinaOur results indicate a remarkable antitumor activity of nelfinavir and nitroxoline in PC cells. Nelfinavir and nitroxoline when used as single agents decreased viability, induced apoptosis, affected the expression of relevant cell cycle proteins, drastically compromised clonogenic activity with distinct effects in the three PC cell lines tested. These effects were enhanced by combined treatments with nelfinavir and nitroxoline. To our knowledge, this is the first study providing evidence that these human approved, non-anticancer drugs, as single agents or in combination, affect several crucial biological processes in different PC cell lines. In the experimental conditions tested in the present study the antitumor activity of these repurposed drugs appears comparable or superior to erlotinib, a targeted agent approved for PC treatment. Therefore, the results obtained with nelfinavir and nitroxoline in PC cell lines suggest that these drugs could be effectively repurposed, as single agents or in combination, for the treatment of the poorly responsive pancreatic cancer.Additional file 1:Table S1. Effects of erlotinib, nelfinavir and nitroxoline as single agents versus drug combinations on cell viability. (XLSX 12 kb)Additional file 2:Table S2. Mean percentages of cells identified as sub-G1 population. (XLSX 11 kb)Additional file 3:Figure S1. Representative images of clonogenic assays. (TIF 13253 kb)


C. mangga against six human cancer cell lines, namely the hormone-dependent breast cell line (MCF-7), nasopharyngeal epidermoid cell line (KB), lung cell line (A549), cervical cell line (Ca Ski), colon cell lines (HCT 116 and HT-29), and one non-cancer human fibroblast cell line (MRC-5) were conducted using an in-vitro neutral red cytotoxicity assay. The crude methanol and fractionated extracts (hexane and ethyl acetate) displayed good cytotoxic effects against MCF-7, KB, A549, Ca Ski and HT-29 cell lines, but exerted no damage on the MRC-5 line. Chemical investigation from the hexane and ethyl acetate fractions resulted in the isolation of seven pure compounds, namely (E)-labda-8(17),12-dien-15,16-dial (1), (E)-15,16-bisnor-labda-8(17),11-dien-13-on (2), zerumin A (3), β-sitosterol, curcumin, demethoxycurcumin and bis-demethoxycurcumin. Compounds 1 and 3 exhibited high cytotoxic effects against all six selected cancer cell lines, while compounds 2 showed no anti-proliferative activity on the tested cell lines. Compound 1 also demonstrated strong cytotoxicity against the normal cell line MRC-5. This paper reports for the first time the cytotoxic activities of C. mangga extracts on KB, A549, Ca Ski, HT-29 and MRC-5, and the occurrence of compound 2 and 3 in C. mangga.Investigations on the cytotoxic effects of the crude methanol and fractionated extracts   Cancer is one of the leading causes of deaths in Malaysia. It is the fourth leading cause of death after diseases of the circulatory and respiratory systems and motor vehicle accidents, and responsible for 10.3% of medically certified deaths. In 2002, about 26,089 cases of cancer were diagnosed in Peninsular Malaysia and an estimated 10,656 cases were not reported. As many as 30,000 new cases are reported yearly [i.e., proliferation, deregulation of apoptosis, angiogenesis, invasion and metastasis), but also synergize with chemotherapy and radiotherapy [Natural products provide a wide variety of phytochemicals which have been shown to have a broad spectrum of activity across multiple signalling pathways. Natural products not only disrupt aberrant signalling pathways leading to cancer , nasopharyngeal epidermoid cell line (KB), lung cell line (A549), cervical cell line (Ca Ski), colon cell line (HCT 116), colon cell line (HT-29) and one non-cancer human fibroblast cell line (MRC-5), using an in-vitro neutral red cytotoxicity assay. We also identified the components in the active hexane and ethyl acetate fractions of C. mangga rhizomes. The chemical investigation led to the isolation of seven compounds, namely (E)-labda-8(17),12-dien-15,16-dial (1), (E)-15,16-bisnorlabda-8(17),11-dien-13-one (2), zerumin A (3), β-sitosterol, curcumin, demethoxycurcumin and bisdemethoxycurcumin. Compounds 2 and 3 are reported for the first time in this plant. To our knowledge, there is no report on the cytotoxic activities of C. mangga on the above mentioned human cancer cell lines, except for the MCF-7 and HCT 116 cell lines [In the present study, we investigated the cytotoxic effects of the crude and fractionated extracts of  line A54, cervicall lines .C. mangga are summarized in The results of preliminary cytotoxic screening of the methanol and fractionated extracts of 50 values ranging from 22.0 to 36.8 μg/mL. The ethyl acetate fraction only showed good activity against HT-29, with IC50 18.5 μg/mL, whilst it showed mild inhibitory activity against the other investigated cell lines. The hexane fraction of C. mangga exhibited good activities against all the cell lines tested (except HCT 116) with IC50 values of 8.1, 15.4, 17.4, 11.4 and 17.9 μg/mL against the MCF-7, KB, A549, Ca Ski and HT-29 cell lines, respectively. Interestingly, the hexane fraction showed no adverse effect on the non-cancer human fibroblast cell line MRC-5 (IC50 >100 μg/mL). If this also occurs in vivo, the hexane extract would be considered safe for human consumption and its non-toxic effect on the MRC-5 cell line merits consideration for further toxicity and clinical studies. It was also observed that the crude methanol and the ethyl acetate extracts caused no damage to the normal human fibroblast cell line. The water extract did not show any cytotoxicity against any of the tested human cancer cell lines (IC50 >100 μg/mL). As the hexane fraction demonstrated good inhibitory activity and the ethyl acetate fraction exhibited mild activity, further chemical investigation was focussed on these two fractions. This led to the isolation of compounds 1-3.The methanol extract exhibited mild cytotoxic effects against all selected human cancer cell lines, with ICE)-labda-8(17),12-dien-15,16-dial (1), (E)-15,16-bisnorlabda-8(17),11-dien-13-one (2) and β-sitosterol. The ethyl acetate fraction afforded zerumin A (3) and the curcuminoids. Compounds 2 and 3 are reported for the first time in this plant. All chemical components . The cytotoxic effect shown by the hexane fraction may be due to the presence of compound 1 which comprises of 1.5% (w/w) in this fraction, whilst compound 3 and the curcuminoids may be responsible for the observed activity of the ethyl acetate fraction.The isolated compounds were further tested on the selected cancer cell lines as the hexane fraction showed strong cytotoxic effect against MCF-7, KB, A549, Ca Ski and HT-29 cells, whilst the ethyl acetate fraction exhibited moderate effects on KB, A549, HCT 116 and HT-29 cells. 50 values from 0.05 to 0.58 μg/mL), but also on the human normal cell line . Due to its high toxicity on human normal cell lines, the continuous use of doxorubicin can cause major adverse effects to human health [In the present study, doxorubicin, used clinically for the treatment of a great variety of cancer diseases, was the positive control ,17,18. Hn health . Even thNMR analysis: 1H- and 13C-NMR spectra were obtained on Bruker Avance III 500 MHz Nuclear Magnetic Resonance spectrophotometers.  GC-MS analysis: GC-MS analysis was performed using an Agilent Technologies 6980N gas chromatograph equipped with a 5975 Mass Selective Detector (70 eV direct inlet); the HP-5ms capillary column (5% phenylmethylsiloxane) with column dimensions 30.0 m × 25 mm × 25 μm was initially set at 150 °C, then the temperature of the oven was increased at 5 °C per minute to 300 °C using helium as carrier gas at flow rate 1 mL/min. The total ion chromatogram obtained was auto integrated by ChemStation and the components were identified by comparison with the supplied mass spectral data database  whenever possible. HPLC analysis: HPLC analysis was performed on a Shimadzu LC system equipped with a Shimadzu LC-10AT VP pump, Shimadzu SCL-10A VP system controller, Shimadzu SPD-M10A VP Photo Diode Array detector, Shimadzu DGU-12A vacuum degasser and Shimadzu LC Solution software. The solvents used for HPLC were of chromatographic grade acetonitrile (J.T. Baker), methanol (J.T. Baker) and ultra-pure water (H2O). The column used was a Chromolith Performance RP-18e (Merck) (100.0 mm × 4.6 mm i.d.) for analytical scale HPLC and semi-preparative column was a Chromolith Performance RP-18e (100.0 mm × 10.0 mm i.d.) for preparative scale separation. In-vitro neutral red cytotoxicity assay: All cancer cell lines were purchased from the American Type Culture Collection , medium like RPMI 1640, McCOY’S 5A, Eagle Minimum Essential Medium (EMEM) from Sigma-Aldrich, USA. The supplements or antibiotics such as foetal bovine serum, penicillin/streptomycin (100 μg/mL), amphotericin B (50 μg/mL), tripsin-EDTA (100X) were purchased from PAA Lab, Austria as well as sodium pyruvate (11 mg/mL) from Sigma-Aldrich, USA. The cells were cultured in a Shel Lab water-jacketed CO2 incubator with 25 cm3 tissue culture flasks , which was observed routinely under inverted microscope  for any contamination and seeded into 96-well flat bottom microtiter plate . The extract or pure compounds were dissolved in dimethyl sulfoxide  from Sigma-Aldrich, USA. The 96-well microtiter plate was agitated rapidly on a microtiter plate incubator shaker (LT Biomax 500) and absorbance of eluted dye measured at 540 nm using a ELISA microplate reader . Cell lines such as MCF-7, A549, Ca Ski and HT-29 were maintained in RPMI 1640 medium, HCT 116 in McCOY’S 5A Medium, KB and MRC-5 cells in Eagle Minimum Essential Medium (EMEM). All of these media were supplemented with 10% foetal bovine serum (FBS), penicillin/streptomycin, amphotericin B and only additional sodium pyruvate in EMEM. The cells were cultured in tissue culture flask in 5% CO2 incubator kept at 37 °C in a humidified atmosphere and observed routinely under inverted microscope from any contaminations. Each fresh medium was replaced every 2 or 3 days until cell confluence was achieved and the cells were detached by using tripsin-EDTA. C. mangga were obtained from Yogjakarta, Indonesia in July 2006. A voucher specimen (voucher number HI 1331) was deposited in the Herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.The authenticated rhizomes of C. mangga (1.0 kg) were soaked in methanol  for three days at room temperature. The solvent-containing extract was then decanted, any traces of water was removed using anhydrous sodium sulphate and evaporated under reduced pressure using a rotary evaporator to give a dark brown crude methanol extract . The crude methanol extract was then extracted with hexane (500 mL) and concentrated in vacuo to obtain a yellowish brown hexane fraction . The hexane insoluble residue was further partitioned using ethyl acetate and water (500 mL:500 mL) to give a dark brown ethyl acetate fraction  and a light brown water fraction . A summary of procedures in the bioassay-guided chemical investigation of C. mangga is shown in The dried, ground rhizomes of 2O) under the following gradient conditions: 0–5 min linear gradient of eluting solvent 45 to 50% ACN; 5–11 min linear gradient of 50 to 65% ACN; 11–21 min linear gradient 65 to 100% ACN; 21–25 min isocratic using 100% ACN; 25–30 min linear gradient solvent 100 to 45% ACN at a flow rate of 1.0 mL/min. Fractions H2 (617.0 mg) and H5 (744.3 mg) were subjected to semi-preparative separation on HPLC using the same method as utilised in the analytical scale separation described above. Fraction H2 gave the following compounds: (E)-labda-8(17),12-dien-15,16-dial  and (E)-15,16-bisnorlabda-8(17),11-dien-13-one . β-Sitosterol  was obtained from fraction H5.The hexane fraction (1.0 g) was subjected to vacuum liquid chromatography  initially eluting with hexane followed by hexane enriched with increasing percentages of acetone and finally with methanol. A total of nine fractions (labelled H1 to H9) were collected. Subsequently, the fractions were subjected to an analytical scale separation on the Shimadzu HPLC system. Each fraction was prepared in 1.0 mg/mL with methanol, filtered and 20 μL was injected into a Chromolith Performance RP-18e analytical column (100.0 mm × 4.6 mm i.d.) and peaks were detected by monitoring the UV absorbance at 214 nm. The mobile phase was a binary eluent of chromatographic grade acetonitrile (ACN) and ultra-pure water . Three curcuminoids were isolated from fraction E8 using preparative thin layer chromatography technique, PTLC : curcumin , demethoxycurcumin  and bis-demethoxycurcumin . All pure compounds obtained were checked by TLC and HPLC to confirm their purity and then subsequently identified through their mass spectral and NMR data which were consistent with published data [One gram (1.0 g) of the ethyl acetate fraction was also subjected to the same VLC setup as for the hexane fraction and a total of eight fractions (labelled as E1 to E8) were collected. The fractions were then prepared and subjected to the same analytical HPLC condition as described for the hexane fraction. Two fractions, E7 (874.4 mg) and E8 (380.0 mg), were subjected to semi-preparative HPLC separation using the same method as utilised in the analytical scale separation. Fraction E7 afforded a colourless oil, zerumin A (hed data ,14.in-vitro cytotoxicity activity was performed using the neutral red cytotoxicity assay based on the initial protocol described by Borenfreund and Puerner, with some modifications [The ications ,19.C. mangga rhizomes are ascribable to the presence of the active compound 1 in the hexane fraction, and compound 3 and the curcuminoids in the ethyl acetate fraction. Even though these compounds are not as effective as doxorubicin against cancer cell lines, in comparison they have low toxicity against human normal cell lines. The present study provides important preliminary data of extracts or isolated compounds with potential anti-neoplastic properties for future work. Since C. mangga is widely used in traditional medicine for the treatment of cancer, the current findings provide scientific validation in the use of the rhizomes for this purpose. As the rhizomes are also widely consumed as salad in food without any known undesirable side effecat, it can be assumed that the plant is safe for consumption at the normal dose as food. Curcuma mangga, is therefore a promising dietary agent that holds great potential for use in chemopreventive and chemotherapeutic strategies. Nevertheless, further investigations are necessary to validate its use and to determine its mode of action.The cytotoxic activities observed for