The primary objectives of this study are to determine the nature of the interactions between BT and cisplatin and to understand the mechanism(s) of action of BT-cisplatin combination.Combination drug therapy appears a promising approach to overcome drug resistance and reduce drug-related toxicities in ovarian cancer treatments. In this in vitro study, we evaluated the antitumor efficacy of cisplatin in combination with Bithionol (BT) against a panel of ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant cell linesThe cytotoxic effects of drugs either alone or in combination were evaluated using presto-blue assay. Cellular reactive oxygen species were measured by flow cytometry. Immunoblot analysis was carried out to investigate changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27. Luminescent and colorimetric assays were used to test caspases 3/7 and ATX activity.The efficacy of the BT-cisplatin combination depends upon the cell type and concentrations of cisplatin and BT. In cisplatin-sensitive cell lines, BT and cisplatin were mostly antagonistic except when used at low concentrations, where synergy was observed. In contrast, in cisplatin-resistant cells, BT-cisplatin combination treatment displayed synergistic effects at most of the drug ratios/concentrations. Our results further revealed that the synergistic interaction was linked to increased reactive oxygen species generation and apoptosis. Enhanced apoptosis was correlated with loss of pro-survival factors , expression of pro-apoptotic markers  and enhanced cell cycle regulators p21 and p27.In cisplatin-resistant cell lines, BT potentiated cisplatin-induced cytotoxicity at most drug ratios via enhanced ROS generation and modulation of key regulators of apoptosis. Low doses of BT and cisplatin enhanced efficiency of cisplatin treatment in all the ovarian cancer cell lines tested. Our results suggest that novel combinations such as BT and cisplatin might be an attractive therapeutic approach to enhance ovarian cancer chemosensitivity. Combining low doses of cisplatin with subtherapeutic doses of BT can ultimately lead to the development of an innovative combination therapy to reduce/prevent the side effects normally occurring when high doses of cisplatin are administered.The online version of this article (doi:10.1186/s12885-016-3034-2) contains supplementary material, which is available to authorized users. Drug resistance to currently used chemotherapies is the fundamental cause of recurrence and poor overall survival in ovarian cancer patients –3. BetweCis-diamminedichloroplatinum (II) (cisplatin) is a platinum-based compound that has clinical activity against a wide array of solid cancers including ovarian, testicular, bladder, colorectal, lung, and head and neck [Platinum-based chemotherapy is still an effective treatment for ovarian cancer in spite of severe side effects and development of resistance associated with its use . Cis-diaand neck . DNA-damand neck , 13. In and neck  via geneand neck . Despiteand neck , 11. Theand neck . In orde50) observed in several ovarian cancer cell lines were well below the reported clinically tolerable levels in humans. Our recent in vivo study did not demonstrate the anti-tumor potential of BT ; however, lack of toxicity at any of the tested doses and the ability of BT to induce apoptosis still make BT a promising candidate to be utilized in therapeutics [In recent years, a number of compounds have been explored in combination with cisplatin. Some of these include N-acetylcysteine , naltrexapeutics . To furtBT and cisplatin combinations were evaluated systematically for drug-ratio dependent interactions in vitro. The nature of the interactions between BT and cisplatin was evaluated by three different approaches – (1) sequential addition of drugs that involves pre-treatment with BT for 24 h followed by cisplatin addition (drugs in non-constant ratio), (2) simultaneous addition of both drugs in non-constant ratio and (3) simultaneous addition of drugs in constant ratio. The combination index was used to evaluate if the interactions are antagonistic, synergistic or additive., we evaluated ROS generation, ATX inhibition, induction of apoptosis and expression of key apoptotic and cell cycle modulators.Cisplatin and other anti-neoplastic agents exhibit cytotoxic effects via elevation of intracellular reactive oxygen species (ROS) that may contribute to their therapeutic effect. BT was shown to induce apoptosis via cell cycle regulation, ROS generation, NF-κB inhibition and autotaxin (ATX) inhibition. To investigate the molecular mechanism(s) of action of BT-cisplatin combination in ovarian cancer cells in vitro2. The cisplatin-resistant variants A2780-CDDP and IGROV-1CDDP cells were treated with 3 μM cisplatin every third passage to maintain cisplatin resistance.Isogenic ovarian cancer cell lines pairs, e.g., A2780 /A2780-CDDP and IGROV-1/, IGROV-1CDDP were received as a generous gift from Dr. Brodsky . The parental cell lines were purchased from Sigma and made resistant in vitro by continuous stepwise exposure to cisplatin to produce the corresponding cisplatin-resistant cell lines. All cell lines were maintained in DMEM media (Sigma) supplemented with 10% heat-inactivated FBS (Hyclone), 100 IU penicillin (Mediatech) and 100 μg/mL streptomycin (Mediatech). All cell lines were cultured at 37 °C in a humidified atmosphere at 5% COCis-diamminedichloroplatinum (II)) were purchased from Sigma . All primary antibodies were purchased from Cell Signaling Technologies, . PrestoBlue™ Cell Viability Reagent and ROS Dye - carboxy-H2DCFDA were purchased from Invitrogen .BT and cisplatin (3 cells/well) were plated into 96-well plates  and incubated overnight. Cells were treated with different concentrations of both drugs either alone or in combination and incubated for 48 h. BT was tested at concentrations ranging from 3.56 μM to 100 μM and cisplatin at concentrations between 1.57 and 200 μM. A minimum of 4–6 h prior to the end of treatment, presto blue reagent was added and incubated for 48 h followed by measurement of fluorescence (540 nm excitation/590 nm emissions). DMSO concentration was corrected to 1% in all wells. All treated cells were compared against control cells (considered as 100% viable) treated with 1% DMSO media. Data were expressed as mean ± SD of triplicate experiments.Cell viability after drug(s) treatment was determined by Presto Blue cell viability reagent (Invitrogen) as descried previously . In brieIn order to determine role of ROS in BT-cisplatin induced cytotoxicity, cell viability assays were performed in the presence the antioxidant ascorbic acid (AA). Cells were pretreated with 1 mM AA for 2 h prior to addition of drugs and further incubated for 48 h. Restoration of cell viability was analyzed.50 values of the individual drugs . Subsequently, this drug mixture was serially diluted to obtain different concentrations of the combination. The dose ranges selected for combination studies were 3.25 μM to 100 μM for BT and 1.56 μM to 200 μM for cisplatin. For non-constant ratio combination, BT and cisplatin were prepared at a series of concentrations that spans the dose–response curves for both drugs. Each concentration of BT was mixed with each concentration of cisplatin, thereby producing a matrix of multiple stock admixtures, containing both drugs together in solution at a variety of concentrations and ratios.To assess combination effects of BT with cisplatin, these drugs were combined in constant and non-constant ratios. In constant ratio combination, BT and cisplatin were combined at a fixed ratio based on the ICThe nature of the interaction between BT and cisplatin was assessed using three different approaches: (1) simultaneous treatment with both drugs in non-constant ratio, where cells were treated with both BT and cisplatin simultaneously combined in a non-constant ratio; (2) simultaneous treatment with both drugs in constant drug ratio, where cells were treated with both BT and cisplatin simultaneously combined in a constant ratio and (3) pre-treatment with BT followed by addition of cisplatin in non-constant ratio, where cells were treated with different concentrations of BT for 24 h after which BT was removed and cisplatin was added for another 24 h. Here also each concentration of BT pre-treatment is followed by each concentration of cisplatin thereby producing a matrix of multiple stock admixtures (non-constant ratio).The tumor growth inhibition obtained for BT-cisplatin combination over a range of concentrations was compared to that obtained for the individual drugs, and a measure of the synergy between the two drugs, referred to as the combination index (CI), was calculated using a median-effect mathematical algorithm . CalcuSy3 cells were plated per well of the 96-well plate and treated with BT and cisplatin either alone or in combination. Following treatment, Caspase-Glo 3/7 reagent was added and incubated for 30 min. at room temperature. The luminescence intensity was measured using a luminometer . Drug-treated cells were compared against cells treated with 1% DMSO media (controls). Data were expressed as mean ± SD of triplicate experiments.Caspase 3/7 activity was measured using Caspase-Glo 3/7 assay kit from Promega, following the manufacturer’s instructions. Briefly, 10 × 105 cells) were seeded into 12-well plate and treated with BT and cisplatin either alone or in combination for 24 h. Following treatment, cells were washed, stained with Hoechst stain (2 drops/mL of media) for 15 min. at 25 °C and observed under a fluorescent microscope. Representative images were taken with an inverted microscope  and 20× objective.NucBlue Live Cell Stain  was used to morphologically assess nuclear condensation indicative of apoptosis. This qualitative test was performed as described previously , 29. In in Situ Colorimetric Apoptosis Detection Kit  following the manufacturer’s instructions. Briefly, cells were seeded at a density of 3 × 104 cells/well, into 96-well flat bottom plates and incubated overnight. Cells were treated with BT and cisplatin either alone or in combination for 24 h. After treatment with drugs, cells were washed and fixed. Subsequently, labeled nucleotides were added and measurements were performed with HRP – HRP substrate (TACS-Sapphire) system. The absorbance was measured at 450 nm using a microplate reader, Multiskan (Thermo Scientifics).DNA fragmentation was detected using the TiterTACS® 2 TdT 6) were seeded in 100 mm2 culture dishes and treated with BT and cisplatin either alone or in combination for 24 h. After treatment, the cells were washed once with PBS, collected by centrifugation after trypsinization, re-suspended in fresh PBS and incubated with 5 μM 5,6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate  for 30 min at 37 °C. The cells were washed twice with PBS, re-suspended in an equal volume of PBS and fluorescence measured with flow cytometry. Data was acquired on a BD Accuri C6 flow cytometer and analyzed using Accuri C6 software . Twenty thousand cells were analyzed for each sample. Subsequent cell viability assay with AA pretreatment was performed.Hydrogen peroxide, hydroxyl radicals and peroxy radicals were detected via carboxy-H2DCFDA using flow cytometry as described previously . BrieflyWestern blotting was performed to evaluate expression of key modulators of apoptosis such as cleaved PARP, XIAP, bcl-2 and bcl-xL. Key cell cycle regulators such as p21 and p27 were also assessed by western blotting. Cell seeding, cell lysis and western botting were done as described previously . In briep-nitrophenylphosphonate (pNppp) at a final concentration of 5 mM prepared in 50 mM Tris–HCl buffer, pH 9.0. After 30 min incubation at 37 °C, the reaction was stopped by the addition of 100 μL of 0.1 M NaOH solution. The reaction product was measured by reading the absorbance at 410 nm. ATX inhibition of treated cells was calculated as the percentage of ATX activity in comparison with untreated cells.The phosphodiesterase activity of ATX was measured as described previously . In briep < 0.05.Comparisons between cisplatin treated and BT/cisplatin combination treated groups were performed by Student’s t–test. The significance level was set at The objective of the present study is to investigate the effects of BT-cisplatin combination in ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant isogenic pair of cell lines. Cells were exposed to different concentrations of BT and cisplatin either alone or in combination. The combination index (CI) value, calculated according to Chou’s methods , was useWhen A2780 cells were pretreated with BT followed by cisplatin addition, there was synergy only at lower BT (3.25 and 6.25 μM) and cisplatin (1.56–6.25 μM) concentrations Fig. . At highIn contrast to A2780 (cisplatin-sensitive), in A2780-CDDP (cisplatin-resistant), BT was synergistic to cisplatin action at most of the drug ratios either when cells were pre-treated with BT or when both BT and cisplatin were added simultaneously Fig. . At highWhen IGROV-1 cells were pretreated with BT followed by cisplatin addition, antagonism was observed at most drugs ratios Fig. . SynergyInterestingly, in the IGROV-1-CDDP (cisplatin-resistant) cell line, BT was synergistic to cisplatin action in a drug concentration dependent manner, either when cells were pre-treated with BT or when both BT and cisplatin were added simultaneously. Synergy was observed at low (3.25 μM) or towards higher (50 and 100 μM) concentrations of BT when combined with cisplatin at all concentrations Fig. . When adIn these cell lines, BT and cisplatin act in general antagonistic, however, synergy was observed at very narrow drugs ratios with slightly better response when both drugs were added simultaneously  staining. As shown in Fig. In order to confirm the results obtained with DNA fragmentation studies, we also assessed other apoptotic markers. As shown in Fig. Similarly, increased PARP protein cleavage product  was observed in cisplatin-sensitive cell lines such as A2780 and IGROV, when treated with either of the drugs alone Fig. . HoweverTo confirm that the potentiation/attenuation of cisplatin-induced cytotoxicity by BT treatment is accompanied by changes in the expression of key regulators of apoptosis, we also assessed the expression of XIAP, bcl-2 and bcl-xL. As shown in Fig. Our results suggest that BT significantly inhibits apoptosis when added in combination with cisplatin in cisplatin-sensitive cell lines (A2780 and IGROV-1) whereas it increases apoptosis in cisplatin-resistant variants (A2780-CDDP and IGROV1-CDDP). Furthermore, these results suggest that cisplatin in combination with BT caused down-regulation of key survival proteins such as XIAP, Bcl-2, and bcl-xL compared to either of the drugs alone, thus resulting in greater apoptosis/cytotoxicity. These results show that the nature of drug interactions depends on the extent of apoptosis that occurs when cells are treated in combination. Synergistic interaction enhanced apoptosis whereas antagonistic interactions reduced the extent of apoptosis.(kip1) and p21 in order to understand their role in causing antagonistic or synergistic effects of BT and cisplatin combination. Figure (kip1) and p21 when used alone. When cisplatin and BT were used in combination, the expression of P27 and P21 was reduced in cisplatin-sensitive cell lines (A2780 and IGROV-1) and enhanced in cisplatin-resistant cell lines (A2780-CDDP and IGROV1-CDDP), compared to cisplatin or BT treatments alone. These results are consistent with the cytotoxicity data, where combination treatment potentiated cytotoxicity in cisplatin-resistant cell lines and attenuated it in cisplatin-sensitive cell lines.We assessed the expression of the cell cycle regulators P27 We measured ROS levels to examine whether ROS are involved in the synergistic/antagonistic interaction between cisplatin and BT. As shown in Fig. ATX inhibition was considered major mechanism of action of BT. In order to determine if ATX inhibition potential of BT has any contribution to the synergy observed in cisplatin-resistant cell lines, we assessed the ATX levels in cell lysates treated with either of the drugs alone or in combination. As shown in Fig. Treatment with of ovarian cancer with a single anticancer agent often cannot provide a satisfactory therapeutic effect in recurrent patients due to drug resistance and dose-limiting side effects . TherefoPreviously, we showed that BT exerts cytotoxic effects on a panel of ovarian cancer cell lines regardless of their cisplatin sensitivities. BT was shown to induce apoptosis both in vitro and in vivo , 27. OurCombination chemotherapy has been in practice for cancer treatments for many decades. The principle underlying this approach so far has been to administer each drug at their maximum tolerated dose (MTD) to achieve maximum therapeutic efficiency. However, clinical data suggested that this approach failed to exploit the full potential of many therapeutic drugs used in combinations and may have resulted in multidrug resistance in tumor cells. The major reason for such a phenomenon is exposure of tumor cells to drugs at antagonistic ratios or concentrations. The nature of the interactions (synergism or antagonism) of anticancer agent combinations against tumor cells in vitro depends on the concentrations of the individual drugs. Ratiometric dosing is an approach to develop drug combinations based on drug ratio–dependent synergy. It is essential to determine optimal drug concentrations/ratios to obtain synergy so as to prevent the exposure of cells to antagonistic ratios. In our study, we employed both constant and non-constant drugs ratios to understand the nature of interactions between our two chosen drugs. BT and cisplatin combinations were evaluated systematically for concentration-dependent interactions in in vitro using the isogenic ovarian cancer cell lines pairs A2780 (cisplatin-sensitive) / A2780-CDDP (cisplatin-resistant) and IGROV-1 (cisplatin-sensitive) / IGROV-1CDDP (cisplatin-resistant). This study shows the differential response of cisplatin-sensitive and cisplatin-resistant cell lines to BT/cisplatin combination treatment. In cisplatin-sensitive cell lines, BT/cisplatin combination was antagonistic at most drug concentrations; however the use of lower concentrations of BT and cisplatin resulted in synergy. In contrast, when tested on cisplatin-resistant cell lines, these agents were synergistic at most of the ratios/concentrations. Our results show that synergy is highly dependent on the concentration of the drugs used. Optimal response was observed when BT and cisplatin were combined at lower concentrations in a non-constant ratio. With regard to sequence of drugs applied, simultaneous treatment with BT and cisplatin appears to be a better option as compared to pretreatment with BT followed by cisplatin treatment. These results support the fact that BT sensitizes cisplatin-resistant cells to respond better to cisplatin treatment.As reported by many, drug ratios identified as antagonistic in vitro provide inferior therapeutic activity in vivo compared with a synergistic drug ratio. Hence, it is very important to determine optimal drug ratios and then translate in vitro information on drug ratio-dependent synergy to in vivo efficacy using various drug delivery technologies. Combination drug delivery systems such as polymeric nanoparticles, dendrimers, liposomes, and water-soluble polymer-drug conjugates can be used to obtain synergistic anticancer effects while reducing concentration-related toxicity of the individual drugs , 32.. Oxidative stress and apoptosis are often the main factors contributing to the anticancer effects of drugs that target DNA [In addition to determining an optimal drug combination, the aim of this study was to investigate molecular mechanism(s) of action of BT/cisplatin combination in ovarian cancer cells in vitrorget DNA , 34. Sinrget DNA , 35, 36.There is a consensus pointing to apoptosis as the principal mechanism by which chemotherapy agents induce cancer cell death. The majority of anticancer drugs induce apoptosis via the intrinsic  pathway , 38. MitSeveral compounds have been shown to alleviate cisplatin-induced nephrotoxicity through the translational regulation of Bcl-2 , 41. BclROS are byproducts of normal cellular metabolism which play a crucial role in cell regulation and signal transduction . Many st(kip1) and p21 in ovarian cancer cell lines in in vitro [(kip1) and p21 in order to understand the role of these cell cycle regulators in causing antagonism or synergy of cisplatin and BT combination. Our results suggest that BT reduced the magnitude of cisplatin-induced cell cycle arrest by inhibiting P27 (kip1) and p21 expression in cisplatin-sensitive cell lines whereas it enhanced their expression in cisplatin-resistant cell lines. With P27 (kip1) and p21 being key inhibitors of cell cycle, our results are consistent with the cytotoxicity data where BT-cisplatin combination treatment potentiated and attenuated cytotoxicity in cisplatin-resistant and cisplatin-sensitive cell lines, respectively.Platinum derivatives have previously been shown to increase the expression of important cell cycle regulatory proteins such as p27 Kip1 and p21 Waf1/Cip1, leading to cell cycle inhibition , 54. Simin vitro . We asseIt is known that the major drawback of current cancer chemotherapy treatments is that the use of different drugs at high doses results in undesired toxicity, drug resistance and lack of expected efficacy (possibly as a consequence of antagonistic interactions between the drugs used). As reported in this study, drugs can be synergistic at certain ratios and antagonistic at other ratios implying the need to administer them at optimal doses. Therefore, there is a need to optimize drug ratios for every combination of drug used. Our results show that the use of cisplatin and BT at lower doses enhanced the efficacy of both drugs in most ovarian cancer cell lines tested. When used in combination with BT, cisplatin can be administered at lower doses, eliminating undesired side effects without compromising its efficacy. In this context, our study provides very significant systematic evaluation of BT-cisplatin interactions for possible application in drug/cisplatin resistance ovarian cancer scenarios.Our results show that BT and cisplatin can be synergistic at certain ratios and antagonistic at other ratios implying the need to administer them at optimal doses. When used at optimal concentrations, BT/cisplatin combination potentiated cisplatin cytotoxicity by increasing the accumulation of ROS and consequently activating pro-apoptotic pathways in cisplatin-resistant cell lines. Our results suggest that repurposing of BT to be used in combination with cisplatin represents an attractive and innovative approach to enhance/restore chemosensitivity of ovarian cancer to cisplatin. Further in vivo experiments may further contribute to our understanding and confirm the therapeutic potential of BT for patients with ovarian cancer.


Clustering of patient-derived glioma stem-like cells (GSCs) through unsupervised analysis of metabolites detected by magnetic resonance spectroscopy (MRS) evidenced three subgroups, namely clusters 1a and 1b, with high intergroup similarity and neural fingerprints, and cluster 2, with a metabolism typical of commercial tumor lines. In addition, subclones generated by the same GSC line showed different metabolic phenotypes. Aerobic glycolysis prevailed in cluster 2 cells as demonstrated by higher lactate production compared to cluster 1 cells. Oligomycin, a mitochondrial ATPase inhibitor, induced high lactate extrusion only in cluster 1 cells, where it produced neutral lipid accumulation detected as mobile lipid signals by MRS and lipid droplets by confocal microscopy. These results indicate a relevant role of mitochondrial fatty acid oxidation for energy production in GSCs. On the other hand, further metabolic differences, likely accounting for different therapy responsiveness observed after etomoxir treatment, suggest that caution must be used in considering patient treatment with mitochondria FAO blockers. Metabolomics and metabolic profiling may contribute to discover new diagnostic or prognostic biomarkers to be used for personalized therapies. Glioblastoma (GBM) is the most aggressive brain tumor in adults with a median survival of 14 months [in vivo) and high resistance to radiation and chemotherapy [in vitro using specific serum-free conditions facilitates the generation of GSCs [in vivo, and expressing an enriched proneural gene expression signature; (b) GSr, with a restricted stem-like phenotype showing expression signatures more similar to commercial cell lines  than to original patient tumors [As other cancers, GBM displays large heterogeneity among patients with relevant differences in genome, transcriptome, proteome, and metabolome features; in addition, it comprises quite different cell populations in the same patient , 3. Bothotherapy –6. Indee of GSCs . Subclas of GSCs  that incs e.g., T8G than tMetabolic reprogramming typical of cellular transformation  is gainiin vitro and in vivo mainly by means of MR techniques, particularly in brain tumors [Literature data on tumors of different origins  make then tumors . These ln tumors . Differen tumors , 17.In cancer cells, FA synthesis is upregulated due to the accelerated cell proliferation , 19. StuThe purpose of this study was to characterize the metabolic phenotypes of a large set of GSC lines. Clustering through unsupervised analysis of MR spectral data allowed us to identify three GSC subgroups with different cellular signatures. The existence of subclones in the same line with different metabolic phenotypes was also found, demonstrating intratumoral heterogeneity of GSCs. Cell energy metabolism was analyzed through combined examination of cells and culture media. A prevalence of aerobic glycolysis in cluster 2 lines was observed, while a role for lipids with contribution of mitochondrial FAO is present in some cluster 1 cells. Different responses to treatment with oligomycin and with the FAO inhibitor etomoxir were observed by both MRS and confocal microscopy and were related to GSC heterogeneity. Treatment with etomoxir produced different effects even in GSCs belonging to the same metabolic/genetic signature cluster that may account for differences of response to therapies.Tumor tissue samples were harvested from 44 patients undergoing craniotomy at the Institute of Neurosurgery, Università Cattolica del Sacro Cuore (UCSC), Rome, Italy. All the patients provided written informed consent according to the research proposals approved by the Ethical Committee of UCSC. Patients were eligible for the study if a diagnosis of GBM was established histologically according to the WHO classification.in vivo tumorigenic potential of GBM neurospheres from 30 out of 37 GSC lines was assayed by intracranial or subcutaneous cell injection in immunocompromised mice. GBM neurospheres were capable to generate a tumor identical to the human tumor both in antigen expression and histological tissue organization. GSC lines were validated by short tandem repeat (STR) DNA fingerprinting. Adult human brain olfactory bulb neural progenitor cells (OB-NPCs) were isolated from surgical specimens as previously described [GSC lines were isolated through mechanical dissociation of tumor tissues and cultured in a serum-free medium supplemented with EGF and basic FGF as described . The neuescribed .Cell proliferation was monitored by counting the cells and confirmed by using the Cell Titer-Blue Viability Assay (Promega) and presented as fold change with respect to control at time = 0.Clones from GSCs were obtained by plating single cells into 96-well plates. After 4 weeks, single clones were mechanically dissociated and replated to expand the culture.4 cells/ml were dispensed in each well of 96-well plates or in 75 cm2 flasks and incubated at 37°C in a 5% CO2 atmosphere. After one week of culture, cells were treated in triplicate with 1 μM oligomycin for 24, 48, and 72 hours or with 200 μM etomoxir for 6 hours and then used for MRS experiments.For oligomycin and etomoxir treatments, 2000 cells in exponential growth phase at a density of 2 × 102O and 2 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) as a frequency standard. A 15 μl aliquot of the suspension was transferred into a 1 mm NMR microtube and centrifuged to obtain a packed cell volume. Conditioned media were collected from cell cultures, added with 20% D2O and 2 mM TMSP, and transferred into a 1 mm NMR microtube.All analyzed cells were removed and washed in PBS and centrifuged at 162 rcf for 3 min. The pellet was suspended in PBS with 20% DAll MRS reagents were purchased from Cambridge Isotope Laboratories, Inc.Male Wistar rats, from Harlan Laboratories, were anesthetized with a mixture of ketamine (90 mg/Kg b.w.) and medetomidine hydrochloride (0.4 mg/kg b.w.) and euthanized by decapitation, and then the brain was removed.All procedures related to animal experiments and care were performed in accordance with the Legislative Decree n.116/92 , which r2O and 2 mM TMSP and transferred into the NMR microtube.The brain was homogenised using a lancet, suspended in PBS, and centrifuged. The pellet obtained was suspended in PBS with 20% DNile red-stained samples  were analyzed by confocal microscopy and flow cytometry, in order to visualize the intracellular presence/distribution of lipid droplets and to indirectly quantify their content, respectively . For con1H MRS experiments were run on a digital Bruker Avance spectrometer at 400.14 MHz, equipped with a 1 mm microprobe. Both one dimensional (1D) and two dimensional correlation spectroscopy (2D COSY) experiments were performed, at T = 298 K.1D 1H MRS spectra of GSCs and culture media were acquired with a 90° RF pulse, and the number of scans (ns) was equal to 1000  for cell spectra while ns = 4000 was used for culture media spectra. When indicated, a Lorentzian-Gaussian function was applied in the time domain, before Fourier transformation.2D COSY spectra were acquired with a 90°-t1–90°-t2 pulse sequence and ns = 32 for cell or ns = 128 for culture media samples. Spectra were acquired as a matrix of 512 × 128 data points in time domain.t-test was performed utilizing XLSTAT software, Addinsoft, version 2012.2.02.Unsupervised agglomerative hierarchical clustering was performed utilizing XLSTAT software, Addinsoft™, version 2012.2.02. Values of log2(FC) ((FC) fold change) resulting from the comparison between the clusters 1a,1b, and 2 were calculated for each metabolite. Student's Forty-four GSC lines, derived from the surgical specimens of newly diagnosed GBM patients, and ten subclones, five from GSC line #1 and five from #83, were profiled by MR spectroscopy to identify tumor groups with different metabolisms. Metabolic profiling of the normal rat brain was also performed in order to compare detectability of brain metabolites and their assignments.Many metabolite signals were detectable in all samples, although with considerable differences of intensity, thus characterizing prevalence of different metabolism.Spectra (low and high field regions) of normal rat brain and of three GSC lines are shown in Supplementary FigureNAA, GABA, Gln, A (ML cross 2D peak), Myo-I, Asp, Glu, glutathione (GSH), and N-acetylgalactosamine  were quantified in 2D COSY spectra, whereas UDP-hexosamine and tCr in 1D spectra as previously described . MoreoveUnsupervised cluster analysis of MRS signal intensities from spectra of forty-four GSC lines, five clonal sublines of line #1 and five of line #83, T98G cells, and OB-NPC cells, all previously profiled , and of Signal intensities of the fifteen metabolites for the three clusters are reported in Clusters 1a and 1b lines were characterized by a prevalent neural fingerprint, with the highest intensities of neural markers NAA and of tCr. Furthermore, cluster 1b when compared to cluster 1a had the highest intensity of the astrocyte marker Myo-I, while cluster 1a showed peaks for Gly and Gln. Cluster 2 lines, with the highest intensity of lipid signals, high GPC, and very low PC, NAA, and tCr signals, were characterized by a prevalent fingerprint of serum-cultured tumor lines .Interestingly, our previous results on gene expression of 35 GSCs , 12 evidPrevalence of different metabolic profiles assigned subclones of line #1 in cluster 1 and subclones of line #83 both into clusters 1 and 2. This observed intracell heterogeneity could be relevant in patient's response to therapies. The line T98G was in cluster 2 while line OB-NPC was classified in cluster 1a .ML signal intensity was one of the parameters that mostly characterized cluster 2 cells compared to cluster 1a and 1b cells. To clarify the origin of ML signals, two GSr-like lines from cluster 2 (lines #61 and #74) and two GSf-like lines from cluster 1a (lines #1 and #163) were examined by MRS, confocal microscopy, and flow cytometry. Lines #1 and #163, although belonging to the same metabolic cluster 1a and to the same GSf-like group , were geConfocal microscopy and flow cytometry showed that LDs were present in the cytoplasm of all examined lines, but their content was higher in lines #61 and #74 compared to lines #1 and #163 Figures . In paraLactate (Lac) extrusion was examined in culture media spectra  to evaluA connection between glycolytic metabolism and lipid droplet content is suggested by comparison of data from lines #1 and #163 with lines #61 and #74. Moreover, Gln consumption increased as a function of cell number during cell growth (data not shown) for all examined lines, thus confirming that these cells were all Gln addicted.Different energy sources may be exploited by cells from clusters 1a and 2, possibly including mitochondrial FAO. Thus, the role of FAO in #61, #74, #1, and #163 cell lines was investigated.The effects of oligomycin, a mitochondrial ATPase inhibitor, were firstly examined. Oligomycin had no effect on cell proliferation in lines #61 and #74 up to 24 and 48 hours of treatment while a proliferative arrest was detected in lines #1 and #163. For longer time intervals, oligomycin was toxic to all cell lines FigureA. MR speConfocal microscopy revealed an increase of LDs after oligomycin treatment in all cell lines even if at different extent Figures  that matOligomycin induced an increase of lipid signal intensities in cluster 1a lines and had no effect on those in cluster 2. At the same time, the intensity from coalescent 1D signal spectra of PC + GPC decreased in lines #1 and #163 and increased or remained unaltered in the other two lines . A sharpcon/Lacoli ratio were stable after 24 hours of treatment  in these cells. The preferred oxidation process in cells is n cancer .β-oxidation to meet the cell energy requests [β-oxidation leads to lipid droplet accumulation after MYC inhibition in NB cells [β-oxidation in VERO fibroblasts [In our view, the high lipid amount stored as droplets plays a role in FAO of some cells. LDs may be associated with mitochondria in many cells by the Perilipin protein family , 36. It requests . This meNB cells . MoreoveNB cells  and LDs roblasts .Present data showed higher accumulation of intracellular LDs when mitochondrial activity was impaired in response to oligomycin treatment in lines #163 and #1, both from cluster 1a, and characterized by lower aerobic glycolysis. In these two lines, the oligomycin treatment induced proliferation arrest, indicating the important role played by TCA cycle.In line #163, cytoplasmic lipids increased after treatment with etomoxir that blocks CPT1, transporter of long FAs into the mitochondrial membrane, thus affecting FAO. Increased LDs observed after mild etomoxir treatment in line #163 reinforce the hypothesis that FAO supports energy requirements of this cell line. This result expands previous evidence on FAO inhibition obtained under metabolic stress  or by MYIn line #1, etomoxir treatment induced ML and LD decrease and proliferation increase differently from line #163 suggesting that the storage lipids are more utilized for membrane synthesis. The different responses to etomoxir treatment showed by the two low lipid-rich cell lines are difficult to explain. Both lines, in fact, belonging to the same metabolic and genetic groups, were characterized by low Warburg and by ML and LD increase after mitochondrial disfunction by oligomycin.After etomoxir treatment of line #1, TCA cycle is not massively altered, as indicated by no increase of fumarate signal intensity, contrary to what happens in this line after oligomycin treatment and in line #163 after both treatments. On the other hand, glucose decrease together with almost absent changes of lactate extrusion in line #1 would be suggestive of an increase of mitochondrial glycolysis to meet energy requirements necessary for proliferation increase. FAs may then be used for membrane synthesis sustaining accelerated proliferation.ω-oxidation, blockage of peroxisomes, and defective activity of succinic dehydrogenase (SDH) were all indicated as possible sources of the anomalous adipate increase. In line #163, adipate mostly derived from lysine catabolism through mitochondria as blockage of this catabolic pathway by oligomycin leads to formation of the intermediate saccharopine while this molecule is almost lacking in line #1 after the same treatment. This could indicate a peroxisome involvement in the lysine degradation, but peroxisomal contribution to FAO is not likely due to failure of lipid increase in line #1 after etomoxir treatment. On the other hand, succinic aldehyde, final product of SDH, is present in MR spectra of both lines #1 and #163 (not shown).A major metabolic difference between the two lines is that line #1, different to line #163, showed almost absent saccharopine formation after oligomycin treatment and belongs to one of the two GSC groups characterized by low/absent adipate content . These tA further hypothesis to explain differences between lines #1 and #163 could be derived by a different presence of the CPT1 isoforms  , 44. RecA different role for CPT1 in line #1 and line #163 could be, therefore, envisaged by present data as well as a role for the adipate source. Although further studies are necessary to clarify this point, the relevant result is that not all GBM cells behave in the same way after treatment that impairs FAO. Caution must therefore be used in considering patient treatment with etomoxir or other mitochondria FAO blockers. Inhibition of CPT1 by etomoxir is proven to suppress cancer growth in glioma cells , and eto


Quadrartus yoshinomiyai, a gall-forming aphid endemic to Japan) have individuals too old to reproduce been shown to be both numerous in natural habitats and able to help close relatives survive or reproduce. Pea aphids, (Acyrthosiphon pisum), common, tractable organisms, frequently outlive their reproductive ages in laboratories, live in tight interacting groups that are often clonal, and therefore should be evaluated as potential model organisms for the study of adaptive post-reproductive life. The first major step in this process is to identify an optimal method for assessing if a parthenogenetic adult is post-reproductive. We evaluated three methods, relying respectively on isolation in clip cages, visual examination for embryonic eyespots, and dissection. In every case each method identified the same individuals as reproductive versus post-reproductive. While the clip-cage method requires a multi-day wait to produce data, and dissection is inevitably fatal, the eyespot method is quick  easy, and non-invasive. This method makes it possible to accurately assess the post-reproductive status of a large number of parthenogenetic pea aphids. We demonstrate the usefulness of the eyespot method in showing that while reproductively valuable adults tend to place themselves near the centers of clonal colonies, less valuable post-reproductive adults are more often at or beyond the edges of colonies. These encouraging early results provide both impetuous and aid for further investigations into the post-reproductive lives of pea aphids.The role of kin-selection in the evolution of post-reproductive life is controversial. While anthropological and demographic studies strongly suggest that humans and a few other species experience kin selection for significant post-reproductive survival, these results are necessarily correlational. Understanding could therefore be advanced by the development of a globally available, field and laboratory tractable experimental model of kin-selected post-reproductive survival. In only one invertebrate ( Humans were long considered to be the only species in which many individuals lived well past the age at which they stopped reproducing . EvolutiA great taxonomic diversity of organisms shown have the capacity for post-reproductive life , but forEven the study of specifically kin-selected post-reproductive life has expanded well beyond female primates. In the last decade, strong arguments for adaptive post-reproductive life-stages has emerged in men  and resiQuadrartus yoshinomiyai, a Japanese gall-forming aphid . Pea aphids are widely available, the subject of a large and diverse body of previous and ongoing research (Acyrthosiphon pisum Harris). Pea aphids are facultative parthenogens with complex life cycles, but clonal colonies are dominated and perpetuated by individuals that reproduce only asexually. These parthenogenetic pea aphids are born with a complete stock of embryos; when it is depleted, they are definitively post-reproductive. For a recent review on the life stages and development of pea aphids, see A likely species to examine in this respect is the pea aphid  that grow through parthenogenetic reproduction. Although in some cases these aggregations may contain more than one clonal lineage, relatedness within pea aphid colonies is generally high . While aAlthough parthenogenetic pea aphids have all of these advantages, it is not yet proven that they have an evolved post-reproductive life stage. Indeed, the behavior and field ecology of post-reproductive pea aphids remain largely unstudied. If post-reproductive individuals make up a significant proportion of natural pea aphid populations and have a meaningful positive net effect on their relatives’ fitness, then they will be an excellent experimental model for studying kin-selected post-reproductive life. The present study aims to lay the groundwork for testing these key points. Our specific goals are twofold. First, we aim to perfect a method for distinguishing post-reproductive parthenogentic pea aphids from younger adults. We test three methods of determining post-reproductive status against each other to determine their accuracy and logistical desirability. We demonstrate a method that is reliable, easy, fast and non-invasive.Q. yoshinomiyai move toward the gall openings, where defense from predators is most often needed, while reproductive members of the same clones move away from the opening into the safety of the gall. Post-reproductive pea aphids could best serve some defense function if they move to the edges of the colony. While evidence for post-reproductive individuals being concentrated away from the center of colony would not prove that they are protecting the colony, knowing that they do so would be a useful early step in exploring possible adaptive roles for post-reproductive pea aphids.We then employ that method to achieve our second goal: testing the prediction that post-reproductive individuals, being of low reproductive value to the group, tend to occupy the edges of colonies, where predation risk is predicted to be higher, and where defensive activities would most likely be needed. This prediction arises from two sources. Acyrthosiphon pisum was obtained from a bean plant at Fresh Start Farms, an organic farm in Lisbon, Maine and allowed to reproduce parthenogenetically in the laboratory on Vicia faba (fava bean plants) grown from seed . The population was founded from one individual reproducing clonally. While pea aphids come in both green and red morphs, this population was entirely green. We kept the population of aphids at 22 ± 3 °C. Temperatures inside clip cages were within 0.4 °C of room temperatures. We placed the plants 20 cm below fluorescent greenhouse lights with a 14/10 light/dark cycle.A single wild All focal individuals in our methods-development experiments were apterous (wingless) parthenogenetic adults. They were gathered from our stock population by gently shaking infested leaves over a petri dish. Care was taken to gather adults from multiple plants. Individual adults were then carefully picked from the dish using a fine paintbrush. Adults were easily distinguished from late nymphs (Instar IV) based on morphology . We mainWe evaluated three separate methods for assessing reproductive status of wingless parthenogentic adult pea aphids, beginning with two pre-existing methods available for determining whether a pea aphid is post-reproductive. The first was to isolate an individual and regularly remove her offspring . It is kThe second method of determining whether an aphid is post-reproductive requires no waiting, but is fatal: dissecting the aphid to observe if she has any developing embryos . ParthenA third, novel method is also possible. Pea aphids, including embryos, have black to red-pigmented eyes. Eyespots of developing embryos can often be seen within the intact abdomen of live parthenogenetic mothers . If thesWe kept every focal individual in a clip cage to determine when she stopped reproducing, then dissected to determine if she had depleted her stock of embryos. After gathering data on the first 19 individuals, we observed that the eyespots of embryos were often visible through the body wall of reproductive adults. For each of 38 further focal individuals we assessed whether eyespots were visible inside each abdomen. This was done after caging but before dissection. We blinded the results between each method, to ensure independence of results. Each method, and blinding procedures, are described in greater detail below.We made clip-cages by cleanly cutting two rings of foam pipe-insulator . One ring had 1 mm meshed fabric glued to it so the aphid could breathe but not escape . We placWe isolated focal individuals in clip cages . Every two days, we removed all nymphs from each cage and resealed the adult in the clip cage. If the leaf was damaged or wilting we moved the clip cages to a new leaf. When we found no offspring within the clip cage for two checks (four days) in a row, that individual was recorded as demographically post-reproductive.After three weeks in clip cages, roughly half of individuals  had been labelled as demographically post-reproductive. At that point each individual was isolated in a covered aliquot tube for further study. Blinding (see below) ensured that each individual’s status was recorded but not known during further examination.To determine if each individual was physiologically post-reproductive, we dissected her under a microscope (Leica Ez4) at 20x magnification to look for embryos in her ovaries. We placed the aphid supine on a rubber tray and anchored her by carefully piercing her head with a size 000 dissecting pin. If necessary, the posterior edge of the abdomen was also pinned down. Next, we peeled the venter of the abdomen off using the tip of another pin or fine forceps shown in . We obseTo check for eyespots we removed each aphid from her test tube and looked at her both supine and prone under 20x magnification . We founIn order to make results from our three methods experimentally independent of each other we used the simple blinding method of placing each individual in an aliquot tube after her removal from the clip cage, writing her data on her tube, and then covering the tube in aluminum foil. After the eyespot method, we wrote our data on the aluminum foil then covered it with another layer of foil. Results from dissections were written on this final layer of foil. At the end of the experiment these layers were peeled off and the data tabulated for comparison. Data analysis consisted of tallying the number of cases in which each method agreed with the others.In order to examine whether post-reproductive individuals are more prone to move away from the center of a pea aphid colony than are reproductive individuals, we established seven colonies, each on a separate fava bean plant. Previously uninfested plants were covered in fine mesh bags (with zippers for experimenter access) and inoculated with six aphids each: two nymphs, two reproductive adults, and two post-reproductive adults . All six were placed on the top of the largest leaf of each plant. These aphids were then given five to seven days to establish themselves on the plant, reproduce, and organize themselves into a colony. At the end of this time, the bag was unzipped and the plant thoroughly examined for aphids. All aphids on the plant were photographed in situ with a 4.5 mm 12 Megapixel iPhone 6S camera and the colonies left intact for further study. Preliminary testing showed that photographs taken in this way consistently revealed intraabdominal eyespots where present. From these photographs, each aphid was classified as being a nymph, reproductive adult or post-reproductive adult. Nymphs were distinguished from adults by observing their cauda (tail-like appendage), and reproductive and post-reproductive adults were distinguished using the eyespot method. Digitally zooming into the photographs was often necessary to observe the eyespots. Separately, each aphid was classified as being internal to the colony, at the edge of the colony, or not on the same leaf as the colony. All plants had a single clearly identified colony and most had a few individuals on other leaves. The ‘center’ was defined as in the colony, surrounded by other aphids on at least three of four sides. The ‘edge’ was defined as any location on the same leaf as the colony, but not surrounded by other aphids on at least three of four sides.In every case, all methods agreed on each individual’s reproductive status. Of the 57 adult apterous aphids that were kept in clip cages, we found 31 to be reproductive and 26 to be post-reproductive. All 57 of these were dissected and 38 were examined for internal eyespots. Of these 38, 23 had spots, and 15 had none. All individuals scored as demographically reproductive were also physiologically reproductive, and all of these examined had embryonic eyespots visible. Likewise, all post-reproductive individuals were identified as such across methods.stats, R 3.3.1, As previously reported, reproductive individuals were mostly positioned within the colony   with theOur fast, easy, accurate, and non-invasive method for identifying post-reproductive pea aphids reveals that they, in contrast to reproductive adults, are generally positioned at the edges of or away from colonies.All three methods we tested identified the same parthenogenetic pea aphids as post-reproductive. However, there are significant logistical differences between these methods. The clip cage method requires waiting at least four days after reproduction ceases before an individual is certainly post-reproductive. While we observed no cases in which an individual had no offspring for four days but later reproduced  we did observe four interbirth intervals of two days. Also, this method is comparatively labor intensive because caging each individual aphid took about three minutes each time and cleaning off any offspring took an additional minute per individual at each check. During checks, care must be taken to avoid allowing the focal aphids to escape. This method is effective at creating a population of post-reproductive individuals for study, but as a diagnostic method requires some days of waiting during which some individuals may die. In contrast, dissecting each individual , while fThe eyespot method is the fastest, easiest, and least invasive method of determining the reproductive state of an aphid in our population. Since sexual reproduction involves the laying of eggs containing early stage embryos unlikely to yet have eyespots, this method likely wouldn’t work beyond the parthenogenetic context. Examination of 10 winged parthenogenetic individuals in our lab revealed eyespots visible in the abdomens of all 10, implying that wingedness does not hinder the use of our method. Red pigment in the mother’s body wall could potentially make seeing the colored eyespots of her embryos more difficult. While we have no data on red morph individuals, review of some published images of red pea aphids reveal that in at least some of them  spots coPost-reproductive pea aphids, unlike younger adults, were most often found at the edges of the colony or on a different leaf entirely. Several mechanisms may explain this difference. Possible adaptive explanations include these post-reproductive individuals helping their kin in the colony by acting as guards or sentinels of some kind, or simply making space within the colony for reproductive adults and nymphs, both of which have higher reproductive value to the clone. Pea aphids, like many other aphids, use an alarm pheromone, and post-reproductive sentinels could potentially alert the colony to an approaching predator or parasite. Non-adaptive explanations, such as decreasing mobility and sensory acuity in senescent adults causing them to separate from the colony, are also possible.Quadrartus yoshinomiyai). It may be that this pattern is found more broadly across aphids. If so, post-reproductive aphids of many species may play some important role in the life of the colony.Post-reproductive individuals are found in the most exposed locations in both pea aphids and Japanese gall aphids  an organism that frequently experiences post-reproductive life in the wild, (b) where post-reproductive life has a strong positive selective value, (c) in which post-reproductive individuals are non-invasively and easily identifiable, (d) that is easy to keep and study in the laboratory, (e) that is available to experimenters all over the world, and (f) which has already been studied by a wide range of other biological disciplines. While there are several candidate species proposed to have (a) and (b), and most common laboratory animals have (d), (e) and (f), we as yet have no organism that combines all of these desirable properties. Whether field population of pea aphids experience significant post-reproductive life is a key question. The measure necessary to answer that question is Post-reproductive Representation (PrR) which is equal to the proportion of the mean individual’s adult life which is lived after the age at which members of her (or his) population have completed most of their fertility. PrR and significance testing thereof are introduced and derived by Levitis and Bingaman Lackey, and its evolutionary use expanded upon by The study of the evolution of post-reproductive life has long focused on humans, alone or in a comparative context. Shifting focus from understanding humans to understanding the evolution of post reproductive life allows for, perhaps requires, examination of post-reproductive life in species that are in many ways dissimilar from humans. Aphids, because of their reproductive physiology, complex life-cycles and sociobiology are appealing targets for investigation of potentially kin-selected post-reproductive life stages. We have shown that post-reproductive pea aphids can easily, accurately, and non-invasively be identified using the eye-spot method, and that these individuals place themselves distal to the rest of the colony. This raises several key questions about post-reproductive life in pea aphids. Is it adaptive, or merely the result of demographic stochasticity? How often do post-reproductive individuals occur in populations outside of the laboratory, and do they behave in the field as we have observed in the lab? Do they help to protect younger kin from predators or parasites? Do they aid in the growth or reproduction of kin? Do they impose costs on kin? In what respects (other than placement and reproduction) does the behavior of post-reproductive individuals differ from that of reproductive? Do many species of aphids have adaptive post-reproductive survival? Answering these questions may advance the study of kin-selected post-reproductive life stages from being largely observational and correlational to having an established experimental model.10.7717/peerj.2631/supp-1Supplemental Information 1The experiment was done in five sessions, each represented by a different box. Each box notes the day of the experiment for that given session when analyzing the clip-cage method. The presence of nymphs was marked every two days for the clip-cage method. If nymphs were present, the corresponding row was marked with “Nymphs” and if no nymphs were present, it was marked with “0.” Below each box, the results for both the clip-cage method and dissection method are labeled with either “R” for reproductive, or “P” for post-reproductive.Click here for additional data file.


In conclusion, while LTT lines displayed diversity in NRF2 activation, NRF2 signalling contributed to cisplatin resistance in LTT lines, albeit in diverse ways. Accordingly, inhibition of NRF2 can be used to resensitise UC cells to cisplatin, but responses in patients may likewise be variable.Therapeutic efficacy of cisplatin-based chemotherapy for advanced-stage urothelial carcinoma (UC) is limited by drug resistance. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway is a major regulator of cytoprotective responses. We investigated its involvement in cisplatin resistance in long-term cisplatin treated UC cell lines (LTTs). Expression of NRF2 pathway components and targets was evaluated by qRT-PCR and western blotting in LTT sublines from four different parental cells. NRF2 transcriptional activity was determined by reporter assays and total glutathione (GSH) was quantified enzymatically. Effects of siRNA-mediated NRF2 knockdown on chemosensitivity were analysed by viability assays, γH2AX immunofluorescence, and flow cytometry. Increased expression of NRF2, its positive regulator p62/SQSTM1, and elevated NRF2 activity was observed in 3/4 LTTs, which correlated with KEAP1 expression. Expression of cytoprotective enzymes and GSH concentration were upregulated in some LTTs. NRF2 knockdown resulted in downregulation of cytoprotective enzymes and resensitised 3/4 LTTs towards cisplatin as demonstrated by reduced IC Bladder cancer is the 9th most common tumour world-wide and the most common cancer of the urinary tract . About 9NFE2L2), a regulator of cytoprotective enzymes, glutathione biosynthesis and metabolism that controls cellular responses towards oxidative stress [Resistance to cisplatin can be due to various mechanisms ,11, incle stress ,15,16,17NRF2 is a cap’n’collar basic-region leucine zipper transcription factor consisting of a large p45 and a small p18 subunit ,22,23,24Constitutive NRF2 activation is common in cancer  and faciNFE2L2 gene, typically missense mutations in the Neh2 domain required for KEAP1 interaction that are expected to cause NRF2 overexpression [As in some other cancer types, a significant fraction of UC contain mutations in the pression ,43. Likepression . NeverthIn order to study cisplatin resistance mechanisms in detail, we have previously established cisplatin-resistant sublines from four different UC cell lines (UCCs) by long-term treatment with escalating cisplatin doses over several months. Development of resistance was accompanied by significant phenotypic changes with increases in markers of epithelial-to-mesenchymal transition (EMT) and canonical WNT pathway target genes ,45. In tWe first evaluated expression of NRF2 and its regulators across a panel of UCCs covering the heterogeneity of the disease and then, in particular, compared the four long-term cisplatin treated LTTs with their parental cell lines. Across the UCCs, NRF2 and KEAP1 protein expression were heterogeneous and tended towards the expected inverse pattern, whereas p62/SQSTM1 expression was more uniform . NRF2 prNRF2 and KEAP1 mRNA expression were both significantly decreased in three LTTs compared to their parental cell lines, whereas p62/SQSTM1 mRNA was upregulated. T-24-LTT was again the exception with no significant difference in NRF2 or p62/SQSTM1 mRNA, but increased KEAP1 mRNA expression . NRF2 transcriptional activity was measured by a reporter assay following transfection with an ARE-luciferase expression construct. Compared to the untreated control, ARE-dependent luciferase activity was increased in RT-112-LTT, J82-LTT, and 253J-LTT, but was significantly decreased in T-24-LTT. Co-transfection with an NRF2 expression construct significantly increased luciferase activity in all four LTTs as well as in the parental lines; however, in T-24-LTT the inducible NRF2 luciferase activity remained lower compared to the parental cell line b. RemarkNFE2L2 and KEAP1 genes. According to the TCGA data on UC [NRF2/NFE2L2 are usually located in exon 2 and were detected in 14 (11%) of 126 sequenced UC. KEAP1 was altered in 10 (7%) of 126 sequenced cases/patients, with 4 missense and 1 truncating mutation , but a non-silent missense mutation in KEAP1 exon 2, namely c. 334A>T (T112S), in RT-112-LTT, which was not present in the parental cell line  urothelial carcinoma cell lines (UCCs), fitting the established notion of NRF2 as a generally important factor in the development of chemoresistance and as a rational target to restore chemosensitivity . Upon cl50 values for cisplatin in RT-112-LTT, 253J-LTT, and T-24-LTT and this sensitisation was paralleled by increased γH2AX foci formation, in line with the presumed functions of NRF2. Unexpectedly, only a minor sensitisation was achieved in J82-LTT by NRF2 knockdown, which, also unexpectedly, instead induced apoptosis in T-24-LTT even in the absence of treatment (see detailed discussion below). Concordantly with our results, NRF2 knockdown sensitised the lung carcinoma cell line A549 to cisplatin, whereas its stable overexpression enhanced cisplatin resistance of breast adenocarcinoma and neuroblastoma cells MDA-MB-231 and SH-SY5Y [In detail, the three resistant cell lines RT-112-LTT, J82-LTT, and 253J-LTT expressed more NRF2 protein, which was localised in the nucleus, and was associated with basal and inducible NRF2 activity in reporter assays. NRF2 protein expression was unchanged in a fourth resistant cell line, T-24-LTT, compared to parental T-24 cells. SiRNA-mediated knockdown of NRF2 decreased IC SH-SY5Y . Similar SH-SY5Y .NFE2L2 exon 2 represents an alternative mechanism for activation of NRF2 in squamous carcinomas [Cip1/Waf1, which competes with oxidised KEAP1 for binding to the NRF2 DLG motif to enhance the stability of the transcription factor [As a possible cause of increased NRF2 expression, we found elevated p62/SQSTM1 protein and mRNA expression in RT-112-LTT, J82-LTT, and 253J-LTT. Analogously, cisplatin-resistant ovarian cancer SKOV3 cells expressed more p62/SQSTM1 and were resensitised upon knockdown of p62/SQSTM1 ,57. The rcinomas . In heparcinomas ,59. Howercinomas . In theircinomas  observedrcinomas . Of notercinomas ,62 couldn factor ,64,65,66Many cytoprotective enzymes inducible by NRF2  were uprIncreased glutathione availability with consequently decreased intracellular ROS formation is another potential consequence of NRF2 activation that protects against cisplatin by forming extrudable conjugates and by ameliorating oxidative stress. For instance, higher GSH levels were observed in cisplatin-resistant A2780 ovarian carcinoma cells and the U373MG glioblastoma cell line ,69. AnotThe diversity of NRF2 regulation is further illustrated by the J82-LTT line, which presented high basal NRF2 protein expression and enhanced reporter gene activity. Nevertheless, efficient siRNA-mediated knockdown of NRF2 had minimal effects on cisplatin sensitivity in this resistant subline. Indeed, expression of cytoprotective enzymes and of genes involved in GSH transport and biosynthesis was largely unchanged in J82-LTT compared to its parental line. As nuclear immunofluorescence staining of NRF2 was not as prominent in J82-LTT as in RT-112-LTT and 253J-LTT, we suspect that NRF2 might be prevented from activating its target genes in a KEAP1 independent manner. An established mechanism that might explain this constellation involves phosphorylation of nuclear NRF2 by GSK3β and recognition of phosphorylated NRF2 by β-TrCP in the ubiquitin E3 ligase complex SCF (Skp1-Cullin-F-box), leading to its poly-ubiquitination and degradation ,73,74,75Whereas NRF2 protein was increased in all other LTTs, it was unchanged in T-24-LTT compared to its parental cell line. Basal NRF2 activity was decreased, but luciferase activity was inducible, albeit to a lesser extent than in its parental cell line. Expression of p62/SQSTM1 remained essentially unchanged, but KEAP1 protein and mRNA were upregulated. However, the low amount of NRF2 present proved essential in this cell line, as siNRF2 knockdown not only sensitised T-24-LTT to cisplatin, but also induced apoptosis and decreased clonogenicity in the absence of cisplatin especially in this cell line. Suppression of cell proliferation by complete siNRF2-mediated knockdown has also been observed in the cholangiocarcinoma cell lines KKU-156 and KKU-100 and was similarly enhanced by cisplatin treatment . The senTwo other potentially interacting pathways, the Hippo and NF-κB pathways ,12, wereIn conclusion, our results indicate that NRF2—in different ways and to different extents—is a key player in the development of cisplatin resistance in UCCs and may constitute a reasonable target to combat chemoresistance in UC. Nevertheless, the diversity in NRF2 activation among the four tested LTTs highlights the complexity of cisplatin resistance even within one tumour entity and predicts that responses to NRF2 inhibition may likewise be highly variable in patients.50 doses were determined after treatment with a single IC50 dose of cis-diamminedichloroplatinum-II  for 72 h by MTT assay. For short-term experiments (STT) a single IC50 dose cisplatin was added for 72 h. As previously reported, long-term treated (LTT) UCCs were generated by adding cisplatin after every passage at escalating doses over months with constant end concentrations of 50, 3.3, 6.6, and 23 µM in RT-112-LTT, J82-LTT, 253J-LTT, and T-24-LTT, respectively [The human UC cell lines RT-112, VM-CUB-1, UM-UC-3, T-24, 639V, 253J, 5637, SW-1710, HT-1376, BFTC-905, and J82, kindly provided by M. A. Knowles , J. Fogh , B. Grossmann , or the DSMZ , were grown in DMEM GlutaMAX-I  containing 10% FCS. All cell lines were recently authenticated by standard DNA fingerprint analysis. ICectively . Reporteectively , and GL3ectively , were trSDHA was used. DNA was extracted using the Blood & Cell Culture DNA Midi Kit according to the manufacturer’s protocol .RNA isolation, cDNA synthesis, and quantitative real-time PCR were performed as previously described . qRT-PCRNFE2L2 and KEAP1 mutation analysis 468 and 842 bp amplicons containing exon 2, respectively, were amplified following PCR from genomic DNA. Primer sequences are detailed in For Cell viability was measured in quadruplicates by means of MTT assay  and CellTiter-Glo assay .2, and 1.6 mM CaCl2. For the quantification of intracellular ROS-concentration, cultured cells were incubated in medium without phenol-red and FCS containing 10 µM 2′,7′-dichlorodihydrofluorescein diacetate  for 30 min in the dark at 37 °C. Following washing with PBS, scratched cells were taken up in PBS. Flow cytometry analyses were performed using the Miltenyi MACSQuant Analyser with MACSQuant analysis software 2.6 .Cell-cycle analyses were performed by staining the attached and supernatant cells with buffer containing 50 µg/mL propidium iodide (PI), 0.1% sodium citrate, and 0.1% Triton X-100 . For assFor staining of NF-κB p65  and pH2A.X Ser139 , cells were fixed on coverslips with 4% formaldehyde for 10 min and permeabilised at room temperature for 10 min in 0.5% Triton-X100 in PBS. For NRF2 staining , cells were fixed with methanol at −20 °C for 10 min and permeabilised at room-temperature for 10 min in 0.5% Saponin in PBS. Cells were blocked for one hour at room temperature in 1% BSA for NF-κB p65 and in 10% goat serum for NRF2 staining or 0.3 M glycine, and 0.1% Triton-X for pH2A.X Ser139 staining. Secondary antibodies Alexa Fluor 488 Goat Anti-Mouse IgG or Alexa Fluor 488 Goat Anti-Rabbit IgG  were added for 1 h in the dark at room-temperature. Cover slips were counter-stained with 0.5 mg/mL DAPI. Quantification of pH2A.X Ser139 foci was performed using the Focinator software tool .Total protein was extracted by lysis for 30 min on ice in RIPA buffer pH 7.6  and 1% phosphatase inhibitor . Determination of protein concentrations and western blot analysis of whole-cell extracts was performed as described previously . PrimaryTotal glutathione was measured enzymatically by the method of Tietze ,90,91 anNRF2/NFE2L2 and KEAP1 mutations in 413 bladder urothelial carcinoma samples (TCGA) were analysed using the cBioPortal for Cancer Genomics ,47.


As a possible viable and non‐invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non‐invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones. CHOChinese hamster ovary CRMConfocal Raman MicroscopyERendoplasmic reticulumFACSfluorescence activated cell sortingPDIprotein disulfide isomerase10.1002/biot.201600412See accompanying commentary by Katja Schenke‐Layland DOI 1Chinese hamster ovary (CHO) cells are the most abundantly used mammalian cells for the production of recombinant biopharmaceuticals Raman spectroscopy relies on the inelastic scattering of light when it interacts with matter. Changes in the polarizability of molecules cause an exchange of energy between the incident and the scattered light due to molecular vibrations (e.g. bond stretching). This energy shift is measured and the resulting Raman spectrum thus provides a ”molecular fingerprint“ with information about specific molecular bonds and their corresponding micro‐environment ex 532 nm) gained from the optical microscope and the sensitive chemical information derived from the Raman spectra Confocal Raman Microscopy (CRM), however, is a non‐invasive and label‐free imaging method that can be performed in situ on single live cells in a non‐destructive manner The large amount of data generated in Raman imaging and the possible presence of overlapping bands in a spectrum require an unbiased way of unmixing the spectral information In the current work, we have used CRM to distinguish different CHO host and producer cell lines in a label free manner, without prior sample preparation. Indeed, we show that subcellular structures that are enriched in specific chemical compounds like proteins, lipids, DNA or RNA and metal ions can be visualized by combining Raman spectroscopy with multivariate analysis using cluster analyses and PCA. Specifically a subcellular structure with high lipid and protein content, resembling the ER, was clearly distinguishable, whose size correlates to the production capacity of a cell line. Our data therefore suggest that CRM might be a valuable analytical tool for identification of cells with biotechnologically relevant characteristics during cell line development or engineering, but potentially also for quality control, proof of identity and stability of production cell lines.22.1®), supplemented with 8 mM L‐glutamine (Merck Millipore) and 0.2% Anti‐Clumping Agent (Gibco®) in shaker flasks at 140 rpm, 37°C, 7% CO2. CHO‐K1‐hDAO medium was in addition supplemented with 10 µM CuSO4 (Sigma Aldrich) and 10 µg/mL Blasticidin‐S‐HCl (Life Technologies). Viability was measured by Trypan Blue and better than 95% during all experiments.Host cell lines included CHO‐S (Invitrogen) and its subclone CHO‐S/4F11, CHO‐K1 (ECACC CCL‐61) and its subclones CHO‐K1/4F10 and 1D9. The subclones were isolated after multiple rounds of cell sorting for increased transient productivity g for 7 min and resuspended in fresh, pre‐warmed growth medium. Cell suspension (15 µL) was applied to a glass microscope slide, covered with a #1.5 glass cover slip  and sealed with nail polish.For CRM analysis, 1.5 mL cell suspension were centrifuged at 170 × 2.2ex = 532 nm . The scattered Raman signal was detected with an optic multifiber (50 nm diameter) directed to a spectrometer UHTS 300  (600 g mm−1 grating) and finally to the CCD camera . Control Four  acquisition software was used for the Raman imaging set up. Spectra were recorded in 1 µm X/Y steps for all samples with the exception of cells used for viable cell analysis, where spectra were acquired every 500 nm. The laser power was set to 30 mW and an integration time of 0.5 s was chosen to ensure fast mapping and to avoid cell damage.Raman spectra of cells were acquired using an upright Confocal Raman microscope  with a 100× oil immersion objective (NA 1.4) . The sample was excited with a linear polarized (0°) coherent compass sapphire green laser λ2.3All spectra were subjected to cosmic ray removal using a cosmic ray detection algorithm  in the software Witec Project Plus 4.0.2.3.1−1), based on the Euclidean distance and follows Ward's minimum variance algorithm: the distance between neighbors is given by heterogeneity . Spectra gathered in the same cluster were used to calculate the average spectra shown in Fig. The Raman spectra of the image scans of the host cell line CHO‐K1 and protein producers CHO‐K1‐hDAO and CHO‐S‐Humira were averaged using a mask filter in order to use only the areas comprising the cells. The average spectra of each cell were baseline corrected and derived  prior to cluster analysis  of individual cells of a total of seven cell lines  were background subtracted  and k‐means cluster analyzed with the following conditions: four clusters, spectral mask 400–1800 cm−1 (fingerprint region), Manhattan normalization mode , no data reduction and no pre‐transformation mode . By this the hyperspectral image is processed to four images presenting the chemically most different four areas (clusters) and the corresponding average spectrum for each distinguished area. One cluster was assigned to ER due to higher intensity of the protein bands, one to background (which was discarded) and the remaining two clusters were more similar and merged as ”rest of the cell“ (RC). The average spectra of ER and RC of each cell, obtained from the clustering, were used in a ”principal component analysis“ (PCA) based on the second derivative of those spectra  and the fingerprint region of 400–1800 cm−1 .Raman images  glass slide, cells were incubated with a monoclonal mouse anti‐PDI antibody , washed and then stained with an Alexafluor 555 donkey anti‐mouse IgG antibody solution . After washing and mounting, images were taken with a Zeiss Axio Observer Z1 fluorescence microscope using a LCI Plan‐Neofluar 63 × 1.3 NA glycerol objective and a DsRed filter set.Cells were fixed for 10 min with 4% formaldehyde‐solution in PBS + Mg33.1−1 were acquired from CHO‐K1 host cells as well as two producer cell lines, one producing hDAO protein, a copper containing amine oxidase; the other, derived from CHO‐S, an alternative host, producing a therapeutic monoclonal antibody as one of the most important recombinant products generated in mammalian cell lines. The CHO‐K1‐hDAO cell line contained a higher concentration of Cu++ to ensure integration of copper into the active site of the enzyme. Hierarchical unsupervised clustering performed on the average Raman spectra of individual cells shows that cells from each cell line cluster together and there is a clear separation of CHO‐K1, CHO‐K1‐hDAO and CHO‐S‐Humira cells  and 1452 (protein bands: CH2 bending), 1585  and 1656 cm−1 (amide‐I of proteins and C=C of lipids), presumably due to differences in the relative protein content Label‐free single cell analysis methods in biotechnology are of high importance, yet still scarce. Here we evaluated the suitability of Raman microscopy for identifying a variety of CHO host and producer cell lines. Raman spectra from 300 to 3050 cm3.22 twisting of lipids) and 1585 cm−1  indicate that there is an enrichment of proteins and lipids in the putative ER. The X‐loadings of the rest of the cell, however, show a quite different pattern of peaks with highest signal coming from 1001 , 1445  and 1665 cm−1 (amide‐I vibration mode of peptide bonds) In order to picture different parts of single cells and identify protein accumulation in producer cell lines, we performed a k‐means cluster analysis 3.3In order to test whether the method is generally suitable to discriminate different CHO cell lines, we included the host cell line CHO‐S as well as additional host cell subclones . These subclones have a phenotype of increased transient productivity relative to the parental cell line −1 was selected for analysis since most of the protein bands occur in this area  discriminates the CHO K1 mother cell line from all others. Interestingly, this cell line is the one with the lowest potential for productivity  and PC4 separate the CHO‐K1‐hDAO from the others, while PC5 versus PC6 isolates CHO‐S/4F11, a host cell line with superior productivity. Other PCs might account for other non‐dominant sample variables. However, the other host cell lines CHO‐S, CHO‐K1/F10, CHO‐K1/1D9 were not clearly separated although cells from the same cell line are close to each other, similar to previously analyzed transcriptome data of these subclones The aim was to reveal spectral features that can separate closely related, but phenotypically distinct cell lines and subclones. PCA was performed with the previous ER and RC clusters obtained by k‐means clustering. After considering different possibilities, the range from 400 to 1800 cmrea Fig. . In PCA,4Confocal Raman microscopy has been introduced recently as a promising tool for label free analysis of cells and 3D tissue cultures In addition, the combination of multivariate analysis and CRM reveals cellular components based on changes in the Raman spectra due to chemical differences, enabling the identification of a subcellular compartment strongly resembling the ER, clearly separated from the remaining cell −1) required 3.4 min. This time would probably be prohibitive for early clone screening during industrial cell line development campaigns. However, by selecting the most informative spectra and limiting the analysis to these, the method might be used, with proper automatization, at the stage of verification of clonality or the stage of first testing for productivity.The ability to distinguish between ER and the remaining cell is likely to be a major contribution towards the discrimination of high or non‐producers or even of cells that have an inherently more efficient production machinery such as the subclones used in this study, as the ER is one of the major bottlenecks encountered in high producing cell lines Finally, another important application of CRM may be as a tool to differentiate between different cell lines that produce specific products, e.g. for confirmation of identity in manufacturing.