Crk and CrkL are cellular counterparts of the viral oncoprotein v-Crk. Crk and CrkL are overexpressed in many types of human cancer, correlating with poor prognosis. Furthermore, gene knockdown and knockout of Crk and CrkL in tumor cell lines suppress tumor cell functions, including cell proliferation, transformation, migration, invasion, epithelial-mesenchymal transition, resistance to chemotherapy drugs, and in vivo tumor growth and metastasis. Conversely, overexpression of tumor cells with Crk or CrkL enhances tumor cell functions. Therefore, Crk and CrkL have been proposed as therapeutic targets for cancer treatment. However, it is unclear whether Crk and CrkL make distinct or overlapping contributions to tumor cell functions in various cancer types because Crk or CrkL have been examined independently in most studies. Two recent studies using colorectal cancer and glioblastoma cells clearly demonstrated that Crk and CrkL need to be ablated individually and combined to understand distinct and overlapping roles of the two proteins in cancer. A comprehensive understanding of individual and overlapping roles of Crk and CrkL in tumor cell functions is necessary to develop effective therapeutic strategies. This review systematically discusses crucial functions of Crk and CrkL in tumor cell functions and provides new perspectives on targeting Crk and CrkL in cancer therapy. The celectively ,5. Crk aectively . Human Cectively  and Birgectively ). Crk anCRK-null and CRKL-null mouse embryos indicate that certain developmental processes require Crk or CrkL, or both [CRK and CRKL resulted in similar developmental phenotypes to those observed in the loss of two alleles from either CRK or CRKL, suggesting shared functions by Crk and CrkL. In addition, both protection of CRK-null fibroblasts from irremediable endoplasmic reticulum (ER) stress and protection of CRK-null or CRKL-null kidney cells from toxic insults suggest that both Crk and CrkL mediate stress-induced cell death, with deficiency of one protein making a difference [Due to structural and functional similarities between Crk and CrkL, overlapping roles of both proteins have been studied extensively in many biological processes. Crk and CrkL play essential overlapping roles in neuronal migration, neuromuscular synapse formation, podocyte morphogenesis, T cell migration into inflammation sites, natural killer cell expansion and differentiation during mouse cytomegalovirus infection, lens fiber cell elongation, and postnatal lens capsule development ,13,14,15 or both ,20. CrkL or both . Moreove or both . Recentl or both . Interesfference ,24.Expression of Crk or CrkL is elevated in multiple human cancers, and the overexpression is positively correlated with poor prognosis. Reduced expression of either Crk or CrkL by RNA interference-mediated gene knockdown has been shown to lead to inhibition of in vivo tumor growth of various cancer cell lines. Unlike the systematic and comprehensive approaches made in the field of development, however, many of the early studies of Crk and CrkL in cancer have lacked thorough and quantitative comparisons among phenotypes that resulted from individual and combined gain or loss of Crk and CrkL . ConsequCytoskeletal elements such as microtubules, actin filaments, and intermediate filaments are crucial in determining cell shape and maintaining cell-cell and cell-matrix interactions. Studies of the role of Crk and CrkL in cytoskeletal network rearrangement have provided insights into alterations of the cytoskeleton in cancer cells. Stable overexpression of CrkI in rat 3Y1 fibroblasts resulted in an alteration of cell morphology into refractile and spindle-like shapes . FibroblOn the other hand, gene knockdown studies have provided different insights into the functions of Crk and CrkL on the cell cytoskeleton. Crk knockdown in NIH3T3 cells resulted in a slower rate of cell spreading, more filopodia formation, and reduced focal adhesion formation in the early stages of cell spreading onto fibronectin-coated surfaces . FurtherThe effect of Crk and CrkL on the cell cytoskeleton has also been demonstrated in cancer cell lines. Crk knockdown resulted in decreased spreading of a breast cancer cell line onto a fibronectin substrate with decreases in focal adhesions and actin stress fibers . In addiUncontrolled cell proliferation is a crucial feature of many tumor cell lines. Crk and CrkL have been studied for their roles in normal and cancer cell proliferation. A close comparison of the Crk- and CrkL-mediated effects between normal and cancer cells may provide valuable insights into therapeutics. Ablation of endogenous Crk and CrkL from immortalized fibroblasts using Cre-loxP recombination-mediated gene knockout caused a blockage of cell proliferation and arrest of the cell cycle at the G1-S transition . While lWhile there are few reports about the effects of Crk and CrkL on cell proliferation in non-transformed cells, Crk and CrkL have been studied extensively for their effects in a number of cancer cell lines. In those studies, Crk and CrkL were ablated by gene knockdown or overexpressed to investigate their cellular functions. Reduction in CrkII and CrkI protein levels by Crk knockdown led to inhibition of cell proliferation in MCAS and SKOV3 ovarian cancer ,29 and sOn the other hand, CrkII overexpression promoted proliferation of Hca-P hepatocarcinoma cells . CrkL ovTaken together, these studies demonstrate essential functions of Crk or CrkL in cell cycle progression and cell proliferation in a variety of cancer cell lines. Since many of these studies addressed either Crk or CrkL, but not both, it is less clear whether the effects on cell proliferation are unique to one protein or shared by both proteins in a given cancer type. Therefore, it is premature to conclude why some cancer cells depend on Crk and others depend on CrkL to proliferate. Systematic comparison among individual and combined knockdown in various cancer cell lines would provide in-depth insights into this question.v-Crk was discovered as an oncogenic fusion protein that lacks catalytic activity but contains the viral gag sequence followed by an SH2 and an SH3 domain . v-Crk wCRKI and CRKII and demonstrated that CrkI overexpression in rat 3Y1 cells induced transforming activities such as spindle-like cell morphology, anchorage-independent cell growth, and in vivo tumor growth in nude mice. However, cells with CrkII expression did not exhibit significant transforming activities except for slight morphological changes. On the other hand, CrkII overexpression induced transformation in different cell types, including Rat-1 [v-fos or v-ras infection was inhibited in mouse fibroblast cell lines lacking endogenous Crk or CrkL [Matsuda et al.  cloned hng Rat-1  and NIH3ng Rat-1  fibroblang Rat-1  and immong Rat-1 . Zheng eng Rat-1  demonstrng Rat-1 . Further or CrkL .Contribution of Crk and CrkL to anchorage-independent growth was demonstrated in several cancer cell lines . Crk knoIn line with their contribution to in vitro cell proliferation and anchorage-independent cell growth, effects of Crk and CrkL on in vivo tumor growth have been studied using the gene knockdown or knockout approach . Crk knoIn addition to suppressing tumor growth at the primary injection sites in nude mice, Crk or CrkL knockdown reduced metastatic tumor burden. Bone metastasis in vivo following intra-cardiac injection of basal breast cancer cells was reduced by Crk knockdown . Crk knoCRK and CRKL was induced in mice to demonstrate that Crk and CrkL play essential overlapping roles in Reelin-dependent neuronal migration in the developing brain [Park and Curran reported that neuronal progenitor cell-specific knockout of ng brain . In thisng brain  reportedng brain ,17. Mousng brain ). In addng brain . In contng brain . Overexpng brain ,92 and Cng brain . Expressng brain ,92. CrkLng brain .Effects on cancer cell migration and invasion are the most extensively studied function of Crk and CrkL . RodriguRecently, Franke et al.  demonstrThe effect of Crk and CrkL on tumor cell migration and invasion has also been studied using overexpression of Crk or CrkL in tumor cells. Transfection of U87MG glioblastoma cells with CrkI, but not CrkII, increased cell migration and invasion . CrkII oThe roles of tyrosine and serine phosphorylation of CrkII in binding to its target proteins in relation to cell motility have also been studied. Noren et al.  reportedWhereas overexpression with CrkII-Y239F, like wild-type CrkII, increased migration of Crk-null fibroblasts and Hs683 glioblastoma cells, CrkII-Y239F, unlike wild-type CrkII, failed to increase migration of 4T1 breast cancer cells . PhosphoAll these results suggest that Crk and CrkL play critical roles in tumor cell migration and invasion in a variety of cancers. In most of these studies, loss of either Crk or CrkL resulted in decreases in tumor cell migration and invasion, suggesting that tumor cell migration and invasion are highly demanding cellular processes that require both Crk and CrkL to reorganize the cellular cytoskeletal network. In particular, both Crk and CrkL have been demonstrated to be important for tumor cell migration and invasion in colorectal cancer, glioblastoma, ovarian cancer, cervical cancer, gastric cancer, and hepatocellular carcinoma . FurtherThe epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain mesenchymal cell properties such as spindle shape, increased survival, and migratory and invasive properties. At the molecular level, EMT is characterized by loss of epithelial markers, including E-cadherin, integrins, and cytokeratins, and gain of mesenchymal markers, including N-cadherin, vimentin, and fibronectin. The EMT switch is mediated through transcriptional reprogramming of crucial transcription factors such as Snail, Slug, TWIST1, ZEB1, and ZEB2 . KnockdoCrkL knockdown also suppressed CCL19/CCR7-induced EMT marker expression and extracellular signal-regulated kinase (ERK) phosphorylation in epithelial ovarian carcinoma cells by decreasing expression of N-cadherin, Snail, and MMP9 and increasing E-cadherin expression . CrkL knTwo in vivo studies have suggested a link between EMT and chemoresistance. Zheng et al.  demonstrSince Nishihara et al.  reportedHigher expression of Crk in cancer tissues has been reported to contribute to poor prognosis and lower survival of patients with oral squamous cell carcinoma , glioblaCRK and CRKL, respectively. The results appear to be in a clear contrast to the results from the numerous studies of Crk and CrkL overexpression in cancer tissues  . Therefore, accumulation of more datasets, together with correlation studies between copy number alterations and protein level changes in cancer tissue, would provide a more concrete explanation of whether copy number alterations of CRK and CRKL contribute to overall patient survival in individual cancer types.Since a majority of these studies conducted immunostaining to examine protein levels in tissues, it is unclear whether the elevated levels of Crk and CrkL proteins represent elevated levels of transcripts. Analysis of the curated set of non-redundant studies in The Cancer Genome Atlas (TCGA) using the cBioPortal platform ,105 indi tissues . Althougd group) . The respression  and to bpression . The toppression , ovarianpression . On the r trends . ConsideCrk and CrkL are known to mediate signal transduction pathways triggered by growth factors, including fibroblast growth factor (FGF) and other extracellular stimuli ,8. StimuInvolvement of the FGF/FGFR receptor signaling pathways in tumor cell functions has been studied in a variety of tumor cell types. Treatment of breast cancer cells with FGF2 increased cell migration by inducing aquaporin 3 expression . TreatmeThese studies together demonstrate that FGF/FGFR signaling pathways promote migration and invasion of various tumor cells. Recent studies using mutant mouse models have demonstrated that Crk and CrkL play essential overlapping roles in FGF-induced lens fiber cell elongation during embryonic eye development  and postThe contribution of Crk and CrkL to various tumor cell functions in vitro and in vivo has been studied extensively for the last two decades. A majority of the studies have focused on either Crk or CrkL and induced decreases or increases in protein levels in cells to study Crk and CrkL functions in vitro. The requirement of Crk or CrkL for cell proliferation in vitro, anchorage-independent cell growth, cell migration and invasion, in vivo tumor growth in mice, and metastasis has been demonstrated in multiple cancer cell lines. Overall, knockdown or knockout of Crk or CrkL suppressed tumor cell growth in vitro and in vivo, whereas overexpression of Crk or CrkL promoted tumor cell growth. Cancer cell migration and invasion were also inhibited by Crk or CrkL knockdown and promoted by Crk or CrkL overexpression. The effects of decreased levels of either protein on cancer cell migration and invasion contrast with the absence of effects from the loss of either protein on neuronal migration in the developing brain and T cell migration to inflamed tissues ,11. InteImmunohistochemical examination of Crk and CrkL expression in tissue specimens from cancer patients revealed that many cancers had elevated protein levels of Crk or CrkL. Ovarian, breast, gastric, and lung cancers exhibited elevated expression of both Crk and CrkL . FurtherFathers et al.  used theAll these studies of Crk and CrkL using cancer tissues and cancer cell lines propose Crk and CrkL as promising therapeutic targets for cancer treatment. Several reviews have previously discussed the critical roles of Crk and CrkL in cancer ,121,122.In addition to genetic modulation of protein levels, activities of Crk and CrkL proteins can be modulated by chemical compounds, peptides, or peptidomimetics. However, it is still unclear what represents the activity of Crk and CrkL. Since Crk and CrkL are adaptor proteins that mediate protein-protein interactions through their SH2 and SH3 domains, their interactions with target proteins may represent the activity. The challenge of this concept is that Crk and CrkL are known to interact with many proteins in various signal transduction pathways. Nevertheless, Posern et al.  has deveMore studies are needed to understand which target proteins are key binding partners in promoting tumor cell functions before assay systems are developed to test the protein-protein interactions. Inhibitors that are specific to an interaction of Crk and CrkL with a binding partner will be critical to understanding how much a given protein-protein interaction contributes to the entire function of Crk and CrkL. In order to test inhibitors, it is necessary to establish measurable, specific cellular responses mediated by Crk and CrkL. In a recent study by Park et al. , commercCrk and CrkL have been reported to play essential overlapping functions in development ,10,12,14


Chronic inflammation causes incessant H2O2 generation associated with disease occurrences such as diabetes, autoimmunity, and cancer. In cancer, conditioning of the tumor microenvironment, e.g., hypoxia and ROS generation, has been associated with disease outcomes and therapeutic efficacy. Many reports have investigated the roles of the action of H2O2 across many cell lines and disease models. The genes predisposing tumor cell lines to H2O2-mediated demise are less deciphered, however. To this end, we performed in-house transcriptional profiling of 35 cell lines and simultaneously investigated each cell line's H2O2 inhibitory concentration (IC25) based on metabolic activity. More than 100-fold differences were observed between the most resistant and sensitive cell lines. Correlation and gene ontology pathway analysis identified a rigid association with genes intertwined in cell cycle progression and proliferation, as such functional categories dominated the top ten significant processes. The ten most substantially correlating genes (Spearman r > 0.70 or < -0.70) were validated using qPCR, showing complete congruency with microarray analysis findings. Western blotting confirmed the correlation of cell cycle-related proteins negatively correlating with H2O2 IC25. Top genes related to ROS production or antioxidant defense were only modest in correlation (Spearman r > 0.40 or < -0.40). In conclusion, our in-house transcriptomic correlation analysis revealed a set of cell cycle-associated genes associated with a priori resistance or sensitivity to H2O2-induced cellular demise with the detailed and causative roles of individual genes remaining unclear.Reactive oxygen species (ROS) such as hydrogen peroxide (H ROS are inevitable in life, and most organisms generate significant amounts of unintentionally or intentionally generated ROS, e.g., NADPH-oxidase (NOX)-produced superoxide and nitric oxide synthase (NOS)-produced nitric oxide, throughout their existence. At physiological concentration, such ROS participate in cellular signaling to respond to moderate extracellular and intracellular perturbations. At supraphysiological concentrations, irreversible modifications of biomolecules contribute to cellular dysfunction and demise. These two modes have been coined oxidative eustress and oxidative distress, respectively [Reactive oxygen species (ROS) such as hydrogen peroxide -mediated conversion of superoxide. Chronic inflammation and H2O2 generation have been linked to both oncogenesis and tumor progression [2O2 have also been implicated in tumor control. Redox-active chemotherapeutic compounds were given a role in tumor therapy [2O2. Among such therapeutic approaches are, for instance, H2O2-guided chemodynamic therapy [2O2-responsive photothermal and photodynamic therapy [2O2 generated via radiotherapy [Being a long-lived non-radical ROS, Hgression . For insgression . At the  therapy , and sev therapy , especia therapy , H2O2-re therapy  and nano therapy , secondaotherapy , and othotherapy . In geneotherapy .2O2, is undisputed in oncology. However, it is less clear which transcriptional profiles are associated with either sensitivity or resistance to H2O2-mediated cytotoxicity. This study used 35 cell lines from various organs and performed in-house transcriptomics and H2O2 inhibitory concentration (IC)25 assessments from the same cultures. A large share of target genes identified to correlate highly (Spearman r > 0.70 or < -0.70) with H2O2-induced cytotoxicity are involved in cell cycle progression and control, and the most relevant genes were validated using qPCR and western blotting, underlining their involvement in translating H2O2-mediated tumor growth control.The importance of ROS, including H22O2-induced cellular demise across 35 cell lines, with 34 of them being tumor cell lines (25) of H2O2 was determined for each cell line. Both datasets were correlated to identify genes with a potentially predictive value on oxidative stress sensitivity. The IC25 values were obtained by measuring the metabolic activity of each of the cell types 24 h after exposure to different concentrations of H2O2. Non-linear curve fitting was employed. Except for the Capan-1 cell lines, all cell lines had at least one H2O2 concentration decreasing metabolic activity by at least 25% to generate valid IC25 values  was high (>0.6) for all cell lines except PaTu-T and SCL-1, where a higher degree of uncertainty of the IC25 can be expected.This study aimed to identify genes associated with the sensitivity and resistance of Hll lines a. To thi5 values b. The re5 values c. The go2O2 IC25 values revealed 171 and 367 to correlate positively  to cellular demise  was correlated against the Hof cells a. Six TFof cells . Cumulatof cells b. Princiof cells c. These ll lines a, and thFinally, the correlation of genes associated with ROS/RNS production, redox signaling, and antioxidant defense was analyzed in our data set . No gene32O2 is the most investigated and best-understood oxidant in redox biology. As a result, much is known about its effects and toxicity. The cellular profiles associated a priori with the sensitivity to H2O2-induced cellular demise are less explored, however. Performing a 35 cell line H2O2 sensitivity screening correlated with culture-matched in-house transcriptomics enabled us to shed light on genes and pathways associated with H2O2 toxicity.H2O2 sensitivity. More than 500 genes correlated to this parameter with high significance, many related to cell cycle progression. This underlines a previous report showing cell cycle phases affecting H2O2-induced apoptosis. Compared to the GO/G1 phase, K562 cells were more or less resistant to H2O2 in the S-phase and G2/M phase, respectively [2O2-mediated toxicity was also found in GO/G1 phase C10 cells [2O2-induced signaling, leading to markedly declined cell death upon pharmacological and genetic augmentation and repression, respectively [2O2 and radiation-mediated cell death [2O2 [2O2 levels might dictate the cell death amplitude towards exogenous H2O2 exposure.The overarching goal of this study was to provide clues on pathways and targets associated with Hectively . The hig10 cells . We did 10 cells , to be l10 cells ,19. In a10 cells  and cycl10 cells  has longectively . Notablyectively , making ectively . Interesll death . Togetheath [2O2 , opening2O2 is a ubiquitously generated and long-lived oxidant with significant contributions to many of these processes via protein thiol-mediated signaling [2O2 sensitivity or resistance directly but rather to be a consequence of individual upstream signaling. Some of the targets, however, have been implicated in cancer and cell death related to oxidants. Tubulin (TUBB) is a druggable target [2O2-treated A549 cell transcriptomes identified DTL as the second top hit regulated by the treatment and confirmed its importance in lung cancer progression [2O2-mediated effects. CDC25A is a nuclear phosphatase regulating several CDK family members to allow cell cycle progression and found to be frequently overexpressed in cancers [2O2 cell lines used in our study remains remarkable, nevertheless. The oxidoreductase biliverdin (BLVRB) is a redox-regulated enzyme involved in heme catabolism and Fe3+ reduction [2O2-resistant cell lines by increasing levels of the free radical scavenger bilirubin [Examining cancer cell sensitivity towards drugs and other effectors is a field of heavy investigation, especially at the genomic level . At the ignaling  employedignaling . Gene exe target  as it hae target  and is ce target . Increase target , presumae target . Stathmie target ,37. Impoe target , which we target . Elevatee target ,41. Albegression . SIRPG igression , a molecgression . Yet, a gression , but lit cancers . It is s cancers . Elevate cancers ,49, but  cancers . The higeduction . BLVRB iilirubin .2O2-mediated toxicity. Three members of the E2F family emerged as top negatively correlating TF. Reduced E2F1 levels or its inactivation leads to reduced cell cycling and inhibition of G1/S transition [2O2-resistant cell lines in our study would be expected to be associated with decreased cell cycling [2O2-mediated toxicity, two KLF (Krüppel-like factor) family members (KLF4 and KLF5) constituted the top genes. Both TFs regulate proliferation, and it was shown that KLF4 and KLF5 overexpression sensitized cells to H2O2-induced toxicity [2O2-sensitive cell lines expressed higher KLF4 and KLF5 levels than resistant cell lines. KLF4 is frequently decreased or lost in epithelial cancers [2O2-mediated cell death [Albeit being overall heterogeneously expressed, several transcription factors (TF) showed a good (Spearman r > 0.50 or < -0.50) correlation with Hansition ,54. Thus cycling . E2F7 ca cycling  as well  cycling . Enhance cycling  and glio cycling , and for cycling . Regarditoxicity . This is cancers , attribu cancers . KLF5, i cancers  and acti cancers . However cancers . NFIL3  [2O2-resistant cells in our study. Inducible NOS (NOS2) negatively correlated in our study and is heavily involved in tumor progression and metastasis and different cancer types due to its regulation by p53 [2O2-mediated cellular demise is unclear. Notwithstanding, its noteworthy mentioning the value of target genes associated with increased sensitivity to stressors as this provides straightforward testable hypotheses to gain a better mechanistic understanding.Among the ROS-related and antioxidant genes, GPX2 and NOXA1 and GPX7 and NOS had a good (Spearman r > 0.40 or < -0.40) positive and negative correlation, respectively, with Htoxicity . GPX2 isn tumors ,71. GPX7 folding . Hence, n cells) , suggestn by p53 . The rea2O2-induced toxicity further. Second, conclusions from our serum-starving approach are limited due to the massive cellular changes that come with starvation. Instead, a thymidine block might be more suitable combined with in depth cell cycle analysis. Yet, it should be kept in mind that different agents, stressors, or inhibitors designed to change the basal expression of genes and proteins may not only have off-target effects but might also be differently effective in different cell types. The approach in our study is simplistic but therefore remains void of additional perturbations. Third, interconnected to the previous points, we did not measure responses to H2O2 but only gene expression of untreated cells. It would be interesting to study such response but many uncertainties are connected with such an approach, e.g., timing of analysis (minutes to hours) interconnected with level of analysis , which is difficult to do across larger panels of cell lines. A fourth limitation is the lack of one or several clear-cut candidates potentially explaining the effects, as even the highly correlating genes had some level of heterogenous expression, and the missing functional verification of the targets. However, keeping the different levels of cellular regulation in mind, we did not aim to proof the highly correlating genes to be causative for the effects observed. Instead, we propose an association of cell cycle signaling pathways being important for cellular survival to H2O2, while the exact genes and proteins crucial to this process might differ for each cell type as seen in the expression heatmaps.Our study had several limitations. First, cells’ states and regulation is governed by many processes, such as thiol oxidation, phosphorylation, epigenetic changes, micro RNAs, oxidative posttranslational modifications, and protein expression that were not covered in our work. Nevertheless, our transcriptomics-based approach identified provides testable hypotheses and appropriate cell lines to elucidate the exact roles of cell cycle control and proliferation and H4a priori associated with H2O2-mediated toxicity in 34 tumor cell lines of different entities. Apart from several pathways identified, many highly correlating genes were significantly related to the cell cycle in demarcating H2O2 sensitivity. As the exact mechanism remains unclear, further understanding of the intertwined relationships between redox signaling and regulated cell death during active or suppressed cell cycling may aid in generating an overarching scheme of how to effectively redox-targeted tumor cells.This study aimed at identifying in-house generated gene expression signatures 55.1Thirty-five cell lines of different origins were used in this study. This included melanoma , leukemia , adenocarcinoma  and squamous cell carcinoma  cell lines. In addition, the neuroblastoma cell lines SH-SY5Y and the non-malignant keratinocyte cell line HaCaT were used. HeLa and its derivative HeLa 2c were kindly provided by Christopher Lillig . All cell lines were maintained in their respective culture media, maintained under standard cell culture conditions, and passaged 2–3x per week.5.24 cells were seeded in 100 μl of fully supplemented cell culture medium  per well in 96-well plates . The plates were equipped with a rim filled with deionized water to provide extra evaporation protection and keep the edge effect at a minimum. Eighteen hours later, the cells received different concentrations of H2O2 . After 20 h of incubation, resazurin  was added to each well, and the cells were cultured for another 4 h. Subsequently, resorufin fluorescence was recorded using a bandpass filter-based multimode plate reader (Tecan F200) at λex 560 nm and λem 590 nm. After background subtraction, data normalization was performed against the untreated controls. The experimental setup for metabolic activity determination was the same for starvation experiments, except that previously expanded fetal bovine serum-starved cells were used. H2O2 concentrations were log-transformed and displayed against normalized metabolic activity rates. IC25 values were calculated using non-linear least squares fit based on asymmetric confidence intervals. The IC25 was chosen over the IC50 for two interconnected reasons. First, we were interested in less drastic concentrations of H2O2 as they appear physiologically or realistically within therapeutic regimens. The maximum concentration of 1000 μM employed in our screening is already beyond this. Second, 34 out of 35 cell lines reached the IC25 based on our measurements, while this was not the case for nearly half the cell lines when testing for the IC50. In the latter case, extensive interpolation of IC50 values would have been needed, potentially introducing additional uncertainty regarding correlation analysis that was to aim to avoid.To determine the metabolic activity, 1 × 105.35 cells were grown in culture flasks and harvested using accutase. Cells were washed, and total RNA was isolated using the Mini RNA purification kit (Bio&Sell). RNA extracts were incubated with RNase-free DNase Ι to remove contaminating DNA. RNA concentrations were then quantified via spectrophotometry (Nanodrop 2000) and stored at −80 °C. Sample preparation and hybridization were performed according to a single-color, chip-based transcriptome microarray gene expression analysis protocol. First, the spike-in mix was added to 100 ng of RNA, and the RNA was transcribed into complementary DNA (cDNA). cDNA was amplified using the Low Input Quick Amp Labeling One Color component. Next, the cDNA was transcribed into complementary RNA while incorporating the fluorescent dye cyanine 3 (Cy3). After purification, the Cy3-cRNA was fragmented, and hybridization onto a microarray chip was performed using the Gene Expression Hybridization Kit  for 17 h at 65 °C. The chips were washed using the Gene Expression Wash Buffer Kit , dried in a dedicated microarray chip oven, and scanned using an Agilent SureScan device. The scan data were extracted using Agilent's Feature Extraction Software and analyzed by the GeneSpring software . Three microarray samples were sampled from three independent biological replicates for each cell line, totaling 105 transcriptomes averaged to 35.For transcriptomic analysis, at least 5 × 105.42O2 IC25 values using TipCo Spotfire 7.8 software (PerkinElmer). Only genes with a Spearman r > 0.50 or r < −0.50 are shown; these genes always correlated significantly. Significantly correlating genes with Spearman r values different from these ranges were not included. Principal component analysis was realized using TipCo Spotfire software as well. Functional gene analysis was done using Gene Ontology (GO) pathway analysis and the Protein Analysis Through Evolutionary Relationships (PANTHER) classification system. Additionally, pathway analysis was performed using ShinyGo (an online open-access software tool). STRING database was used to identify co-expressed targets and shared networks. In this work, ‘target’ refers to genes identified from our analysis and not the protein being directly oxizided by H2O2.Transcriptomic microarray raw data files were loaded into GeneSpring and RMA-normalized before normalizing expression against RPL13A as housekeeping gene . Hierarc5.5Cells were fixed in ice-cold methanol, washed, and stained with 4′,6-diamidino-2-phenylindole  for 1 h. After washing, cells were resuspended in PBS with DAPI and measured using flow cytometry . Analysis was performed using Kaluza software 2.1 (Beckman-Coulter) using the Michael H. Fox algorithm to identify cells in the G1 phase of the cell cycle.5.6ΔΔCT method was applied to calculate relative expression intensities where treated and untreated samples were compared. For assessment of baseline gene expression, reciprocal ΔCT values were calculated.Total RNA (1 μg) in a total volume of 20 μl of buffer was reverse-transcribed, and qPCR was done using a QuantStudio 1 device (Thermo Fisher Scientific). GAPDH  was used as a housekeeping gene and normalization control. Cycling parameters were: 95 °C for 1 min to activate the DNA polymerase, then 40 cycles of denaturation for 10 s at 95 °C, annealing for 15 s at 60 °C, and extension for 20 s at 72 °C. Single product formation was confirmed by melting point analysis. Samples were run in duplicate, and the 5.7WES system (ProteinSimple) according to the manufacturer's instructions. Band intensities were quantified using Compass for Simple Western Software and normalized to housekeeping control protein expression (GAPDH) for six cell lines  investigated.Cells were pelleted and lysed in RIPA buffer containing protease and phosphatase inhibitors . Protein expression levels of β-tubulin, SIRP-γ, DTL, NCAPG, MCM10, CDC25A, and ME1 were determined using appropriate monoclonal antibodies  and capillary-based gel electrophoresis performed by the 5.825 was done using nonparametric, two-tailed Spearman correlation with 95% confidence intervals. Comparison between two groups for qPCR results was made using unpaired, two-tailed t-test. Statistically significant results were indicated as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).The experiments were repeated three independent times. Statistical analysis was performed using prism software 9.2. Analysis for gene and protein expression in relation to IC10.13039/501100002347German Federal Ministry of Education and Research (BMBF), grant numbers 03Z22DN11, 03Z22DN12, and 03Z22Di1.Funding was received by the The authors declare no conflict of interest.


Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of  Prostate cancer is the second most frequently diagnosed cancer and the fifth leading cause of cancer-related death among men in 2020 ,2. The dHistopathologically, prostate cancer can be classified into prostate adenocarcinoma (PAC) or transitional cell carcinoma (TCC) taking place in the bladder neck, urethra, or periurethral duct ,5,6,7,8.Since therapeutic options for CRPC and canine prostate cancer are limited to cytotoxic chemotherapy with unsatisfactory results, the development of novel treatments addressing specific molecular targets is required for both dogs and humans. In this context, different components of the cellular tight junctions have been moved in the spotlight ,16.Among promising tight-junction molecular targets for cancer diagnosis and therapy, claudins (CLDNs) are proteins abnormally regulated in different human and animal neoplasms affecting the mammary gland, prostate, pancreas, and colon ,21,22,23Clostridium perfringens enterotoxin (CPE) [The second extracellular loop of CLDN-3, -4, and -7 acts as a receptor for the in (CPE) ,28,29,30in (CPE) ,32,33.In recent years, laser therapy using gold nanoparticles (AuNPs) has rapidly evolved as a noninvasive thermotherapy for cancer as it enables hyperthermia of tumor tissues ,36,37,38Although AuNPs can passively accumulate in cancer cells , they haThis study aimed to evaluate the elimination of stably transfected canine PAC and TCC tumor cell lines using C-CPE-AuNPs complex and GNOME-LP, and characterize, if the red fluorescent cell lines emission interferes with conventional laser ablation and optimized the therefore required parameters in vitro. Further, to investigate if the C-CPE binding affects the cell viability, a transcriptome analysis of the cells after treatment with C-CPE was performed.CLDN-3, -4, and -7 in transfected cell lines were examined by quantitative real-time RT-PCR and compared to the native cell lines. The level of CLDN-4 expression in 0840-FusionRed was significantly lower in comparison to the reference cell line  < 0.05. The comparison analysis of 0840-C-CPE vs. 0840 showed only 11 DEGs. Contrarily, 0840-FusionRed-C-CPE and 0840-FusionRed revealed 1070 DEGs.A total of seven DEGs were common among 0840-C-CPE vs. 0840 and 0840-FusionRed-C-CPE vs. 0840-FusionRed a.Further, the comparison analysis of 0846-C-CPE to 0846 demonstrated 601 DEGs. In total, 569 genes were extracted from the comparison of 0846-FusionRed-C-CPE to 0846-FusionRed. Additionally, the comparison between 0846-C-CPE vs. 0846 and 0846-FusionRed-C-CPE vs. 0846-FusionRed revealed 280 overlapping genes b. Only oIn order to evaluate the effect of C-CPE on the gene expression independent from cell lines, GO and KEGG pathways analyses were carried out for overlapping DEGs between 0846-C-CPE vs. 0846, 0846-FusionRed-C-CPE vs. 0846-FusionRed, and 0840-FusionRed-C-CPE vs. 0840-FusionRed cells.The analyses could not be performed for 0840-C-CPE vs. 0840 as only 11 DEGs were detected.GO enrichment analysis revealed that the overlapping DEGs are involved in a number of biological processes (BP) including inflammatory response, immune response, and cellular response to interleukin-1. In terms of cellular components (CC), DEGs were mostly enriched in extracellular space, integral component of plasma membrane, and external side of plasma membrane. Molecular functions (MF) analysis indicated that the overlapping DEGs were only associated with chemokine activity . SubsequLaser exposure of 0840 native and transfected CLDN expressing cells in combination with C-CPE functionalized AuNPs significantly reduced number of vital cells down to 32.73% and 26.86%, respectively, in comparison to untreated cells . GNOME-LIn vivo models are the key to understanding the pathogenesis of cancer and the development of novel therapeutic approaches . AlthougCLDN-3, -4, and -7 expressions in generated fluorescent cell line 0840-FusionRed revealed no significant difference in comparison to native 0840 for CLDN-3 and -7; however, CLDN-4 was significantly decreased. An analysis of CLDN-7 in 0846-FusionRed showed no difference in expression, whereas CLDN-3 and -4 were even higher expressed after transfection. Additional immunofluorescence staining confirmed strong expression of all three CLDN proteins in all used cell lines. Therefore, 0840-FusionRed appears as a sufficient model for further experiments despite significant decrease in CLDN-4 mRNA level measured by qPCR. Interestingly, immunostaining revealed that CLDN-3, -4, and -7 in 0840 cells, as well as CLDN-4 in 0846, were punctually located in the cytoplasm. Such apparent miss-localizations were also described for the CLDN-4 protein in human prostate cancer-derived cell lines and may be related to the loss of cellular organization due to a defect in tight-junction formation or cell polarity—features common in tumor cells [It is well documented that CPE receptors CLDN-3, -4, and/or -7 are abnormally regulated in many tumor types including prostate cancer ,19,20,48or cells .The binding of CPE to CLDN-3 and -4 can trigger cell death ,52,53,54To confirm the specific binding of the functionalized AuNPs, scanning electron microscopy was performed in the present study. These images indicated that the C-CPE conjugated AuNPs retain the affinity to its receptors  on 0846 and 0846-FusionRed cell lines. The GO term and KEGG pathways analyses of DEGs demonstrated significant differences between C-CPE-treated and nontreated cell lines. These changes were mostly related to the cell surface/membrane as expected. C-CPE binding can disrupt the tight-junctional barrier but does not have a cytotoxic effect . FurtherThe GNOME-LP technology has been used for the cellular introduction of dyes as well as siRNA into different cell types via transient cell permeabilization ,56,57,583 and a scanning speed of 0.5 cm/s in combination with C-CPE-AuNPs reduced cell survival to less than 30% of claudin expressing cell lines [3) on native and fluorescent cell lines. Using the new setting, GNOME-LP in combination with C-CPE functionalized AuNPs reduced cell survival to down to 30% in 0840 and less than 10% in 0846 (native and fluorescent) cells.A previous study of our group showed that the energy power of the applied laser at 60 mJ/cmll lines . In a fiThe significant killing of 0840 (native and transfected) and native 0840 cells treated with nonfunctionalized AuNPs may be related to endocytosis activity, allowing them to internalize the AuNPs. This interpretation is supported by SEM analysis showing the presence of many uncoupled AuNPs that are bound nonspecifically on the cell surface microvilli even after three hours of incubation whereas fewer C-CPE functionalized AuNPs are present on the cell surface, mostly restricted along cell–cell borders. This suggests that C-CPE-AuNPs efficiently bind to their protein targets and are rapidly internalized into the cells as it can be traced through SEM see . HoweverThe results of this study confirm for the first time that the therapy concept of C-CPE functionalized AuNPs can be used efficiently against PAC and TCC cell lines. By using GNOME-LP system and C-CPE functionalized AuNPs, an irreversible laser ablation of prostate tumor cells was achieved in vitro. Cells, which were irradiated with maximal laser power without C-CPE-AuNPs, maintained viability. Likewise, cells incubated with C-CPE and irradiated with the maximal laser fluence maintained viability as well. A combination of laser treatment and C-CPE-AuNPs, however, reduced tumor cell viability down to less than 10% in 0846.To further extend the presented in vitro findings, in vivo studies need to be carried out as the next step. The same cell lines used for the in vitro findings could be detectable in vivo through deep tissue imaging, thereby enabling one to observe tumor growth and subsequently possible tumor ablation through C-CPE treatment in a living animal. In vivo studies could allow the characterization if the C-CPE complex is able to diffuse through the extracellular matrix and bind to tumor tissues. If successful, a combination between GNOME-LP and functionalized AuNPs may establish a treatment option for canine PAC and TCC cancer.Canine tumor cell lines TihoDTCC0840 (0840) and TihoDProAdCarc0846 (0846) were previously derived by our group from canine prostate carcinomas ,60. BothTransfected cell lines 0840-FusionRed and 0846-FusionRed were generated and characterized by our group . Cell li2 cell culture flasks in medium 199 , supplemented with 10% fetal calf serum  and 2% penicillin/streptomycin , and incubated in a humidified incubator maintained at 37 °C with 5% CO2. Cultivation medium was replaced twice per week.The cells were cultivated separately in 25 cm®Mini Kit RNA Purification  according to the manufacturer’s instruction. RNA isolation was performed three times per cell line. DNase digestion was carried out with RNase-Free DNase Set (Qiagen) to avoid genomic DNA contamination. Subsequently, cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit , 1000 ng of total RNA, and anchored-oligo (dt)18 primer according to the manufacturer’s instructions.Total RNA was isolated from transfected and native prostate tumor cell lines using the RNeasyCLDN genes after transfection process, transfected and native prostate tumor cell lines were comparatively analyzed by quantitative PCR. Primer pairs for CLDN-3, CLDN-4, and CLDN-7 were designed according to the mRNA sequences given by the National Center for Biotechnology Information (NCBI) (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The experiment was performed three times.To verify expression of n (NCBI) . Real-tiImmunofluorescence was performed for native and transfected cell lines to further confirm CLDN expression. The cells were cultivated on rat collagen type I  coated glass coverslips. Thereafter, cells were washed with PBS, fixed wit (1:1) Acetone/Methanol for 5 min at −20 °C and blocked for 30 min with 1% BSA  in PBS at 37 °C. CLDNs were stained with primary antibodies  diluted C. perfringens enterotoxin C-terminal fragment (C-CPE) with an N-terminal Strep-tag II was prepared as described previously [The eviously . MDA-MB-® Chromeo 488 dye . The complex was freshly generated before usage, by mixing 2.5 μL Strep-Tactin® Chromeo 488 (0.5 mg/mL) as recommended by the manufacturer with C-CPE dissolved in elution buffer. The mix was incubated overnight at 4 °C to allow binding of C-CPE with Strep-Tactin® Chromeo 488. To reach a final concentration of 20 μg/mL C-CPE, the mixture was diluted with 250 µL culture medium. Cells were cultured in a monolayer and stained for 3 h at 37 °C with 20 µg/mL C-CPE-Chromeo 488 complex. For nuclei staining, 1 µM Hoechst 33258 (Sigma-Aldrich) was used. Thereafter, cells were fixed with 4% formaldehyde for 10 min at room temperature and stored in PBS at 4 °C. The cells were imaged with a Nikon Eclipse TE2000-E confocal laser scanning microscope (346 nm for Hoechst 33258 and 488 nm for Chromeo 488) with a 60× water immersion objective and software EZ-C1 3.80 (Nikon).For C-CPE-CLDN binding visualization, the C-CPE was conjugated to green-fluorescent Strep-Tactin2 as an intermedium . The coverslips were flat-mounted on SEM-stubs with adhesive carbon tape  and coated with a carbon layer . Specimens were analyzed in a field-emission SEM  equipped with HE-SE and in-lens-Duo detectors operated at 5 kV and images with a size of 1024 × 768 pixels were recorded at different steps of magnification.To examine the binding of AuNPs and C-CPE-AuNPs on cell surfaces, the 0846 and transfected 0846-FusionRed cell lines were analyzed with SEM. Confluent cells were treated with AuNPs and C-CPE-AuNPs for 3 h in a cell culture incubator to allow complex adhesion to the cells. The cells were exposed to a pulsed laser with 60 mJ/cm² at a scanning speed of 0.5 cm/s. Subsequently, the cells were fixed with 4% formaldehyde, washed with PBS, and stored for further processing. For SEM preparation, the coverslips were dehydrated with a graded series of ethanol completed with an acetone step prior to critical point drying with CO5 cells in 6-well plates 48 h to reach monolayer. C-CPE with a concentration of 20 µg/mL was added, and the cells incubated for 3 h to allow binding of C-CPE to CLDNs. In the next step, culture medium was removed, and cells were washed with 5 mL phosphate buffered saline (PBS). TrypLETM Express  was used for detaching cells, centrifugation at 1000 rpm for 10 min followed for pelleting. Pellets were stored at −80 °C and followed by RNA-isolation for transcriptome analysis.C-CPE was prepared as described previously . Native ®Mini Kit RNA Purification  according to the manufacturer´s instruction. RNA isolation was performed three times per cell line. On-column DNase digestion was carried out with RNase-Free DNase Set  to avoid genomic DNA contamination.Total RNA was isolated from transfected and native prostate tumor cell lines with C-CPE treatment and without C-CPE treatment using the RNeasyThe RNA quality was assessed using an Agilent RNA 6000 Nano kit and 2100 Bioanalyzer (Agilent). Samples with RNA integrity number (RIN) > 8 were used for the DNA library preparation using a TruSeq Stranded mRNA Sample Preparation kit according to the manufacture’s protocol (Illumina). In brief, 1 µg of total RNA was used as input for an mRNA enrichment using poly-T oligo coated magnetic beads and chemically fragmented under elevated temperature. The fragmented RNA was then reverse-transcribed into the first- and second-strand cDNA using random hexamers and Superscript II reverse transcriptase. Double-stranded cDNA fragments were ligated with TruSeq RNA adapters with a unique DNA sequencing index and PCR-amplified. The DNA libraries were quality-controlled using an Agilent Technologies 2100 Bioanalyzer and Agilent DNA-1000 Chip kit.cDNA library concentration was quantified using a Qubit dsDNA HS Assay kit  and normalized to 2 nM prior to multiplexing. The DNA libraries were sequenced at a final concentration of 13 pM for 125 bp single-end reads using the high-output mode on a HiSeq2500 (Illumina) at the sequencing facility of Genome Biology Institute, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 1 May 2021) and preprocessed by filtering out low-quality reads with a mean Q-score < 20 and trimming adapter-like sequences using TrimGalore version 0.6.5. High-quality reads were aligned to the reference genome CanFam2 (Ensembl release 100) using Hisat2 version 2.2 [The raw fastq reads were quality-checked using FastQC (version 0.11.5) . A list including all sequenced genes was used as background.GO and KEGG analyses were applied for the functional annotation and pathway analysis using the Database for Annotation Visualization and Integrated Discovery   were mixed and incubated overnight at 4 °C. The concentration was adjusted to 20 μg/mL C-CPE and 2.5 × 1010 AuNPs/mL with cell culture medium.For tumor cell killing using a laser beam, confluent cells in 96-wells were treated with the C-CPE-AuNPs complex for 3 h in a cell culture incubator to allow adhesion of the complex onto the cells. The C-CPE-AuNPs complex was generated as previously described ,47. ShorIn addition to nontreated cells, cells incubated with either nonfunctionalized AuNPs or C-CPE alone were used as controls. Cells were exposed to a pulsed laser with 72 mJ/cm² at a scanning speed of 0.5 cm/s. Laser-treated cells were incubated for 30 min with 1 µM Hoechst 33258 (Sigma-Aldrich) and 10 min with SYTOX green  in a cell incubator. Under the Ti-E inverted fluorescence microscope , images were taken with 4x objective and Nikon Software Nis-Elements 4.4 (346 nm for Hoechst 33258 and 488 nm for SYTOX green). Vital cells were indicated by Hoechst uptake, whereas dead cells were referred by SYTOX and Hoechst uptake. Mean values of three wells were used per experiment. The experiment was performed three times. For cell survival quantification, Hoechst and SYTOX stained cells were counted with the image processing software ImageJ/Fiji V2.0.0 .t-test. Statistical analysis of cell killing using GNOME-LP was performed using Dunnett´s multiple comparison test and Student´s two-sided t-test. Differences were considered statistically significant for p < 0.05.The results are given as the mean of at least three independent experiments for quantitative real-time RT-PCR and cell killing using GNOME-LP. Statistical analysis was performed using SAS software 7.1 . Significant differences in gene expression of CLDN-3, -4, and -7 were calculated using Student’s two-sided In summary, the fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft in vivo studies.Since dogs represent a model for prostate cancer, the development of therapeutic strategies provides an important contribution to translational research directed to treat humans, thus providing a benefit for both species.


MET gene locus; we studied the influence of HGF on the autocrine secretion and migration behavior, as well as protein expression and phosphorylation. Two MET/ALK inhibitors were investigated for their effects on cell viability, cell cycle, cyclin alterations, apoptosis, and downstream signaling pathways. Moderate and strong expression of membrane and cytoplasmic cMET in chordoma patients and cell lines used, as well as concentration-dependent increase in phospho cMET expression after HGF stimulation in all four chordoma cell lines was shown. U-CH2, MUG-Chor1, and UM-Chor1 are polysomic for MET. Chordoma cell lines secreted EGF, VEGF, IL-6, and MMP9 upon HGF-stimulation. Sacral cell lines showed a distinct HGF-induced migration. Both inhibitors dose-dependently inhibited cell growth, induce apoptosis and cell-cycle arrest, and suppress downstream pathways. Heterogeneous responses obtained in our in vitro setting indicate that cMET inhibitors alone or in combination with other drugs might particularly benefit patients with sacral chordomas.Chordomas are rare slow growing, malignant bone tumors of the axial skeleton with no approved medical treatment. As the majority of chordomas express cMET and its ligand, HGF, and crosstalks between EGFR and MET-signaling exist, we aimed to explore cMET activity in chordoma cell lines and clinical samples. We investigated nine chordoma patients and four chordoma cell lines for cMET expression. Two clival and two sacral chordoma cell lines were tested for chromosomal abnormalities of the  Several authors have suggested an interaction between hepatocyte growth factor (HGF)/cMET and epidermal growth factor receptor (EGFR) signalling4. Although chordomas have a very heterogeneous geno- and phenotype, gains can be observed on chromosome 7q31.2, the site of the cMET receptor5. cMET is activated upon binding of its sole ligand HGF, which is either released by fibroblasts within the tumor-microenvironment or by tumor-cells themselves in an autocrine manner6. MET-signaling regulates cell survival, migration, and proliferation7. Changes in cMET expression levels due to mutations and amplifications comprise well-known resistance mechanisms in other types of cancer8. Strong HGF and MET expression has been reported in a majority of clinical chordoma samples10.Chordomas are rare, malignant, locally destructive, slowly growing bone tumors that show an invasive growth behavior; prognosis is poor with a median survival of seven years after diagnosis. Current treatment concepts include surgical en-bloc resection with negative margins and high-dose radiation therapy as well as chemotherapy for the extremely rare dedifferentiated chordomasThe present study aims to further investigate the role of cMET in chordomas in vitro: two sacral  and two clival  chordoma cell lines were karyotyped and then tested in functional studies. To mimic physiological conditions, we stimulated tumor cells with HGF. Protein expression, phosphorylation, cytokine secretion and migration potential were analyzed. Furthermore, the effects of the MET/ALK inhibitors crizotinib and cabozantinib were investigated in terms of viability assessment, cell cycle arrest, cyclin alterations, apoptosis and analyses of downstream signaling pathways. These studies contribute to a better understanding of cMET and HGF in chordoma biology.To figure out the cMET expression, immunohistochemical staining was performed on different chordoma cell lines as well as on patient samples. All four chordoma cell lines showed a moderate or strong expression of membrane and cytoplasmic cMET immunostaining Fig. A. cMET wMET locus specific and centromeric FISH signal were detected, which confirmed the polysomy 7 from karyotyping and sWGS  technique Fig. A. While WGS Fig. B. In U-CTo determine the appropriate concentration for HGF stimulation, MUG-Chor1 cell line was treated with 0, 1, 5, 10, 25, and 50 ng/ml HGF. A concentration dependent increase of phospho-cMET expression was observed Fig. A. Based To assess the role of HGF on chordoma cell migration, we studied the cell lines´ migration behaviour upon stimulation with HGF using the real-time xCELLigence system. We found that HGF distinctly enhanced the migration of two sacral chordoma cell lines MUG-Chor1 and U-CH2 cells, moderately attracted the clival UM-Chor1 cells, and did not affect MUG-CC1 cells Fig. C.Stimulation with HGF leads to secretion of the epidermal growth factor (EGF), the vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and the matrix metalloproteinase 9 (MMP9).To visualize the HGF/cMET axis in chordoma cells, all four chordoma lines were stimulated with HGF and the expression of angiogenesis modulating enzymes such as VEGF and MMP9, pro-inflammatory IL-6, and EGF, the synergistic growth factor to HGF, was measured by multiplex analysis. Samples were analysed in duplicates and related to the cell amount used. Data were presented in the mean fluorescence intensity (MFI) Fig. D. MUG-Ch50 values after crizotinib treatment were 189 nM MUG-Chor1, 314 nM U-CH2, 442 nM MUG-CC1, and 663 nM UM-Chor1, respectively.Stimulation with 10 ng/ml HGF for 1 h lead to a significant increase of cell viability in chordoma cells after 72 h. Subsequent treatment with crizotinib for 72 h decreased the viability of all four chordoma cell lines highly significant in a concentration dependent manner Fig. A, left. 50 values after cabozantinib treatment were 433 nM MUG-Chor1 and > 10 µM for the other three cell lines. Due to the minor effects it was not possible to fit the IC50 values.  and cabozantinib (XL), respectively, resulted in a cell cycle shift. Both inhibitors led to a decrease in cells in the G1 phase and an associated G2 arrest. The protein expression of the corresponding G2/M checkpoint proteins cyclin B, CDK1, and CDK2 were significantly decreased  Akt, Erk, and Stat Fig. C by prot12. HGF, the only known ligand for the cMET receptor, is mainly expressed in cells of mesenchymal origin, although some epithelial cancer cells appear to express both HGF and MET14.In the pathogenesis of various epithelial tumors, receptor tyrosine kinases such as cMET or EGFR, have been implicated and molecular-targeted therapies are being administeredcMET gene-locus was polysomic or amplified in two sacral and two clival chordoma cell lines via single cell COBRA FISH analysis and copy number profiling. Comparing the number of allele/chromosomes within these cell lines, MUG-CC1 presented with 33 chromosomes, which comprises a nearly-haploid (< 30 chromosomes) karyotype; this cell line showed monosomy of chromosome 7. In contrast, the other chordoma cell lines displayed a varying polysomy of chromosome 7. Thus, heterogeneity between the karyotypes of these cell lines existed, which particularly affected chromosome 7, on which both, the cMET and HGF genes, are located15. Comparing chordoma cell lines after HGF stimulation, U-CH2 showed the strongest phospho-cMET—which is the activated form of cMET-expression, and MUG-CC1 the lowest. We found that sacral chordoma cell lines showed a stronger migration towards an HGF-gradient compared to clival cell lines. Cell migration and chemotaxis towards an HGF gradient have been described previously in the chordoma cell line CCL316.To better understand the role of cMET in chordomas, we studied whether the MET gene alterations have been shown to benefit from cMET inhibitors17.Although the overall results for cMET inhibitors in phase III clinical trials in non-small cell lung cancer are disappointing, subgroups of patients with 50 of 5–10 µM18, which is similar to the IC50 obtained in our HGF-stimulated, crizotinib-treated chordoma cells. Yakes et al. tested the effect of cabozantinib, a small molecule tyrosine kinase inhibitor, on the proliferation behavior of different tumor cell lines and achieved different effects with respect to cMET amplification and IC50 concentrations19. Cell lines with cMET amplifications presented an IC50 between 10 and 19 nmol/L, cells without cMET amplification only an IC50 of 1–5 µmol/L. Our viability data clearly showed that, after HGF-stimulation, sacral chordoma cell lines responded better to treatment with cMET inhibitors than clival cell lines. In accordance with these data, treatment with cMET inhibitors caused a reduction of cells in the G1 phase, resulting in G2/M arrest of the cell cycle. It is well known that the cyclin B1/CDK1 complex together with cyclin A/CDK2 promotes G2/M transition in the eukaryotic cell cycle20. Compared to control cells, the expression of cyclin B1 and its corresponding cyclin-dependent kinase CDK1 was clearly reduced in MUG-Chor1 and U-CH2 cells. Thus, the checkpoint for the transition between the cell cycle phases was not being formed and the cells were arrested. Aberrant HGF/cMET axis activation, which is closely related to cMET gene polysomy and amplification, promotes tumor development and progression by stimulating the MAPK and other signalling pathways22. Our data revealed that the sacral chordoma cell lines showed a significant reduction in phosphorylation of Akt, Erk, and Stat3 after treatment with cMET inhibitors. Remarkably, UM-Chor1 showed no changes in these phosporylation patterns.Therefore, we investigated whether sacral and clival chordoma cells respond differently to a treatment with cMET inhibitors. The dual MET/ALK inhibitor crizotinib showed a dose-dependent reduction of viability in various breast cancer cell lines after 48 h of treatment, with an ICFor a future treatment of chordoma patients the origin of the primary tumor seems to play a crucial role. A high grade of heterogeneity between individual cell lines was observed. cMET inhibitors effected the growth behavior and cMET phosphorylation state of chordoma cells in different ways. Based on our in vitro cell culture analyses, it can be concluded that eventual treatment with cMET inhibitors would be more appropriate in sacral patients than in clival patients. Further investigation into chordoma heterogeneity will allow for a more precise treatment of individual patients and will pave the way towards a personalized chordoma treatment management.The authors confirm that all used methods were performed in accordance with the relevant guidelines and regulations.Patient data and histological specimens were collected from the databases of the Diagnostic and Research Institute of Pathology, Medical University of Graz. The study protocol and the informed consent of patients were approved by the ethics committee of the Medical University of Graz (vote #18-192ex06/07). The informed consent was obtained from all participants. Nine formalin-fixed paraffin-embedded chordoma specimens  were analysed. IHC stains were conducted with antibodies against S-100, CK, Vimentin, EMA, and brachyury . Only brachyury-positive samples (nuclear expression), in combination with a characteristic morphology were included in the study. cMET staining was performed with the mouse monoclonal MET antibody on the Leica Bond-III detection platform, using the Bond Polymer Refine Detection system (Leica).23 and U-CH2 and the clival chordoma cell lines UM-Chor1  and MUG-CC124 were cultured in IMDM/RPMI 4:1  containing 10% fetal bovine serum , 1% insulin, transferrin, sodium selenite , 2 mM glutamine, and 1% penicillin/streptomycin . Incubation was carried out at 37 °C in a humidified atmosphere of 5% CO2. All cell cultures were periodically checked for mycoplasma by PCR, cell line identification was performed by STR analyses .The sacral chordoma cell lines MUG-Chor125. Slides with metaphase chromosomes were hybridized using a multicolor FISH approach, known as COBRA-FISH. The 43-color FISH staining of every chromosome arm in a different color combination, digital imaging and analysis were performed as described26. Chromosomal breakpoints were assigned using inverted images counterstained with 4',6-diamidino-2-phenylindole  together with the information derived from the short- and long-arm specific hybridization during COBRA-FISH. Karyotypes were described according to ISCN 2009. Two-color Fluorescence In Situ Hybridization (FISH) was performed as previously described27. BAC clone (BACPAC Resources Center) RP11-95I10 was selected to analyze the cMET locus. As reference DNA of centromere 7 was used. RP11-95I10 and DNA of centromere 7 were labeled Cy3-dUTP and Fluorescein-12-dCTP respectively using a nick translation labeling reaction28.Cells were harvested using the chemically induced chromosome condensation technique published29.Genome wide copy number alterations (CNA) were established using shallow whole genome sequencing (sWGS). Shotgun libraries were prepared using the TruSeq DNA LT Sample preparation Kit  following the manufacturer´s instructions. Briefly, after fragmentation and concentrating the volume to 50 µl end repair, A-tailing and adapter ligation were performed following the manufacturer’s instructions. Libraries were quality checked on an Agilent Bioanalyzer using a DNA 7500 Chip  and quantified using qPCR with a commercially available PhiX library (Illumina) as a standard. Libraries were pooled equimolarily and sequenced on an Illumina MiSeq in a 150 bp single read run. Copy number profiling from sWGS data was performed using the ichorCNA algorithm applying 1 Mb bin, a probabilistic Hidden Markov Model (HMM) model for the estimation of tumor fraction, roughly equivalent to tumor purity from bulk tumor analyses. Focal amplifications were called from 50 kb bins according to the criteria published in Ulz et al. 2016For immunoblotting, whole cell protein extracts were prepared with RIPA lysis buffer including protease inhibitors, subjected to SDS-PAGE and blotted onto PVDF membrane . Primary antibodies against cMET/phospho-cMET, Akt/phospho-Akt, Erk/phosphor-Erk, and Stat3/phosphor-Stat3 were purchased from Cell Signaling Technology  and β-actin from Sigma-Aldrich . Blots were developed using horseradish peroxidase-conjugated secondary antibodies  at room temperature for 1 h and the SuperSignal West Pico Chemoluminescent Substrate , in accordance with the manufacturers’ protocol. All original membranes can be found in the supplementary data.Real-time monitoring of cell migration was performed using the xCELLigence system with the CIM-Plate 16 . When cells migrated through the microporous membrane into the bottom chamber they contacted and adhered to the electronic sensors, resulting in an increase in impedance. The cell-index showed the impedance changes and cell migration were automatically and continuously recorded every 15 min for 30 h. Acquisition and analysis was performed with the RTCA 2.0 software (OLS).Cells were starved (without FBS) overnight and treated with 10 ng/ml HGF for 1 h. After incubation supernatant was stored at -80 °C for further analysis. The epidermal growth factor (EGF), the vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), and the matrix metalloproteinase 9 (MMP9) levels were determined using the commercially available Affymetrix eBioscience lot number (ln) 107311000  on a Bioplex200 system  in combination with Bio-Plex Manager software, version 4.1 , with 5-parametric curve fitting. Standard range and sensitivity for the respective cytokines: EGF (ln 95592000) 2.93–12000; VEGF-A (ln 107198005) 5.37–22000 pg/ml; HGF (ln 99618101): 10–41700 pg/ml; IL-6 (ln 102903000) 9.57–9800 pg/ml; MMP9 (ln 97975000) 1.06–4350 pg/ml. Complete growth medium was used as background control.CellTiterGlo cell proliferation assay (Promega) was performed according the manufacturer's instructions. Cells were pretreated with 10 ng/ml HGF for 1 h and treated with the selective cMET inhibitors crizotinib (PF-02341066) or cabozantinib (XL184)  for 72 h.After incubation with 5 µM crizotinib or 5 µM cabozantinib for 72 h, cells were fixed with 70% ice cold ethanol for 10 min at 4 °C. After washing, the cell pellet was resuspended in propidium iodide (PI) staining buffer . Cell cycle distribution was analyzed by FACSCalibur  using ModFit software.5 cells in 100 µl Annexin V Binding buffer containing 5 µl Annexin V-APC and 5 µl PI for 15 min at room temperature. Flow cytometry analysis was performed with BD FACS LSR II (BD Biosciences). The experimental design of Annexin evaluation comprised the factors cell line, time, dose and treatment. The measurements from FACS represent relative frequencies which are binomially distributed with mean between 0 and 1 and variance depending on the mean. Taking the odds pertaining to a cell type of a cell culture with dose zero at time zero and cell line MUG-Chor1 as a reference, any factor combination can be predicted by multiplying the odds ratios of the individual factors. The odds ratios of time and dose were exponentiated by time and dose, respectively. This logistic model was implemented as a generalized linear mixed model  using the R  and the package MASS 7.3–51.1.The APC Annexin V Apoptosis Detection Kit  was performed following the manufacturer’s instructions. Apoptotic cells were identified by the incubation of 1 × 10Supplementary Information.


Amyloidosis is a common pathological event in which proteins self-assemble into misfolded soluble and insoluble molecular forms, oligomers and fibrils that are often toxic to cells. Notably, aggregation-prone human islet amyloid polypeptide (hIAPP), or amylin, is a pancreatic hormone linked to islet β-cells demise in diabetics. The unifying mechanism by which amyloid proteins, including hIAPP, aggregate and kill cells is still matter of debate. The pathology of type-2 diabetes mellitus (T2DM) is characterized by extracellular and intracellular accumulation of toxic hIAPP species, soluble oligomers and insoluble fibrils in pancreatic human islets, eventually leading to loss of β-cell mass. This review focuses on molecular, biochemical and cell-biology studies exploring molecular mechanisms of hIAPP synthesis, trafficking and degradation in the pancreas. In addition to hIAPP turnover, the dynamics and the mechanisms of IAPP–membrane interactions; hIAPP aggregation and toxicity in vitro and in situ; and the regulatory role of diabetic factors, such as lipids and cholesterol, in these processes are also discussed. E. coli, plays a role in biofilm formation and mediates infection. Chaplin, found in Streptomyces, plays a role in protection against water surface tension. URE2p, found in S. Cerevisiae, plays a role in nitrogen catabolism. Pmel17, found in humans, plays a role in melanin synthesis [Amyloid formation or amyloidogenesis, an intrinsic property of all polypeptides , is a prynthesis ,4,5. On ynthesis ,7.The onset of T2DM is characterized by three determining factors: the insufficient ability of pancreatic β-cells to secrete insulin, decreased insulin sensitivity of peripheral tissues, and the deposition of hIAPP-derived aggregates, or amyloid ,8,9,10. In view of this, here we summarize findings of cellular and reconstituted studies that explored roles of biosynthetic/degradation pathways and cellular constituents, such as cholesterol, lipids and biological metals in hIAPP turnover and aggregation in the pancreas that have important relevance for etiology of islet amyloidosis and T2DM. The clinical implications and molecular mechanisms of hIAPP–lipid and hIAPP–metal interactions in respect to its aggregation and toxicity in pancreas is also reviewed. In this review, we discuss an intriguing idea and evidence for possible crosstalk between hIAPP and other amyloid proteins and their possible relevance for brain and islet amyloidosis. Finally, advancements in small-molecule-amyloid-inhibitor therapy relying on natural products is also reviewed.INS) located on chromosome 11p15.5 and consists of several exons [The two major glucose-regulating pancreatic hormones are hIAPP and insulin ,24,25. Tal exons . Similaral exons ,30,31  showed that, in rat pancreatic islets, the steady-state level of IAPP mRNA is 10-fold less than the steady-state insulin mRNA levels . Interestxnip promoter is enhanced in high-glucose and ER-stressed β-cells, thereby augmenting TXNIP transcription under these adverse conditions [A study by Jing et al. (2014) demonstrated that, in the β-cells, the major glucose-induced gene, a thioredoxin-interacting protein (TXNIP), stimulated IAPP transcription by increasing the expression of transcription factor FoxA2, thereby revealing a novel β-cell-signaling cascade under physiological conditions  Figure B. Intrignditions ,48,49. HIn addition to biosynthetic pathways, studies suggest an important contribution of degradation (proteolytic) pathways to IAPP transcription in the pancreas. For instance, impaired activity/function of the ubiquitin–proteasome system (UPS) in human islet β-cells, elicited by diabetogenic stress conditions, resulted in downregulated IAPP promoter activity and decreased IAPP mRNA and protein levels . FoxA2 bIncreasing evidence suggests the contribution of hIAPP-derived toxic oligomers and aggregates toward the progressive loss of functional β-cell mass during T2DM ,25,55,56In addition to its 18–29 aa amyloidogenic segment, various IAPP molecular conformations seems to be important for its aggregation and toxicity. It has been suggested that pre-fibrillar low-MW oligomeric species, rather than the mature fibrillar forms of hIAPP, induced membrane damage and β-cell death ,65,66,67Another important question besides the role of proteasome on hIAPP degradation is whether hIAPP may modulate proteasome’s proteolytic function in the beta-cell. Casas et al. (2007), using mouse clonal beta-cell MIN6 and primary human islets, showed that hIAPP, but not rIAPP, treatment leads to a significant decrease of proteasomal activity  Figure . In partThe other important cellular degradation system besides the ubiquitin/proteasome system is the autophagy/lysosomal system (ALS). Costes and colleagues, using mouse genetics, showed that UCHL1 deficit/dysfunction resulted in increased β-cell apoptosis by increased accumulation of autophagosomes, SQSTM1/p62-positive cytoplasmic inclusions and polyubiquitinated proteins with lysine 63-specific ubiquitin chains in hIAPP transgenic mice, indicating a defect in the autophagy/lysosomal pathway  Figure . ImportaIn addition to the UPS and ALS, studies have also revealed the important contribution of insulin degrading enzyme (IDE) in the degradation and clearance of synthetic IAPP in cell-free systems and immortalized cell lines ,79,80 F. AlthougTogether, these data indicate the existence of complex and independent but not mutually exclusive proteolytic pathways that regulate hIAPP intracellular and extracellular levels, thereby protecting cells from amylin accumulation and aggregation. Thus, any dysregulation of proteolytic pathway(s) may potentially disrupt the fine balance between hIAPP turnover and global proteostasis in β-cells and accelerate the progression of diabetes and its pathogenesis .In addition to cell’s diverse proteolytic system, a myriad of reconstituted and cellular studies show that membrane-associated lipids and cholesterol act as potent regulators of hIAPP aggregation and toxicity . Reconsta) of hIAPP aggregation by ΔEa = −3.7 kJ/mol, which, in turn, increases the rate of its aggregation by ~4 folds [a = 845 J/mol [Furthermore, hIAPP is a positively charged (cationic) peptide. Therefore, it is conceivable that it may interact with negative charged lipids from plasma and endo-membranes, such as phosphatidylserine (PS). This idea was first tested by Miranker and colleagues , and lat~4 folds . Convers45 J/mol . Importa45 J/mol , indicat45 J/mol , a commo45 J/mol .Similar molecular impact of lipids and cholesterol on the structure and organization of hIAPP aggregates at the membrane interface was observed in high-resolution optical and force microscopy studies. Using time-lapse AFM followed by single-particle analysis , the invA two-fold symmetrical organization of hIAPP and other amyloid proteins that was first incorporated into liposomes and subsequently into planar membranes has also been reported . These fm) of hIAPP particles was larger on lipid membranes than on lipid-free mica surface. A further, much larger increase in size of hIAPP particles was detected on PS-membranes containing cholesterol. Importantly, the authors reported a decrease in total volume (Vt) of hIAPP aggregates on the cholesterol-containing membranes, as compared to the cholesterol-free membranes, due to a large decrease in the hIAPP seeding capacity. Overall, reconstituted studies suggest that cholesterol restricts hIAPP aggregation and accumulation on planar membranes to discrete sub-micron membrane areas, which serve as templates for the ongoing hIAPP binding and deposition  of hIAFM studies not only revealed polymorphism of hIAPP aggregates A–C, but Cholesterol is an important controller of membrane fluidity. It is found in lipid rafts of plasma and many endo-membranes, where it establishes a platform for assembly of various protein–lipid complexes important for cell signaling, endocytosis and other essential physiological processes . It is po and ld domains [In regards to potential amylin toxicity, reconstituted studies revealed a complex modulatory action of cholesterol on hIAPP-induced membrane damage . The aut domains . CollectTo confirm that hIAPP may also interact with native membranes, which, in turn, may affect its aggregation, and toxicity in situ, cellular and in vivo studies have been undertaken ,108,109.Variations in PM cholesterol levels also seems important for hIAPP toxicity in rat and human pancreatic islet cells. Studies showed that the combination of cholesterol-depleting agents, BCD and lovostatin, reduced PM cholesterol and, in turn, increased hIAPP toxicity as compared to cells with normal membrane cholesterol content . A combiIn addition to membrane cholesterol, free lipids were recently suggested as potent modulators of aggregation and toxicity of hIAPP and other amyloid proteins . PreviouBased on these findings, a common molecular mechanism of membrane disruption by amyloidogenic proteins called the lipid-chaperone hypothesis was recently proposed . This geAs mentioned earlier, fully processed hIAPP has tendency to aggregate in a concentration and time-dependent manner. However, hIAPP-derived aggregates are rarely observed in non-diabetic individuals, and hIAPP’s aggregation potential in the pancreas is strongly linked with clinical manifestation of T2DM. Namely, ~90% of diabetic patients develop islet amyloidosis . Thus, pIn addition to the cell’s native constituents, a number of natural products and derivates, such as small-molecule polyphenol compounds isolated from plants, showed real promise to inhibit hIAPP aggregation in vitro and in cells . EGCG, aT2DM is a progressive metabolic disease, and together with late-onset neurodegenerative disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), it falls under the category of “protein misfolding diseases”. While the primary misfolded protein linked with T2DM is pancreatic hormone hIAPP, the distinctive deposition of misfolded proteins, such as tau and β-amyloid (Aβ) , and α-sIt has been suggested that an increased level of hIAPP is a contributory but not decisive factor for pancreatic amyloid deposition and progression of diabetes . SimilarDrosophila melanogaster expressing hIAPP/Aβ to show heterologous interactions between hIAPP and Aβ, and co-deposition of these amyloid-forming peptides in animal brains, resulting in reduced fly longevity [Along this notion, several epidemiological studies have insinuated crosstalk between brain and islet amyloid proteins, as well as a common ethological link between T2DM and neurodegenerative diseases, such as AD and PD ,146,147.ongevity . Similarongevity . From thongevity ,146,147.ongevity . Accelerongevity .It has been reported that α-Syn strains are determined by species of misfolded seeds, intracellular environments and protein–protein interactions ,152. AccIn summary, molecular and cellular studies spanning the last two decades provided novel mechanistic insights into structure, function and toxicity of amyloid proteins, such as hIAPP. Studies have revealed that natural polyphenolic products and essential cellular constituents, such as metals, membrane-associated lipids and cholesterol, potently modulate aggregation and cytotoxicity of hIAPP, and this, in turn, may affect broad cellular and systems functions, such as glucose homeostasis. Besides unravelling the important roles of metal–amyloid and lipid–amyloid complexes in the etiology of amyloid diseases, new emerging evidence points to synergistic actions, interactions and cross-seeding of hIAPP and other amyloid proteins at various locations in human body, most notably the brain and the pancreas. An intriguing idea that awaits verification is that these co-precipitation factors may work together to worsen pathology and clinical outcome of amyloid-laden tissues and organs. In addition to natural products, cholesterol-modifying and metal-chelating agents, the use of synthetic small-molecule activators and inhibitors of cell proteolytic system, proteasome, lysosome and soluble or membrane-associated proteolytic enzymes offers exciting opportunities for the development of novel approaches and drugs that may halt or slow down the progression of amyloid-associated diseases.In spite of the significant progress and conceptual advancements in regards to general mechanisms of hIAPP aggregation and toxicity, there is still a gap in understanding cellular pathways and mechanisms that prevent hIAPP from aggregating and forming amyloid in cells and organs. Particularly challenging and lacking are studies that will identify transient cytotoxic hIAPP structural intermediates in vivo and the inherent molecular mechanisms involved in cell/tissue protection. To capture formation and recycling/degradation of hIAPP transient toxic and non-toxic molecular species, a real-time single-molecule resolution technique that is compatible with intact cellular analysis will be required. Emerging evidence of the important inhibitory role of natural products and essential cellular constituents opens opportunities for early targeted intervention during hIAPP-induced islet amyloidosis and possibly other amyloid diseases. Finally, recent technological advancements in computational biology, structural biology and synthetic chemistry have opened a new frontier and opportunities for designing novel small-molecule drugs that will selectively and efficiently stimulate cellular process and pathways involved in hIAPP clearance and detoxification in pancreatic and other tissues. This gain-of-function approach can be utilized alone or in conjunction with amyloid inhibitors designed to directly target and disrupt cytotoxic amyloid aggregates.


In the absence of suitable molecular markers, non-small cell lung cancer (NSCLC) patients have to be treated with chemotherapy with poor results at advanced stages. Therefore, the activity of the anticancer marine drug fascaplysin was tested against primary NSCLC cell lines established from pleural effusions. Cytotoxicity of the drug or combinations were determined using MTT assays and changes in intracellular phosphorylation by Western blot arrays. Fascaplysin revealed high cytotoxicity against NSCLC cells and exhibit an activity pattern different of the standard drug cisplatin. Furthermore, fascaplysin synergizes with the EGFR tyrosine kinase inhibitor (TKI) afatinib to yield a twofold increased antitumor effect. Interaction with the Chk1/2 inhibitor AZD7762 confirm the differential effects of fascplysin and cisplatin. Protein phosphorylation assays showed hypophosphorylation of Akt1/2/3 and ERK1/2 as well as hyperphosphorylation of stress response mediators of H1299 NSCLC cells. In conclusion, fascaplysin shows high cytotoxicity against pleural primary NSCLC lines that could be further boosted when combined with the EGFR TKI afatinib. Approximately 80% of all lung cancers are of the Non-small Cell Lung Cancer (NSCLC) type that is often detected at an advanced stage and portends a dismal prognosis . The staThe first-generation EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib bind reversibly to the kinase domain of the receptor, but second-generation drugs such as the pan-ErbB inhibitor afatinib show irreversible inhibition of the kinase activity . In NSCLFascaplysinopsis spp. with fascaplysin  as the most important agent [50 350 nM) and by intercalation into DNA [A range of antitumor compounds has been extracted from the sponge nt agent . Fascaplinto DNA , 11. Furinto DNA .50 0.89 µM) and against SCLC Circulating Tumor Cell (CTCs) lines (mean IC50 0.57 µM) [50 of 1.15 µM for fascaplysin and the compound showed an additive cytotoxic effect with cisplatin. Available permanent cancer cell lines have been adapted for vigorous in vitro growth and may not be truly representative of the in vivo situation in patients. Acquisition of NSCLC cells for tests is possible by routine thoracentesis in patients with advanced NSCLC. Malignant pleural effusion (MPE) is observed in half of advanced NSCLC cases and is associated with a short survival [Our previous studies revealed that fascaplysin exhibited high cytotoxicity against Small Cell Lung Cancer (SCLC) cell lines (mean IC0.57 µM) , 16. Selsurvival . MPE samsurvival –20. In tCell Culture and reagents Unless otherwise noted, all chemicals were obtained from Sigma-Aldrich . Dulbecco’s phosphate buffered saline (PBS) was purchased from Gibco/Invitrogen . Compounds were prepared as stock solutions of 2 mg/mL in either DMSO or 0.9% NaCl for cisplatin and aliquots stored at − 20 °C. Equivalent concentrations of DMSO were supplemented to medium controls. Established permanent cell lines were obtained from the American Type Culture Collection  and primary lung cancer lines were established in our lab. Collection of pleural effusions of lung cancer patients, isolation of tumor cells and generation of cell lines was done according to the Ethics Approval 366/2003 by the Ethics Committee of the Medical University of Vienna, Vienna, Austria. In brief, pleural effusions were centrifuged and the tumor cells washed with tissue culture medium consisting of RPMI-1640 medium, supplemented with 10% FBS  and antibiotics . When required, erythrocytes were removed by Histopaque®-1077 (Sigma-Aldrich) gradient centrifugation. Primary NSCLC cell lines were established in tissue culture medium and cultures split by trypsination. All cell lines showed an EGFR del19 deletion, with exception of BH1059/RET mutation, BH419 BRCA1 mutation and three lines, namely BH482, BH583 and BH827, with ALK rearrangements.Phosphokinase Array Relative protein phosphorylation levels of 38 selected proteins were obtained by analysis of 43 specific phosphorylation sites using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003B/C  in duplicate tests carried out according to the manufacturer’s instructions. Briefly, cells were rinsed with PBS, 1 × 107 cells/mL lysis buffer were solubilized under permanent shaking at 4 °C for 30 min, and aliquots of the lysates were stored frozen at − 80 °C. After blocking, membranes with spotted catcher antibodies were incubated with diluted cell lysates at 4 °C overnight. Thereafter, cocktails of biotinylated detection antibodies were added at room temperature for 2 h. Phosphorylated proteins were revealed using streptavidin-HRP/chemiluminescence substrate  and detection with a Molecular Imager ChemiDoc MP imaging system . Images were quantified using Image J  and Origin  software. The different Western blot membranes were normalized using the 6 calibration spots included.Cytotoxicity Assay Aliquots of 1 × 104 cells in 200 µL medium were treated for four days with twofold dilutions of the test compounds in 96-well microtiter plates in quadruplicate . The plates were incubated under tissue culture conditions and cell viability was measured using a modified MTT -2,5-diphenyltetrazolium bromide) assay . Optical density was measured using a microplate reader at 450 nm and values obtained from control wells containing cells and media alone were set to 100% proliferation. For the assessment of the interaction of the test compounds, tests were performed comprising the individual drugs alone and in combination, followed by analysis using the Chou-Talalay method with help of the Compusyn software .Statistics Statistical analysis was performed using Student’s t test for normally distributed samples . Values are shown as mean ± SD.50 values for fascaplysin varied from 0.48 – 1.37 µg/ml, with 8/17 cell lines exhibiting high chemosensitivity . The difference in fascaplysin sensitivity of the permanent cell line H23, H1299, PC-9 and A549 versus primary NSCLC lines is not statistically different.Cytotoxicity of fascaplysin, cisplatin and afatinib were determined in MTT assays employing primary NSCLC cell lines and the permanent NSCLC cell lines H23, H1299, PC9 and A549 , with 13/17 cell lines exhibiting chemosensitivity below clinical achievable peak plasma concentrations (PPCs) of 3 µg/ml  and, thus, completely different chemosensitivities of the primary NSCLC cells to these cytotoxic drugs.The IC50 values for afatinib range from 2 µM to approximately 8 µM indicating relatively low sensitivity for these primary NSCLC cell lines with exception of BH584 and BH659 that have revealed a NSCLC-SCLC transformation  are shown in Fig. Combination experiments employing fascaplysin and AZD7762 showed synergistic effects with CI values ranging from 0.35 – 1.13 , except of the BH419 BRCA1-mutated NSCLC cell line. The combinations of cisplatin with AZD7762 showed synergy in 3/13 cell lines  with 10/13 cell lines being significantly different from fascaplysin-AZD7762 combinations  . However50 values of 0.5, 14, and 1 nM, respectively [50 afatinib values for the whole primary NSCLC cell line panel of 4.81 ± 2.05 µM is a typical result for cell lines not dependent on mutated EGFR, such as breast cancer cell lines T47D and BT20, whereas IC50 values for afatinib and cell lines addicted to mutated EGFR may be as low as 6–10 nM [50 values of approximately 50 µM. A pharmacokinetics analysis revealed that plasma concentrations of afatinib peaked at 3 – 4 h after administration and decreased with a half-life of 37 h at steady state [50 values. Although afatinib is the standard drug for the treatment of lung squamous cell carcinoma (SCC) with EGFR overexpression, attempts have been made to use this irreversible blocker for other EGFR expressing tumors. Advanced head and neck squamous cell carcinoma (HNSCC) hold a poor prognosis and tumor progression is associated with overexpression of EGFR [R and A549R, and the cisplatin/gefitinib combination applied promoted apoptotic cell death [The EGFR TKI afatinib is a second generation, irreversible ErbB family blocker, that exhibits inhibitory activity against EGFR, human EGFR 2 (HER2) and 4 (HER4), with ICectively , 24. The 6–10 nM . At extrdy state . Afatini of EGFR . Afatinill death . Anotherll death . In detall death . In genell death .50 5 nM) [The induction of the DNA repair system involves sensing of the damage by ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-Related) kinases and activation of Chk1/2 downstream kinases . The ove50 5 nM) . AZD776250 5 nM) . The sen50 5 nM) . Surpris50 5 nM) . Fascapl50 5 nM) .Investigation of fascaplysin-induced changes in protein phosphorylation in H1299 NSCLC cells was assessed using Western blot arrays, as previously demonstrated for the A549 cell line . The PI3In summary, fascaplysin promotes cell death of NSCLC cell line in a manner different from the standard platinum drugs. This marine drug induces a DNA repair response, syngergizes with the Chk1/2 inhibitor AZD7762 and with the EGFR TKI afatinib.