In addition many ixodid and argasid tick cell lines and, by inference the parent ticks from which they were derived, harbor endogenous viruses (ETV) of which almost nothing is known. In general, low level persistent infections with viral pathogens (arboviruses) are not known to have a deleterious effect on tick survival and fitness, suggesting that they can strike a balance with the tick innate immune response. This tolerance of arbovirus infection may be modulated by the permanent presence of ETV in the host cell. In mosquito cells, temporary or permanent silencing of the genes of an endogenous virus by RNA interference can result in changes in replication rate of a co-infecting arbovirus. We propose that tick cell lines offer a useful model system for  Flaviviridae, Bunyaviridae, and Reoviridae, including the causative agents of tick-borne encephalitis, Crimean-Congo hemorrhagic fever and Kemerovo tick-borne viral fever infections in humans, and louping ill and Nairobi sheep disease in domestic ruminants , is transmitted by ticks of the genus Ornithodoros  and 702 to the Ixodidae (hard ticks) .The first continuous tick cell lines were established from developing adult Ixodes scapularis  decoloratus and Ixodes ricinus, but were unable to amplify a larger section of a different bacterial gene from the same samples. DNA sequences matching Rickettsia africae and Francisella-like endosymbionts were amplified from cell lines derived from, respectively, Amblyomma variegatum and three different Dermacentor species, although no actual bacteria were seen by light or electron microscopy (EM)  remains the only identified and sequenced “tick virus”  and mammalian Vero, BHK-21, BSR, BGM, KB, HELA, Hep2 cells (authors' unpublished results), indicating that it is highly unlikely to be an arbovirus. On the other hand, SCRV was passaged successfully alongside the bacterial pathogen Ehrlichia ruminantium from IDE8 , was detected in the Nuttall, . PhylogeNuttall, . When otBunyaviridae) sequences were amplified by PCR from several of these argasid lines, as well as from nine of the ixodid cell lines  and 3 non-structural proteins (NS1–NS3). Belhouchet et al. (in vitro have a tendency to be strongly phagocytic (authors' unpublished observations) and could actively take up virus particles.The genus t et al.  identifit et al. . Immunoft et al. , NS4 is t et al. , though t et al. . Core pat et al. . MoreoveSince tick cell lines are increasingly being used for isolating and propagating viruses and bacteria, the possible consequences of the presence of endogenous tick viruses (ETV) on co-infection with exogenous microorganisms must be considered.In nature, ticks may be infected with more than one pathogen, but little is known about the interactions between different pathogens co-infecting the same tick, nor about the interactions between co-infecting pathogens and endosymbiotic bacteria , Nudivirus (dsDNA), Totivirus (dsRNA), Partitivirus (dsRNA), Seadornavirus (dsRNA), Cripavirus (+ssRNA), Flavivirus (+ssRNA), Phlebovirus (−ssRNA), Nairovirus (−ssRNA), Lyssavirus (−ssRNA), and Thogotovirus (−ssRNA) , cell fusing agent virus (CFAV)  and Aedes pseudoscutellaris reovirus (ApRV)  in mosquito cell lines, SCRV  in tick cell lines, and Drosophila melanogaster totivirus  in a Drosophila cell line , while many other densoviruses persistently infect specific mosquito cell lines  17D, relatively low copy numbers of YFV could be detected. Ultimately, YFV replication was “silenced” (authors' unpublished data). However, if the co-infection with both viruses was realized simultaneously, the effect of CFAV on YFV 17D was found to be minimal.An endogenous arthropod virus may or may not be species-specific. For example, a densovirus (ssDNA) derived from 4 viral particles/cell. Moreover, an exogenous virus may have unexpected effects on replication of an endogenous virus as seen with infection of Aedes albopictus cell lines. Following Liaoning virus (LNV) infection of C6/36 cells, significantly large amounts (milligram quantities) of the C6/36 densovirus were generated alongside similar quantities of LNV  was used to infect AP61, relatively low copy numbers of SABV were detected (authors' unpublished observation). Like endogenous viruses, endosymbiotic bacteria may also interfere with the replication of an exogenous virus. Dengue virus replication, dissemination and transmission were suppressed in Aedes aegypti mosquitoes infected with Wolbachia . It will be interesting to apply this methodology to tick cell lines and attempt to derive ETV-free tick cells. The well-characterized SCRV represents an ideal target given the availability of the entire genome sequence (Attoui et al., Ultimately, silencing replication of an endogenous virus may be a practical way to generate a virus-free cell line. The replication of ApRV in AP61 cells was silenced by continuously feeding AP61 cells with ApR specific siRNA derived, E. ruminantium in IDE8 cells (Figure Comparison of ETV-infected and ETV-free tick cell lines would be an ideal model system to study the effect of the ETV on replication of various exogenous pathogens, for example SCRV and s Figure , and theThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs  in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict  Small cell lung cancer (SCLC) accounts for about 10–20% of lung cancer cases and is notorious for its aggressiveness and poor survival rate MicroRNA expression correlates with biological characteristics of cancer, such as cell differentiation, aggressiveness, invasion, angiogenesis, and metastatic behavior We recently demonstrated, by using a real-time polymerase chain reaction (PCR) -based plateform, that expression profiles of a panel of seven cancer-associated microRNAs  are neither predictive nor prognostic in NSCLC patients receiving platinum-based adjuvant chemotherapy in situ hybridization of miR-34b in a SCLC tumor specimen and observed cytoplasmic expression of miR-34b and nuclear expression of nucleolar RNA U6 as expected . We observed that the expression of the 7 microRNAs was not associated with chemosensitivity of SCLC cells to either agent .50 values upon cisplatin treatment were 0.9±0.02 µM, 1.19±0.3 µM, 0.83±0.15 µM, and 0.79±0.13 µM, respectively (p = 0.1 by one-way ANOVA)     . The IC5 = 0.24) . For GLC = 0.24) , the IC5 = 0.14) ; the IC5 = 0.08) . In summWe evaluated the expression of selected miR-34a target-genes in SCLC and NSCLC cell lines. Genes selected were METHere we showed the expression of 7 known cancer-associated microRNAs in SCLC tumors and cell lines as well as in NSCLC cell lines. We observed that the expression of these microRNAs was unrelated to clinical characteristics and outcome of SCLC patients. Expression of these microRNAs was unrelated to the sensitivity of SCLC cells to cisplatin or etoposide. Moreover, overexpression or downregulation of miR-34a did not influence SCLC cell viability or sensitivity to cisplatin and etoposide. We further observed that the expression of miR-34a target genes may differ among SCLC and NSCLC cell lines.et al. compared microRNA expression between SCLC cell lines and tumor-free lung tissue from patients with chronic obstructive pulmonary disease et al. compared the microRNA expression pattern between 9 SCLC and 7 NSCLC cell lines, and one mesothelioma cell line et al. compared a SCLC cell line, NCI-H69, with its doxorubicin-resistant sub-line et al. reported the only study using SCLC tumor specimens Although microRNAs have been widely studied in several cancer types, there are only a few publications addressing the role of microRNA in the malignant behavior of SCLC in vitro, in vivo, and in tumor specimens, they have not been evaluated before in relation to patient survival in SCLC. Although our study is retrospective and the patient cohort is small, this is by far the largest SCLC patient cohort and collection of SCLC cell lines investigating microRNA expression in this tumor type. In line with our previous report, a posthoc analysis of a large prospective randomized trial in NSCLC Although expression of the 7 microRNAs in our study was evaluated in many cancer types et al. et al. showed that overexpression of miR-34a does neither affect cell proliferation, cell cycle distribution, nor apoptosis in a hepatocellular carcinoma cell line et al. demonstrated that overexpression of miR-34a has no effect on proliferation of cervical cancer cell lines Hela and SiHa as well as choriocarcinoma cell line JAR miR-34a is a widely studied tumor suppressor microRNA that mediates apoptosis, cell cycle arrest, inhibition of proliferation, senescence, and inhibition of invasion and migration in various cancer types  tumor samples from patients who were included in a phase III trial of the North-Holland Oncology group http://p53.free.fr/), the IARC TP53 Database (http://www-p53.iarc.fr/), and the COSMIC database (http://www.sanger.ac.uk/genetics/CGP/cosmic/).A panel of 14 SCLC and 26 NSCLC cell lines were studied and are listed in the Total RNA was extracted from FFPE samples using the RecoverAll Total Nucleic Acid Isolation kit  and total RNA from cell lines was extracted with Trizol reagent , in accordance with the manufacturer's instructions.Expression of mature miRNAs, including miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a, was evaluated by qRT-PCR analysis as described previously Expression of AXL gene was performed by using Taqman gene expression assay (Applied Biosystems) and was reported as delta Ct value. GAPDH gene was used as endogeneous control.NeoFX™ Transfection Agent (Ambion) following manufacturer's recommendation at a final oligonucleotide concentration of 30 nM.miR-34a precursor and inhibitor as well as Cy3-labeled control miR precursor and inhibitor were purchased from Ambion. Transfection of miR-precursor or inhibitor was carried out using siPORT™ To determine the effect of transfection on cell growth, 5,000–10,000 cells transfected with the indicated microRNA precursor or inhibitor were plated in 96-well plates. Cell growth was determined by CellTiter 96 AQueousOne Solution Cell Proliferation Assay  every 24 hours for 5 successive days. Results were presented as the optical density  absorbance at 490 nm.100,000 NCI-N592 SCLC cells were plated into 24-well plates and transfected with miR-34a precursor or control precursor. Cells were harvested, counted, and transfected again with the respective miRNAs every 72 hours.50) was determined.Cisplatin and etoposide were obtained from Sigma-Aldrich . 10,000 cells were seeded into 96-well flat-bottomed plates, transfected with indicated precursor or inhibitor microRNAs, cultured for 24 hours before the addition of various concentrations of cisplatin and etoposide, and incubated for an additional 72-hour. The cytotoxic effects of cisplatin and etoposide were determined by CellTiter 96 AQueousOne Solution Cell Proliferation Assay. Experiments were performed in triplicate. The percentage of cell viability was determined by dividing the absorbance values of the treated cells by those of the untreated cells, and the concentration of cisplatin or etoposide resulting in 50% growth inhibition . Cell cycle fractions were analyzed using the Modfit v3.0 software.In situ hybridization of miR-34b was performed as described previously Statistical analysis was performed using SPSS version 17.0 . Survival was calculated using the Kaplan-Meier method. The comparison of survival between different groups was determined by the log-rank test. The differences in microRNA expression between groups were analyzed by the Wilcoxon Rank-Sum test. The correlation between drug sensitivity and microRNA expression was analyzed by Spearman rank correlation. Comparison of drug sensitivity of cells transfected with various oligonucleotides was carried out by one-way analysis of variances (one-way ANOVA). All p-values were two-sides and p-values less than 0.05 were regarded significant.Figure S1Expression of microRNAs in SCLC versus in NSCLC cell lines. A delta Ct value of −15 indicates that the expression of the microRNA in the cell is too low to be detected.(TIF)Click here for additional data file.Figure S2in situ hybridization of microRNAs in a SCLC tumor. Nucleolar RNA U6 and miR-34b were detected using a biotinyl tyramide-based system with Vector NovaRed as a substrate (brown). Specimens for scramble negative control and miR-34b were co-stained with Mayer's hematoxylin (blue).(TIF)Click here for additional data file.Figure S3Correlation of expression of p53 and (A) miR-34a, (B)miR-34b, and (C) miR-34c, and (D) correlation of expression of miR-34a and bcl-2. Expression of p53 and bcl-2 were presented as percentage of stained cells by immunohistochemistry. Correlation coefficient (r) and p-value were determined by Spearman method.(TIF)Click here for additional data file.Figure S4NCI-H82 cell transfected with (A) Cy3-labeled microRNA inhibitor and (B) Cy3-labeled microRNA precursor.(TIF)Click here for additional data file.Figure S5Cell growth assay of NCI-H1299 NSCLC cells transfected with miR-34a or control precursor. Error bars refer to standard deviations.(TIF)Click here for additional data file.Figure S6Growth inhibition assay of the GLC4 cells treated with (A) cisplatin and (B) etoposide. Cells were transfected with the indicated oligonucleotides. Error bars indicate standard deviation of assays in triplicate.(TIF)Click here for additional data file.Figure S7Protein expression of c-MET and bcl-2 in 5 SCLC and 3 NSCLC cell lines. (B) Correlation between miR-34a and c-MET expression as well as bcl-2 in 5 SCLC cell lines.(TIF)Click here for additional data file.Table S1MicroRNAs and clinical covariables.(DOC)Click here for additional data file.Table S2Effect of clinical covariables on progression-free survival (PFS).(DOC)Click here for additional data file.Table S3Effect of microRNA expression on progression-free survival  in SCLC patients stratified by limited disease or extensive disease.(DOC)Click here for additional data file.Table S4TP53 status and miR-34a expression in SCLC cell lines.(DOC)Click here for additional data file.Materials S1Cancer cell lines.(DOC)Click here for additional data file.


CTNNB1 will respond to trametinib and other MEK inhibitors, and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR, ABL1 and BRAF(V600E)-driven cell growth, and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors, we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action.The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013), and for six of these drugs, the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers, suggesting that cancers dependent on mutant  Targeted therapies significantly increase the efficiency of cancer therapy. They bring great benefit to patients because they improve survival rates with much less side effects than traditional cytotoxic therapies. Small molecule inhibitors of protein kinases are a prime example of the success of targeted therapy. There are currently (Nov. 2013) twenty-five kinase inhibitor drugs approved for clinical use, all except two for cancer . In 2012Crucial to the development of targeted therapies is the ability to couple drug response to a genetic marker such as mutation, translocation or overexpression of a cancer gene To better predict patient responder populations at an early stage in drug development, and to better understand kinase drug action, we have established a cancer cell line panel of forty-four cell lines that have been derived from a wide diversity of human tumours . The canRecent studies have demonstrated that cell line panels can be used to identify new markers of drug sensitivity by coupling drug response to the presence of cancer gene mutations To compare the anti-proliferative activities of all kinase inhibitor drugs that have been approved for clinical use, we have profiled them in our forty-four cell line panel. We correlated drug activities to cancer gene mutations and identified new drug sensitivity markers for MEK and EGFR inhibitors. In addition, the cell panel data were used to quantitatively compare the relative targeting efficacy of drugs designed to inhibit the same kinase.To further study biochemical origins of differential effects in cellular targeting we profiled all kinase inhibitor drugs on a panel of enzyme activity assays of more than 300 wild-type and mutant kinases 50s fell within the testing range, it was extended to subnanomolar concentrations in the case of very potent compounds. To preserve cell line characteristics, the cell lines were cultured in the media recommended by the original investigators who deposited the cell lines, and by ATCC. All cells used were within nine passages from the original ATCC vial.A panel of forty-four human cancer cell lines was assembled from the American Type Culture Collection (ATCC). The cell lines were selected to represent both a wide range of different tissue tumour types  and many50 shift of a factor 2 and a standard deviation of 0.07 on 10logIC50 values (a factor of 1.17), based on multiple independent measurements of the same compounds across the panel  mutation. ABL1 inhibitors and EGFR inhibitors preferentially inhibited cell lines that are dependent on ABL1 and EGFR oncogenes, respectively  mutant and the Ephrin receptors, which can suppress EGFR by cross-talk EGFR inhibitors are one of the earliest examples of targeted therapies . Gefitinhibitors . Also EGhibitors , which hhibitors . Its supi.e., BRAF(V600E), has led to the development of the RAF inhibitors vemurafenib and dabrafenib (BRAF(V600E), followed at a distance by vemurafenib (50 than non-mutant cell lines. For vemurafenib the difference was 3-fold. There was no correlation between the cellular activity of sorafenib and BRAF(V600E). Targeting efficacy of the RAF inhibitors is related to the enhanced biochemical potency of dabrafenib compared to vemurafenib, as dabrafenib is less selective . Imatinib, nilotinib, dasatinib, ponatinib and bosutinib are approved drugs for this indication. However, also many growth factor kinase inhibitors such as crizotinib, vandetanib, axitinib and sunitinib are potent ABL1 inhibitors  of the association is low. The most targeted ABL1 inhibitor in Another important class of kinase inhibitor drugs are those targeting ABL1, of which a re-arranged form, hibitors . Anova aosutinib . Dasatin50 shift (ΔIC50) of a compound in the Anova analyses was taken as basis, as it indicates the difference in potency of a compound between sensitive (mutant) and insensitive (wild-type) cell lines. Another important parameter is the remaining variance between IC50s in the wild type or oncogene-carrying group of cell lines (σmutwt or ), which is indicative of additional effects on cell growth besides the main inhibitor mechanism. To combine both values we selected the standardized mean difference (SMD) as a quantitative tool, which is calculated as ΔIC50/. For the clinically used EGFR, ABL1 and BRAF kinase inhibitor drugs this quantity clearly shows that dabrafenib and imatinib are exceptionally targeted and that gefitinib and erlotinib are near-equivalent  as significant side-activity . Inactivation of SMAD4 cooperates with KRAS to enhance EGFR expression levels SMAD4 mutation may therefore be further explored as a candidate marker for the selection of patients eligible for EGFR-inhibitor therapy.Another new drug sensitivity marker was identified for EGFR inhibitors, which were not only active in utations . The assBRAF, ABL1 and EGFR. Our data demonstrate that biochemical potency (illustrated by comparing dabrafenib to vemurafenib) and biochemical selectivity (illustrated by comparing the EGFR inhibitors) are both important for effective targeting of EGFR, BRAF and ABL1 inhibitors to EGFR, BRAF(V600E) and ABL1 transformed cell lines (ABL1(T315I) resistance mutation  mutant melanoma more effectively in vivo than current available BRAF inhibiting drugs.Next our data shows that there is still potential to increase the targeting to highly validated cancer driving genes such as ll lines . These imutation . A compoin silico identification of such fortuitous polypharmacology. This revealed that ABL1 inhibitors benefit from a secondary ABL2 activity in the targeting of ABL1-driven cell growth  and were authenticated by ATCC. Master and working cell banks and assay-ready stocks were prepared by subculturing in ATCC-recommended media and freezing according to ATCC recommended protocols. Master cell banks, working cell banks and assay stocks were prepared between 3, 6 and 9 passages from the original ATCC vial, respectively. A full list of the cell lines in the Oncolines panel is available in −5 M to 3.16·10−9 M in duplicate. In case a compound showed low nanomolar or subnanomolar activity, it was further diluted 100 times and a new dose-response curve in duplicate was measured. The final DMSO concentration during incubation was 0.4% in all wells.All compounds were obtained from commercial suppliers  and disswww.fda.gov).Information on the therapeutic application of compounds was obtained from the label information of the US Food and Drug Administration  solution was added to each well. Luminescence was recorded on an Envision multimode reader .An assay stock was thawed and diluted in the media as recommended by ATCC, and dispensed in a 384-well plate, depending on the cell line used, at a concentration of 400–1600 cells per well in 45 μl medium. For each used cell line the optimal cell density was used and the number of cells per well was optimized to obtain a maximum assay window. Plated cells were incubated in a humidified atmosphere of 5% CO50 of the reference compound doxorubicin was measured on a separate plate. The IC50 was trended. If the IC50 was out of specification (i.e. <0.32 or >3.16 times deviating from historic average) the assay was invalidated. Fourth, the cellular doubling times of all cell lines were calculated from untreated wells. If the doubling time was out of specification  the assay was invalidated.Firstly, the cell signal at the start of incubation was recorded separately in order to distinguish between cell population growth and cell death. Secondly, for each cell line, the maximum growth was measured by incubation of a duplicate without compound in the presence of 0.4% DMSO. Third, as a control for compound dilutions, the IC50s were fitted by non-linear regression using IDBS XLfit5 using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50. In addition, using the initial cell counts, measures for cell death (LD50) and growth inhibition (GI50) were fitted 50s were measured within an enveloping concentration range.Percentage growth was used as the main y-axis signal. IC10logIC50s were used as unscaled data to preserve their physical meaning and to compare absolute compound potencies. Biochemical %-inhibition data at 1 μM (see below) were restricted to values between 0% and 100% before clustering. For both datasets we applied unsupervised hierarchical clustering in the program R Cellular ABL1 transformation on basis of the literature ABL1-transformed in the CCL database. Mutations were treated as factors and the 10log IC50 as signal and analysed by n-way Anova in the statistical program R 50 data were not normalized, but taken as-is, as this was giving good results for known associations. No interactions between factors were allowed. The minimum number of mutants for each gene to be incorporated in the test was 2, consistent with earlier analyses 10log IC50 between mutant and wild-type cell lines. To correct for multiple testing, p-values were subjected to a Benjamini-Hochberg correction Anova  was used to test if there is a statistical correlation between a particular genetic change in the panel of cell lines and drug sensitivity. In principle, any number of genetic markers can be queried but because the number of hypotheses influences the reliability of associations in Anova analysis, we selected for our analysis the most frequent and most-studied oncogenes, as assessed from their frequency in a large cell line panel 10logIC50 was subsequently analysed with type II Anova as above.For gene expression and copy number analysis, we limited the amount of hypotheses in Anova testing and reduced multiple-testing artefacts, by only considering the validated oncology targets EGFR, ERBB2, Kit, Met, PDGFRα and PDGFRβ . Gene exM,ATP for every individual kinase (KM bin). All 1 μM full profiles are given in 50s were determined  were calculated for each kinase inhibitor drug as outlined above . Each kiFigure S1Characteristics of the CCL (COSMIC) panel and the NCI-60 panel.(DOCX)Click here for additional data file.Figure S2Reproducibility of the cell panel data.(DOCX)Click here for additional data file.Figure S3Volcano-analysis of nutlin 3a.(DOCX)Click here for additional data file.Figure S4Volcano-analysis of drug sensitivity of twenty-five approved kinase inhibitors and seven cytostatic therapies to twenty-three common genetic changes.(DOCX)Click here for additional data file.Figure S5Volcano-analysis of drug sensitivity of twenty-five approved kinase inhibitors and seven cytostatic therapies to genetic changes in growth factor signalling.(DOCX)Click here for additional data file.Figure S6Volcano-analysis of the MEK inhibitors PD-0325901 and selumetinib (AZD-6244).(DOCX)Click here for additional data file.Figure S7Volcano-analysis of the EGFR inhibitors pelitinib (EKB-569) and neratinib (HKI-272).(DOCX)Click here for additional data file.Figure S8sel) can be estimated on basis of single concentration data.Selectivity entropy (S(DOCX)Click here for additional data file.Table S1Names and sources of approved kinase inhibitor drugs used in this study.(DOCX)Click here for additional data file.Table S2Frequency of point mutations in validated cancer genes in the cell panel.(DOCX)Click here for additional data file.Table S3List of genes mutated in the Oncolines panel that have been used to investigate drug sensitivity.(DOCX)Click here for additional data file.Table S4Statistical power analysis to determine cutoff levels for significance in the cell line panel.(DOCX)Click here for additional data file.Table S550s of 25 kinase inhibitors on 44 cell lines.IC(XLSX)Click here for additional data file.Table S6Biochemical inhibition of more than 300 kinases by 25 kinase inhibitors .(XLSX)Click here for additional data file.Table S7List of all cell lines in the Oncolines panel.(DOCX)Click here for additional data file.


In the present study, we wished to examine CAP selectivity in HNSCC in vitro models, and to explore its potential for use as a minimally invasive surgical approach that allows for specific cancer cell or tumor tissue ablation without affecting the surrounding healthy cells and tissues. Four HNSCC cell lines  and 2 normal oral cavity epithelial cell lines (OKF6 and NOKsi) were subjected to cold plasma treatment for durations of 10, 30 and 45 sec, and a helium flow of 20 l/min−1 for 10 sec was used as a positive treatment control. We showed that cold plasma selectively diminished HNSCC cell viability in a dose-response manner, as evidenced by MTT assays; the viability of the OKF6 cells was not affected by the cold plasma. The results of colony formation assays also revealed a cell-specific response to cold plasma application. Western blot analysis did not provide evidence that the cleavage of PARP occurred following cold plasma treatment. In conclusion, our results suggest that cold plasma application selectively impairs HNSCC cell lines through non-apoptotic mechanisms, while having a minimal effect on normal oral cavity epithelial cell lines.The treatment of locoregional recurrence (LRR) of head and neck squamous cell carcinoma (HNSCC) often requires a combination of surgery, radiation therapy and/or chemotherapy. Survival outcomes are poor and the treatment outcomes are morbid. Cold atmospheric plasma (CAP) is an ionized gas produced at room temperature under laboratory conditions. We have previously demonstrated that treatment with a CAP jet device selectively targets cancer cells using  Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide with an approximate 5-year survival rate of 50% . The proThe standard of care for advanced tumors is surgery combined with adjuvant radiation therapy and/or chemotherapy. Survival outcomes are poor , and the treatment leads to morbidity  were cultured in RPMI-1640 cell culture medium  supplemented with 10% fetal bovine serum (Sigma) and Pen/Strep (100 units/ml penicillin and 100 μg/ml streptomycin) . The 2 normal oral cavity epithelial cell lines (OKF6 and NOKsi) were grown in Keratinocyte-SFM (1X) supplemented with Keratinocytes Supplements (both from Gibco/Life Technologies). All cells were obtained from the Johns Hopkins University Head and Neck Cancer Division cell bank and incubated at 37°C in an atmosphere of 5% CO−1, with a distance of 3 cm from the plasma source to the cells, and treatment durations of 10, 30 and 45 sec.The CAP device, created in the School of Engineering and Applied Science of The George Washington University, contains 4 blocks. Block 1 is a DC power supply. Block 2 is a centrally powered electrode with a ground outer electrode wrapped around a quartz tube, which is part of the cold plasma production. Block 3 consists of a capacitor, a transistor and a timer; and block 4 is the helium gas supply, as previously described . Cold pl−1 for 10 sec as a positive treatment control  was performed on the plated cells 48 h after cold plasma application, according to the manufacturer’s instructions and the absorbance at 570 nm was measured. Clonogenic or colony formation assays were performed 7 days after treatment with cold plasma; colonies were visualized by staining with crystal violet (Sigma).Western blot analysis of PARP cleavage was performed 48 h after cold plasma treatment as follows: cell lysates were separated by SDS-PAGE on Tris-glycine gels and transferred to PVDF membranes  The membranes were blocked with TBS-T + 5% non-fat dry milk and incubated overnight at 4°C with an antibody specific for PARP . The membranes were washed and incubated with horseradish-peroxidase conjugated secondary antibodies. Protein detection was performed by enhanced chemiluminescence.The results from MTT assay revealed that cold plasma selectively diminished the viability of the SCC25 and JHU-O28 HNSCC cells in a dose- response manner . The JHUThe main purpose of this study was to assess the selectivity of cold plasma in HNSCC cell lines and the mechanisms underlying this selectivity. Our results suggest that cold plasma application selectively impairs some HNSCC cell lines through non-apoptotic mechanisms, as the cleavage of PARP was not significantly altered in the treated cells, while having a minimal effect on normal oral cavity epithelial cell lines.in vitro mechanisms have been suggested to be associated with a decrease in the expression of cell-surface proteins, such as integrins and FAK: cell detachment, the induction of apoptosis, the induction of senescence and the generation of ROS  represent extremely difficult management problems . SalvageTP53 mutations in their tumors, but the cell cycle is still deregulated in these patients, as the E6 HPV protein silences TP53  cells, while minimally affecting healthy human bone marrow mesenchymal stem cells (MSCs)  . SimilarCold plasma treatment represents an alternative means to selectively targeting HNSCC cells, while having a minimal effect on the normal adjacent tissue and a feasible therapeutic strategy if coupled with endoscopic technology. It could also be potentially used in the first-line eradication of small malignant growths and as an adjuvant irradiation treatment of malignant tissue prior to surgery and surgical margins after surgery. Cold plasma represents an alternative adjuvant therapy that may lead to a reduction in LRR, particularly in HPV-negative patients and, as such, it warrants further investigation.


Primary mediastinal B cell lymphoma (PMBL) is characterized by specific molecular hallmarks including the expression of B Cell Lymphoma factor 6 (BCL6) and the presence of the activated Signal Transducers and Activators of Transcription factor 6 (STAT6). Recently we have shown that combined targeting of BCL6 and activated STAT6 by specific chemical inhibitors in PMBL resulted in additive efficacy regarding their negative effects on cell viability. Given that despite general efficient immuno-chemotherapy in PMBL the delayed treatment-related sequelae remains still a main challenge, we analyzed the role of BCL6 and activated STAT6 in the sensitivity of PMBL cells to the current treatment components. We found that the knock-down of BCL6 / STAT6 by siRNA sensitized the PMBL cells to the effects of R-CHOP components in two of three PMBL cell lines. In one cell line, MedB-1, which is marked by less expression of BCL6 and mutated STAT6, the knock-down of BCL6 / STAT6 did not enhance the efficiency of Doxorubicin, Rituximab, and Vincristin.Thus, the targeting of BCL6 and STAT6 in addition or prior to the treatment with components of the current immuno-chemotherapy may sensitize the PMBL tumor cells for drug effects, at least in parts of PMBL cases. Primary mediastinal B cell lymphoma (PMBL) is a distinct subtype of aggressive B cell lymphoma and meanwhile belongs to the most curable lymphoma subtypes . Since rOne of specific molecular characteristics of PMBL is the presence of the anti-apoptotic and pro-proliferative factors BCL6 in about 90% -5 and pSHere we used knock-down approach to evaluate the role of BCL6 / STAT6 in the modulation of the PMBL cell sensitivity to the commonly used therapeutic agents, such as Doxorubicin, Rituximab, and Vincristin which represent major components of the current standard therapy for PMBL, R-CHOP . We found in 2/3 PMBL cell lines that downregulation of BCL6 / STAT6 sensitizes the tumor cells for treatment with immuno-chemotherapeutic agents.Therefore, our study suggests that targeting BCL6 / STAT6 additionally to standard therapy in PMBL may further improve responsiveness of these tumors.In order to investigate the potential role of BCL6 and pSTAT6 in the modulation of PMBL sensitivity to the anti-tumor effects of Doxo, RmAb and VC we followed the experimental scheme shown in Figure After 72h of post-transfection the therapeutic agents, Doxorubicine (Doxo), Rituximab (RmAB), and Vincristin (VC) were added and cells were incubated for further 24h. In each cell lines the efficacy of BCL6 or /and STAT6 downregulation mediated by siBCL6 or siSTAT6 relative to control sample (siCtr) was monitored by western blot Figure .The cell viability analysis was performed by using a luminescence method based on quantitation of the ATP as the measure for metabolically active cells.As showen in Figure Next, we analysed the cell viability by a luminescence assay, based on the quantitation of the ATP as a measure of metabolically active cells. We observed that downregulation of BCL6 or STAT6 in K1106 and U2940 sensibilized the cells to the anti-tumor effects of Doxo, RmAB, and VC by impairing cell viability, although some differences between cell lines were detected Figure . NotablyThese results underline our recently published data regarding the new therapeutic approaches using chemical inhibition of BCL6 and /or STAT6 . Thus, tUnexpectedly, in MedB-1 cells neither the knock-down of BCL6 nor of STAT6 induced any additional anti-tumor effects to the threatment with Doxo, RmAB, and VC Figure . MoreoveThe expression of BCL6 in MedB-1 cells is less frequent then in the other two PMBL cell lines  . Of noteOnly in MedB-1 cells the administration of current therapy components in combination with STAT6 downregulation did not lead to any advantage with respect to the drugs efficiency. This suggests a specific molecular background of this particular cell line. In fact, MedB-1 showed the highest amount of pSTAT6 within the three PMBL cell lines   and harbThe inactivation of STAT6 in PMBL by using selective JAK2 inhibitor, TG101348, reduced the growth of PMBL cells ,10. TakeIn sum, targeting of STAT6 / BCL6 by siRNA in two of three cell lines sensitizes the PMBL cells to the anti-tumor effects mediated by treatment with Doxo, RmAb, and Vincristin. Thus, the sensitisation of PMBLs to the current chemo-immunotherapy by the additional targeting of BCL6 / STAT6 may be useful and may help to reduce drugs toxicity and/or relapse in PMBL patients.6) were transfected with 0.2nmol/sample of control siRNA  or siRNA targeting STAT6  or BCL6 . After the incubation for 72h several components of current R-CHOP were added or not to the cells.Three established human PMBL cell lines MedB-1, K1106, and U-2940 were maintained as described . All celWestern blots were performed as described  by usingPMBL cell lines were treated for 24h with Doxo , VC (1μg/ml) and RmAB (1μg/ml). According to manufacture's protocol the cell viability analysis was performed using the CellTiter-Glo® Luminescent (Promega, Mannheim, Germany.p <0.05.T-tests were used for statistical comparisons and the statistical significance was defined as


Donor Lymphocyte infusion (DLI) is a therapy for the patients with relapsed hematological malignancy after allogeneic hematopoetic stem cell transplantation. However, the development of Graft-Versus-Host Disease (GVHD) is a serious adverse event and the efficacy is limited when one needs to control the GVHD. To inhibit the development of GVHD with increased tumor-specificity of transferred allogeneic lymphocytes, here we demonstrate the development of DLI with lymphocytes engineered to express tumor-specific T cell receptor (TCR) in combination with decreased GVHD-inducing potential utilizing the novel retrovirus vector (siTCR vector) that specifically silences endogenous TCR in gene-engineered T cells.3H uptake proliferation assay. Immunodeficient NOG mice were inoculated with a NY-ESO-1-expressing human melanoma cell line NW-MEL-38, received TCR gene-transduced T cells, and monitored for tumor growth and the development of GVHD.Human PBMC were transduced with a high affinity TCR specific to a cancer/testis antigen, NY-ESO-1, by the retrovirus vector with siRNA specific to the endogenous TCR. Resulting TCR gene-transduced T cells were examined for their reactivity to allogeneic LCL by Human lymphocytes that were genetically engineered to express a high affinity NY-ESO-1-specific TCR with siTCR vector showed reduced expression of endogenous TCR associated with the dramatically diminished reactivity to allogeneic lymphocytes. When administrated into NOG mice, these TCR gene-transduced T cells induced tumor regression without the development of GVHD.The results here suggest that the allogeneic T cells transduced with a tumor-specific TCR by siTCR vector showed diminished GVHD potential. These T cells will be applicable to the donor lymphocytes infusion therapy after allogeneic stem cell transplantation for the treatment of hematological malignancy, providing diminished GVHD and increased tumor eradication.


Amyloid depositioninvolves the islet amyloid polypeptide (IAPP), a neuropancreatic hormone cosecretedwith insulin by β-cells. IAPP is physiologically involved in glucose homeostasis,but it may turn toxic to β-cells owing to its tendency to misfold giving rise to oligomers and fibrils.The process by which the unfolded IAPP starts to self-assemble and the overall factors promoting thisconversion are poorly understood. Other open questions are related to the nature of the IAPP toxic speciesand how exactly β-cells die. Over the last decades, there has been growingconsensus about the notion that early molecular assemblies, notably small hIAPP oligomers, are the culprit of β-cells decline. Numerous environmental factors might affect the conformational, aggregation, and cytotoxic properties of IAPP. Herein we review recent progress in the field, focusing on the influences that membranes, pH, and metal ions may have on the conformational conversion and cytotoxicity of full-length IAPP as well as peptide fragments thereof. Current theories proposed for the mechanisms of toxicity will be also summarized together with an outline of the underlying molecular links between IAPP and amyloid beta (Aβ) misfolding.Pancreatic islets in type 2 diabetes mellitus (T2DM) patients are characterized byreduced  Type 2 diabetes mellitus (T2DM) accounts for about 90% of all the diabetes cases [β-cell death indicates the possible contribution of islet amyloidosis in the increase of β-cell failure in T2DM. IAPP is a member of the calcitonin like family of polypeptide and it is synthesized as an 89-residue preprohormone. Removal of the 22-residue signal sequence leads to the 67-residue pro-IAPP, which is then processed in the Golgi and in the insulin secretory granule to give the 37-residue mature hormone [Diabetes is a complex metabolic disorder characterized by chronic hyperglycemia and associated with macrovascular and microvascular complications . It is ees cases , and amyes cases –5. Thesees cases . The strThe physiological roles of IAPP are receptor mediated. The IAPP receptor is formed by a complex of the calcitonin receptor with a receptor activity modifying protein (RAMP) , 11. IAPThe affinity of IAPP for the CTR-RAMP complex is higher, with an IC50 reported to be in the order of 8 nM for the CTR-RAMP-1 complex .Circulating concentrations of IAPP have been reported to range between 3 and 5 picomolars in rats, rising to 15–20 picomolars with increased blood glucose levels . However in vitro, while the rat IAPP (rIAPP) is not, even though the two polypeptides differ at only six positions located in the region 18–29. Notably, rIAPP contains three proline residues within the 20–29 sequence and these are believed to be responsible for its inability to form amyloid. Much attention has been focused on the sequence within the 20–29 region and the role it plays in controlling amyloid formation. This portion of the polypeptide chain is considered to be one of the major determinants of the ability of IAPP variants to form amyloid [IAPP is phylogenetically well preserved and found in all mammals, but not all species form amyloids –25. Mice amyloid –28. in vitro amyloidogenicity, mutations outside of this region can eliminate amyloid formation, indicating that it cannot be the sole factor controlling IAPP's amyloidogenicity [β-cell cultures as well [While there is a strong correlation between the primary sequence of the 20–29 segment andgenicity . Moreove as well , 33.β) misfolding.In the present review we summarize recent progress in the field focusing on the influences of membranes, pH, and metal ions on the aggregation and cytotoxicity of IAPP and related peptide fragments. The current theories proposed for the mechanisms of toxicity will be also illustrated together with a brief account of the molecular links between hIAPP and amyloid beta  until a critical concentration of nuclei (oligomers) is reached, at which point fibrils growth proceeds exponentially. The kinetic of assembly is typically intricate and involves a variety of on- and off-pathway intermediates found among all amyloidogenic proteins [l growth . In facthIAPP fibrils are stable structures not easily degraded by proteases. Generally harsh conditions, such as the use of concentrated formic acid or organic solvents, are needed to obtain their depolymerization . The staβ-sheet structure running perpendicular to the length axis of the fibril [β-sheet structure with a loop region located between them, whereas residues 1–7 are largely unstructured. Another model of the hIAPP fibril suggests that the fibril is composed of three protofilaments, each based on stacking of single hIAPP molecules in which residues 9–37 participate in a planar S shaped fold forming the β-sheet structure [On the contrary, the study of the monomeric and oligomeric structures of hIAPP in physiological buffers has been troubled by their instability and fast “spontaneous” aggregation of the species. The most recent and most detailed model for the structure of hIAPP fibrils suggests that they are composed of stacked layers of two symmetric hIAPP molecules. These molecules form a parallel e fibril . Residuetructure , 39.β-cells, but the molecular mechanisms of hIAPP deposition are poorly understood.Substantial evidence indicates that the membrane-mediated aggregation and subsequent deposition of hIAPP are linked to the dysfunction and death of pancreatic  in vitro studies have shown that early oligomers of hIAPP formed during the aggregation process are the principal cytotoxic species and their toxicity is exerted through interactions with cell membranes. However many different hypotheses have been reported [Manyreported , 40.Two general theories exploring membrane disruption by hIAPP or other amyloid proteins have been proposed to date, even though the mechanisms are not exclusive and both may be operative at the same time. The first, known as the “pore theory,” suggests that amyloid proteins form discrete pores within the membrane, either through a traditional ion channel type (“barrel stave”) or in localized ruptures of the membrane that resemble discrete pores in some respects , 41.In fact hIAPP has been found to increase the conductance of planar phospholipid bilayers . The secβ-cells contain 2.5–13.2 mol% of anionic lipids and raft components (cholesterol and sphingomyelin) [ in vivo environment are useful for the understanding of amyloid deposition by hIAPP and for the development of effective therapeutically strategies [omyelin) –46. Thusrategies . Recent rategies –49. Interategies , 50. Therategies .The N-terminal part of hIAPP, whilst not significantly involved in hIAPP fibril growth, was proposed to be important in the light of hIAPP membrane interactions –53. Howeα-helical structure of hIAPP might play an important role in IAPP fibrillation in vivo. This is probably because of the exposure of the 20–29 region to an aqueous environment [It is suggested that a transient membrane-bound ironment , 56, eveironment . The expironment , 58, 59.Support to the importance of the helix intermediate on IAPP aggregations is given by the effect of helix-inducing solvent HFIP on aggregation. A low percentage of HFIP (1%) has a strong catalytic effect on aggregation while higher percentages trap the protein in a helical monomeric state , 39.Since low percentage of HFIP affects the aggregation process it is important prior to starting any biophysical or biological measurements to exclude any trace of HFIP from the sample solution.For these reasons it becomes important to adopt suitable procedures for sample preparation in order to achieve reproducible results .α-helical or β-sheet conformations into insoluble, aggregated β-pleated sheet structures. The conformational changes of IAPP are high conditional and even slight changes in parameters, such as pH, peptide concentration, presence of preformed seeds, or metal ions concentration, may lead to different forms of aggregates that can be either fibrillar or amorphous.Fibrillization involves the conversion of soluble random coil, β-cells [As reported above, hIAPP is stored at extremely high concentrations in the secretory granules of pancreatic β-cells , togetheβ-cells –63.Conversely, the physiological pH of 7.4 for the extracellular matrix, into which amylin is secreted as the mature active hormone, favors fibril formation .α-amino group at the N-terminus and His18, because the C-terminus is naturally amidated in the mature hormone and it does not titrate with pH [Amylin is a relatively simple system for the study of the effects of pH on fibrillization, because it has only two ionizable groups near neutrality, the  with pH , 63.α-amino group leads to a relatively modest inhibition of fibrillization; this slight effect of the α-amino group is consistent with its location in a disordered part of the amylin fibril structure model [β-sheet of amylin fibrils, causes a stronger inhibition of fibrillation as shown in different works. This is due to electrostatic repulsion between His18 stacked along the fibril axis [It has been shown that protonation of the re model . Ionizatril axis , 63. As ril axis , 64.While the effect of pH on the morphology of the hIAPP aggregates has been reported by several groups , 65, 66, in vivo [Literature work on IAPP's aggregation process focuses also on biophysical studies on IAPP related shorter sequences, derived from fibrillogenic regions of the full length parent peptide. Among these, the hIAPP17–29 peptide has been proposed as a useful model able to reproduce some of the key features of the whole hIAPP1–37 , 67. In  in vivo .β-amyloid and prions were found to interact with copper, affecting the redox cycle of the metal to generate ROS in vitro [β-cell toxicity is still controversial. The majority of the studies dealing with the significance of Cu(II) and IAPP interactions emphasized the formation of H2O2 by human IAPP in vitro, the production of which is modestly elevated in the presence of Cu(II) [2O2 was found to directly contribute to the toxicity of the β-amyloid peptide in primary neuronal cell cultures [β-cells as copper compounds increased hIAPP-induced cytotoxicity by facilitating the apoptotic effect of hIAPP. Furthermore, Cu(II) promoted ROS overproduction and mitochondrial disruption which might be the main reason for the enhanced apoptosis [β-amyloid and prions, were consistent with these findings. It has been reported that, similar to human amylin, these two misfolded proteins form Cu(II) complexes with physiologically relevant affinities. Chelation of Cu(II) by these two amyloids greatly diminished the production of ROS by Cu(II) and reduced their toxicity, which further corroborates the hypothesis that hIAPP/Cu(II) complex may have a protective role in cells [β-cells from IAPP toxicity by increasing the activation energy needed for IAPP aggregation and by inhibiting IAPP-induced stress-kinase activation and signalling. Interestingly, the interaction of Cu(II) with hIAPP works as an antioxidant in cells rather than prooxidant activity as observed in vitro. This study further shows that both the hIAPP-induced ROS generation and the hIAPP-dependent activation of stress kinases (JNK) and caspases were blocked by preincubating hIAPP with Cu(II) [Metals ions, notably Cu(II) and Zn(II), and metal ions dyshomeostasis have been linked to the progression of several pathologies, including Alzheimer's and T2DM diseases , as theiin vitro –72. The in vitro , 74 havef Cu(II) , 76. Remcultures . A very poptosis . Previouin cells , 80. Othh Cu(II) .Determinations of hIAPP fibrillization with ThT, AFM, and EM have revealed that Cu(II) inhibits the formation of mature hIAPP fibrils . HoweverConformational changes can also explain the highest level of toxicity that has been observed after cell culture treatment with the hIAPP 17–29 fragment preincubated in the presence of Cu(II) and the absence of effects of copper on hIAPP1–37 spontaneous evolution towards toxic species . Moreoveβ-cell granules contain millimolar concentrations of Zn(II), one of the highest levels in the body, and diabetics often exhibit zinc deficiency [β; however, the physical basis of the effect is complex [β-sheets, this would disfavour the formation of mature fibrils [The effect of Zn(II) on hIAPP fibrillogenesis and toxicity is of particular interest as ficiency , 86, 87. complex . Zn(II)  complex , 68. Sin fibrils , 89.β-cells zinc was proposed to be required for multiple steps in insulin synthesis crystallization and release [β-cells, the ZnT8, were shown to become glucose intolerant when fed with a high fat diet [Another study shows that zinc binds and stabilizes prefibrillar aggregates of amylin during the lag-phase . Zinc is release , 92, butfat diet .β-cell apoptosis are two key determinants of islet dysfunction in T2DM [β-cells is nowadays acknowledged, but neither the precise nature of the harmful IAPP species nor the mechanisms of cytotoxicity have been clarified. Most studies carried out during the last decade have pointed out that hIAPP amyloid fibrils do not contribute directly to IAPP cytotoxicity, since there is no strong correlation between the extent of amyloid deposits and the disease severity [β-cell death. Konarkowska and colleagues found that mature hIAPP fibrils were not cytotoxic in vitro; rather the transition from random coil to β-sheet, coupled with de novo aggregation, was necessary for the induction of β-cells death [β-cells from apoptosis induced by either overexpression or application of hIAPP [β linked to Alzheimer's disease and α-synuclein linked to Parkinson's disease [β-cells of patients with T2DM and increase in abundance with obesity. Recently, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, have been exclusively identified in diabetic patients and have been shown to neutralize the apoptotic effect induced by these oligomers [Current studies performed in primates strongly support the concept that islet amyloidosis and  in T2DM –96. The severity . A growils death . In linels death  which isls death  did not of hIAPP . Similar disease , 103. hIligomers . The preligomers .β-cells or transgenic animal model susceptible to hIAPP-evoked diabetes, β-cell apoptosis may be activated by hIAPP oligomers originating either on the surface of cells or within them [Another big unsolved issue is whether the hIAPP-induced apoptosis is triggered by species formed outside or inside cells. Based on studies carried out using cultured hin them , 106–110hin them  implyinghin them , 113. Rehin them , 33. Sevhin them ).α-helical structure upon binding to anionic membranes and forms β-sheet-rich amyloid fibers during the progression of type II diabetes. Interaction between IAPP and cellular membranes was proposed as the cause of IAPP cytotoxicity and β-cell death in T2DM, since detailed ultrastructural investigations have revealed that islet amyloid was often in contact with β-cell membranes [ in vitro studies noting that toxic oligomers of amyloidogenic proteins induce membrane permeability and membrane disruption [β-cell death, particular T2DM-related surrounding conditions should exist that initiate hIAPP-induced membrane damage. An increase in the level of hIAPP, which is coproduced and cosecreted with insulin, in a state of insulin resistance, could initiate hIAPP oligomerization. More specifically, an altered ratio of insulin to hIAPP, as observed in diabetic patients [ in vitro studies [β-cells, in particular an increase in negatively charged lipids, as inferred from studies with mouse and rat models for T2DM [ In vitro studies have shown that negatively charged lipids increase the rate of hIAPP fibril formation [ in vitro [β peptide, it has been suggested that not a particular species but the ongoing amyloid fibrillogenesis is the true cause that is responsible for membrane damage [IAPP is natively unstructured but gains embranes  and becaembranes . An impoembranes , 117–119sruption , 118–120patients , could l studies , 123. Onfor T2DM  could trormation , 126 andormation , 128. Thormation  or a dicormation , can indin vitro . These re damage . Altogete damage , 118.β-cells in T2DM in analogous of neurons in neurodegenerative diseases. Some of them will be discussed more in depth in the following sections.A number of factors related to the formation of membrane-permeant oligomers might contribute to the dysfunction of pancreatic The delivery of toxic oligomers to the extra- or intracellular membranes might be expected to significantly affect signalling pathways involved in cell survival and cell death.2+ concentration has been described as being provoked by amyloid oligomers in neurons and astrocytes [2+ concentration has been suggested to be an important cofactor able to modulate the mechanism of membrane destabilization, favouring one mechanism rather than another. In particular the presence of Ca2+ seems to enhance membrane disruption induced by hIAPP fibers through detergent like mechanism [2+ sensitive calpain might contribute to impaired intracellular vesicle trafficking [Calcium homeostasis and signalling not only is important for cell survival but also underpins the intracellular trafficking and regulated discharge of secretory and synaptic vesicles at the cell membrane. Remarkably, an increase in the intracellular Catrocytes , 134. Noechanism . Yet, unfficking , resulti2+ mobilization in pancreatic islets. They also suggested that this could explain the failure in GSIS. Soty and coworker overexpressed hIAPP in rat pancreatic islets using lentiviral infection, which allows long term hIAPP overexpression, and demonstrated that hIAPP overexpression altered the activity of the ATP-sensitive potassium channels (KATP), leading to defects in insulin and IAPP secretion in response to glucose. Moreover, authors reported an associated increase in ROS production and mitochondrial activity interpreted as a compensatory mechanism for counteracting these defects. Elevated ROS levels can, in turn, lead to the progressive loss of β-cell function therefore contributing to the further worsening of the T2DM pathological condition [2+ permeable pores by the amyloid oligomers themselves and/or the activation of VCC [β-cell death since oligomeric hIAPP has been detected in ER membranes and exogenously added hIAPP has been reported to induce ER stress [ in vivo is controversial, since ER stress is important in transgenic models that overexpress hIAPP at high levels but is not detected when lower levels of IAPP are produced in cultured islet [2+ concentration, and an oxidative environment. Mature insulin secretory vesicles should also be relatively protected from hIAPP aggregation because of the acidic pH and the high insulin concentration, both factors being known for their ability to inhibit IAPP aggregation [β-cells, bearing a high burden of protein synthesis and folding, the disposal of misfolded or denatured proteins, and particularly those with a potential to form toxic oligomers, is essential. Therefore the detected increased toxic IAPP oligomers in T2DM may be due to defective oligomer removal [β-cells to hIAPP increases autophagosome formation and that the inhibition of autophagy increases the vulnerability of β-cells to the cytotoxic effects of hIAPP. In mice with β-cells-specific expression of hIAPP, a deficiency in autophagy resulted in the development of overt diabetes, which was not observed in mice expressing hIAPP alone or lacking autophagy alone. Furthermore, lack of autophagy in hIAPP-expressing animals resulted in hIAPP oligomerization and amyloid accumulation in pancreatic islets, leading to increased β-cells death [Several studies have reported that hIAPP overexpression or exposure induces defects in glucose stimulated insulin secretion (GSIS), possibly due to its capacity to aggregate and form intracellular oligomers , 138. Zhondition . Other mn of VCC  not onlyR stress , 142. Hoed islet –143. Indregation , 145. It removal . The aut removal , 148 It  removal . Hence, ls death . Also trls death .β-cells apoptosis, suggesting that ROS mediate in IAPP toxicity [β-cells in T2DM and neurons in neurodegenerative diseases [1F0ATP synthase (complex V), the oxygen consumption , and the ROS levels. This demonstrates that different regulatory systems of the cells, associated with mitochondrial function and metabolism, are deregulated by hIAPP emphasising a role for oxidative stress in T2DM. Accordingly, cryoimmunogold labelling has also been used to directly reveal that the toxic oligomers of IAPP form within the secretory pathway, escaping and disrupting cellular membranes including mitochondrial membranes [α-helical aggregates endowed with cell penetrating features [Oxidative stress, which is an imbalance between the generation of ROS and the cells antioxidant defence capacities provided by glutathione and other reducing molecules, has been associated with a number of diseases, including cancer, heart diseases, Alzheimer's disease (AD), and T2DM . At hightoxicity , 155. Bediseases , 156–158diseases  demonstrembranes , 113. A features . It has features . Therefofeatures . Disrupt2-terminal kinase (JNK)-1/c-Jun and p38 kinase pathways [β-cell membranes in a specific manner that activates death receptor signaling to trigger apoptosis. Several death receptor-signalling/death receptor-inducing molecules, including Fas and Fas-associated death domain (FADD), have been implicated in β-cell destruction in various cellular and animal models of immune-mediated diabetes and type 1 diabetic individual. Increased β-cell Fas expression has also been reported in type 2 diabetic individuals [β-cell apoptosis [Several groups have described activation of multiple apoptosis pathways following hIAPP exposure, including the sequential activation of caspase-8 and caspase-3 and the Jun NHpathways . Oligomeividuals . Cell-supoptosis .β-cell apoptosis induced by both exogenous and endogenous hIAPP. Islet amyloid increased mRNA levels of both the extrinsic  and the intrinsic (Bim) apoptotic pathways markers, caspase-3, and the antiapoptotic molecule Bclxl in a JNK-dependent manner [Numerous studies have also indicated that the c-Jun N-terminal kinase (JNK) pathway has also been reported as a critical mediator of t manner .β-cells [β-cells. For instance, rIAPP was found that to produce comparable H2O2 concentrations, indicating that amylin aggregation and amylin-induced ROS are unrelated processes [ in vitro evidence that no aggregated forms could be detected for rIAPP by using CD and DLS experiments, β-cells treated with rIAPP1 were not reactive when immunostained with the A11 antibody, suggesting that oligomers formation is not a stringent prerequisite for the observed rIAPP cytotoxicity. At the moment the hypothesis which seems more appropriate is that rIAPP toxicity might relate to a different mechanism compared to hIAPP. As a matter of fact fluorescent rat IAPP was detected in the lysosomal/endosomal compartment, whereas fluorescent hIAPP was seen in mitochondria [Unlike hIAPP, rat IAPP (rIAPP) is a nonamyloidogenic form that does not accumulate and does not induce oligomer and/or fibril deposition in β-cells –28. Howerocesses . Remarkarocesses . Similarrocesses , 165. Yerocesses , 166 togchondria . As Magzchondria . In linechondria .β are more than unrelated molecules: both can populate several conformational states ranging from random coil to α-helix or β-sheet structures depending on the environmental surroundings. Similar to Aβ, IAPP is mainly unstructured in the monomeric state, but it can readily adopt α-helical conformation in the presence of helix-inducing fluorinated alcohols (TFE and HFIP) [α-helical and β-sheet conformations in a membrane mimicking environment [β, and induction of nonselective ion channels and/or disruption of membranes of the secretory pathway is also consistent with cytotoxicity mediated by increased cytosolic Ca2+ in AD [β and hIAPP that induce oxidative stress in neurons and β-cells, respectively, as well as elevated tissue copper levels found in the brain and pancreas of the subjects harbouring amyloid plaques, are also common features shared by T2DM and AD pathologies. Despite the absence of a large sequence homology between Aβ and hIAPP (about 25% sequence identity and 50% sequence similarity) both aggregated oligomeric forms of these peptides display a common conformation structure [β and hIAPP are substrates for insulin degrading enzyme (IDE) a metalloprotease involved in the homeostasis of these two peptides in the brain [A variety of interconnecting factors links T2DM with Alzheimer's disease AD –169. Thind HFIP)  or both ironment , 172. Am2+ in AD , 174. Oltructure . In bothtructure . Finallyhe brain –179.β-metal ion interaction has been extensively investigated, since the recognition of the fundamental role played by transition metal ions dyshomeostasis in the etiopathology of AD [β, metal ion interactions occur mainly within the 1–16 N-terminal region where a variety of metal coordinating functional groups, from the side chains of the His, Asp, or Glu aminoacid residues, in addition to the N-terminal free amino group, are present. By contrast in hIAPP the imidazole moiety of the histidyl residue at position 18 and the N-terminal amino group are the only physiologically relevant anchoring sites for metal coordination. A lower affinity for these metal ions is therefore expected for hIAPP in comparison with Aβ.Disturbance of Cu(II) and Zn(II) physiological levels is another factor joining T2DM and AD. Whereas Agy of AD –184, lesβ-sheet rich fibrillar aggregates of IAPP or Aβ peptides in the pancreas and brain, respectively. IAPP aggregation is associated with pancreatic β-cell loss, whereas clumps of Aβ lead to neuronal cell loss. The recent identification of deposits of amylin in the brains of people with Alzheimer's disease, as well as combined deposits of amylin and Aβ plaques, strengthens evidence on the relationship between T2DM and AD indicating also that amylin is a second amyloid and, potentially, a new biomarker for age-related dementia and Alzheimer's [β seems to behave as a brain protective factor able to regulate synaptic activity and to activate insulin/IGF-1 receptor signalling. Thus it has been suggested that depletion of β-amyloid monomers, occurring in the preclinical phase of AD, might be the cause of early insulin/IGF-1 signalling disturbances that anticipate cognitive decline [The similar pathological parallels between AD and T2DM have allowed recent hypotheses suggesting that AD might be considered as a neuroendocrine-like disorder, in other words a sort of “type 3 diabetes” . Epidemiheimer's . Finally decline , 190.According to major research, amylin or IAPP is a pancreatic peptide hormone naturally produced with insulin and appears to be involved in the cause of both main types of diabetes. Current efforts are focused on understanding the local environmental conditions and interactions with metal ion that can directly influence IAPP aggregation process. The molecular mechanisms underlying amyloid formation are not completely known and particularly their temporal sequence, the hierarchy, and the threshold dividing physiology from pathology are unknown.β in the brain. There is clear evidence that amylin may be associated with Aβ, but the nature of their relationship remains unclear. In any case, the consciousness of such a high connection may support the idea that beneficial therapeutic strategies against T2DM might also be beneficial against AD. Thus research efforts at preventing oligomers and fibril formation by IAPP, together with a clear elucidation of the role played by metal ions in T2DM, might represent a new research field attempting to develop drugs as a cure for both pathologies [That T2DM causes severe brain injury with time is already known, and although there has been a lot of speculation about why that occurs, no conclusive evidence has been found until now. Amylin can readily cross the blood brain barrier (BBB) and presumably interact with Ahologies –195.


Tumour heterogeneity and resistance to systemic treatment in urothelial carcinoma (UC) may arise from cancer stem cells (CSC). A recent model describes cellular differentiation states within UC based on corresponding expression of surface markers (CD) and cytokeratins (CK) with CD90 and CK14 positive cells representing the least differentiated and most tumourigenic population. Based on the fact that this population is postulated to constitute CSCs and the origin of cisplatin resistance, we enriched urothelial carcinoma cell lines (UCCs) for CD90 and studied the tumour-initiating potential of these separated cells in vitro.+ and CD90− UCCs. Distribution of cell surface markers CD90, CD44, and CD49f and cytokeratins CK14, CK5, and CK20 as well as the effects of short- and long-term treatment with cisplatin were assessed in vitro and measured by qRT-PCR, immunocytochemistry, reporter assay and flow cytometry in 11 UCCs.Magnetic- and fluorescence-activated- cell sorting were used for separation of CD90+ cells by immunomagnetic sorting or following cisplatin treatment. Enriched CD90+ cells did not exhibit CSC-like characteristics like enhanced clonogenicity and cisplatin resistance. Moreover, selection of cisplatin-resistant sublines by long-term drug treatment did not result in enrichment of CD90+ cells. Rather, these sublines displayed significant phenotypic plasticity expressing EMT markers, an altered pattern of CKs, and WNT-pathway target genes.We observed cell populations with surface markers according to those reported in tumour xenografts. However, expression of cytokeratins did not concord regularly with that of the surface markers. In particular, expression of CD90 and CK14 diverged during enrichment of CD90Our findings indicate that the correspondence between CD surface markers and cytokeratins reported in xenografts is not maintained in commonly used UCCs and that CD90 may not be a stable marker of CSC in UC. Moreover, UCCs cells are capable of substantial phenotypic plasticity that may significantly contribute to the emergence of cisplatin resistance.The online version of this article (doi:10.1186/s13046-015-0259-x) contains supplementary material, which is available to authorized users. Bladder cancer (BC) is the 9th most common tumour world-wide and the most common cancer of the urinary tract . In deveResistance to cisplatin in UC is brought about by a variety of mechanisms . In partIn addition to CSC, urothelial tissue hierarchy and the mechanisms regulating its normal or aberrant differentiation are incompletely understood. Previously, all cell types of the normal urothelium were thought to originate from the least differentiated basal cells, positive for both cytokeratin 5 (CK5) and cluster-of-differentiation-44 (CD44) , 16. CK1de novo expression of CK14 in a so-called ‘basal’ subtype was generally indicative of unfavourable prognosis . Di. Di10]. + CD44+ CD49f+) cells. Intriguingly, this cell line did not express CK14 at detectable levels. With the exception of HT-1376, all cell lines contained a high fraction of CD44+ cells. Similarly, almost all UCCs were CD49f+, only the cell lines SW-1710 and J82 contained few CD49f+ cells . The efficiency of immunomagnetic enrichment of CD90+ cells was monitored by triple staining  via flow cytometry. After enrichment the abundance of CD90-positive cells was significantly increased up to 50 % .Next, we investigated whether CD90+ magnetically enriched and FACS sorted cell fractions were examined for their cisplatin sensitivity. CD90+ enriched cell fractions from RT-112, J82, and HT-1376 cell lines were no more resistant to cisplatin than the corresponding CD90 depleted fractions . Across the cell lines, no correlation was obvious between the abundance of CD90+ cells  with cisplatin at IC50 doses for 72 h, the abundance of CD90+ cells increased significantly in 6/11 UCCs, particularly in cell lines with originally low abundance of CD90+ cells  and J82 (CD49flow).To investigate the relation between the abundance of CD90try Fig. . Based o μM Fig. . Across lls Fig.  and senslls Fig. . For ins+ cells after cisplatin treatment for cytokeratin RNA expression. Again, cytokeratins generally expressed in uroepithelial cells, like CK7 and CK13, remained mostly unchanged and no increase in CK14 expression was observed  over several months with escalating doses, following a protocol similar to the Resistant Cancer Cell Line (RCCL) collection [50, determined after 72 h treatment of LTT cell lines, increased significantly compared to the corresponding parental cells. In RT-112-LTT IC50 for cisplatin was >50 μM compared to 12.5 ± 2.7 μM in the parental cells and IC50 for J82-LTT increased from 1.5 ± 0.48 μM to 9.2 ± 4.2 μM. The RT-112-LTT cells grew much slower than the parental line, with population doubling times of 32.7 and 25.3 h, respectively. Similar results were found in J82-LTT and untreated parental J82 with doubling times of 33.1 and 24.5 h, respectively . We further demonstrated that WNT-signalling activity could be significantly inhibited in HCT-116 cells by the applied doses of the compound in the TOP-FOPflash-reporter assay  to enrich or deplete CD90LTT-UCCs were established by continuous exposure to increasing cisplatin concentration as we were interested in selection for highly resistant cells and to characterize them rather regarding stem cell properties than comprehensively for resistance mechanisms. Thus, underlying mechanism of acquired resistance might differ from resistant cell lines established by intermittend protocols. However, to our knowledge, to date no conclusive data has been published on resistance acquired by different treatment protocols. Further, our observations were in accordance with cell culture models with acquired resistance from the RCCL . Like doAXIN-2, CCDN1, c-MYC, and PITX2 were induced, suggesting activation of this pathway subsequent to long-term treatment of UCC with cisplatin. However, a reporter assay for active WNT-signalling revealed only moderate stimulation of activation and a WNT-signalling inhibitor did not revert resistance [Like acquisition of EMT, activation of WNT-signalling was previously observed in cisplatin resistant non-small cell lung cancer and osteosarcoma cell lines , 46. WNTsistance . We reposistance . Thus, oDespite the canonical WNT-pathway, signalling via β-catenin, WNT can also signal via secondary pathways, as such as protein kinase A and C, calcium-dependent signalling, JNK or Rho-like GTPases. WNT-5a can activate some of those, while inhibiting the canonical WNT-signalling in parallel . Thus, aIn conclusion, the postulated differentiation markers CK14, CK5, and CK20 as well as CD90, CD44, and CD49f were neither sufficient to define a CSC population in UCCs nor to define UCCs with an increased resistance to cisplatin treatment. In addition, cisplatin did not enrich for a UC population defined by these markers, but rather induced phenotypic plasticity likely to be associated with EMT and enhanced the WNT-signalling activity. Thus, in the future LTT-UCCs need to be characterized by expression profiling regarding the mechanisms of cisplatin resistance  and furt


SLC26A4) was initially identified through the positional cloning in patients with Pendred syndrome, a heritable recessive genetic disorder (MIM #274600) characterized by deafness and goiter . As predicted from the linkage between pendrin gene and characteristics of Pendred’s syndrome, pendrin is highly expressed in the inner ear and thyroid gland, where it controls the volume and pH of the endolymphatic fluid, and the transport of iodide required for organification of thyroid hormones, respectively. Pendrin is also highly expressed in the kidney where it controls the final excretion of bicarbonate , one of the key mediators in inflammatory response in asthma, and analyzed the ASL thickness, expression and activities of selected ion transporters expressed on human nasal epithelia, as well as status of goblet cells, a specialized mucus-secreting cells.While the results from mouse models suggest that abnormal overexpression of pendrin in airway epithelia could lead to severe airway inflammation, for clinical purposes it is imperative to test the role of pendrin in airway epithelia directly using human tissue. To address this point, the present study by Lee et al. , ANO-1 (Ca2+-dependent Cl− channel) and cystic fibrosis transmembrane conductance regulator (CFTR) between the two cultures under the baseline condition. The only significant difference observed in this study was reduction in mRNA expression of CFTR in the pendrin-null cell culture. Overall, pendrin is likely to be the major mediator of Cl−-HCO3− exchange activity in nasal epithelia following IL-13 treatment, but at the same time, the loss of pendrin had very little effect on the expression of other transporters.Because nasal epithelia express a variety of transporters, one important question is to determine the relative role of pendrin in electrolyte transport in these cells. At mRNA level, Lee et al. detected 9 anion exchangers in addition to pendrin. Among those genes, only SLC26A3 was significantly elevated in response to IL-13 treatment, and the magnitude of increase was much lower than that of pendrin . Furthermore, following the IL-13 treatment, no increase in ClOn the other hand, the loss of pendrin led to major physiological changes in nasal epithelia. Unlike results from mouse models, the ASL was significantly thicker in pendrin-null cell culture even in the baseline condition, and the IL-13 treatment significantly increased the ASL thickness in both types of cell cultures. In addition to abnormal ASL thickness, in the pendrin-null cell culture, the IL-13 treatment did not stimulate the expression of Muc5ac, or induce hyperplasia in goblet cells as visualized by Periodic-acid Schaff stain.Taken together, results of Lee et al. show that previous results obtained from mouse models are largely applicable to human nasal epithelia. That is, the loss of pendrin reduces mucus secretion and increases the ASL thickness at the same time, and likely to alleviate the hyperreactivity of lung airway. This makes pendrin a potential therapeutic target for asthma. However, several questions remain to be addressed. For example, what would be the role of pendrin in controlling ASL under basal condition, especially in comparison to other ion transporters expressed on the epithelia? Determining if the loss of pendrin has an effect on ASL in vivo, as well as in vitro, in the human airway would be relevant to understand the ASL homeostasis regulation. Furthermore, there still is a very simple mechanistic question—“how exactly does the inhibition of pendrin affect the ASL thickness?” Because the ASL thickness is controlled by the solute transport and ASL osmolarity, one possibility is that loss of pendrin is simply affecting overall osmolarity of the ASL, as described in the schematics presented by the authors of the article. An alternative, although speculative, possibility is that the loss of pendrin alters pH of the ASL (Garnett et al. In conclusion, Lee et al. provides additional support for the linkage between pendrin and lung epithelial pathophysiology. Future studies should emphasize on clarifying the molecular basis for the linkage between pendrin function and lung epithelial physiology. Insights from such studies would be invaluable to solidly establish pendrin as a therapeutic target of asthma.


Resistance to chemotherapy is common in gastroesophageal cancer. Mechanisms of resistance are incompletely characterised and there are no predictive biomarkers in clinical practice for cytotoxic drugs. We used new cell line models to characterise novel chemotherapy resistance mechanisms and validated them in tumour specimens to identify new targets and biomarkers for gastroesophageal cancer.Cell lines were selected for resistance to oxaliplatin, cisplatin and docetaxel and gene expression examined using Affymetrix Exon 1.0 ST arrays. Leads were validated by qRT-PCR and HPLC of tumour metabolites. Protein expression and pharmacological inhibition of lead target SPHK1 was evaluated in independent cell lines, and by immunohistochemistry in gastroesophageal cancer patients.Genes with differential expression in drug resistant cell lines compared to the parental cell line they were derived from, were identified for each drug resistant cell line. Biological pathway analysis of these gene lists, identified over-represented pathways, and only 3 pathways - lysosome, sphingolipid metabolism and p53 signalling- were identified as over-represented in these lists for all three cytotoxic drugs investigated. The majority of genes differentially expressed in chemoresistant cell lines from these pathways, were involved in metabolism of glycosphingolipids and sphingolipids in lysosomal compartments suggesting that sphingolipids might be important mediators of cytotoxic drug resistance in gastroeosphageal cancers . On further investigation, we found that drug resistance (IC50) was correlated with increased sphingosine kinase 1(SPHK1) mRNA and also with decreased sphingosine-1-phosphate lysase 1(SGPL1) mRNA. SPHK1 and SGPL1 gene expression were inversely correlated. SPHK1:SGPL1 ratio correlated with increased cellular sphingosine-1-phosphate (S1P), and S1P correlated with drug resistance (IC50). High SPHK1 protein correlated with resistance to cisplatin (IC50) in an independent gastric cancer cell line panel and with survival of patients treated with chemotherapy prior to surgery but not in patients treated with surgery alone. Safingol a SPHK1 inhibitor, was cytotoxic as a single agent and acted synergistically with cisplatin in gastric cancer cell lines.Agents that inhibit SPHK1 or S1P could overcome cytotoxic drug resistance in gastroesophageal cancer. There are several agents in early phase human trials including Safingol that could be combined with chemotherapy or used in patients progressing after chemotherapy.The online version of this article (doi:10.1186/s12885-015-1718-7) contains supplementary material, which is available to authorized users. The clinical outcomes for gastroesophageal cancer are poor. One year survival is only 40–50 % and 5 year survival 10–20 % , esophageal adenocarcinoma  and esophageal squamous cell carcinoma [OE21OX4 (n = 859)] cell lines compared to the sensitive parental line.Gene expression was performed using RNA isolated from AGS, AGSCIS5, AGSOX8, AGSDOC6, OE33, OE33CIS4, OE33OX4, OE21, OE21OX4 Fig. , with 3 p < 0.05) identified pathways that were over-represented among each of these gene lists  compared to their parental wild type lines .SPHK1 and SGPL1 mRNA levels measured on microarrays were validated by qRT-PCR, with strong correlations between gene expression measured with each assay  using a pre-determined semi-quantitative Quick-score. SPHK1 IHC Quick-score = intensity x proportion: intensity scored as 0 = negative, 1 = weak, 2 = moderate and 3 = strong SPHK1 staining in tumour cells; positive proportion scored as 0 = 0 %, 1 = 1-10 %, 2 = 11-50 %, 3 = 51-70 %, and 4 = > 70 % tumour cells positive for SPHK1 staining. In the parental and drug resistant cell lines a strong correlation between SPHK1 mRNA expression and SPHK1 protein expression was observed  between SPHK1 staining as negative or weak versus moderate or strong and any of the clinco-pathological variables listed in table n = 67) or the neo-adjuvant chemotherapy and surgery group (n = 36), nor the surgery alone group (n = 31). Higher expression of SPHK1 correlated with poor survival in patients treated with cisplatin based combination chemotherapy before surgery, but not those who received surgery alone without prior cisplatin based chemotherapy and SGPL1 (decreased) in resistant cell lines and we propose this leads to increased cellular S1P and cytotoxic drug resistance.S1P is a phospholipid with many functions, formed intra-cellularly through the phosphorylation of sphingosine by sphingosine kinases (2 isoforms SPHK1 and SPHK2) . S1P is Accordingly S1P could lead to cytotoxic drug resistance in gastroesophegal cancer acting in an autocrine or paracrine manner via cell surface S1P receptors following transportation out of the cytosol. Alternatively S1P may mediate cytotoxic drug resistance acting intracellularly by counteracting apoptosis mediated by its pro-apoptotic precursor ceramide or interaction with known intracellular targets involved in cancer pathogenesis and cytotoxic drug resistance such as Histone deacetylase 1 (HDAC1) and Histone deacetylase 2 (HDAC 2) to which S1P directly binds and inhibits, and TNF Receptor-Associated Factor 2 (TRAF 2), or Protein Kinase C (PKC) . FurtherRex et al. demonstrated no effect on the viability of cancer cell lines (cultured with and without serum starvation), nor on the growth of xenografts, with the use of highly specific SPHK1 and SPHK2 inhibitors . HoweverOur finding of an inverse relationship between SPHK1 and SGPL1 expression in gastroesophageal cancer associated with increased levels of S1P and cytotoxic drug resistance is consistent with a recent report in prostate cancer, where a similar inverse relationship between expression of SPHK1 and SGPL1 was noted leading to increased production of S1P and an association with resistance to docetaxel . In addiOur data suggest that increased SPHK1 expression in gastroesophageal cancers has predictive impact indicating chemotherapy resistance in gastroesophageal cancer patients, consistent with our cell line findings, but not therapy independent prognostic value. A previous study by Li et al., demonstrated that high SPHK1 expression in resected gastric cancers was associated with worse survival . Li et aHere we have investigated the relationship between resistance and benefit from neo-adjuvant chemotherapy in gastroesophageal cancer patients. It would be valuable to investigate the relationship between SPHK1 expression and response to palliative chemotherapy to determine if there is any differential impact according to the stage of the disease. This would be useful in planning future clinical trials of therapies targeting SPHK1. The small numbers of patients in our surgical cohort who would have recurred and received palliative chemotherapy mean that such an analysis in our cohort would not be informative. In addition there may be a change in the SPHK1 expression from the primary tumour that was resected and the recurrent disease at a later date when palliative chemotherapy might be administered. Investigation in a large cohort of patients with advanced gastroeosphageal cancer that have received palliative chemotherapy would be worthwhile. Further investigation of the relationship between the ratio of SPHK1 and SGPL1 protein expression determined by immunohistochemistry or other methods, and the response/benefit from both neo-adjuvant and palliative chemotherapy in clinical cohorts of patients would also be valuable.in vitro by examining the effect of safingol on cisplatin resistance in gastric cancer. We chose safingol (L-theo dihydrospingosine) rather than a more specific SPHK1 inhibitor or SPHK1 knock down by siRNA mediated knock down of SPHK1, since it is the most developed SPHK1/S1P inhibitor as an anti-cancer agent and in particular safingol has demonstrated safety in combination with cisplatin in a phase I clinical trial and lead to decreased serum S1P in treated patients [CIS4 and the gastric cancer cell line N87 we used safingol at concentrations ranging from 0.375–12 μM, which is well within the readily achievable serum plasma concentrations of safingol in cancer patients and at which significant decreases in serum S1P are reported [There are a number of SPHK1 and S1P inhibitors in clinical development as anti-cancer agents that confer increased sensitivity to cytotoxic drugs, targeted agents or radiotherapy and/or have single agent activity in preclinical cancer models , 44–49 . Therefopatients . Accordipatients –53. In apatients . In our reported . As wellIn conclusion, we have demonstrated for the first time in gastroesophageal cancer that increased SPHK1 occurs in combination with decreased SGPL1 and increased S1P, and cytotoxic drug resistance, and that a SPHK1 inhibitor safingol can reverse cisplatin resistance. Given the demonstrated clinical safety and tolerability of safingol in combination with cisplatin, as well as the availability of several other SPHK1 and S1P inhibitors in stage clinical development, we suggest that combining such agents with cytotoxic chemotherapy or investigating their activity in patients who have developed secondary resistance to chemotherapy is a promising clinical strategy and that a high tumour SPHK1:SGPL1 ratio could provide a predictive biomarker to select patients for this therapeutic approach.