These hyperploid cell lines form xenografts in mice and show metabolically active and necrotic areas on fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging. WES of ITOC cell lines recapitulate the genomic tumor profile of ICGC GB-SCC database. We further identified smokeless tobacco associated genetic alterations  and oncogenic PIK3CA mutation in GB-SCC cell lines. Transcriptome profiling identified deregulation of pathways commonly altered in cancer and down-regulation of arachidonic acid metabolism pathway, implying its possible role in GB-SCC. Clinical application of high throughput sequencing data depends on relevant cell line models to validate potential targets. Extensively characterized, these oral SCC cell lines are particularly suited for mechanistic studies and pre-clinical drug development for smokeless tobacco associated oral cancer.Smokeless tobacco associated Gingivobuccal squamous cell carcinoma (GB-SCC) is a major public health problem but available oral cancer cell lines are mostly from smoking associated tongue SCC raising the need for pertinent GB-SCC cell line models. As part of the International Cancer Genome Consortium (ICGC) Project, 4 novel cell lines, namely, Indian Tata Memorial Centre Oral Cancer (ITOC) −01 to −04 were established and characterized with conventional methods, karyotyping, ultrastructure,  Smokeless tobacco is consumed in various forms with areca nut, lime or betel leaves as quid, pan, masheri or gutka4. Most oral cancers are squamous cell carcinomas (SCC) and arise from the lip, tongue, gingivobuccal mucosa, retromolar trigone, floor of mouth or the hard palate. The form of tobacco use not only influences the site of cancer within the oral cavity but also its biology and mutational profile. In western countries where smoking is the predominant form of tobacco use, tongue cancer is the most common oral cancer while in south Asian and east Asian countries where tobacco chewing is more prevalent, Gingivobuccal SCC (GB-SCC) is the most common oral cancer. GB-SCC differs from the tongue SCC in its clinical behaviour5 as well as molecular8 and immunoproteome profile9, yet the treatment modalities are identical. Previous studies have revealed that as compared to tobacco smoking, the smokeless tobacco associated cancers have predominance of specific transversions or transitions in TP53 and the genome12.Advanced stage oral cancer with poor clinical outcome is a major public health problem in south Asian and east Asian countries where smokeless tobacco use is prevalent6 and The Cancer Genome Atlas (TCGA) project in USA for diverse HNSCC13. Large number of novel and recurrently mutated genes and deregulated pathways in HNSCC, with some being unique to GB-SCC were identified. Mechanistic studies on the role of an increasing number of genetic variants being identified in different cancers and their translational exploitation requires cell lines from same organ and histology with full molecular genetic characterization.Comprehensive molecular genetic characterization using whole genome, exome and transcriptome analysis has been reported in over 400 Head and Neck SCC (HNSCC) tumours. These include GB-SCC by our group as part of the International Cancer Genome Consortium (ICGC) India Projectin vitro models due to the ease of performing large scale experiments, genetic manipulation and reproducibility14. Cell lines maintained using appropriate conditions retain parent tumor features and phenotype, making them preferred tumor representative models in laboratory studies16. The dearth of commercial cell lines for tumours which are uncommon in large parts of the world necessitates the need to establish and characterize novel cell lines from primary tumor17.Primary cultures and immortalized cell lines are indispensible source for conducting translational cancer research. Tumor-derived cell lines are widely used 20 and none from the Indian subcontinent. As part of our systematic efforts to establish relevant cell lines in the ICGC India oral cancer project, we have established and performed comprehensive molecular genetic characterization of 4 oral squamous cell carcinoma (OSCC) cell lines from tumour tissues obtained from tobacco chewing Indian patients. These cell lines are named ITOC-01, 02, 03 and 04, after the ethnic origin, institute and cancer site - Indian Tata Memorial Centre Oral Cancer (ITOC). These cell line models established from smokeless tobacco consuming patients provide a platform to carry out wide spectrum of assays spanning from functional genomics to drug response studies. To the best of our knowledge this is the first study from Indian subcontinent characterizing smokeless tobacco GB-SCC derived cell lines.Of the several hundred HNSCC cell lines reported worldwide, very few are from GB-SCC in patients with smokeless tobacco useDetails of the patients from whom these cell lines were derived are provided in Table Classical epithelial morphology and characteristic SCC features were seen in all four parent tumors Fig. . Detaile21.Genotyping confirmed the human origin and unique genotype of each cell line  with GeneJuice transfection reagent. EGFP expression observed after 48 hours of transfection revealed low transfection efficiency in ITOC-01 (1%) and ITOC-02 (6%) but high efficiency in ITOC-03 (30%) and ITOC-04 cell lines (20%)  and Bonferroni post-tests showed high significance between control and time points of 24 and 48 hours  mice in 3, 2 and 2 weeks respectively Fig. . GrosslyTP53, FAT1, CASP8, NOTCH1, KMT2B, PCLO, UNC13C, SMG1, FAT3, EP300, KMT2D, SYNE2, TRPM3, PIK3CA and NSD1), 12 were mutated in one or more of the ITOC cell lines  and inflammatory pathways , implying their role in cancer progression29. Mutational signature in the 4 cell lines were similar with C > T transitions (44%) being most common, followed by T > C (20%), C > A (21%) and C > G (15%) transversions  was done at a mean depth of 155x, 112x, 128x and 113x for ITOC-01, -02, -03 and -04 cell line respectively with mean mapping quality of 58.12. A total of 24150, 21921, 22576 and 22018 exonic variants were identified in the ITOC-01, 02, 03 and 04 cell lines respectively, of which, 369, 265, 343 and 312 somatic variants were considered deleterious. Each cell line had a mutational profile matching with the GB-SCC or HNSCC mutational profile reported in the ICGC GB-SCC and TCGA HNSCC database , cell lines at late passage  and xenograft tumors was performed by evaluating RNA-seq of three normal buccal mucosa tissues and four OSCC cell lines generated ~65 million reads each. Compared to the 3 normal buccal mucosa tissues, 661, 811, 885, 907 genes were upregulated and 1627, 1997, 2343, 1950 genes down-regulated in ITOC-01, -02, -03 and -04 cell lines respectively  and down-regulation of Vimentin validates the epithelial origin of these cell line and also corroborates with the earlier result of expression of keratin 8 in OSCC cell lines  were down-regulated in ITOC cell lines.RNA-seq data was validated by quantitative polymerase chain reaction (qPCR) for 30 genes which are known to be deregulated in cancer and also in these cell lines Fig. . Gene exnes Fig. . The AAM18. The Expert protein analysis system (ExPASy) – Cellosaurus database has reported over 150 SCC cell lines derived from tongue cancer but very few from buccal mucosa32 or gingiva35. Surprisingly, no GB-SCC cell lines are reported in literature or databases from the Indian subcontinent where it is the most common oral cancer. One possible reason may be the difficulty in culturing and establishing cell lines from heavily infected locally advanced gingivobuccal cancers.Human cancer cell lines are indispensable to functionally characterize new or previously uncharacterized genetic alterations identified by large scale genomic studies. In the precision medicine era, the utility of a cell line for identifying clinically relevant targets requires these cell lines to closely recapitulate the molecular genetic profile of specific human cancers. Despite the common SCC histology, prior tobacco exposure and several common genetic alterations, the carcinomas arising at different primary sites within the Head and Neck region have several distinct featuresTP53 mutations across parent tumor, cell line at early and late passage and xenograft tumors affirms the genomic characteristics of primary tumor being preserved throughout the cell line cultivation and further in the xenograft model as well. The genomic and histopathological analyses assure the OSCC cell lines are representative models of the parent tumors. Through exome and transcriptome analysis we confirm that ITOC cell lines recapitulate the mutational and gene expression profile of GB-SCC and HNSCC in the ICGC and TCGA studies13. Of the 15 most recurrently mutated HNSCC or GB-SCC genes, 8 genes  were mutated in >10% of smokeless tobacco associated GB-SCC tumours of ICGC6 but not in the cigarette smoking associated HNSCC of TCGA study13 and may be considered as smokeless tobacco associated genes  had deleterious mutation in GB-SCC ITOC cell lines  but not in the Tongue-SCC ITOC-02 cell line. This highlights the need for establishing, characterizing and using cell lines from tumours of the same subsite within HNSCC that have developed after similar form of tobacco exposure. Interestingly, the tongue SCC ITOC-02 cell line had truncating mutations in a few genes which are rarely associated with some cancers, PYROXD2, PCMTD1 reported to be mutated in hepatocellular cancer37 and PABCP1 reported to be upregulated in gastric carcinoma and targeting miR-34c38.We have successfully established and characterized 4 OSCC cell lines including 3 from GB-SCC derived from tobacco chewers. We demonstrate these cell lines to be of human origin with characteristic features of SCC. Identification of identical nes Fig. . Three oPIK3CA mutation (H1047R). It would be particularly useful to study PIK3CA inhibitors like Alpelisib (BYL719) which has been evaluated in only 2 PIK3CA mutated HNSCC cell lines39. Encouraging results from first in human use of Alpelesib in diverse tumours with oncogenic PIK3CA mutations has been reported40 and merits further evaluation in HNSCC including GB-SCC.Moreover, ITOC-01 is the only GB-SCC cell line harbouring an oncogenic 10, however, this was not observed in any of the 4 ITOC cell lines. Similar to our finding, Fadlullah et al.20 had also not observed any correlation between the patient habits and mutation signatures. It is important to note that smokeless tobacco is used in many forms with or without lime, areca nut and other potentially carcinogenic ingredients. Hence there is a need to re-examine signature 29 and its association with smokeless tobacco and forms of smokeless tobacco use.Earlier studies have proposed genetic signature 29 with abundance of C > A transversion for smokeless tobacco associated cancers41. For the ITOC cell lines we used stringent bioinformatic methods to deduce somatic mutations. This seems to be a valid approach since the known mutational profile of HNSCC and GB-SCC is recapitulated in the ITOC cell lines. Since gene expression is tissue dependent, to interpret deregulated pathways in specific cancers, it is important to use representative normal tissues. In the TCGA HNSCC study with 16 GB-SCC tumours, their head neck region normal tissue dataset did not have any normal buccal mucosa sample for gene expression analysis. Similarly, the oral cancer cell line panel study used cultured keratinocytes in lieu of normal oral mucosa tissue20. Pure normal cell population in culture may be used as a convenient surrogate for normal tissues. However it is well known that isolation of specific cell populations, immortalization of normal or tumour cells, and the culturing conditions could all result in major alterations in gene expression as compared to their counterpart normal tissue or primary tumor42. This may result in misidentification of altered genes and pathways leading to false discovery. Possibly as a result of using normal buccal mucosa for determining differential gene expression, the deregulated genes in ITOC cell lines show excellent correlation with the qPCR data 43, epigenetic markers ; ECM receptor interaction molecules (laminin - LAMC2) and IL23A. A recent study has shown up-regulation of LAMC2 as a marker of cancer invasion in oral leukoplakia44. We propose a similar role could be observed in OSCC-GB. ITOC cell lines also show up-regulation of ECM receptor interaction and focal adhesion pathway, similar to previous large scale transcriptomic analysis of 326 oral cancer tissues45. Previous report by ICGC India team has shown loss-of-function somatic mutations in AAM pathway leading to better patient survival46. Considering the identification of somatic mutations and down-regulation of gene expression in AAM pathway components of these novel cell lines it would be interesting to further elucidate the role of AAM pathway in OSCC.The ITOC cell lines show up-regulation of inducers of epithelial to mesenchymal transition  (Gibco) supplemented with antibiotic] at 37 °C in a humidified atmosphere containing 5% CO2. Culture medium was replaced every 48 hours and the epithelial pool of cells was enriched by differential trypsinization and subsequent removal of fibroblasts. Cells at 80% confluency were split in the ratio of 1:2 twice a week. Four cell lines were established – 3 from GB-SCC  and 1 from tongue SCC (ITOC-02).This study was approved by Tata Memorial Centre (TMC) – Advanced Centre for Treatment, Research & Education in Cancer (ACTREC) Institutional Ethics Committee III (Project No. 3). This study was conducted at TMC, ACTREC and all experiments involving human tissue were performed in accordance with relevant guidelines and regulations. Tumor tissues were collected at the time of surgery after obtaining informed consent from patients with smokeless tobacco associated oral SCC. Tumor tissues were transported in 5 mL Plain Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco) with double strength antibiotic mixture of Penicillin 400 U/mL, Streptomycin 40 µg/mL, Gentamycin 50 µg/mL and Mycostatin 10 U/mL (PSGM). Tissues were processed within half an hour of resection and were rinsed twice with sterile 1X phosphate buffered saline (PBS) followed by 10% Povidone-iodine (Wockhardt) and PBS wash. Tumor tissues were minced finely to small pieces of approximately 1–2 mmMorphological characteristics of the cell lines were studied using Olympus IX51 phase contrast microscope and ultra structure analysis was performed with Transmission Electron Microscope  at 120 kV .Cells were harvested and arrested with colcemide (0.05 µg/mL) for 1 hour at 37 °C. Mitotic cells were centrifuged at 2000 rpm for 5 minutes followed by hypotonic KCl treatment for 30 minutes at 37 °C. Cells were fixed in fresh methanol: glacial acetic acid  for 15 minutes at 4 °C. G-banding was performed by treating the slides with trypsin followed by Giemsa staining (GTG). Metaphases were captured using Olympus BX61 microscope  and analyzed using Applied Spectral Imaging (ASI) GenASIs software version 7.0 .http://strdb.cogcell.org) and ATCC STR database (http://atcc.org/STR_Database) of cell lines using match threshold of ≥80%.Genomic DNA of cell lines was used for STR analysis of 8 autosomal markers  and 1 gender marker, Amelogenin. PCR products were analyzed with 3500 Genetic Analyzer (Applied Biosystems) and compared with COG STR database -2,5-diphenyltetrazolium bromide (MTT) solution was added at a final concentration of 0.5 mg/mL and incubated for 4 hours at 37 °C, 5% CO2. Purple formazan crystals were dissolved by addition of 10% SDS. Absorbance was measured at 540 nm with a spectrophotometer (BMG Labtech). Cell proliferation was assayed at different time intervals - 0, 24, 48, 72 and 96 hours. MTT readings were recorded in triplicates and the overall mean plotted against time. Standard error of the mean (SEM) were calculated for each day and the exponential growth curve plotted. Doubling time calculated using the equation ln(2)/K, where K is the growth rate constant.Cells were seeded at a density of 5 × 10Lymphocytes isolated from fresh blood of a healthy donor were used as control for ploidy studies. Cells fixed in 70% ethanol for 60 minutes were incubated with 100 µg/mL RNase A (Sigma-Aldrich) and stained with 50 µL propidium iodide (Sigma-Aldrich) for 30 minutes at 37 °C. Acquisitions were performed on BD FACSCalibur (Becton Dickinson) and data analyzed using Modfit software (version 2.0).Cells were grown on sterile coverslips for 48 hours, fixed in ice-cold methanol at −20 °C for 10 minutes. Coverslips were rinsed with PBS and permeabilized using 0.3% Triton X and ice-cold methanol at room temperature (RT). Blocking was carried out using 3% bovine serum albumin (BSA) in PBS for 15 minutes at RT. Cells were incubated with primary antibodies for 1 hour at RT; p53 , Cytokeratin 8  followed by 1 hour incubation with fluorescein isothiocyanate (FITC) secondary antibody , Alexa Fluor 568 goat anti-mouse IgG (H + L), Molecular Probes). Coverslips were mounted using Vectashield (Vector Labs) mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) and viewed under confocal microscope .5 in 35 mm cell culture dish were transfected with 1 µg of pEGFP-C1 plasmid using GeneJuice (Merck) transfection reagent as recommended by the manufacturer’s instructions. Cells were incubated at 37 °C for 48 hours and transfection efficiency was evaluated by counting 200 cells from five different fields under a fluorescent microscope.Cells seeded at a density of 3 × 106 in 6-well plate and on reaching 100% confluency a wound was inflicted with a pipette tip and images were captured every 24 hours on Olympus IX51 microscope and analyzed using ImageJ software. The relative cell free area at respective time points was calculated by considering the inflicted wound at 0 hours as 100%. One-way analysis of variance (ANOVA) and Bonferroni post-tests were performed to analyze effect of wound healing with respect to time.Cells were seeded at a density of 2 × 105 cells/mL were suspended in 0.2% agar in IMDM with 10% FBS and overlaid onto 0.5% basal agar coat in 35 mm plates. The plates were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 21 days and colonies formed were visualized under an inverted microscope. Images were captured using Axiovert 200 inverted microscope (Carl Zeiss).1 × 107 cells were injected subcutaneously in nude (ITOC-01) and NOD-SCID mice . All experiments were performed in accordance with relevant guidelines and regulations of Institutional Animal Ethics Committee. The mice were housed in sterile pathogen-free environment and observed daily for appearance of tumors. FDG PET studies were performed for the xenografts established from these cell lines in nude and NOD-SCID mice . Tumors from sacrificed mice were stained with hematoxylin and eosin (H&E) and images captured using Axio Imager.Z1 upright microscope (Carl Zeiss).For animal study approved by Institutional Animal Ethics Committee (Project no. 05/2012), 1 × 10Genomic DNA libraries were prepared using Nextera® Rapid Capture Enrichment Kit (Illumina) as per the manufacturer’s instructions and paired-end reads were generated using Illumina HiSeq 1500 platform. Approximately 37 Mb region of human genome comprising of 214,405 exons was captured. Detailed analysis provided in Supplementary Materials and Methods.47. Mutational analysis was carried out by bidirectional Sanger sequencing of PCR products on ABI 3730 capillary sequencer (Applied Biosystems).Genomic DNA was isolated from cells , fresh frozen tissue  and formalin-fixed paraffin-embedded (FFPE) tissue  as per the manufacturer’s instructions. Primer sequences used for amplification of Exon 1–11 of TP53 gene were as described earlierTotal RNA was isolated from the 4 tumor cell lines and 3 normal buccal mucosa tissues using miRNeasy mini kit (Qiagen). RNA libraries were prepared using TruSeq RNA sample preparation kit (Illumina) as per the manufacturer’s instructions and paired-end reads were generated using Illumina HiSeq 2500 platform. Please refer to Supplementary Materials & Methods for detailed RNA-seq analysis. RNA-seq data was validated by qPCR . Primer sequences are specified in Supplementary Table Supplementary InformationDataset 1Dataset 2Dataset 3


RIL/PDLIM4 gene was identified as a tumor suppressor, its expression is frequently altered in various types of malignancies. The product of RIL/PDLIM4 gene is an adapter protein involved in the actin cytoskeleton remolding and assembly of stress fibers crucial for cell motility and epithelial-mesenchymal transition. Although the exact mechanism tethering RIL to cancer development remains unknown some pieces of evidence suggest that RIL may act by suppressing activation of the proto-oncogene tyrosine-protein kinase Src. To further explore this issue we tested how different expression levels of RIL affected the activity of Src in breast cancer cell lines. RIL was ectopically overexpressed in the cell cultures with its relatively low endogenous level, or, otherwise, was downregulated by RNA interference. Whereas we observed no correlation between expression levels of RIL and activity of Src we found that in several cell lines elevated levels of RIL were associated with higher cell migratory activity along with the increased incidence of breast xenograft formation and metastasizing. The obtained data suggest that in some breast cancer models RIL may not act as Src kinase inhibitor, but rather play the role of a potential oncogene that promotes cell motility and contributes to cancer cells spreading. RIL gene  encodes a highly conserved adapter protein, the member of the ALP/Enigma family , 2. The Conversely, a growing body of data suggests that RIL may also act as an oncogene –18. The In order to find a correlation between the level of RIL expression and the activity of Src in the breast cancer model we tested a panel of eight human breast carcinoma cell lines. Expression levels of RIL were measured by qPCR, the proportion of active Src (phosphorylated at Y419) was determined by ELISA .Quantitative evaluation of the relationship between the two estimated parameters was performed by the Pearson bivariate correlation test. The calculated coefficient stood at -0.172 suggesting no evident correlation between the expression level of RIL and the activity of Src.The absence of the expected pattern could be explained by the fact that the activity of Src is controlled by numerous tyrosine kinases and phosphatases varying depending on the cell line. The analysis of the panel of different breast cancer cell lines fails to take into account the individual characteristics of each cell culture, which may affect the obtained results.In order to eliminate the impact of cell cultures heterogeneity a panel of breast cancer cell lines with artificially induced and suppressed level of RIL was established. RIL was upregulated in the relatively RIL-deficient cell lines by the introduction of a lentiviral expression construct. To downregulate the expression of RIL in breast carcinoma cell lines with the relatively high levels of endogenous RIL we used RNA interference mediated by the introduction of lentiviral constructs expressing RIL-specific shRNAs . The traThe created panel of transgenic breast cancer cell lines was used to measure the effect of suppression and overexpression of RIL on the activity of Src .According to the results, the influence of RIL on Src activity varies distinctly between cell lines. While for the T47D and HCC1395 cell cultures the low level of RIL correlates with the considerable inactivation of Src, its activity in BT549 and MDA-MB-468 does not significantly respond to RIL overexpression. Meantime, in MDA-MB-231 and MDA-MB-436 cells the high level of RIL is associated with the increased Src kinase activity, demonstrating the positive correlation between the studied parameters.The observed RIL-dependent activation of proto-oncogene Src may increase the malignant properties of cells, providing evidence for the oncogenic activity of RIL. To test this effect, we compared the phenotypic characteristics of breast cancer cells within the established panel.Migratory activity is considered among critical parameters that determine the aggressiveness of the tumor. The migration level of the RIL (+) and RIL (-) transgenic breast cancer cell lines was evaluated by the Boyden Chamber assay .in vivo could favor a metastatic spread of cells and a poor outcome of the disease.According to the data obtained, in two (MDA-MB-231 and HCC1395) of the six cell lines the expression of RIL demonstrates a statistically significant association with the increased migratory activity. The other two cell cultures (MDA-MB-436 and BT-549) show a similar trend. In these cell lines RIL acts as an oncogene stimulating the invasive behavior, which in vitro, we attempted to confirm the observed effect in vivo by evaluating the impact of RIL on tumor formation in the orthotopic xenograft mouse model. The transgenic cells derived from MDA-MB-231 demonstrating the most pronounced positive correlation of RIL expression with the cell migration rate were used within this experiment  group , 5C. RILin vitro results obtained for the correspondent breast cancer cell culture , but also for HCC1395 cell line  which shThis assumption is consistent with the results of our previous study, which revealed that RIL overexpression correlated with the unfavorable prognostic parameters of tumors, namely an increased CD74 level, reduced expression of E-cadherin, and inactivated intracellular tumor suppressor protein Scribble. Although the RIL-dependent mechanisms of tumor formation remain elusive, RIL may be involved in breast cancer development through the CD74-mediated mechanism typical of malignant cells  where inAlong with the previous evidence of RIL being a tumor suppressor, the present results reveal its more ambiguous role in breast cancer development. As a scaffold protein, RIL could participate in a wide range of protein-protein interactions, whereby its contribution to malignant phenotype could be primarily affected by the cellular context and the spectrum of abnormalities accumulated at the preceding steps of carcinogenesis.Summing up, RIL should not be considered a canonical oncogene or tumor suppressor. The assessment of the role of RIL in cancer development should be carried out taking into consideration the epigenetic features of the tumor or the cell line. Revealing mechanisms by which RIL affects positively or negatively malignant phenotypes of cancer cells could contribute to future personalized approaches to cancer therapy.Breast cancer cell lines were obtained from the American Type Culture Collection  and maintained according to the ATCC’s instructions.The cell lines used for the artificial modulation of RIL expression were selected within a single molecular subtype of breast cancer (triple-negative subtype).To upregulate RIL the selected cell lines were transduced with the lentivirus expression construct pLM-CMV-FLAG-RIL. The recombinant RIL was fused with the Flag epitope and placed under the control of CMV promoter. As a control, the corresponding cell lines were transduced with a similar lentiviral construct in which RIL was replaced with a Red fluorescent protein (pLM-CMV-RFP).To downregulate RIL the pLSLPw-shRIL lentiviral construct was used that contained shRNA expression cassette driven by the H1 promoter within the 3′-LTR. As a negative control we used the pLSLPw-shE6 construct that expressed a shRNA specific for the human papillomavirus E6 protein absent in the human genome.The corresponding genetic constructs were introduced into breast cancer cell lines by lentiviral transduction .To obtain the lentivirus vector particles, HEK293T cells were co-transfected with four plasmids: the lentivirus vector itself, two helper plasmids (pGag1 and pRev2), and pVSV-G. The transfection was performed via the liposome method with the use of LipofectAmine and Plus-reagents (Invitrogen) according to the conditions provided by the manufacturer. The medium containing recombinant virions was collected from the transfected cells and used to infect the target breast cancer cells.Proteins from whole-cell lysates were separated by 10% SDS–PAGE and analyzed by Western blotting for the expression of RIL ; GAPDH  was used as a loading control.Quantitative analysis of RIL expression was carried out by TaqMan probe-based Real-time PCR; GAPDH was used as the housekeeping gene. Primers and TaqMan probes were designed using Primer-BLAST tool; a comparative Ct method was used to determine the relative expression level of target genes.Bipartite Transwell chambers with 8-μm diameter pore  were used to assess cell migration rate in accordance with the Corning Cell Migration Assay. The migratory activity was estimated as the ratio of the number of cells on the lower side of the membrane to the number of non-migrated cells remaining on the upper side of the membrane; the proposed method allowed to avoid the cell count error which occurs when only the migrated cells were taken into account.® Phospho-SRC (Y419) and Total SRC ELISA kit was used to assess the activity level of Src, according to the manufacturer’s recommendations. Fluorometric analysis of breast cancer cell lysates was performed at 450 nm using Microplate Reader . The relative activity of Src was estimated as the ratio of the optical absorption value obtained for the Phospho-SRC (Y419) to the total amount of endogenous Src.RayBioSCID Beige mice (CB17. Cg-PrkdcscidLystbg-J/Crl) were purchased from Charles River Laboratory and kept under conditions of the SPF vivarium at the Institute of Bioorganic Chemistry (IBCH RAS); the experiments were performed in accordance to the guidelines recommended by the Bioethics Commission of IBCH RAS.Eight-week old female mice were injected unilaterally with 1 × 10^6 cells in 50 µL of PBS into the fourth abdominal fat pad by subcutaneous injection at the base of the nipple. Tumor growth was monitored externally using Vernier calipers for up to 30 weeks; the animals were euthanized when the tumors reached 10% of body weight.Lymph nodes, liver, heart, and lungs of the each euthanized mice were dissected, homogenized under cryogenic conditions, and used for the extraction of total RNA. The presence of the transgenic human cells (corresponding to the secondary sites of tumor development) in the analyzed organs was defined by RT-PCR. Puromycin-resistance gene presented in the transplanted cell lines (MDA-MB-231 shRIL and MDA-MB-231 E6) was chosen as a marker of metastases.p-value ≤ 0.05 was considered to be statistically significant.Statistical differences between groups of nominal, independent samples of small size were determined by Fisher’s exact test; the Bivariate (Pearson) correlation test was used as a measure of strength of the association between two continuous independent variables. All data were analyzed using GraphPad Prism, error bars representing standard deviation of the mean. A


As a result, the modified LLO (LLO@rGO) cathode exhibits superior electrochemical performance including cycling stability and rate capability compared to the pristine LLO cathode. In particular, the LLO@rGO with a 0.5% rGO content can deliver a high discharge capacity of 166.3 mAh g−1 at a 5C rate. The novel strategy developed here can provide a vital approach to inhibit the undesired side reactions and structural deterioration of Li-rich cathode materials, and should be greatly useful for other cathode materials to improve their electrochemical performance. Layered lithium-rich manganese oxide (LLO) cathode materials have attracted much attention for the development of high-performance lithium-ion batteries. However, they have suffered seriously from disadvantages, such as large irreversible capacity loss during the first cycle, discharge capacity decaying, and poor rate performance. Here, a novel method was developed to coat the surface of 0.4Li Nowadayvehicles . Howevervehicles ,5,6. In vehicles ,8,9, metvehicles ,11,12 anvehicles ,14,15. Avehicles . By far, oxides , fluoridGraphene is a two-dimensional, macromolecule carbon atom sheet with a honeycomb structure. Owing to its excellent electronic conductivity and mechanical properties, graphene can serve as an ideal conductive additive for hybrid nanostructured electrodes ,20,21,222MnO3∙0.6LiNi1/3Co1/3Mn1/3O2) was synthesized by using a simple and feasible carbonate co-precipitation method in our work. Then, the LLO surface was modified with an alkyl-bromide surfactant, which has been successfully applied in other fields [Therefore we developed a novel and robust method to coat thermally-reduced graphene oxide (GO) uniformly on the LLO surface, mainly based on electrostatic forces. Specifically, LLO , shows a far higher positive potential. Obviously, the surfactant greatly changes the surface charge state. Such a phenomenon is due to a bilayer surfactant structure formed on the LLO surface. Previous study showed that the alkyl-bromide surfactant can be adsorbed on a cathode oxide based upon the interaction between the cationic heads of the surfactant, and the surface O of the cathode oxide [To verify the working mechanism of the surfactant, the zeta potentials of pristine LLO, surfactant-modified LLO in aqueous solutions, were measured as shown in de oxide . Thus, ide oxide . Hence, the final charged state of the surfactant-modified LLO will be determined by the outside cationic heads of the out-layer surfactant, and it is highly positive , which c38H80BrN-GO mixture shows a positive potential. This is because the negatively-charged GO sheets are fully covered by the surfactant molecules, and are reversed to a positively-charged state. The results prove the strong interaction between the surfactant and the GO via the electrostatic interaction. No doubt, GO will coat tightly on the surfactant-modified LLO surface when they are mixed. Moreover, after the GO coating, the surface state of the LLO@rGO particles should be dominated by the coating nanosheets, which is negative, as shown in To confirm the strong electrostatic interaction between the GO sheets and the surfactant, their zeta potentials were also measured. The zeta potential of GO sheets is presented in 2 (R3m) phase, a typical α-NaFeO2-type structure. The weak peaks at 20–25° belong to a monoclinic Li2MnO3 (C2/m) phase [The effect of rGO coating on the crystal phase evolution of LLO was first determined. m) phase ,33,34. Bm) phase ,36. Thesm) phase . HoweverNext, the effect of rGO coating on the morphology of LLO were also checked to see if the rGO nanosheets could wrap the LLO particles well, as shown in To further exam the details of the morphology of rGO-coated LLO, transmission electron microscopy (TEM) images of LLO and LLO@rGO are shown in It is obvious that the rGO wraps the LLO particles tightly, owing to the addition of the surfactant. We believed that such kind of structure is beneficial for the improvement of the electrochemical properties of the LLO material.X-ray photoelectron spectroscopy (XPS) was used to monitor the evolution of the chemical states of the particle surface to further ensure the coating of rGO. + ion is extracted from the active LiMO2 phase, since the voltage gradually increases up to 4.50 V from 4.45 V, accompanied by the oxidation of Ni2+ to Ni4+ and Co3+ to Co4+. Then, the voltage maintains a long plateau at 4.50 V, during which Li+ ions are further removed from the Li2MnO3 component (TM layer) [+ ion transport.Electrochemical properties of the modified LLOs and pristine LLO were side-by-side compared to determine the effect of rGO coatings with the aid of the surfactant. The first cycle voltage profiles of LLO and LLO@rGO electrodes obtained at 0.1C are shown in M layer) . Figure −1 at the 5C rate, which is far higher than that of the LLO electrode (122.8 mAh g−1). This high-rate capability is attributed to the highly conductive ultrathin graphene (0.5%) around LLO particles. Moreover, the rGO coatings can also reduce the electrolyte decomposition and serve as a fast pathway for electron transportation during the charge/discharge process [−1 when the discharge rate is back to 0.1 C. Cycling stability is also an important issue for high-performance materials. To further elucidate the changed electrochemical performance of the LLO by the rGO modification, extended rate capability testing was implemented, as presented in  process ,44. MeanIt can be seen that the discharge capacity of the pristine-LLO electrode is going to decay from 40 cycles. Surprisingly, the LLO@rGO electrode manifests a stable capacity retention after 100 cycles. The improved cycling capacity is owed to the physical protection layer provided by a proper amount of rGO wrapping layers. The rGO layer can separate active material and electrolyte away, while suppressing the attack from HF and inhibiting the surface side reaction ,46. s, Rct, Q and W represent the ohmic resistance of the electrode, charge transfer resistance at the electrode/electrolyte interface, double layer capacitance, and Warburg impedance of the Li+ diffusion in the electrode, respectively. It can be seen that the values of Rs of various cells are almost the same. Rct shows big difference in different cells. The LLO@rGO-0.5%-based cell displays the lowest value of Rct compared with those of other cells, as shown in To clarify the kinetic features of pristine-LLO and LLO@rGO-based cells, EIS measurement was also performed. As shown in 4·6H2O, MnSO4·H2O and CoSO4·7H2O (Ni:Co:Mn = 0.6:0.2:0.2) was stirred at room temperature to achieve a precursor solution. Then, an adequate amount of precipitant (NH4HCO3) was added to the above solution, which was kept stirring at room temperature for 2 h. Next, the solution was filtered, washed and dried. The dried powder was mixed with LiOH·H2O, and then calcined at 400 °C for 2 h, 600 °C for two more hours, and 800 °C for a final five hours in air, to obtain the pristine LLO.The layered lithium-rich manganese oxide (LLO) cathode material was synthesized by a carbonated co-precipitation method. An aqueous solution of NiSO38H80BrN) was added into the LLO solution under continuous stirring. Next, different amounts of GO solution (12 mg/mL) were added to the modified-LLO solution at different weight ratios , and were stirred at 60 °C overnight. The mixed solutions were centrifuged and then dried at 60 °C for 12 h. Finally, the as-prepared powders were annealed at 400 °C for 5 h in an Ar atmosphere to obtain the LLO@0.5% rGO, LLO@1.0% rGO and LLO@2.0% rGO.GO was prepared by the Hummer’s method. To wrap reduced graphene-oxide (rGO) onto the LLO surface, 0.15 g LLO powders was first dispersed in 10 mL deionized water. Then, 3 mg dimethyldioctadecylammonium bromide  (ESCALAB 250Xi) . Morphological observations were carried out on a Zeiss Supra 55 field emission scanning electron microscope (FE-SEM) . The transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM) were used for morphology and micro-crystal observation with a JEM-2100 transmission electron microscope . The weight contents of rGO in the LLO@rGO composites were determined using thermo-gravimetric analysis  .X-ray diffraction (XRD) patterns of the samples were obtained by a Rigaku D/max 2500 diffractometer with Cu Kα radiation at V = 4 0 kV and I = 150 mA in a 2θ range of 10–80°, and the scanning rate was 10° minN-methyl-2-pyrrolidone, was prepared and then coated on an aluminum foil to fabricate working electrodes. The cells were assembled in an Ar-filled glove box with water and oxygen contents less than 0.1 ppm, using polypropylene film (Celgard 2400) as the separator. The electrolyte was a solution of 1 M LiPF6 in a 1:1 (v/v) mixture of ethylene carbonate (EC)/dimethyl carbonate (DMC). The cells were galvanostatically charge-discharged at various current rates (1C = 250 mA g−1) between 2.0 and 4.8 V at room temperature. The cyclic voltammetry (CV) tests were conducted between 2.0 V and 4.8 V at a scan rate of 0.1 mV s−1 on an electrochemical workstation. Electrochemical impedance spectroscopy (EIS) was also performed on an electrochemical workstation . The frequency of EIS ranged from 100 kHz to 10 mHz with an amplitude of 5 mV.All the electrochemical tests here were carried out on CR2032-type coin cells with lithium metal as their anodes. The details are described as follows. A slurry, with 75 w% active materials, 15 w% acetylene black, and 10 w% polyvinylidene fluoride dispersed in −1 at 5C rate. The results show that the rGO coating enables a greatly enhanced rate performance as well. Such improvement is due to the enhanced interfacial charge transfer, the reduced side reactions and the inhibited structural deterioration of Li-rich cathode materials. Using a proper surfactant to enhance the coating of graphene derivatives on different surfaces is demonstrated to be very promising to realize outstanding energy storage devices. In conclusion, we have successfully fabricated a high performance rGO-coated LLO cathode material. A surfactant was proved to be beneficial for the robust coating of rGO nanosheets on the LLO powder surface. The rGO-coated LLO cathode material exhibited outstanding rate capability as 166.3 mAh g


BRAF-mutant (n = 11), NRAS-mutant (n = 10), BRAF/NRAS wild type (n = 10), and GNAQ/GNA11-mutant uveal melanomas (UVMs) (n = 8). Cell surface expression of established IFNγ downstream targets PD-L1, PD-L2, HLA-A, -B, and -C, HLA-DR, and nerve growth factor receptor (NGFR) were analyzed by flow cytometry. Basal cellular expression levels of HLA-A, -B, -C, HLA-DR, NGFR, and PD-L2 predicted the levels of IFNγ-stimulation, whereas PD-L1 induction was independent of basal expression levels. Only 13/39 (33%) of the melanoma cell lines tested responded to IFNγ with potent induction of all targets, indicating that downregulation of IFNγ signaling is common in melanoma. In addition, we identified two well-recognized mechanisms of immunotherapy resistance, the loss of β-2-microglobulin and interferon gamma receptor 1 expression. We also examined the influence of melanoma driver oncogenes on IFNγ signaling and our data suggest that UVM have diminished capacity to respond to IFNγ, with lower induced expression of several targets, consistent with the disappointing response of UVM to immunotherapies. Our results demonstrate that melanoma responses to IFNγ are heterogeneous, frequently downregulated in immune checkpoint inhibitor-naïve melanoma and potentially predictive of response to immunotherapy.Immune checkpoint inhibitors that block the programmed cell death protein 1/PD-L1 pathway have significantly improved the survival of patients with advanced melanoma. Immunotherapies are only effective in 15–40% of melanoma patients and resistance is associated with defects in antigen presentation and interferon signaling pathways. In this study, we examined interferon-γ (IFNγ) responses in a large panel of immune checkpoint inhibitor-naïve melanoma cells with defined genetic drivers;  The identification of checkpoint signaling pathways that dampen anti-cancer immune responses and the subsequent development of checkpoint inhibitors have transformed the treatment of patients with metastatic cancer. Antibodies blocking immune checkpoints such as the cytotoxic T-lymphocyte-associated protein-4, the programmed cell death protein 1 (PD-1), and its ligand PD-L1 induce durable anti-tumor immune responses in many advanced malignancies, including melanoma, non-small-cell lung cancer, and renal cell carcinoma. PD-1 inhibition in melanoma promotes tumor regression and prolonged overall survival in 30–40% of patients with advanced disease (Recent studies have shown that resistance to immune checkpoint blockade involves defects in the interferon-γ (IFNγ) signaling pathway –9. Once B2M) gene, the structural component of MHC class I complexes, is enriched in pre-treatment tumor samples from melanoma patients with innate and acquired resistance to checkpoint inhibitor therapy , HLA-A, -B, -C, and HLA-DR] could serve to assess the integrity of IFNγ signaling in melanoma. We also examined the potential influence of melanoma driver oncogenes on IFNγ signaling activity and found that uveal melanoma (UVM) cells show evidence of diminished IFNγ pathway activity with minimal baseline and IFNγ induction of HLA-DR, NGFR, and PD-L2. Importantly, nearly 70% of melanoma cells included in this study showed incomplete responses to IFNγ stimulation, indicative of pre-existing resistance to immunotherapy. Furthermore, our data confirm that measuring IFNγ output with a select number of targets may be useful for detecting intrinsic defects in the IFNγ/JAK/STAT pathway, including JAK and STAT mutations which are associated with PD-1 inhibitor resistance , 13.A total of 39 cell lines were included in this study. Oncogenic driver mutation status is shown in Table 2. For IFNγ treatment, 7 × 104 melanoma cells per well were plated in complete media in six-well plates. After an overnight incubation, the complete media was replenished, and cells treated for 72 h with 1,000 U/ml IFNγ  or vehicle control [0.1% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline ]. Cells were collected, washed with PBS, and analyzed by flow cytometry.Cell lines were cultured in Dulbecco’s Modified Eagle Medium or Roswell Park Memorial Institute-1640 media supplemented with 10 or 20% heat inactivated fetal bovine serum , 11.25 mM glutamine , and 10 mM HEPES (Gibco) and were maintained at 37°C in 5% CO5) were incubated for 30 min on ice with mouse anti-human antibodies against HLA-ABC (clone W6/32), HLA-DR (clone L243), CD271/NGFR (clone ME20.4), CD273/PD-L2 (clone 24F.10C12) , and CD274/PD-L1  conjugated to phycoerythrin (PE), fluorescein isothiocyanate, PE-cyanine (Cy)7, allophycocyanin, and brilliant violet 421, respectively. All antibodies were titrated prior to experiment to ensure optimal concentrations were used. Fc block (BD Biosciences) was used to prevent non-specific staining due to antibody binding to Fc receptors. Fluorescence minus one controls  were included with each experiment. Prior to acquisition, cell viability was determined by staining cells with either 5 µM DAPI , Zombie Yellow dye (BioLegend), or Live Dead near-IR fixable dye . For the analysis of interferon gamma receptor 1 (IFNGR1) and B2M expression, cells were first stained with a fixable viability dye and either PE-conjugated anti-CD119 (clone GIR-208) or PE-Cy7 conjugated anti-B2M (clone 2M2), both from BioLegend. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm kit and stained intracellularly with the same antibody that was used for cell surface stain.Staining was performed in flow cytometry buffer . Cells (2 × 10Samples were acquired on BD LSRFortessa X20 flow cytometer (BD Biosciences) and the FlowJo software  was used for data analysis. At least 10,000 live events were acquired. General gating strategy included forward and side scatter area to exclude cell debris, time parameter to exclude electronic noise, forward scatter area and height to exclude doublets and gating on viable cells . Relative marker expression levels were calculated by dividing the geometric mean fluorescence intensity (MFI) of the antibody-stained sample by the FMO control MFI  human skin cutaneous melanoma (SKCM) and UVM datasets using single sample gene set enrichment analysis (ssGSEA) . RNA cou package , 18. The package .http://www.ingenuity.com) was used to identify mutations in genes associated with the JAK-STAT (KEGG) signaling pathway  using BWA . V600BRAF-mutant, 10 NRAS-mutant and 10 BRAF/NRAS wild type (BRAF/NRASWT) cutaneous melanoma cell lines, and 8 GNAQ/11-mutant UVM cell lines , and HLA-DR molecules was examined in a panel of 39 human melanoma cell lines with defined oncogenic driver mutations  and the uveal MP46 cells (relative MFI = 2.3) . PD-1 ligands PD-L1 and PD-L2 were expressed at comparably low levels in our panel of melanoma cells  BRAFV600-mutant, 1/10 (10%) NRAS-mutant, and 7/8 (87.5%) uveal cell lines  revealed a broad range of expression for all five markers Figure ; Table 1p < 0.01) and NGFR expression  . The cell surface protein expression patterns of these markers in our melanoma panel did not precisely reflect their transcript expression patterns in the human SKCM dataset of TCGA  and HLA-DR (HLA-DRA) were correlated  and PD-L2 (PDCD1LG2) transcripts were correlated (Spearman’s rank = 0.793 p < 0.01) in the TCGA SKCM dataset, although we did not observe any correlation in their cell surface protein expression  transcript expression was also different between the TCGA uveal and cutaneous datasets, whereas HLA-A transcript expression was indistinguishable between the TCGA uveal and cutaneous tumor groups   because PD-L1 and PD-L2 showed disparate expression at baseline  .Comparison of all five target molecules also showed positive correlation between IFNγ-induced expression of PD-L1, PD-L2, and HLA-DR. In particular, post-stimulation levels of PD-L1 and PD-L2 were correlated  demonstrated diminished response to IFNγ stimulation, usually manifested as no induction  of one or more markers in response to IFNγ stimulation. HLA-ABC expression was absent in the 7% demonsWe also examined the impact of IFNγ treatment on cell cycle progression in our panel of melanoma cells using flow cytometry. Of the 38 melanoma cell lines tested, three showed increasing cell death in response to IFNγ, with greater than 10% increase in sub G1 Table . Of thesvia alternate pathways or downstream elements.Analysis of the IFNγ target proteins, HLA-ABC, HLA-DR, NGFR, PD-L1, and PD-L2, in a panel of 39 melanoma cell lines revealed that IFNγ stimulated cell surface expression of all five markers in only 13 melanoma cell lines tested. The degree of IFNγ-mediated induction was highly variable for all five markers but closely reflected the corresponding basal expression levels for HLA-ABC, HLA-DR, PD-L2, and NGFR. By contrast, PD-L1 expression was frequently absent at baseline (relative MFI < 1.5) but was still induced to high levels after IFNγ treatment in the majority of cell lines. Consequently, although the JAK/STAT/IRF1 pathway is critical for the IFNγ-mediated induction of HLA-ABC, HLA-DR, and the two PD-1 ligands , 23, theHLA-DRA, NGFR, CD274 (PD-L1), and PDCD1LG2 (PD-L2) transcripts, and this was associated with reduced transcript expression of the IFNγ master transcription factors STAT1, STAT3, and IRF1 and with reduced IFNγ transcriptome signatures. It is worth noting that although transcriptome data are derived from high quality tumor samples with at least 60% tumor nuclei, they do contain variable levels of tissue-infiltrating immune and stromal cell populations that may influence the level of transcript expression  was compared. This may reflect disparate baseline expression levels due to IFNγ-independent regulatory influences but also the complexity and redundancy of the IFNγ signaling pathway. For instance, whereas the JAK–STAT1/2/3–IRF1 axis is critical for PD-L1 regulation, the JAK–STAT3–IRF1 node is important for PD-L2 stimulation . We alsopression . NeverthIncomplete responses to IFNγ-stimulation, usually manifested as lack of induction of one or more markers were evident in 26 of 39 (67%) melanoma cell lines. Although it is still not clear whether incomplete IFNγ stimulation in melanoma cells has significant impact on patient responses to immunotherapy, it is evident that this pathway is important for response to PD-1 blockade. In particular, nuclear expression of the IFNγ transcription factor IRF1  is assocIt is worth noting that of the 26 melanoma cell lines displaying incomplete induction of the 5 target proteins, 8 showed cell cycle distribution changes in response to IFNγ treatment. Importantly, 5/7 melanoma cell lines with no IFNγ-mediated PD-L1 induction showed no cell cycle profile changes after treatment with IFNγ. This may reflect the critical role of the STAT1 transcription factor in promoting PD-L1 expression and mediating IFNγ-induced cell cycle effects , 32. FivB2M gene  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Royal Prince Alfred Hospital Human Research ethics committee.SA, ES, and AS performed the experiments. ES, HR, and SL wrote the manuscript. SA, SL, ES, and HR analyzed and interpreted the data. SA, AS, RK, SL, ES, and HR read, revised, and approved the final manuscript.RK served on advisory boards for Roche, Amgen, BMS, Merck, Novartis, and TEVA and has received honoraria from Merck, BMS, and Novartis. The remaining authors declare no conflict of interest.


EC50 value in PANC-1 cell line dropped from 4.04 ± 0.046 to 1.68 ± 0.004 µM. The ICPD47 and ICPD62 under the same conditions could act synergistically with GEM, 5-FU and DOX and is worth of further investigations, and studies of synergistic effect is a promising path for more efficient anticancer therapies.Heat shock protein 90 (Hsp90) is a widely investigated target for anticancer therapy. The experimental Hsp90 inhibitors ICPD47 and ICPD62 demonstrated anticancer activity against colorectal, osteosarcoma and cervical cancer cell lines. However, their anticancer activity has not been investigated against pancreatic cancer cell lines yet, and there are no data about synergistic activity of these compounds in combination with clinically used anticancer agents. Pancreatic cancer cell lines, MIA PaCa-2 and PANC-1 were exposed to ICPD47 and ICPD62 alone and in combinations with antimetabolites gemcitabine (GEM), 5-fluorouracil (5-FU) and topoisomerase inhibitor doxorubicin (DOX). Effects on cell viability were determined by MTT assay. The synergistic activity was evaluated using Chou-Talalay method. Also, 3D cell cultures were formed using 3D Bioprinting method and the activity of each compound and their combinations was examined by measuring the size change of spheroids. The strongest synergistic activities were determined in combinations using all ratios of ICPD47 with GEM and ICPD62 with GEM in MIA PaCa-2 cell line (combination index <0.5). The combinations of ICPD47 with 5-FU and ICPD47 with GEM in a ratio of 1:5 showed the greatest effect on tumour spheroid growth in both cell lines. The ICPD47 in combination with mild hyperthermia showed significant results, where the  In addition, the increasing resistance of pancreatic cancer to available chemotherapy is a serious problem.Pancreatic cancer is one of the most aggressive and deadliest cancer types. After diagnosis, the patients have a 5-year survival rate of only 8.5%. Such a low survival rate is mainly because the disease is diagnosed at late stages and it progresses fast to lymphatic system and local organs3. Therefore, there is a huge need for novel therapeutic agents that could act on multiple targets and enhance the success of pancreatic cancer therapy.There are seventeen registered drugs for pancreatic cancer chemotherapy, but statistics show that the current treatment is not effective. Recent studies show, that the resistance for 5-fluorouracil (5-FU) and gemcitabine (GEM) is increasing5. Since this molecule regulates multiple signalling cascades, the inhibition of Hsp90 has a suppressive effect on many client proteins and biochemical pathways5. Because of this characteristic and the fact that in cancer tissues Hsp90 is expressed in higher levels, the inhibition of Hsp90 can overcome the redundancies of signals and drug resistance in many cancers5. Recent scientific and clinical data show that Hsp90 inhibitors strongly affect cancer cell survival and using them in combinations with anticancer agents, it is possible to postpone cell resistance to chemotherapy7.Heat shock protein 90 (Hsp90) is a chaperone protein of 90 kDa. Hsp90 is required for the folding and stability of proteins, which are critical for cell growth, differentiation and survival8. To determine the combination effect, the Chou-Talalay method is often used, when the combination index (CI) is calculated. This method is the most accurate to evaluate the type of combinatorial effect of different compounds. CI values below 1 (CI < 1) describe synergistic effect, values equal to 1 (CI = 1) describe additive effect, and values over 1 (CI > 1) show antagonistic effect of the compounds8.Combinatorial chemotherapy is often used for the treatment of cancers. Synergistic effect of different drugs in combinations can increase its therapeutic effect, decrease dosage and toxicity, compared to individual drugsP values, rather than Chou-Talalay method, when CI is calculated; (b) using randomly chosen concentrations of compounds in the combination instead of using constant EC50 ratios of compounds in the combination8. Some studies have shown that the expression of Hsp90 in pancreatic cancer tissue and in tissues adjacent to cancer is higher up to five fold, compared to healthy pancreatic tissue10. With this study, we wanted to obtain new fundamental insights on the effects of Hsp90 inhibitors in combinations with already used anticancer agents  against pancreatic cancer cell lines. Therefore, for the first time, we tried to determine the combination effect of experimental resorcinol-like Hsp90 inhibitors (ICPD47 and ICPD62) and clinically used anticancer agents in two-dimensional (2D) and three-dimensional (3D) models of different pancreatic cancer cell lines. In addition, we aimed to determine whether a mild hyperthermia could increase the activity of Hsp90 inhibitors.There are many scientific manuscripts published on the combinations of Hsp90 inhibitors and anticancer agents in various cancers except for pancreatic cancer. Never the less, credible data on synergistic activity of Hsp90 inhibitors and anticancer agents are limited or falsely determined. The main mistakes in determining the combinatorial effects of the drugs are: (a) using statistical analysis and calculating p < 0.05). The ICPD62 had higher effect on cell viability than ICPD47, almost nine times in MIA PaCa-2 cell line, half maximal effective concentration (EC50) values of ICPD62 and ICPD47 after 72 h were 0.45 ± 0.06 µM and 3.91 ± 0.25 µM. And in PANC-1 cell line ICPD62 inhibited cell viability six times stronger than ICPD47, as EC50 values of ICPD62 and ICPD47 after 72 h were 0.41 ± 0.08 µM and 2.38 ± 0.25 µM .Hsp90 inhibitors and anticancer agents demonstrated different effects on cell viability Fig. . All comSimilar to DOX, both Hsp90 inhibitors showed cytotoxic activity, as they totally inhibited cell viability in higher concentrations Fig. . The GEMCI value, when fraction affected (fa) equal to 0.5, was 0.16 ± 0.05) in ratio 1:5 in MIA PaCa-2 cell line  in PANC-1 cell line  in all concentrations (fa range from 0 to 1) in MIA PaCa-2 cell line , PANC-1 cells formed relatively small and compact spheroids (401 ± 13.8 μm in diameter) Fig. . The effp < 0.05), and 5-FU reduced the size of the spheroids by 1.5 times (p < 0.05) . The least effective compounds were ICPD62 and DOX, as they reduced the size of the spheroid by 1.17 and 1.25 times respectively  Fig. . Howeverp < 0.05) and 1.35 (p < 0.05) times smaller compared to control group. However, the difference between the effects of the combination and GEM alone was not significant (p > 0.05). ICPD62, DOX and 5-FU had almost no effect on the growth of PANC-1 spheroids  and in MIA PaCa-2 - 1.2 times, from 3.71 ± 0.01 to 2.48 ± 1.65 µM (p > 0.05) , and in PANC-1 - 1.8 times from 0.39 ± 0.01 to 0.22 ± 0.02 µM, but the results were not of exceptional significance (p > 0.05)  Fig. . ICPD62 05) Fig. .Figure 8et al. study, where ICPD62 showed almost 10 times higher activity than ICPD47 in U2OS and HeLa cell lines11. Such results can be explained by the differences in compound structure12.In our study, ICPD62 showed from 6 to 9 times greater effect on cell viability than ICPD47, in PANC-1 and MIA PaCa-2 cell lines, respectively. Similar results were demonstrated in Cikotiene 14. Also it is possible, that some antimetabolites, such as cisplatin, may inhibit Hsp90 by binding to its’ C-domain, and therefore using it in combination with Hsp90 inhibitors can enhance the inhibition of Hsp9015. Kaiser et al. in their study determined that DOX in combination with resorcinol-like Hsp90 inhibitor could have synergistic effect on cell viability, at higher concentrations of compounds when higher concentrations of compounds are used. However, due to variable conditions, such as incubation plan and time with individual compounds, the effect can vary from synergism to antagonism16.The strongest synergistic activity was determined by using combinations of Hsp90 inhibitors with GEM and 5-FU. According to different studies of Fallahi-Sichani and Arlander, the Hsp90 inhibitors may slow down the cell metabolism, and therefore making them more sensitive to antimetabolitesEC50 ratio, can have different effect, even though, both inhibitors have the same mechanism of action. It is possible that such differences in action can be influenced by different structures and activities of both compounds. Lee et al. in their study determined synergistic activity of combinations of Hsp90 inhibitor NVP-AUY922 and 5-FU against gastric cancer cells17. Researchers explained that NVP-AUY922 inhibited the expression of thymidylate synthase (TS), which is the main reason of increased resistance of cancer cells to 5-FU. Therefore, decreased level of TS, makes gastric cancer cell more sensitive to 5-FU17. It is possible, that pancreatic cancer cells ICPD47 interacts with 5-FU in similar way. In different study, Liu et al. determined that 5-FU in combination with another Hsp90 inhibitor, SNX-2112, showed an antagonistic effect on human oesophageal cancer cell viability4. In their opinion, 5-FU could compete with SNX-2112 in binding to Hsp90 N-domain, or induced the cancer signal paths that were inhibited by SNX-2112.In this study, it was determined that different Hsp90 inhibitors in combination with the same anticancer agent exhibiting the same p < 0.05). The ICDPD47 combination with GEM had greater effect on PANC-1 spheroid growth (p < 0.05) than individual compounds. According to scientific literature, pancreatic cancer cell lines are different in genotype and phenotype, so their aggregation qualities and spheroid formation abilities are different. Therefore, it could determine different spheroid growth kinetics between MIA PaCa-2 and PANC-1 cell lines. Overall, the combinations of compounds had greater potency in 2D monolayer cultures than in 3D spheroid cultures. The diffusion of the compounds to inner layers of spheroid could be limited due to high density of the cells and the increased levels of structures of extracellular matrix, which makes a natural barrier similar in solid tumors, therefore only the outer layers could be affected18. Also, the genes related to increased resistance to anticancer agents could be more activated in spheroids than in monolayer cells19.To our knowledge, no studies have been conducted to evaluate combination effects of Hsp90 inhibitors and anticancer agents in pancreatic cancer spheroids. The combination of ICPD47 with 5-FU had a greater effect on MIA PaCa-2 spheroid growth than individual compounds , and DOX hydrochloride and 5-FU were purchased from Sigma-Aldrich Co. .Hsp90 inhibitors ICPD47 and ICPD62 were synthesized at Vilnius University, Lithuania as previously described2 at 37 °C.Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were obtained from the American Type Culture Collection . Human foreskin fibroblast cells HF were provided by prof. Santos A. Helder . All cell lines were cultured in Dulbecco’s Medium GlutaMAX (DMEM Glutamax) medium. The medium was supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 10% fetal bovine serum. The medium and supplements were purchased from Gibco . Cells were maintained in humidified atmosphere containing 5% COEC50  of the compounds were calculated.Compound effect on cell viability was established using 3--2,5-diphenyltetrazolium bromide  assay. The MIA PaCa-2 and PANC-1 cells were seeded in 96-well plates in a volume of 100 µL . After 24 h preincubation, the cells were treated with 100 µL of different concentrations of Hsp90 inhibitors and anticancer agents. The medium without cells was used as a positive control, and the medium with 0.25% DMSO (Sigma-Aldrich Co.) served as a negative control. After 72 h, the cells were incubated with 20 µL 5 mg/mL MTT solution (Sigma-Aldrich Co.) for 4 h under the same conditions. The absorbance was measured at wavelengths of 570 and 630 nm, and the EC50 of each compound was determined as described previously. To evaluate the effect of combination treatment, different combinations of Hsp90 inhibitors and anticancer drugs in three constant ratios  of EC50 were prepared and serial dilutions were made. The combinations of the compounds were added to the 96-well cell plates with cells after 24 h preincubation and incubated for 72 h. After incubation the MTT assay was performed. To calculate the combination indices (CI), the CompuSyn software was used  that is based of Chou-Talalay method8.The 23. The pancreatic cancer cells were mixed with human fibroblasts in a ratio of 1:1 in order to better represent the tumour microenvironment24. The cells were incubated with nanoparticles NanoShuttle  for 8 h. The cells were resuspended and seeded into ultra-low attachment 96-well plates in a volume of 100 µL . The plate was placed on magnetic drive and incubated in humidified atmosphere containing 5% CO2 at 37 °C for 72 hours until spheroids were formed. The formed spheroids were imaged using light microscopy (Olympus TH4-200). Then, the medium was replaced with the fresh one, containing different concentrations of separate compounds and their combinations in a ratio of 1:5 at the concentrations when fa was 0.5. The photographs of spheroids were taken every 48 h, using Olympus cellSens Standard 1.16 software . The medium was replaced every 96 h25.Spheroids were formed from MIA PaCa-2 and PANC-1 cells using 3D Bioprinting methodThe effect of Hsp90 inhibitors, anticancer agents, and their combinations on spheroid growth was examined by measuring the size change of spheroids using ImageJ software .EC50 values of single Hsp90 inhibitors and the combination of inhibitors and hyperthermia.The cell suspension was prepared and seeded as described previously in cell viability assay in two 96-well plates. After 24-hour preincubation, one plate was heated in a water bath at 43 °C temperature for 30 minutes. Then the cell viability assay (as described previously) was performed to establish p < 0.05 was considered as the level of significance.Statistical analysis was performed using Microsoft Office Excel 2007 software . All experiments were performed in at least triplicate independent measurements and the obtained values were reported as mean ± standard deviation. Student’s t-test was used and p-values were calculated. A value of This article does not contain any studies with human participants or animals performed by any of the authors.For this type of study, formal consent is not required.Supplementary data


The dysfunction of salivary glands commonly induces dry mouth, infections, and dental caries caused by a lack of saliva. This study was performed to determine the genetic and functional changes in salivary glands using a klotho (-/-) mouse model. Here, we confirmed the attenuation of KLF4 expression in the salivary glands of klotho (-/-) mice. Soluble klotho overexpression induced KLF4 transcription and KLF4-mediated signaling pathways, including mTOR, AMPK, and SOD1/2. Silencing klotho via siRNA significantly down-regulated KLF4 expression. Additionally, we monitored the function of salivary glands and soluble klotho and/or KLF4 responses and demonstrated that soluble klotho increased the expression of KLF4 and markers of salivary gland function  in primary cultured salivary gland cells from wild type and klotho (-/-) mice. In a 3D culture system, cell sphere aggregates were observed in soluble klotho- or KLF4-expressing cells and exhibited higher expression levels of salivary gland function-related proteins than those in nontransfected cells. These results suggest that activation of the klotho-mediated KLF4 signaling pathway contributes to potentiating the function of salivary glands. Salivary gland dysfunctional changes occur with reduced salivary flow and dry mouth (xerostomia) and commonly involve oral dysfunction, tooth structure deterioration, and infection through reduced salivation , 2. AnatKlotho is a transmembrane protein and a putative antiaging agent. Klotho-knockout mice demonstrate age-related phenotypes, such as a short lifespan, growth retardation, infertility, skin atrophy, hypoglycemia, hyposalivation, hyperphosphatemia, ectopic calcification, osteoporosis and pulmonary emphysema , 8.2O2-induced apoptosis and senescence [The membrane klotho type acts as a coreceptor of fibroblast growth factor 23 (FGF23) and is capable of regulating phosphate and vitamin D metabolism , 10. FGFnescence . Additionescence , 16.+/K+-ATPase (Atp1a2), Ca2+-ATPase (Atp2a1), epidermal growth factor (EGF), and nerve growth factor (NGF), which have been suggested to regulate submandibular salivary gland function, were significantly decreased. In a network constructed by the differentially expressed genes induced by klotho depletion, proliferator-activated receptor-γ (PPARγ), which regulates energy homeostasis and insulin sensitivity, was located at the core [In our previous study, we analyzed the gene expression profiles of salivary glands from klotho-deficient mice (klotho -/-). Microarray analysis showed that the expression levels of alpha2-Nathe core . RecentlTherefore, this study aimed to evaluate salivary gland functional changes by the soluble klotho/KLF4 signaling pathway, and to provide further baseline information for preclinical research on salivary gland dysfunction related to aging.To determine the tissue level expression of KLF in klotho (-/-) mice, we analyzed KLF4 expression in 8 organs  harvested from four-week-old klotho (-/-) mice. Quantitative RT-PCR revealed that the mRNA levels of KLF4 were decreased to different extents—from 2- to 7-fold—in several organs. The salivary glands, skin, brain, and spleen exhibited significantly decreased KLF4 expression in klotho (-/-) mice compared to that in these organs of wild-type mice . SimilarTo elucidate the relationship between klotho and KLF4 gene expression, we evaluated KLF4 mRNA expression using real-time quantitative RT-PCR in wild-type and klotho (-/-) mouse embryonic fibroblasts (MEFs). Depletion of klotho led to significantly diminished mRNA and protein levels of KLF4 –2C, and To directly assess the functional contribution of soluble klotho to KLF4 signaling, we investigated the expression changes in KLF4-related genes upon the overexpression of soluble klotho in MEFs. We first evaluated the overexpression of soluble klotho in soluble klotho-transfected MEFs by real-time quantitative RT-PCR and observed abundant KLF4 mRNA expression –3D. As sWe next assessed the effect of soluble klotho depletion on KLF4-related protein expression. The inhibition of soluble klotho by siRNA was detected by real-time PCR and Western blot analysis in HEK293 cells, and reduced expression of KLF4 and FOXO1 was observed. KLF4 protein expression was also inhibited in siRNA soluble klotho-transfected cells –4D.To further clarify whether KLF4 depletion modulates soluble klotho-induced KLF4 signaling, we knocked down KLF4 by siRNA in HEK293 cells. As shown in Single-cell suspensions obtained by mechanical and enzymatic dissociation of klotho (-/-) mouse salivary glands were grown in our culture system as described in the Materials and Methods. In klotho (-/-) PSGCs, we examined the expression of KLF4 by real-time quantitative RT-PCR and Western blot. Similar to the results shown in To confirm the function of PSGC populations, we examined the expression of salivary gland functional markers, such as α-amylase (AC secretion product), aquaporin 5 (water channel protein), and ZO-1 (tight junction protein). Quantitative reverse transcription PCR (qRT-PCR) was used to validate the altered expression of these genes. Klotho (-/-) PSGCs showed reduced expression of α-amylase and aquaporin-5 compared to that in klotho (+/+) PSGCs. The tight junction protein ZO-1 was also inhibited in klotho (-/-) PSGCs –5E. In cTo determine whether PSGCs induced by soluble klotho or KLF4 transfection could differentiate into functional activating cells, such as salivary gland ACs, we utilized a 3D culture system. First, we observed the induction of typical mesenchymal stem cell (MSC) surface antigen markers, such as CD44 and CD90, which are known to interact with the extracellular matrix, in soluble klotho- or KLF4-expressing wild-type PSGCs . In addiβ-induced renal fibrosis and metastasis [α and IFNγ. In addition, soluble klotho maintained a low plasma phosphate concentration by inhibiting both renal (NaPi-IIa) and intestinal (NaPi-IIb) phosphate transporters [Soluble klotho has been suggested to function as a humoral factor that targets multiple tissues. A recent study demonstrated that soluble klotho attenuated the generation of reactive oxygen species by insulin and IGF-1 signaling , 19. Soltastasis , 21 and sporters , 23. TheIn a previous study, we performed a transcription factor (TF) activation plate array to profile the TF activities affected by soluble klotho expression. TFs play essential roles in regulating gene expression and act as sensors to monitor cellular changes and convert the signals into altered gene expression. We first found that KLF4 expression was significantly increased after soluble klotho transfection (unpublished data). In this study, KLF4 expression was clearly highly abundant and diverse in the salivary gland, followed by the lung, skin, brain and spleen. The high KLF4 expression in salivary glands is consistent with the immunoblot results. Based on this analysis, KLF4 expression was significantly downregulated in the salivary glands of klotho-deficient mice compared with that in wild-type mice. We confirmed that the expression of KLF4 was decreased in klotho (-/-) MEFs relative to that in wild-type cells. Moreover, using gene transfection and RNA interference techniques, we demonstrated that soluble klotho regulated the expression of KLF4. Interestingly, the KLF4 promoter activity was significantly increased in soluble klotho-overexpressing cells. These results strongly indicate that soluble klotho is involved in the regulation of KLF4 expression.KLF4, an essential regulator of stem cells, was found to be specifically expressed in several normal tissues, especially the salivary gland. KLF4 is downregulated during parotid AC terminal differentiation . We defiCip1/Waf1 by recruiting p53 [A previous study suggested that KLF4 primarily contributes to p53-dependent cell cycle arrest by DNA damage , 26. KLFting p53 , 28. ThuWe recently showed that submandibular salivary glands in klotho-deficient mice show a loss of granular ducts and mucous acini compared to those of wild-type mice . Loss ofin vitro characteristics of PSGCs isolated from klotho wild-type and klotho (-/-) mice. The PSGCs of klotho (-/-) mice showed low KLF4 expression compared to those of wild-type mice and inhibited the expression of salivary gland functional markers, such as α-amylase, aquaporin 5, and ZO-1. However, overexpression of soluble klotho induced KLF4 and salivary gland functional markers in PSGCs. In addition, small colonies of epithelial-shaped cells were observed on the culture substrate within 5 days under soluble klotho or KLF4 transfection conditions. These spherical cells were positively labeled with AQP-5 and α-amylase. Notably, strong staining of ZO-1 was observed. The expression of salivary gland functional markers in soluble klotho- or KLF4-overexpressing ACs is shown in Therefore, we examined the critical role of soluble klotho/KLF4 in salivary gland dysfunction. In this study, we investigated the in vitro will express CD44 and CD90 and retain these morphological and phenotypical characteristics after 3D culture. These results showed that soluble klotho-induced cells retain their ability to self-renew as stem cells and may restore the function of the salivary gland.In addition, we demonstrated the expression of stem cell markers (CD44 and CD90) in soluble klotho-induced PSGCs. CD44, a stem cell marker, has previously been reported in serous ACs of human salivary gland tissue , 30. FurIn summary, soluble klotho directly induces cyclin D1/B1 and AMPK and regulates p53/p21 and SOD1/2 signaling by inducing KLF4. Our findings suggest a critical role for soluble klotho/KLF4 in the functional restoration of the salivary gland and selectively inducing the proteins necessary for salivary gland function. Soluble klotho/KLF4 signaling in PSGCs induced the expression of salivary gland functional markers . Additionally, 3D culture analysis showed that spherical cells were formed in soluble klotho- or KLF4-induced PSGCs. We predict that the expression of the progenitor cell markers CD44 and CD90 in soluble klotho-induced PSGCs may at least partially explain their regenerative potential. We expect that our results will contribute to better understanding soluble klotho signaling in the salivary gland and the development of potential cell therapies for patients with irreversible salivary gland function loss.2/95% air atmosphere at 37°C. Immortalized human salivary gland ACs were cultured on keratinocyte serum-free medium  containing 100 U/ml penicillin and 100 μg/ml streptomycin.MEFs were derived and cultured from SAMP1/kl+/+ and SAMP1/kl-/- 13-day-old embryos that were obtained by mating SAMP1/kl+/- mice with SAMP1/kl+/- mice  and main2 (6.25 mM) at 37°C for 40 min. Dissociated cells were centrifuged at 1000 rpm for 5 min, and the cell pellet was resuspended in the culture medium. The cells were plated in a 12-well plate at a density of 5 × 104 cells/well in DMEM/F-12 (Gibco) medium supplemented with 100 U/mL penicillin G, 100 μg/ml streptomycin, 20 ng/mL EGF (Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (Gibco), 1/100 N2 supplement (Gibco), 10 μg of insulin-transferrin-selenium (Gibco), and 1 μM dexamethasone (Sigma-Aldrich) and incubated in 5% CO2 at 37°C. Fresh medium was added every three days. After 4-5 passages, the expression of salivary gland functional markers, such as α-amylase (AC secretion product), aquaporin 5 (water channel protein), and ZO-1 (tight junction protein), was examined.Submandibular gland tissues were harvested from 4- or 8-week-old wild-type and klotho (-/-) mice. The tissues were dissociated from cervical fascia and connective tissues under a dissecting microscope and gently collected in phosphate-buffered saline (PBS). Freshly dissociated tissues were washed twice with DMEM and Ham’s F-12 medium  supplemented with 100 U/mL penicillin G and 100 μg/ml streptomycin (Gibco), minced with scissors, and incubated with DMEM/F12 medium containing collagenase type II (0.025%), hyaluronidase (0.04%) and CaCl(target gene) − CT  and ΔΔCt = ΔCt  − ΔCt (control group). The relative expression levels of related genes were determined by the 2−ΔΔCT method, and the experiments were repeated three times.Total RNA was extracted with TRIzol reagent (Invitrogen). Reverse transcription was performed on 1 μg of total RNA using oligo dT primers and M-MLV Reverse Transcriptase (Invitrogen) in a final volume of 20 μl for 5 min at 65°C followed by 1 h at 37°C. Real-time quantitative PCR was performed using the SYBR Green kit  according to the manufacturer’s instructions. The expression levels of soluble klotho, KLF4, and FOXO1 mRNA were normalized to those of GAPDH for each sample. The following primers were used for real-time quantitative PCR detection: soluble klotho, 5′-GCTCTGCTTCCGCC ACTT-3′ (forward) and 5′-GCATGAGCCAGGAGGAG G-3′ (reverse); KLF4, 5′-TTCCAACTCGCTAACCCAC C-3′ (forward) and 5′-GCTCGGGACTCAGTGTAG-3′ (reverse); FOXO1, 5′-AGACCCGGGTTCTTTGACAC-3′ (forward) and 5′-TTGTCATAGGCTTCCCACCA-3′ (reverse); amylase, 5′-GGTGCAACAATGTTGGTGTC-3′ (forward) and 5′-ACTGCTTTGTCCAGCTTGAG-3′ (reverse); Aqua5, 5′-CGACCGTGTGGCTGTGGTCA-3′ (forward) and 5′-GTGCCGGTCAGTGTGCCGTC-3′ (reverse); ZO-1, 5′-CGAGGCATCATCCCAAATAAGA AC-3′ (forward) and 5′-TCCAGAAGTCTGCCCGATC AC-3′ (reverse). The samples were quantified based on the threshold cycle by interpolation from the standard curve. The reliability of the PCR results was evaluated by a solubility curve as follows: ΔCt = CT KLF4 siRNA was obtained in the form of Silencer Select Validated siRNA (Applied Biosystems). The sense sequence of the KLF4 siRNA was 5′-CUCGUUCUCG GCUUGUCUA (dTdT)-3′, and the antisense sequence was 5′-UAGACAAGCCGAGAACGAG (dTdT)-3′. The cells were transfected with siRNA (20 nM) using Lipofectamine 2000 siRNA Transfection Reagent  according to the manufacturer’s instructions. The cells were harvested 48 h after transfection. Total cell lysates were separated by SDS-PAGE and analyzed by Western blot as described above.Cells were lysed, and proteins were extracted in a radio immunoprecipitation assay buffer  and complete protease inhibitor cocktail (Sigma-Aldrich). The lysates were obtained after centrifugation, and the protein concentration was measured using the BCA protein assay kit . The lysates were separated by 12% SDS-PAGE. After transfer to PVDF membranes , the membranes were blocked for 1 h at room temperature with 5% nonfat dry milk in TBS-T buffer. The blocked membranes were then incubated with primary antibodies (1:1000 dilution) against klotho (10E2), KLF4 (3F3), FOXO1 (7G6C5), phospho-mTOR (D15C3), phospho-AMPK (D2E5), and p21 (D1F2), which were purchased from Cell Signaling Technology. The cyclin D1, cyclin B1, SOD1, SOD2, p70S6K, and actin antibodies were purchased from Santa Cruz Biotechnology . The membranes were washed 3 times with TBS-T buffer and then incubated with secondary antibodies conjugated to HRP  for 1 h at room temperature. The immunoreactive proteins were visualized with ECL detection reagents (Thermo).The pGL4-KLF4 promoter (-2.046 bp from the start codon) was generously provided by Dr. Yoshida . HEK293 cells were transfected with 1 μg of the pGL4-KLF4 Luc plasmid and 1 μg of Tk-Renilla by Fugene 6 HD (Promega) reagent and harvested after 48 h. Additionally, the cells were transfected with pcDNA3-sKL and/or pcDNA3-KLF4 using Fugene 6 HD reagent for 48 h. Transfected cells were lysed and measured with the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions. Relative luciferase units were normalized by the Tk-Renilla luciferase value in each sample. Each experiment was performed in triplicate and repeated twice.4 cells/well were seeded on Matrigel, and 3D spheroids were grown at 37°C in 5% CO2. For the generation of single-cell suspensions from 3D spheroids, spheres were isolated by removing media and adding 0.5 ml of Dispase (1 mg/ml) directly to the Matrigel followed by incubation for 1 h at 37°C in 5% CO2 to digest the Matrigel. The spheres were washed in PBS, centrifuged at 1200 rpm for 5 min, dissociated with TrypLE Express (Gibco) and passed through a 40 μm filter to remove clumps and obtain single-cell suspensions. The cells were then plated and grown as monolayers in either serum-free medium (SFM) or serum-containing medium (SCM).One hundred microliters of Matrigel  in 48-well chambers was allowed to solidify in a 37°C incubator for 1 h. Upon solidification, 5 x 10+ confocal microscope (Japan). The average intensity value of the nonspecifically bound secondary antibody was subtracted from all images using the ImageJ program.The presence of aquaporin-5 and ZO-1 in 3D cultured cells was confirmed by indirect immunofluorescence staining as described previously . BrieflyP-values <0.05 were considered statistically significant.Data are presented as the mean ± SEM. Statistical significance between groups was determined by Student’s t test and one-way analysis of variance (ANOVA). Supplementary Figures


While some studies confirmed a (near)‐essential role for BUB1 in the SAC . To this end, we performed live cell imaging of cells treated with a high dose of nocodazole to trigger a full SAC response. We observed that both ∆BUB1 clones were able to establish a functional SAC as cells displayed a prominent arrest in response to nocodazole treatment, not significantly different from WT HAP1 cells or our previously published BUB1 KO cells (∆BUB1 “Ex3”), that were generated by the stable integration of a Blasticidin resistance cassette in exon 3  have been unsuccessful. Although generating knockouts in these cell lines can be technically more challenging due to the presence of multiple alleles, this result could indicate that the loss of BUB1 is not well tolerated in all cell types. This possibly reflects a difference in the relative contribution of BUB1 to the SAC between cell lines or a difference in the tolerance toward mild chromosome segregation errors. Thus, although the most reliable way to fully resolve the exact contribution of BUB1 to the SAC in different genetic backgrounds would be to stably eradicate the full gene, this is only feasible if this is a tolerated condition.AppendixClick here for additional data file.Review Process FileClick here for additional data file.


There are errors in the Abstract. The correct Abstract is as follows: Anthropogenic infrastructure can negatively affect wildlife through direct mortality and/or displacement behaviors. Some tetranoids (grouse spp.) species are particularly vulnerable to tall anthropogenic structures because they evolved in ecosystems void of vertical structures. In western North America, electric power transmission and distribution lines (power lines) occur in sagebrush (Artemisia spp.) landscapes within the range of the greater sage-grouse . The U.S. Fish and Wildlife Service recommended using buffer zones near leks to mitigate the potential impacts of power lines on sage-grouse. However, recommended buffer distances are inconsistent across state and federal agencies because data are lacking. To address this, we evaluated the effects of power lines on sage-grouse breeding ecology within Utah, portions of southeastern Idaho, and southwestern Wyoming from 1998–2013. Overall, power lines negatively affected lek trends up to a distance of 2.3 km. Power lines did not affect lek persistence. Female sage-grouse avoided transmission lines during the nesting and brooding seasons at distances up to 1.1 and 1.2 km, respectively. Nest and brood success were negatively affected by transmission lines, however no maximum effect distance was identified. Distribution lines did not appear to affect sage-grouse habitat selection or reproductive fitness. Our analyses demonstrated the value of sagebrush cover in mitigating potential power line impacts. Managers can minimize the effects of new transmission power lines by placing them in existing anthropogenic corridors and/or incorporating buffers at least 2.3 km from active leks. Given the uncertainty we observed in our analyses regarding sage-grouse response to distribution lines coupled with their role in providing electric power service directly to individual consumers, we recommend that buffers for these power lines be considered on a case-by-case basis. Micrositing to avoid important habitats and habitat reclamation may reduce the potential impacts of new power line construction.


Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC50 value of 0.81µg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo. Dihydroactinidiolide ( Pereskia bleo (Kunth) DC. (Cactaceae) are used traditionally in Malaysia for the treatment of cancer, high blood pressure, diabetes and diseases associated with rheumatism and inflammation. They are also used as remedy for the relief of gastric pain, ulcers and for revitalizing the body  and cytotoxic investigation on all isolated compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5).In the present study, we report further progress in ongoing research on 4), 2,4-di-tert-butylphenol (5), α-tocopherol (6) and phytol (7) were obtained from Pereskia bleo as previously described by Sri Nurestri et al.  + (15), 137 (8), 111 (100), 109, 67. Compound 1 was identified by comparison of its mass spectral data with NIST mass-spectral library  with reported data . In contrast, the cytotoxic effect of the crude methanol extract on the MCF7 cell line gave IC50 of 39.0 µg/mL (mild) whilst two isolated compounds 2,4-di-tert-butylphenol (5) and α-tocopherol (6), showed good inhibitory activities with IC50 values of 5.75 and 7.5 µg/mL, respectively.However, the observed activity might be due to synergism between compounds present in the plant extract. The synergism among these compounds which contribute to the cytotoxic activity, is not only dependent on the concentration of the compounds, but also on the structure and interaction(s) between the compounds . This ca5) displayed very remarkable cytotoxic activity against KB cells with an IC50 value of 0.81 µg/mL and strong cytotoxicity against MCF7 (IC50 5.75 µg/mL), A549 (IC50 6 µg/mL) and CasKi cells (IC50 4.5 µg/mL). This in vitro data of 2,4-di-tert-butylphenol (5) support the findings that phenolic antioxidants exert cytoctoxic activity against cancer cells  did not display cytotoxic effects against the tested cell lines. The results obtained here were in agreement with published data  and finally MeOH (7.6 L). The CHCl3 fraction was concentrated to give a dark brown residue (3.47 g). The brown residue was subjected to a Si-gel CC initially eluting with a gradient of hexane followed by hexane enriched with increasing percentages of CH2Cl2, monitoring with TLC. The volume of each fraction was 25 mL. The mixture of sterols (20.5 mg) was obtained from the fraction upon elution with CH2Cl2-hexane (3.5: 6.5). Further elution with CH2Cl2 yielded a mixture (206.7 mg) containing 1. Purification of 1 was obtained through preparative-TLC using CHCl3 as the developing solvent to yield pure compound 1 (5.4 mg). β-Sitosterol  kept at 37°C in a humidified atmosphere. Human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7), human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5) were purchased from the American Tissue Culture Collection . KB cells were maintained in Medium 199 (Sigma), CasKi, A549 and MCF7 cells in RPMI 1640 medium (Sigma), HCT 116 in McCOY’S 5A Medium (Sigma) and MRC-5 cells in EMEM  (Sigma), supplemented with 10% foetal bovine serum , 100 µg/mL penicillin or streptomycin  and 50 µg/mL of fungizone . The cells were cultured in a 5% CO4/well) were seeded in 96-well microtiter plates (Nunc) and allowed to grow for 24 hours before treatment. After 24 hours of incubation, the cells were treated with six different concentrations (0.1-100 µg/mL) of test compounds, in three replicates. The plates were further incubated for 72 h at 37°C in a 5% CO2 incubator. A stock solution was initially obtained by dissolving the test compounds in DMSO. Further dilution to different tested concentrations were then carried out ensuring that the final concentration of DMSO in the test and control wells was not in excess of 1% (v/v). No effect due to the DMSO was observed. Doxorubicin was used as the positive control. The well containing untreated cells was the negative control. At the end of the incubation period, the media were replaced with medium containing 50 µg/mL of Neutral Red. The plates were incubated for another 3 hours to allow for uptake of the vital dye into the lysosomes of viable and injured cells. After the incubation period, the media were removed and cells were washed with the neutral red washing solution. The dye was eluted from the cells by adding 200 µL of Neutral Red resorb solution and incubated for 30 minutes at room temperature with rapid agitation on a microtiter plate shaker. Dye absorbance was measured at 540 nm using a spectrophotometer ELISA plate reader. The average data from triplicates were expressed in terms of killing percentage relative to negative control. The percentage of inhibition (%) of each of the test samples was calculated according to the following formula:The Neutral Red cytotoxicity assay is based on the initial protocol described by Borenfreund and Puerner  with som50 value. The IC50 value is the concentration of test compounds that cause 50 % inhibition or cell death, averaged from the three experiments, and was obtained by plotting the percentage inhibition versus concentration of test compounds. According to US NCI plant screening program, a plant extract is generally considered to have active cytotoxic effect if the IC50 value, following incubation between 48 to 72 hours, is 20 μg/mL or less, while it is 4 μg/mL or less for pure compounds [50 value corresponds to a significant or non-significant cytotoxicity depends on the sensitivity of the cell line. Cytotoxicity of each sample is expressed as ICompounds ,13,22,23Pereskia bleo [1, 5, 6 and 7. Although the cytotoxicity of these compounds and mixture are not as effective as doxorubicin, in comparison to the latter they have low toxicity against normal MRC5 cell line. The cytotoxicity assay used in the present study could only provide important preliminary data to help select plant extracts or isolated compounds with potential antineoplastic properties for future work. A detailed investigation on the mechanism of cell death would provide more convincing evidence. An investigation into this phenomenon is now underway and will be reported in due course. The resulting information will certainly contribute to a better understanding of the anti-carcinogenic activity of the natural constituents in Pereskia bleo. In conclusion, and depending on the cell lines used, the cytotoxic activities observed for kia bleo  are ascrPereskia bleo has been traditionally used for the treatment of cancer and the findings of the current study thus provide scientific validation on the use of the leaves of Pereskia bleo. In view of the increasing popular consumption of medicinal plants as alternative therapy, it is therefore necessary to conduct serious research to support the therapeutic claims and also to ensure that the plants are indeed safe for human consumption.