CLDN11) is silenced in gastric cancer via hypermethylation of its promoter region.Claudins are membrane proteins that play critical roles in tight junction (TJ) formation and function. Members of the claudin gene family have been demonstrated to be aberrantly regulated, and to participate in the pathogenesis of various human cancers. In the present study, we report that claudin-11  and quantitative reverse transcriptase-PCR (qRT-PCR), respectively. Analyses of paired gastric cancers and adjacent normal gastric tissues revealed hypermethylation of the CLDN11 promoter region in gastric cancers, and this hypermethylation was significantly correlated with downregulation of CLDN11 expression vs. normal tissues. The CLDN11 promoter region was also hypermethylated in all gastric cancer cell lines tested relative to immortalized normal gastric epithelial cells. Moreover, CLDN11 mRNA expression was inversely correlated with its methylation level. Treatment of CLDN11-nonexpressing gastric cancer cells with 5-aza-2′-deoxycytidine restored CLDN11 expression. Moreover, siRNA-mediated knockdown of CLDN11 expression in normal gastric epithelial cells increased their motility and invasiveness.Levels of CLDN11, leading to downregulated expression, contributes to gastric carcinogenesis by increasing cellular motility and invasiveness. A further understanding of the mechanisms underlying the role of claudin proteins in gastric carcinogenesis will likely help in the identification of novel approaches for diagnosis and therapy of gastric cancer.These data suggest that hypermethylation of  Gastric cancer (GC) remains the second most-common cause of cancer deaths globally. It is one of the most lethal malignancies and a leading cause of cancer deaths in developing countries, with overall 5-year survival rates below 20% Studies of GCs and their preneoplastic precursor lesions have identified several genetic and epigenetic alterations, including microsatellite instability, point mutations, and loss of heterozygosity (LOH) affecting tumor suppressor genes (TSGs) Epigenetic alterations are extremely important in cancer development and progression Previously, studies have been published on promoter hypermethylation in GCs and their premalignant precursors CLDN11), a tight junction (TJ) protein, as a potential target of epigenetic inactivation in gastric cancers (unpublished data).A pilot microarray-based genome-wide search conducted by our group, performed to discover novel epigenetically silenced genes in gastric carcinogenesis, identified claudin-11  were obtained from Dr. Duane T. Smoot (Howard University) and GC cell lines AGS, SIIA, MKN28, KATOIII, and SNU-1 were obtained from ATCC. All cell lines were cultured and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (Invitrogen).Paired primary gastric normal and tumor tissues were collected at the Johns Hopkins Hospital (JHH). Specimens were snap-frozen immediately after resection.Johns Hopkins University (JHU) Institutional Review Board (IRB) approval was obtained for all the cases included in the study. Cases were obtained from JHU surgical pathology under JHU IRB-approved exemption 02-07-19-05e under rule 45 CFR 46.101(b), which waives the requirements for obtaining patient consent.Genomic DNA was extracted from snap-frozen tissue samples or cell lines using a DNeasy Blood & Tissue Kit  according to the manufacturer's protocol. Total RNA was extracted with Trizol reagent (Invitrogen). Extracted DNA and RNA were quantified using a NanoDrop ND-1000 Spectrophotometer .CLDN11 gene. CLDN11-specific primers and probe sequences used were: forward primer 5′ CGCGATTGGTCGGCGCGTTTC 3′; reverse primer 5′ GACGAAAACAACAACGCTACT 3′; TaqMan probe 5′TCGGAGTCGCGGGGTTTAAAGAG 3′. CpGenome Universal Methylated DNA  served as a positive control, and serial dilutions of it were used to plot a standard curve. qMSP with TaqMan probe were performed on an iQ5 thermal cycler  using iQ Supermix (ibid.). Fifty cycles of PCR amplification starting with 50 ng of bisulfite-treated genomic DNA were performed in triplicate, according to the manufacturer's protocol. Duplex PCR with β-actin (ACTB) primer and probe sequences containing no CpGs were performed for normalization. The primer and probe sequences used were as published previously CLDN11-S/CLDN11-FM)/(ACTB-S/ACTB-FM) * 100, where CLDN11-S and CLDN11-FM represent CLDN11 methylation levels in the sample and fully methylated DNAs, respectively, while ACTB-S and ACTB-FM correspond to β-actin in the sample and fully methylated DNAs, respectively. Whole-genome amplified DNA (WGA) was used as an unmethylated negative control.Genomic DNAs obtained from 36 patient samples comprising 18 GC and 18 matched noncancerous stomach mucosal (NS) tissues, as well as from various gastric cell lines, were subjected to qMSP. qMSP was performed as described previously, with minor modifications CLDN11 RT-PCR amplicon was designed to overlap an intron-exon boundary in order to exclude genomic DNA (gDNA) amplification. Primer sequences were as published previously CLDN11expression was normalized to β -actin expression. Total RNA from HFE145 cells was used for the standard curve. (CLDN11-S/CLDN11-C)/(ACTB-S/ACTB-C), where CLDN11-S and CLDN11-C represent CLDN11 mRNA expression levels in the test sample and control mRNAs, respectively, while ACTB-S and ACTB-C correspond to β-actin expression levels in the test sample and control mRNAs, respectively.The CLDN11 promoter in conjunction with absent CLDN11 mRNA expression. 5-aza-2′-deoxycitidine (5-aza-dC) treatment of cells was performed as described previously 5 cells/ml in a T-75 flask. Twenty-four hours later, cells were treated with 1 µM 5-aza-dC for 72 hours. Media containing 5-aza-dC was replaced with freshly prepared media every 24 hours. Cells were then harvested for total RNA extraction. Total RNAs from AGS cells before and after 5-aza-dC treatment were subjected to quantitative real-time RT-PCR analysis.AGS is a GC cell line manifesting hypermethylation of the CLDN11-specific siRNA oligos were purchased from Ambion, Inc. . The HFE145 cell line, which is CLDN11 positive, was selected to study the effect of claudin-11 knockdown on migration and invasion properties of gastric cells. Experiments were conducted as described previously . Cells cultured in 6-well plates were transfected with siRNA duplexes using LipofectAMINE 2000 (Invitrogen), following the manufacturer's instructions. Mock-transfections and nonspecific siRNA duplexes were used as negative controls. Cells were treated for 48 to 72 hours to allow maximum knockdown, after which they were either harvested for Western blot analysis or used for migration and invasion assays.Confluent cell cultures were washed with HBSS (Invitrogen) and whole cell lysates were made using lysis buffer: 62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, and 2% SDS. Protein concentration was determined using a bicinchoninic acid (BCA) assay kit . Twenty micrograms of total proteins were separated by 10% to 20% SDS-PAGE on Tris-Glycine gels (Invitrogen) and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% nonfat dry milk, washed in TBST buffer, and probed with an anti-claudin-11 antibody . Blots were then washed and incubated in horseradish peroxidase-conjugated secondary antibody . For detection, chemiluminescence was carried out using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).i.e., the well beneath the filter) to act as a chemoattractant. Assay plates were incubated at 37°C for up to 48 hrs. Cells that did not migrate or invade through the pores of the Transwell inserts were manually removed with a cotton swab. Cells present at the bottom of the membrane were fixed in cold methanol for 10 min and then stained with 0.01% crystal violet in 20% ethanol. After 10 min incubation, the filters were washed thoroughly in water and suspended in 200 µL of 5% acetic acid and 5% methanol. Colorimetric readings were taken at OD595. Experiments were repeated at least three times, with triplicates in each experiment. To assess cell migration, assays were carried out essentially as above, except that cells were plated on top of uncoated (Matrigel-free) inserts.Invasiveness of siRNA-transfected cells was determined using Matrigel-coated invasion chamber inserts  using a modified Boyden chamber assay p<0.05 considered statistically significant.Statistical analyses were performed using Student's t-test , with CLDN11 promoter is hypermethylated and that this hypermethylation correlates inversely with CLDN11 mRNA expression in primary GC tissues. Paired primary gastric normal and tumor tissues were collected at the Johns Hopkins Hospital (JHH). Specimens were obtained immediately after resection and snap-frozen until use. Only cases obtained with informed consent as approved by the Institutional Review Board (IRB) were included in this project. Genomic DNAs were obtained from 36 clinical specimens, comprising 18 GC and 18 matched noncancerous gastric mucosal (NS) tissues. CLDN11 promoter methylation levels in these samples were analyzed using quantitative real-time methylation-specific PCR (qMSP). i.e., the ratio of methylated value of the CLDN11 promoter region in each specimen to a fully methylated control DNA. CLDN11 NMVs were significantly higher in GC specimens than in their matching NS tissues (P<0.001). To assess whether CLDN11 promoter hypermethylation in GCs was associated with silencing of CLDN11 expression, CLDN11 mRNA levels were measured using quantitative real-time (qRT–PCR) in RNAs extracted from the 36 specimens used for methylation analysis. As demonstrated in CLDN11 in GC specimens were significantly lower than in paired NS samples (P<0.001). This data establishes that CLDN11 promoter hypermethylation correlates with diminished or silenced CLDN11 mRNA expression in primary GCs.We first tested our hypothesis that the CLDN11 promoter methylation and expression in gastric cell lines. Immortalized human normal gastric epithelial cells (HFE145) and GC cell lines AGS, SIIA, MKN28, KATOIII, and SNU-1 were studied. All GC cell lines tested  demonstrated promoter hypermethylation, whereas no methylation was observed in immortalized normal HFE145 cells . CLDN11 mRNA levels were assessed using qRT-PCR on RNAs purified from the cell lines, while claudin-11 protein expression was analyzed by Western blotting. As shown in CLDN11 mRNA or protein, while HFE145 cells manifested high CLDN11 expression levels. This finding establishes that CLDN11 is coordinately hypermethylated and downregulated in GC cell lines relative to normal gastric epithelial cells (NGECs).Next, we evaluated 45 cells . CLDN11 CLDN11 expression by hypermethylation, we treated the GC cell line AGS with the demethylating agent, 5-aza-2-deoxycytidine , for varying time intervals. Total RNAs extracted before vs. after treatment were subjected to qRT-PCR for CLDN11. As can be seen in CLDN11 mRNA became re-expressed upon treatment with 5-aza-dC, confirming that CLDN11 is silenced by promoter hypermethylation in AGS GC cells.To further validate silencing of CLDN11 influences cell motility or invasiveness. HFE145 cells, which express abundant CLDN11, were chosen to study effects of CLDN11 knockdown on the invasive and migratory properties of gastric epithelial cells. Transient siRNA transfections were carried out using CLDN11-specific siRNA duplexes. Transfection with CLDN11-specific siRNA duplexes efficiently repressed claudin-11 protein levels by>90%, whereas expression remained unchanged in mock- or control siRNA-treated cells . This scan identified putative binding sites for Sp1 and GATA-1 and GATA-2. Previous studies have shown that hypermethylation of promoter DNA contributes to silencing of CLDN3 and CLDN4 in ovarian cell lines, in part via methylation-induced disruption of binding of Sp1 to its cognate binding site . In addition, members of the GATA family have been shown to be positive regulators of CLDN11 transcription CLDN11 gene transcription.Promoter hypermethylation is known to be associated with transcriptional silencing of certain genes. DNA methylation can interfere with binding of transcription factors whose binding sites contain CpG dinucleotides. Using the TFSEARCH software program, we scanned the Tumor cells typically exhibit structural and functional deficiencies in their TJs Modulations in expression of TJ-associated proteins, particularly claudin proteins, have been shown in a number of cancers. Recent studies by us and others have shown alterations in claudin protein regulation in various epithelial cancers CLDN18 gene as downregulated in GCs possessing an intestinal phenotype CLDN23 gene is also downregulated in intestinal-type GCs et al. reported CLDN4 expression to be increased in intestinal metaplasia and gastric epithelial dysplasia, identifying this gene as a marker of GC precursor lesions CLDN11 expression to be downregulated or silenced in GC via hypermethylation of its promoter region. Moreover, we found that unlike CLDN18 and CLDN23, CLDN11 expression was downregulated in GC patient specimens as well as in cell lines, irrespective of diffuse vs. intestinal subtype.Studies have previously reported changes in expression profiles of members of the claudin family to be associated with GC. Serial analysis of gene expression (SAGE) identified the CLDN11 from the blood–testis barrier in Sertoli cells to be associated with testicular intraepithelial neoplasia in men CLDN11 is expressed in normal gastric tissues and immortalized NGECs. However, its complete functions in normal stomach as well as in gastric carcinogenesis remain unclear.Claudin-11, also known as oligodendrocyte-specific protein, was first identified to be specifically expressed in the tight junction strands of oligodendrocytes in brain and in Sertoli cells of rats and mice CLDN11 in GC, we studied phenotypic changes associated with silencing of CLDN11 expression in CLDN11-expressing gastric epithelial cells (HFE145). Very interestingly, siRNA-mediated knockdown of CLDN11 resulted in increased cell motility and invasion.In an attempt to explore the possible involvement of Previous studies have reported cancer-specific phenotypic changes to be associated with modulations in claudin expression in various cancer types. Overexpression of claudin-3 and claudin-4 proteins in ovarian cancer cell lines results in increased invasion by these cells Although in recent years it has become clear that TJs and TJ-associated proteins have extensive and diverse functional and physiological activities in normal and cancerous conditions, molecular mechanisms underlying these activities remain unclear. TJs are sophisticated intercellular apparatuses capable of recruiting signaling proteins, thereby regulating various cellular processes including cell growth, differentiation, and tumorigenesis CLDN11 as a target of epigenetic modification, as well as a promising biomarker for gastric cancer early detection, diagnosis, and therapy. These findings also suggest involvement of CLDN11 downregulation in carcinogenesis via promotion of cell invasion and motility. The mechanisms for this phenomenon are subject to further investigation.In summary, data from the current study identify


The results of cytotoxicity trials against a panel of seven human cell lines for a series oftriorganophosphinegold(I) 3- and 4-mercaptobenzoates are reported. While the new compounds showmoderate to high toxicity, their potencies are inferior to those reported previously for their isomeric 2-mercaptobenzoate derivatives. The results therefore suggest a structure-activity relationship in that the 2-isomeric species are more active, particularly against the non-small cell lung cancer and renal cancer celllines, results that may indicate some selectivity in their cytotoxic profile.


The relationship between ageing and transformation has been investigated by a serial study of the changes in cell-surface morphology as normal and carcinogen-treated cells progressed in culture. A progressive increase in the density of cell surface microvilli occurred in association with the adoption of a more rounded profile and concomitant increase in the rate of cell detachment. These changes occurred earlier after carcinogen treatment, which appeared to indicate a carcinogen-induced acceleration of ageing. The alterations have also been described as characteristic of the transformed state. The observations suggest that the expression of in vitro transformation may be the result of continuous selection from a population with genetic instability and variable morphology.


Fascin is overexpressed in many cancers, including colorectal, but its role in the malignant transformation of benign colorectal adenomas is unclear.Immunohistochemical analysis of fascin expression was carried out in resected human colorectal adenoma specimens. The effects of forced overexpression of fascin on adenoma cell motility were also analysed.We show fascin overexpression in adenomas increasing with tumour size, histological type, and degree of dysplasia and increased cell motility in adenoma cell lines following fascin transfection.These data suggest an important role for fascin in the malignant progression of colorectal tumours. Colorectal cancer development is widely accepted to involve the multifaceted progression of a benign lesion (adenoma) to invasive adenocarcinoma (hereafter referred to as ‘carcinoma’) by acquisition of a migratory, and ultimately invasive phenotype . Two recent studies have demonstrated that fascin expression correlates with a poor prognosis in colorectal cancers . A total of 64 surgically resected colorectal adenomas were categorised on the basis of polyp size, histological type and the degree of dysplasia as outlined in Fascin was detected using a mouse monoclonal antibody  as previously described (Stained samples were scored by two independent observers (DQ and NB) in terms of the proportion of epithelial cells staining positive  and also the intensity of epithelial staining relative to that observed in adjacent endothelial cells . The adenomas were also scored for focal fascin intensity around the tumour stalk.The adenoma cell lines AA/C1, AN/C1, BH/C1 and RG/C2 have been described previously . All of 6 cells were prepared for western blotting as described by α-tubulin  to show equal sample loading.Samples of 2 × 102 flasks and grown to 90% confluency. Cells from each cell line were then transfected with either 10 μg pcDNA3  or 10 μg pcDNA3/human fascin-1 , 77% of medium (05–3.5 cm) and 90% of large (>3.5 cm) adenomas. Statistical analysis of the data shows a significant correlation between polyp size and both the proportion of positive epithelial cells and the intensity of fascin immunoreactivity (scored relative to the endothelium), see Observation of the adenomas at higher magnification showed a cytoplasmic subcellular distribution as shown in The adenoma samples studied were also graded according to histological type (tubular or tubulovillous) with a villous component observed in 17, 64 and 100% of small, medium and large adenomas, respectively . PositivThe most intense epithelial staining for fascin, particularly in the medium and large adenomas, tended to be focussed towards the stalk of the polyp . Only 14Western blotting was used to assess the native levels of fascin expression in adenoma-derived cell lines compared with widely used carcinoma-derived cell lines . These cFascin expression was not clearly detected under these conditions in BH/C1 and RG/C2, even though the blots shown represent a high level of exposure. However, the AA/C1 and AN/C1 adenoma cell lines showed readily detectable levels of fascin expression but much lower than those observed in the carcinoma-derived cell lines HT29 and SW480.in vitro model to determine whether fascin increases migration of benign colorectal adenoma cells.Fascin expression in colorectal adenomas was found to relate to polyp size, histology and dysplasia in a manner consistent with a role in increasing malignant potential. Malignant transformation of tumours requires increased cell motility and we therefore used an In vitro migration assays were carried out on three adenoma- and two carcinoma-derived cell lines 24 h after transient transfection with either fascin or an empty vector control , with a comparative response in the carcinoma-derived cell line HT29.Despite the continued broadening of our knowledge base, tumour spread results in 90% of all cancer deaths, and around half of patients presenting with colorectal cancer have advanced disease at clinical presentation . TherefoThe dynamic rearrangements required in the cytoskeleton to allow cell motility and ergo tumour invasion require the action of multiple proteins including actin cross-linkers, such as fascin. The expression of fascin has been shown to be low or absent in the normal epithelium of a number of tissues, but deregulated in cancer . These fOur data not only show a consistent overexpression of fascin in adenomas compared with normal tissue, but also significant positive correlations between the expression of fascin with tumour size, histology, dysplasia, both in terms of the proportion of the epithelium staining positively and the intensity of immunoreactivity. The strongest correlation was observed between fascin expression and the degree of dysplasia.in vivo. If graded by the size of the original polyp from which they were derived, the BH/C1 (<0.5 cm), RG/C2 (1–2 cm), AN/C1 (1–2 cm) and AA/C1 (3–4 cm) cell lines (Analysis of fascin expression in adenoma-derived cell lines could also be considered to show the same relationship with adenoma size seen ll lines  show a dThe observation of fascin immunoreactivity focussed around the stalk of the polyps augments the idea that fascin could be important in malignant progression, as this is the potential site for malignant invasion. This localisation of fascin at the point of maximal contact with the underlying mesenchyme also allows us to speculate on the mechanism of fascin regulation, raising the possibility of epithelial–mesenchymal crosstalk directly affecting fascin expression. If true, this regulation may occur through alterations in the extracellular matrix deposited by the epithelium and the underlying mesenchyme, or through the secretion of paracrine factors affecting fascin transcription in the epithelial cells.in vitro cell motility assays. The adenoma cell lines represent benign tumours and are anchorage-dependent and non-tumorigenic. The role of fascin in the pre-malignant stages of colorectal tumorigenesis has not previously been studied in vitro. Fascin overexpression promoted cell motility in all the cell lines tested. The acquisition of motile behaviour is necessary for localised invasion and therefore malignancy. Therefore, the observed focal expression of fascin around the stalk of large adenomas in vivo, combined with the promotion of cell motility in vitro, implies that fascin overexpression may be important in the malignant progression of colorectal tumours.The functional consequence of fascin expression in adenomas was demonstrated through the use of


The taxanes are a promising family of anti-tumour drugs that block cell cycle replication by interfering with the microtubule network. The clinical use of these drugs involves some problems related to their low solubility and occurrence of resistance, which is mainly dependent on the multidrug-resistant (MDR) phenotype. To investigate the possible interaction between docetaxel and tamoxifen (TAM), three oestrogen receptor-negative cancer cell lines, MDR- MDA-MB 231, MDR + CEM-VBLr and MCF-7 ADRr, were used. In all three cell lines, the combination of docetaxel and TAM was more effective in terms of growth inhibition than single drug exposure. Isobolic analysis confirmed the presence of synergism in all cell lines when docetaxel was used at 0.2 microM and TAM at a dose equal to or higher than 1 microM. Flow cytometric DNA analysis performed on the three cell lines showed that TAM was able to increase the G2/M blocking activity of docetaxel. This blocking activity was followed by an increased flow cytometric DNA fragmentation suggestive of the presence of apoptosis, which was confirmed by DNA gel fragmentation and morphological analysis. While an antagonistic effect on P-glycoprotein (P-gp) activity may contribute to the synergistic effect of tamoxifen and docetaxel on CEM-VBLr and MCF-7 ADRr, other mechanisms must be involved, as the synergistic effect is also apparent with a P-gp-negative cell line.


There have been many reports that high soya-based diets reduce the risk of certain types of cancer. This effect may be due to the presence of high levels of isoflavones derived from the soya bean, particularly genistein which has been shown to be a protein tyrosine kinase (PTK) inhibitor and have both oestrogenic and anti-oestrogenic properties. We have examined the effect of genistein and a number of novel synthetic analogues on both normal  and transformed  intestinal epithelial cell lines. Responses were compared to those elicited by oestradiol, the anti-oestrogen tamoxifen, and the tyrosine kinase inhibitor tyrphostin. Genistein and tamoxifen were potent inhibitors of cell proliferation. Of seven novel isoflavones tested, none were more potent inhibitors than genistein, and all displayed similar relative activities across the different cell lines. In addition to inhibiting cell proliferation, cell death via apoptosis was observed when the cells were exposed to the isoflavones and all but one exhibited PTK inhibitory activity. These data suggest that by reducing proliferation and inducing apoptosis, possibly due in part to PTK inhibition, isoflavones may have a role in protecting normal intestinal epithelium from tumour development (reducing the risk) and may reduce colonic tumour growth. © 1999 Cancer Research Campaign


Recently, it has been shown that both complexes bindspecifically to human serum apotransferrin and the resulting adducts have been studiedthrough spectroscopic and chromatographic techniques with the ultimate goal of preparingadducts with good selectivity for cancer cells due to the fact that tumour cells express highamounts of transferrin receptors on their cell surface.Two ruthenium(III) complexes, namely         2Cl4] and HIm[RuIm2Cl4] with its apotransferrin- and transferrin-bound formin the human colon cancer cell line SW707 using the microculture tetrazolium test (MTT).In order to investigate whether the cellular uptake of the complexes was mediated byapotransferrin or transferrin, we compared the antiproliferative efficacy ofHInd[RuInd        Our results show that especially the transferrin-bound forms exhibit high antiproliferativeactivity, which exceeds that of the free complex, indicating that this protein can act as acarrier of the ruthenium complexes into the tumor cell.


The growth inhibitory effects, the reduction of [3H]-TdR incorporation and the perturbation of the cell cycle induced by the new agent mitozolomide on the M14 human melanoma cell line and on the SW626 human ovarian cancer cell line were compared to those produced by BCNU. Flow cytometry showed an interesting difference: at the high concentration mitozolomide induced an accumulation of cells in S middle and S late-G2-M phase of the cell cycle whereas BCNU caused only a block in S late-G2-M. Further studies were aimed at investigating the susceptibility of freshly isolated human ovarian cancer cells to pharmacologically reasonable mitozolomide concentrations. Only in one out of 16 primary cultures of human ovarian cancers was mitozolomide able to induce cell cycle perturbation, suggesting that ovarian carcinoma cells may not be sensitive to this drug.


Roquinimex (Roq) is an immunomodulator known to stimulate cellular immune responses. It is currently used for immunotherapy after bone marrow transplantation (BMT). One of the major features of this compound is an enhancement of natural killer (NK) cell activity and numbers. We studied the in vitro effect of Roq on human peripheral blood NK and adherent lymphokine-activated killer cell (ALAK) activities. In cultures supplemented with recombinant interleukin 2 (rIL-2) (1000 U ml-1) and Roq a significant increase in NK and LAK function was observed without a parallel increase in cell numbers. We also examined the generation of NK cells from human bone marrow (BM) immature progenitors, obtained by purging with 4-hydroperoxycyclophosphamide (4HC). NK cell numbers and activity were both increased when cultures with rIL-2 (10 U ml-1) were supplemented with Roq. These results confirm findings obtained in vivo and in vitro in the murine system and suggest that Roq is an active agent on these lymphoid populations. These properties and good tolerability make Roq an attractive tool for immunotherapy.


In two recently described human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50, derived from one tumour, we detected identical E-cadherin and β-catenin gene mutations as in colon carcinoma cell line HCT 116. We demonstrate by HLA-typing, mutation analysis and microsatellite analysis that cell lines JROECL 47 and JROECL 50 are admixtures of the human colon adenocarcinoma cell line HCT 116. © 2000 Cancer Research Campaign


TP53, PTEN and BRAF but not CDKN2A or KRAS cancer-related genes was found to be associated with changes in expression of specific microRNAs. The microRNA dataset generated here should be valuable to those working in the field of microRNAs as well as in integromic studies of the NCI-60 panel.The NCI-60 panel of 60 human cancer cell-lines of nine different tissues of origin has been extensively characterized in biological, molecular and pharmacological studies. Analyses of data from such studies have provided valuable information for understanding cellular processes and developing strategies for the diagnosis and treatment of cancer. Here, Affymetrix® GeneChip™ miRNA version 1 oligonucleotide microarrays were used to quantify 847 microRNAs to generate an expression dataset of 495 (58.4%) microRNAs that were identified as expressed in at least one cell-line of the NCI-60 panel. Accuracy of the microRNA measurements was partly confirmed by reverse transcription and polymerase chain reaction assays. Similar to that seen among the four existing NCI-60 microRNA datasets, the concordance of the new expression dataset with the other four was modest, with mean Pearson correlation coefficients of 0.37–0.54. In spite of this, comparable results with different datasets were noted in clustering of the cell-lines by their microRNA expression, differential expression of microRNAs by the lines’ tissue of origin, and correlation of specific microRNAs with the doubling-time of cells or their radiation sensitivity. Mutation status of the cell-lines for the  Systematic studies of sets of cell-lines have provided insights on biological processes and their mechanisms, and have proved useful for devising diagnostic and therapeutic approaches. For instance, phosphatase and tensin homologue (PTEN)-dependent and -independent tumor suppressor mechanisms have been identified using a panel of melanoma cell-lines MicroRNAs, short non-coding RNAs, play significant roles in cellular processes by targeting protein-encoding mRNA transcripts to inhibit translation or cause their degradation. In humans, 2,042 species of mature microRNAs have been identified as per the latest version  of the miRBase microRNA sequence repository The expression of microRNAs in the NCI-60 cells were first quantified in 2007 by Gaur, et al. 6) in cell-line-specific culture medium containing 10% dimethylsulfoxide was obtained from the Developmental Therapeutics Program (DTP) of the Division of Cancer Treatment and Diagnosis of the NCI. Immediately prior to RNA isolation, cells were thawed in a water-bath at 37 °C for five minutes and collected by centrifugation at 200 g for five minutes at room temperature. Isolation of total RNA from cells using the miRVana™ kit  was done in randomized batches of 12–24 cell-lines over four days within 10 days of obtaining the cells. RNA concentration was measured by absorbance spectrophotometry on a NanoDrop™ 2000 instrument .For each of the 60 cell-lines of the NCI-60 panel , one ml miR-16, −21, −30a-5p, −106a and −200b in 50 ng total RNA were measured using TaqMan™ microRNA reverse transcription kit and RT-PCR assays  as per protocols suggested by the manufacturer. Identification numbers of the microRNA assays were 391, 397, 417, 578, and 1800, respectively. RT and PCR reactions for all RNA samples were performed together for each microRNA assay. Cycle quantification (Cq) values were calculated as the average of Cq values identified by SDS software  for triplicate 40-cycle PCR reactions that were performed in a 7900HT thermocycler (Applied Biosystems®).Levels of mature microRNAs The GeneChip™ miRNA array  platform was used to quantify microRNAs in total RNA isolated from the NCI-60 cells. One µg RNA was polyadenylated and ligated with biotinylated 3DNA™ dendrimers using the FlashTag™ Biotin HSR RNA Labeling Kit . The labeled RNA, in a volume of 80 µl, was heated to 99°C for 5 minutes and then to 45°C for 5 minutes for loading on an array using material and methods provided with the Affymetrix® GeneChip™ Hybridization, Wash and Stain kit. RNA-array hybridization was performed with agitation at 60 rotations per minute for 16–18 hours at 48°C on an Affymetrix® 450 Fluidics Station, after which arrays were washed and a streptavidin-phycoerythrin conjugate was used for the detection of bound RNAs on the arrays with a GeneChip™ Systems 3000Dx2 (Affymetrix®) confocal laser-scanning microscope. Signal values were computed using the Affymetrix® GeneChip™ Command Console software. Data from all the arrays had low background noise, a similar distribution of signals for endogenous RNAs, and a similar and concentration-dependent signal distribution for specific RNAs that were spiked in the RNA samples before they were labeled. The raw signal data has been deposited with accession number E-MTAB-327 in the ArrayExpress database of the European Bioinformatics Institute The GeneChip™ miRNA version 1.0 array has 11 µm-sized 46,228 probe-spots for 7,815 DNA oligonucleotide probes including those against 922 non-microRNA human RNAs, 847 human microRNAs, and 5,856 microRNAs of 70 other species. Probes to detect microRNAs are spotted on the array in quadruplicate. The array also has 95 non-specific probes, each spotted up to 94 times, that are binned by GC content. Signals from these background probes are used to make ‘absent’ or ‘present’ detection calls. In total, 72 array hybridizations were performed in nine batches, each with eight arrays, over three days, with two, three and four batches used on the first, second and third day, respectively . QuadrupRaw data files from the 72 array hybridizations were processed together using Affymetrix® miRNA QC Tool software (version 1.0.33) with the following steps in order as suggested by Genisphere®: background detection, background correction with the robust multichip average (RMA) method Pre-processed microRNA expression datasets generated by Blower, et al. http://www.targetscan.org. The mRNA expression data was pre-processed using the RMA method with the Affy Bioconductor package 2-transformed microarray signal values. A filter was then applied to retain values for only those probes for which the sample range of signal values was ≥1. Of the 495 microRNAs considered expressed in this study, 192, belonging to a total of 121 conserved microRNA families, were identified as targeting 6,465 of the 10,392 mRNAs. Gene symbols of the 6,465 mRNAs were converted to 10,612 Affymetrix® probe identifiers using the biomaRt Bioconductor package Raw gene expression data acquired using Affymetrix® GeneChip™ HG-U133A DNA oligonucleotide microarrays for the expression of mRNAs in 57 of the 60 NCI-60 cell-lines of this study could be obtained using the CellMiner™ web application. A list of 10,392 conserved mRNAs (unique gene symbols) predicted by the TargetScan 5.1 algorithm 2-transformed expression values. Statistical analyses and graphical plotting were done using Excel™ , Prism™ , and R (version 2.12.1). TM4 The limma (version 3.10.0) Bioconductor package 6 cells of the cell-lines ranged from 11 to 60 ug .GeneChip® miRNA 1.0 microarrays from Affymetrix® were used to examine the expression of 847 microRNAs in total RNA of 60 cell-lines of the NCI-60 cancer cell-line panel . This wo2-transformed microarray signal values for the quadruplicate sets for the 1,779 probes (847 for microRNAs) annotated as human-specific were 3.58 (0.07), 3.61 (0.12), 3.62 (0.04) and 3.50 (0.10), respectively. For the 95th percentiles, the values were 9.60 (0.10), 9.43 (0.09), 9.66 (0.11) and 9.60 (0.21), respectively. Mean (and SD) of the 12 self-self Pearson correlation coefficients for the four cell-lines were 0.98 (<0.01), 0.97 (0.01), 0.98 (<0.01) and 0.98 (0.01), respectively. For each cell-line, the quadruplicate hybridizations were performed on different days except that for OVCAR-8, one pair of the quadruplicates was assayed in the same batch  of the 75th percentiles of logme batch . The PeaAt least one of the 60 cell-lines expressed 523 (63.4%) of the 847 microRNAs detectable by the microarray platform as indicated by the significantly higher signal values from the microRNA-specific probes compared to non-specific mismatch probes of similar GC content. Although the inter-replicate correlations described above appear excellent, a closer examination revealed that correlations were not good at low microarray signal values. This is shown in miR-16, −21, −30a-5p, −106a, and −200b in 50 ng each of the RNA preparations for 12 cell-lines. The cell-lines were randomly selected and the microRNAs were arbitrarily picked from those for which at least half of the cell-lines had log2-transformed microarray signal value >5. As shown in −0.40 (miR-16) to −0.93 (miR-21) for four microRNAs, indicating better detectability of the microRNAs with the RT-PCR assay. For miR-200b, this is also notable with the five RNA samples with poor microarray signal values (21.2–21.7) but detectable RT-PCR Cq values in a good range (27.8–34.3). For miR-30a-5p, the slope of the regression line was −2.2, suggesting better detectability with the microarray platform.To at least partially confirm the accuracy of the array-based microRNA expression measurements, RT-PCR was used to quantify levels of microRNAs Of the 495 microRNAs considered expressed in the set of 60 cell-lines in this study, 135 (27%), 144 (29%), 299 (60%) and 359 (73%) were also quantified in 59 of the 60 cell-lines in the studies of Blower, et al. 2-transformed microarray signal values for the 495 expressed microRNAs , colon cancer (n = 7), leukemia (n = 6), melanoma (n = 9), ovarian cancer (n = 7) and renal cancer (n = 8), respectively, showed differential expression of 83 (17%), 37 (7%), 137 (28%), 74 (15%), 14 (3%) and 3 (0.6%) of the 495 microRNAs present in the full dataset. No differentially expressed microRNAs could be identified in the groups of cell-lines from breast (n = 6), NSCLC (n = 9) or prostate (n = 2) cancer though the groups had 10 (2%), 24 (5%) and 4 (0.8%) microRNAs for which the P values were <0.05 without adjustment for false discovery. The heat-maps in 2 transformation and z-score normalization. A bimodal distribution with similarly-sized sensitive and resistant groups was not observed for any of the seven radiation survival parameters , 27 (5%) and 33 (7%) of the 495 expressed microRNAs of this study for SF2, SF5 and SF8, respectively , KRAS, PTEN, and TP53– oncogenic mutations exist in >10 of the cell-lines, which allows for a comparison of microRNA expression levels between the groups of mutant and wild-type cell-lines. Using empirical Bayes moderated t-statistics and Benjamini-Hochberg correction for FDR <5%, microRNAs miR-29b and miR-769-3p were found to be the only two microRNAs that were differentially expressed between the 47 mutant PTEN and 12 wild-type PTEN lines (P<0.01 for both microRNAs). In a similar analysis, microRNAs miR-34a and miR-34a* (miR-34a-3p) were found to be the only two microRNAs that were differentially expressed between the 43 mutant TP53 and 16 wild-type TP53 lines (P<0.01 for both microRNAs). The differential expression of the four PTEN- or TP53-associated microRNAs is depicted in BRAF gene, for which 11 cell-lines carry mutations, 40 microRNAs were differentially expressed, with miR-146a and miR-509-3p overexpressed and miR-149 and mR-192b underexpressed the most  or KRAS (11 mutant cell-lines).The mutation status of 24 oncogenes is known for 59 of the 60 cell-lines of this study the most . This hi−16) from those obtained with any of the 10 permutated datasets.Significant correlations between expression of microRNAs and mRNAs were detected for 3,483 (3%) of 117,004 possible pairs of microRNAs and their target mRNAs as predicted by the TargetScan algorithm. The 3,483 pairs consisted of 167 microRNAs and 1,232 target mRNAs with unique gene symbols. Among the 3,483 significant correlations, 1,997 (57%) were negative and 1,486 (43%) were positive . In contThe expression of 847 microRNAs in the NCI-60 panel of human tumor cell-lines was examined in this study using Affymetrix® GeneChip™ miRNA 1.0 DNA oligonucleotide microarrays to generate a dataset of 495 microRNAs identified as expressed in at least one of the 60 lines of the panel. Thus, the study expands the microRNA coverage of three of the four existing NCI-60 expression datasets. These sets were independently generated using different high throughput platforms – custom 40-mer DNA microarray, TaqMan™ RT-PCR, Agilent® 60-mer DNA microarray, or Exiqon® locked nucleic acid miRCURY™ microarray – to assess levels of 321, 241, 723 or 955 microRNAs, respectively miR-192 and miR-194 in colon cancer cell-lines, and of miR-708 and miR-886-3p in renal cancer cell-lines  and miR-768-3p, all microRNAs differentially expressed between the mutant and wild-type lines were also differentially expressed between the eight melanoma and 52 non-melanoma cell-lines. Nevertheless, the observed associations may indicate a universal role of the BRAF pathway in regulation of certain microRNAs. This is supported by the observation that miR-146a, whose levels are higher in BRAF mutant cell-lines  and inter-replicate Pearson correlation coefficients, r (lines) for 1779 human RNA-specific probes. A rolling window of width 99 along the × axis was used to calculate r at the mid-window abscissa. Horizontal gray lines represent Y = 0.5 along the right Y axis. Names of the cell-lines and the batches of microarray hybridizations are noted.(TIF)Click here for additional data file.Figure S2Correlations between different NCI-60 cell-line microRNA expression datasets. Cumulative frequency distributions are shown for Pearson correlation coefficients (r) with a bin-size of 0.025 for microRNAs quantified in the study of Sokilde, et al. left) or that of Liu, et al. right) and in other similar studies including this one (Patnaik) and those of Blower, et al. (TIF)Click here for additional data file.Figure S3Gaussian kernel density estimates of seven radiation survival parameter values of 59 NCI-60 cell-lines. Data on the parameters SF2, SF5, SF8, D0, n, Casp8 and Casp16 were obtained from the study of Amundson, et al. 2-transformed and then z-score-normalized. The ks package (version 1.8.3) for R was used for density estimation with the package’s default settings. The short gray ticks along the × axis indicate parameter values of the 59 cell-lines.(TIF)Click here for additional data file.Figure S4Distribution of Pearson coefficients in correlation analyses of microRNA expression and radiation sensitivity of 59 NCI-60 cell-lines. Data on the radiation sensitivity parameters SF2, SF5, SF8, D0, n, Casp8 and Casp16 were obtained from the study of Amundson, et al. Patnaik), and the study of Sokilde, et al. 2-transformed before correlation analyses. Pearson coefficients (r) are binned with a width of 0.02. Curve smoothing was done using four neighboring values and a zero-order polynomial.(TIF)Click here for additional data file.Figure S5Distribution of Pearson coefficients for significant correlations of expression levels of microRNAs and their target mRNAs in 57 NCI-60 cell-lines. Pearson coefficients (r) are binned with a width of 0.02. Curve smoothing was done using four neighboring values and a zero-order polynomial.(TIF)Click here for additional data file.Figure S62-transformed microarray signal values of the 60 cell-lines for the sets of eight microRNAs each with lowest P values in tests of differential expression in cell-lines of a specific tissue of origin compared to all the other cell-lines.Heat-map, with its pseudo-color scale underneath, of log Both cell-lines and microRNAs are grouped by tissue of origin.(TIF)Click here for additional data file.Table S1The 60 NCI-60 cell-lines examined in this work listed along with the day and batch of microarray hybridizations.(PDF)Click here for additional data file.Table S2Differential expression of microRNAs in NCI-60 cell-lines by tissue of origin.(PDF)Click here for additional data file.Table S3MicroRNAs whose expression correlates with an absolute Pearson correlation coefficient >0.4 for at least one of the radiation sensitivity parameters SF2, SF5 or SF8 in 53 non-leukemia NCI-60 cell-lines.(PDF)Click here for additional data file.Table S4MicroRNAs whose expression correlates with doubling-time of 59 NCI-60 cell-lines.(PDF)Click here for additional data file.Table S5MicroRNAs differentially expressed between NCI-60 cell-lines with and without BRAF gene mutations.(PDF)Click here for additional data file.


Ovarian cancer remains an on-going challenge mainly due to the development of drug resistance and also because the cancer is likely to have metastasized at the time of diagnosis. Currently, chemotherapy based on platinum drugs such as cisplatin is the primary treatment for the disease. Copper transporter 1 is involved in the transport of cisplatin into the cell, but is also down-regulated by the drug. Bortezomib, a proteasome inhibitor, has been reported to block this platinum-induced down-regulation of CTR1, so that in the presence of bortezomib, the cellular uptake of platinum drugs may be increased. Increased platinum accumulation may result in increased platinum − DNA binding so that the platinum drug in combination with bortezomib may produce enhanced cell kill.trans-platinum compound coded as CH1 with BORT on the human ovarian A2780, A2780cisR, A2780ZD0473R and SKOV-3 cancer cell lines was evaluated. The levels of cellular platinum accumulation and platinum-DNA binding were determined following the treatment with these combinations. In order to investigate the effect of the combinations of the formation of ROS, the total and oxidized glutathione levels were also determined.In this study the efficacy of the sequential combinations of carboplatin, oxaliplatin and a Prevention of copper transporter 1 degradation by bortezomib is found to enhance the cellular accumulation of platinum, the level of Platinum − DNA binding and increases oxidative stress especially in the resistant cell lines.The results suggest that the prevention of CTR1 degradation by bortezomib may be playing a major role in increasing the cellular uptake of platinum drugs and platinum-DNA binding level. Furthermore, the generation of oxidative stress appears to be a major contributor to the enhanced cell kill. Although platinum drugs cisplatin (CS), carboplatin (CB) and oxaliplatin (OX) are widely used alone and in combination with other drugs such as paclitaxel for treatment the of various cancers, their uIn line with the idea that copper transporter 1 (CTR1) is a carrier for CS into the cell-10, it hHowell and co-workers have demonstrated that although CS is transported into the cell by CTR1, the drug triggers the proteasomal degradation of the carrier thereby limiting its own uptake, a proceProteasome inhibition represents a unique approach to anticancer therapy as it targets the key regulator of intracellular protein degradation. In vitro studies have shown that the inhibition of the proteasome leads to the accumulation of inhibitor of κB (IκB) causing the down-regulation of the anti-apoptotic transcription factor NF-κB. It alsotrans-planaramineplatinum(II) CH1[trans-planaramineplatinum(II) coded as CH1 with BORT in ovarian tumour models[Human hCTR1 contains two methionine-rich motifs (hCTR1 Met) and two histidine-rich motifs (hCRT1 His) on its extracellular N-terminus that are thought to be essential for the function of the transporter. It has been shown that the interaction of CS, CB and OX with synthetic peptides corresponding to hCTR1Met motifs that contain three or more methionines result in the removal of the carrier ligands in the case of CS and CB whereas (II) CH1 and even(II) CH1,27. The r models.Trans-bis(3-hydroxypyridine)dichloroplatinum(II) coded as CH1 was synthesized in the host laboratory as described by Chowdhury et al.[cisR, A2780ZD0473R and SKOV-3 ovarian cancer cell lines were gifts from Ms. Mei Zhang, Royal Prince Alfred Hospital, Sydney, Australia. Stock solutions (1 mM) of CB and OX were prepared in mQ water, that of CH1 prepared in 1:4 DMF to mQ water and that of BORT was made in ethanol. The solutions were sterilised by filtration.CB and OX were purchased from Sigma Aldrich, Sydney, Australia. BORT was purchased from LC Laboratories Woburn, MA, USA. The y et al.. Foetal cisR, A2780ZD0473R and SKOV-3  values i.e. drug concentrations required for 50% cell kill.Stock solutions of CB, OX, CH1 and BORT were subjected to serial dilutions to give final concentrations ranging from 0.0008 to 250 μM, made. The dilutions were made using 10% RMPI-1640 medium without serum and were added to equal volumes of cell culture in triplicate wells. Cells were treated with the drugs for 72 h in the incubator. Single drug treatments against each cell line were carried out to determine the  at three different concentration. Three modes of administration: 0/0 h, 0/2 h and 2/0 h were used, where 0/0 h indicates that both the compounds were added simultaneously, 0/2 h means that the platinum drug was added first followed by BORT 2 h later and 2/0 h means that the platinum drug was added 2 h after the addition of BORT. The period of drug treatment was 72 h counted from the time of addition ofthe first compound. Cell growth inhibition was determined using the MTT reduction assay. Combination index values (CIs) were used as measures of synergism, additiveness or antagonism calculated using the program CalcuSyn-31. The 1 and D2 respectively represent mean doses of compounds 1 and 2 in combination required to cause x% inhibition, whereas D1× and D2× represent the doses of compounds 1 and 2 respectively required to cause x% inhibition when present alone. Dx is calculated from the following median effect equation, where Dx denotes the dose of drug, Dm is the median-effect dose, fa is the fraction of cells affected so that fu = 1-fa and m is the exponent defining the shape of the dose effect curve. CI values of <1, =1 and >1 indicate synergism, additivity and antagonism in combined drug action, respectively.Where DcisR cell lines were determined. Combinations of the drugs at their IC50 values were added to culture plates containing exponentially growing A2780 and A2780cisR cells in 10 mL 10% FCS/RPMI 1640 culture medium with cell density of 5 × 106 cells mL–1 and incubated for 24 h. The cells were scraped off the culture plates and transferred to 10 mL centrifuge tubes and spun at 3500 rpm for 2 min at 4°C. The cells were washed thrice with ice-cold phosphate-buffered saline (PBS) and the pellets were stored at −20°C until assayed. A minimum of three independent experiments were performed.The cellular accumulation of platinum and platinum − DNA binding levels from the 0/0 h and 0/2 h combinations of CB and OX with BORT in A2780 and A2780Following drug treatments and collection, the cell pellets were resuspended in 0.5 mL 1% triton-X and sonicated for 30 min on ice. The total intracellular content of platinum was determined by graphite furnace atomic absorption spectrophotometry.260/A280 nm ratios were between 1.75 and 1.8 for all samples indicating high purity of the DNA.The DNA isolated from cell pellets using JETQUICK Blood DNA Spin Kit/50 Astral Scientific Pty Ltd were analysed for it platinum bound content by graphite furnace AAS. AcisR cell lines were determined for the 0/0 h and 0/2 h sequenced combinations of CB and OX with BORT. Drugs made in 10% RMPI-1640 serum free medium were added to equal volumes of cell culture wells of a white wall clear bottom 94 well plate containing exponentially growing A2780 and A2780cisR cells (cell density = 12 × 103 cells/well). Cells were left to incubate for 24 h. The media was aspirated out of the treatment wells with minimal disturbance of the cell pellets and cells were washed with 200 μL of PBS following which the levels of glutathione were determined using the GSH/GSSG-Glo™ Assay kit (Promega). The plate was read in a LUMIstar Omega luminometer .As a measure of the redox state of the cells, the levels of total glutathione (GSH and GSSG) as well as oxidised glutathione (GSSG) in A2780 and A2780cisR, A2780ZD0473R and SKOV-3. The parent A2780 cell line was most sensitive to the drugs. A2780ZD0473R was the most resistant to CB whereas SKOV-3 was the most resistant to CH1, OX and BORT. The IC50 values of the compounds against the cell lines are presented in Table 50 values for CB and OX are found to be greater in the resistant A2780cisR, A2780ZD0473R and SKOV-3 cell lines with OX having very high value in SKOV-3. The activity of CH1 on the other hand is found to be comparable against all the cell lines so that it has the lowest resistance factors as compared to CB and OX. The IC50 values for BORT against A2780cisR and A2780ZD0473R are found to be nearly the same as that against the parent A2780 cell line and slightly greater against SKOV-3 cell line. Furthermore, BORT is found to be significantly more active than CB, OX and CH1 against all four human ovarian cancer cell lines. This work does not require any ethical approval as it does not involve animals and humans.Figure cisR, A2780ZD0473R and SKOV-3 cell lines. Combinations of CB with BORT were found to be synergistic in A2780, A2780ZD0473R and SKOV-3 cell lines irrespective of the sequence of administration with the greatest cell kill resulting from the 0/2 h sequence. In the CS resistant cell line A2780cisR, all combinations of CB and BORT produced pronounced cell death. The SKOV-3 cell line also responded well to the combination of OX with BORT with greatest synergism being observed with the 0/2 h sequence of administration. The bolus administration of OX and BORT resulted in synergism in A2780 whereas 0/2 h and 2/0 h sequences of administration were slightly antagonistic. On the other hand, the bolus and 2/0 h sequence of combinations of OX with BORT caused synergism in A2780ZD0473R cell line while 0/2 h sequence of administration was found to be antagonistic. The combinations of the trans-platinum CH1 with BORT were also found to be synergistic in A2780ZD0473R, SKOV-3 and A2780cisR cells except for the 0/2 h sequence of administration in A2780cisR. The 0/2 h sequence of administration was also antagonistic in the parent A2780 cell line.Figure cisR, A2780ZD0473R and SKOV-3 cell lines were determined after 24 h treatment for each drug combination  for both 0/0 h and 2/0 h sequences of administration (more so for the 2/0 h sequence of administration). The presence of BORT was also found to increase cellular accumulation of CB in SKOV-3 cell line but more so for the 0/0 h sequence of administration than 2/0 h sequence of administration. The presence of BORT was not found to have significant on cellular of CB in A2780ZD047R cell line. As applied to the parent A2780 cell line, on face value, served to decrease rather than increase the cellular accumulation of CB although uncertainty remains because of large error. A further point to note that increase in accumulation of CB in A2780cisR cell line, did not result in any increase in the cell kill. The cellular accumulation of OX was found to be highest in the resistant A2780cisR cell line but lower than that of CB in all the four cell lines. As applied to the combination of OX with BORT, 0/0 h sequence of administration resulted in highest platinum accumulation in the resistant A2780cisR cell line whereas 2/0 h sequence of administration resulted in highest platinum accumulation in the parent A2780 cell line.To determine whether the presence of BORT led to an enhancement in the uptake of CB and OX, the level of intracellular platinum in A2780 and A2780 Figure . It was cisR, A2780ZD0473R and SKOV-3 cell lines were determined for the 0/0 h and 2/0 h combinations of CB and OX with BORT. Figure cisR, A2780ZD0473R and SKOV-3 cell lines resulting from CB and OX alone and from the 0/0 h and 2/0 h combinations of CB and OX with BORT. Platinum − DNA binding level from CB alone was found to be highest in the resistant A2780cisR cell line and from OX alone it was highest in the parent A2780 cell line.As the action of platinum drugs is associated with their binding with DNA, platinum − DNA binding levels in A2780 and A2780cisR cell lines. The levels in A2780ZD047R and SKOV-3 cell lines were found to be much lower from the drugs alone as well as their combinations with BORT. A more careful analysis shows that 0/0 h combination of CB with BORT resulted in a significant increase in platinum − DNA binding level in both A2780ZD047R and SKOV-3 cell lines. As applied to combination of OX with BORT, both the sequences of administration resulted in increase in platinum − DNA binding in all the four cell lines A2780, A2780cisR, A2780ZD047R and SKOV-3.Platinum − DNA binding levels from the combinations of CB and OX with BORT were found to be greater than those from CB and OX alone in both the parent A2780 and the resistant A2780cisR and SKOV-3 cell lines after treatment of cells with combinations of BORT with CB and OX, administered using 0/0 h and 0/2 h sequences. The level of glutathione in A2780ZD0473R cell line was not determined to minimise cost. Table As both platinum drugs and BORT are able to induce oxidative stress in the cells that may also lead to apoptosis, the effect of the drug combination on cellular glutathione levels was investigated. Figures cisR cell line and lowest in the parent A2780 cell line. Treatment with BORT alone also caused a significant decrease in GSH level in A2780, A2780cisR and SKOV-3 cell lines, thus indicating the heightening of oxidative stress.Both total and oxidised glutathione levels were found to be highest in the CS-resistant A2780cisR and SKOV-3 cell lines than in the parent A2780 cell line.It can be seen that treatments with BORT and its combinations with CB and OX have served to decrease values for GSH/GSSG ratio relative to the control – more so in the resistant A2780cisR, A2780ZD0473R and SKOV-3 cell lines was determined, as differences in mechanism of action suggest that the drugs might act synergistically in combination. Besides being a highly potent anticancer drug on its own right, BORT can also enhance the activity of platinum drugs by counteracting platinum-induced loss of CTR1 expression[cisR and A2780ZD0473R cell lines. Although both CB and CS form intrastrand bifunctional adducts with DNA (that are followed by protein recognition and cascade of events leading to programmed cell death), the two compounds differ in their leaving groups (chloride in CS and cyclobutanedicarboxylate (CBDCA) ligand in CB) and consequently in their reactivity. The ligand exchange reactions with carboxylate groups are much slower than those with the chloride ligand so that CB is significantly more stable than CS[50 values of the two compounds[In this study, efficacy of sequenced combinations of CB, OX and CH1 with proteasome inhibitor BORT in human ovarian A2780, A2780pression. Amongst than CS. NMR stu than CS. The low than CS. Consequompounds. Howeverompounds.cisR and A2780ZD0473R cell lines may be due to differences in their structures in terms of both the leaving groups  and the carrier ligands . Although OX, having a cis-geometry like CS, can form intrastrand 1,2-(GpG) adducts with DNA, several conformational differences exist in the intrastrand 1,2-(GpG) adducts formed by CS and OX[trans-platinum compound CH1 has a relatively lower activity than cis-platinums against all four ovarian cancer cell lines, it has lower resistance factors, indicating that at the level of its activity CH1 has been better able to overcome mechanisms of resistance operating in A2780cisR, A2780ZD0473R and SKOV-3 cell lines.Much greater activity of OX than CS against A2780, A2780S and OX,38. ThesS and OX. Much loS and OX. In a paS and OX. FurtherBORT has shown remarkably high activity against all the four human ovarian cancer cell lines. Inhibition of proteasome leads to the up-regulation of pro-apoptotic proteins such as inhibitor of κB (IκB), p53 and NOXA and down-regulation of anti-apoptotic proteins such as MCL1, IAP,20, thusZD0473R and SKOV-3 cell lines irrespective of sequence of administration but antagonistic in A2780cisR cell line. The synergism in activity from 0/0 h and 2/0 h combinations of CS with BORT in A2780 and A2780cisR cell lines is in line with the increased cellular accumulation of platinum and increased level of Pt − DNA binding. In a phase I clinical trial, the combination of CB with BORT has shown promising results[cisR cell lines. It is likely that much higher activity of BORT against both A2780 and A2780cisR cell lines but much lower activity of CB against A2780cisR cell line than the parent A2780 cell line, has served to dampen the effect of increase in Pt − DNA level that results from the protective role played by BORT against CTR1 degradation. As applied to the combinations of BORT and OX also, both cellular accumulation of platinum and the level of Pt − DNA binding were found to be greater than those from OX alone in both the cell lines. The increase in platinum uptake and the level of Pt − DNA binding from OX in the presence of BORT suggests that CTR1 can also serve as a carrier for the much larger molecule OX. In the case of the much larger molecule CH1, it appears that this compound also acts synergistically in combination with BORT in A2780cisR, A2780ZD0473R and SKOV-3 cell lines  suggesting that BORT may be acting as a carrier for OX and CH1 as well. This is not unexpected as the association between CTR1 and platinum drugs does not involve tight fit into a small pocket. Surprisingly, the SKOV-3 cell line that showed marked resistance to OX was most responsive to the combination of OX with BORT, indicating that the presence of BORT had served to greatly sensitize the SKOV-3 cells to cell kill due to OX. The cellular accumulation of platinum from combinations of OX with BORT are found to be higher  in all the four A2780, A2780cisR, A2780ZD0473R and SKOV-3 cell lines as applied to the 0/0 h sequence of administration and in A2780, A2780cisR and SKOV-3 cell lines as applied to 2/0 h sequence of administration and the levels of platinum − DNA binding are greater in A2780, A2780cisR, A2780ZD0473R and SKOV-3 cell lines (more so in A2780 and A2780cisR cell lines ) for both 0/0 h and 2/0 h sequences of administration. The results can be seen to be in line with synergistic nature of the combinations. Finally, the results indicate the combinations of CB, OX and CH1 with BORT generally serve to enhance cell kill especially in the resistant cell lines.Combinations of CB with BORT were found to be synergistic in A2780, A2780 results. BORT de results. In the cisR and SKOV-3 ovarian cancer cells with BORT alone and its combinations with CB and OX significantly reduced the total glutathione (GSH and GSSG) levels in all the three cell lines – more so from BORT alone than from the combinations. The results indicate that the proteasome inhibitor BORT induces a greater oxidative stress in cancer cells than platinum drugs CB and OX although all the three compounds − BORT, CB and OX − can induce oxidative stress in the cells. The change was found to be more significant for the reduced form GSH than the oxidized form GSSG so that treatments with BORT and its combinations with CB and OX have served to decrease the values for GSH/GSSG ratio (relative to the control) and more so in the resistant A2780cisR and SKOV-3 cell lines than in the parent A2780 cell line. The results indicate that treatment of the cells with BORT and its combinations with CB and OX have served to heighten oxidative stress in the cells.As BORT and platinum drugs are known to cause oxidative stress in cancer cells, the level of cellular glutathione was determined for the combinations of BORT with CB and OX. It was found that the treatment of A2780, A2780Contrary to the common observation that the oxidised form of glutathione (GSSG) is elevated following oxidative stress, in the present study it was found that the level of GSSG decreased  after the drug treatments. It is important to note that GSSG may either recycle to GSH or exit from the cells, leading to the overall depletion of cellular glutathione content. The facIn summary, the key points in regard to the combinations of platinum drugs and BORT administered to ovarian tumour models are: i. Proteasomal degradation of CTR1 induced by CS and possibly by other platinum drugs so that CS serves to decrease its own uptake, ii. BORT plays a protective role against CS-induced proteasomal degradation of CTR1 so that in presence of BORT cellular accumulation of platinum and the level of platinum-DNA binding is enhanced, iii. Protein recognition of platinum − DNA lesions may initiate a cascade of events leading to apoptosis or repair of the DNA lesions causing drug resistance, iv. BORT causes death of cancer cells through proteasome inhibition, v. both platinum drugs and BORT cause apoptosis through oxidative stress, and vi. the much greater activity of BORT as compared to platinum drugs, especially in the resistant tumour models, masks the effects of CTR1-prtoection. Figure The increase in cellular accumulation of platinum and the level of Pt − DNA binding associated with combination of BORT with CB and OX in ovarian tumour models indicate that BORT may serve to protect CTR1 from CS-induced proteasomal degradation. However, the effect on the cell kill appears to be less significant due to much lower activity of platinum drugs as compared to BORT.Trans-bis(3-hydroxypyridine)dichloroplatinum(II); CI: Combination index; CS: Cisplatin; CTR1: Copper transporter 1; Cu: Copper; DACH: Diaminocyclohexane; FCS: Foetal calf serum; GSH: Glutathione; GSSG: Oxidised glutathione; His: Histidine; IκB: Inhibitor of Kappa B; Met: Methionine; MRP: Multidrug resistance-associated protein; MTT: 3--2,5-diphenyltetrazolium bromide; NF-κB: Nuclear factor Kappa B; OX: Oxaliplatin; PBS: Phosphate buffered saline; Pt: Platinum; ROS: Reactive oxygen species.BORT: Bortezomib; CB: Carboplatin; CBDCA: Cyclobutanedicarboxylate; CH1: The authors declare that they have no competing interests.ZA developed the methodology, acquired and interpreted data, and drafted the manuscript. PB, CC and JQY aided in study design. FH conceived and designed the study, developed the methodology, interpreted data, edited the manuscript, and oversaw the study. All authors have read and approved the final manuscript.


XMRV is a gammaretrovirus first identified in prostate tissues of prostate cancer(PC) patients and later in the blood cells of patients with Chronic Fatigue Syndrome (CFS). Since its discovery there have been controversial reports of detection in both PC and CFS. Although XMRV is thought to use XPR1 for cell entry, it infects A549 cells that do not express XPR1, suggesting usage of other receptors or co-receptors. The aim of the study was to determine receptor and co-receptor usage to elucidate the mode of entry, transmission, infectivity of XMRV in different cell types and to determine cellular tropism.To study the usage of different receptors and co- receptors that could play a role in XMRV infection of lymphoid cells and GHOST (GFP- Human osteosarcoma) cells expressing CD4 along with different chemokine receptors including CCR1, CCR2, etc., were infected with XMRV. Culture supernatants and cells were tested for XMRV replication using real time quantitative PCR.Infection and replication of XMRV was seen in a variety of GHOST cells, LNCaP, DU145, A549 and Caski cell lines. The levels of XMRV replication varied in different cell lines showing differential replication in different cell lines. However, replication in A549 which lacks XPR1 expression was relatively high. XMRV replication varied in GHOST cell lines expressing CD4 and each of the co- receptors CCR1 – CCR8 and Bob.XMRV replication was observed in GHOST cells that express CD4, and each of the chemokine receptors ranging from CCR1- CCR8 and Bob suggesting that infectivity in hematopoietic cells could be mediated by use of these receptors. Infection of Lung epithelial cell A549 lacking XPR1 expression clearly indicates usage of other receptors by XMRV for entry into susceptible cells.


We have found that the ANN models correctly classified more than 95% of the cell lines, while the LDA models with the same data sets could classify 85% of cases. In the process of different ranges of spectra, the best classification of data set in the range of 1000-2000 cm-1 was done using ANN model, while the data set between 2500-3000 cm-1 was more correctly classified with the LDA model. PCA of the spectral data also provide a good separation for representing the variety of cell line spectra. Our work supports the promise of ANN analysis of FTIR spectrum as a supervised powerful approach and PCA as unsupervised modeling for the development of automated methods to determine the resistant phenotype of cancer classification.Drug resistance enables cancer cells to break away from cytotoxic effect of anticancer drugs. Identification of resistant phenotype is very important because it can lead to effective treatment plan. There is an interest in developing classifying models of resistance phenotype based on the multivariate data. We have investigated a vibrational spectroscopic approach in order to characterize a sensitive human ovarian cell line, A2780, and its cisplatin-resistant derivative, A2780-cp. In this study FTIR method have been evaluated via the use of principal components analysis (PCA), ANN  and LDA . FTIR spectroscopy on these cells in the range of 400-4000 cm There is an increasing interest in the use of molecular data from human resistant neoplasm for chemotherapy purposes  and artificial neural networks (ANN) are trained using data that is labeled with the correct answer . In this study, we attempt to apply three different methods of PCA, LDA and ANN in the discrimination of FTIR spectroscopic results from sensitive human ovarian cell line, A2780, and its resistant derivative, A2780-CP. We are presenting the success of mixed classification methods in the characterization of differences between the spectra of resistant and sensitive phenotypes.Cell lines 2.A2780 (human ovarian carcinoma-sensitive to cisplatin) and A2780-CP (human ovarian carcinoma-resistant to cisplatin) cell lines were obtained from Pasture Institute National Cell Bank of Iran . All cell lines were grown in RPMI-1640 medium and supplemented with 10% heat inactivated fetal bovine serum, antibiotics: penicillin, streptomycin . Cells were maintained at 37 °C in humidified atmosphere containing 5% COCell preparation for spectroscopy 2 flasks with fresh medium to reach the logarithmic phase of growth curve. After that, cells were washed twice in saline (0.9% NaCl), suspend and centrifuged at 1000 rpm for 5 min, then resuspended in saline to obtain a concentration of 1 × 105 cells. 10 μL of each cell suspension was placed on a zinc selenide sample carrier which was dehydrated in a vacuum cabin (0.8 bar) for approximately 4 min. These plates were then used for FTIR spectroscopy.The following procedure was similarly applied for both sensitive and resistant cell lines. Cells were trypsinized from the original flask and seeded in 25 cmFTIR spectroscopy -1 for every wave number between 400 and 4000 cm-1. These experimental conditions for each cell line and situation were kept constant for all the measurements. Each single spectrum was baseline corrected and then normalized in order to have the range spanning from 0 to 1.Thin dried films of cell suspensions on the Zinc selenide window were used for FTIR spectroscopy using a WQF-510  spectrometer, equipped with a KBr beam splitter and a DLaTGS (deuterated Lantanide triglycine sulphate) detector. The whole system was continuously purged with N2 (99.999%). In each spectrum, 100 scans were collected at a resolution of 4 cmData analysis -1 have been used in this study as the dataset. Distribution of different FTIR spectra was equal for types of A2780 and A2780-CP cell lines.A total of 60 FTIR spectrums between 1000-3000 cmLinear discriminate analysis (LDA)The basic theory of LDA is to classify the dependent variable by dividing an n-dimensional feature space into two regions that are separated by a hyper plane which is denoted by a linear discriminate function. In LDA, observations from each case are compared with others to provide models for groups of data . This prβ0 + β1 X1 + β2 X2 +· · ·+ βn Xn,(1)D = β0 is the intercept term, and βn  represents the β coefficient associated with the corresponding explanatory variable Xi   .The data were analyzed by multivariate (LDA) linear discriminate analysis tool of MATLAB software using cell types as the dependent variable and the absorbance of FTIR spectra as independent variables. The same data sets used for ANN were used for the LDA analysis.Artificial neural networks analysis Artificial neural networks (ANN) are computerized mathematical models designed to mimic the architecture of the brain. They are able to detect nonlinearity, making them capable of learning and adaptability . NeuronsPrinciple component analysisPCA is a well-known method of dimension reduction. The basic idea of PCA is to reduce the dimensionality of a data set, while retaining as much as possible the variation present in the original predictor variables. In mathematical terms, PCA sequentially maximizes the variance of a linear combination of the original predictor variables . The samSpectrum alteration-1 in Figure 2. The normalized FTIR spectra in this region showed alterations in different spectrum areas. Comparison between spectra showed at least three areas of variation: Spectral features of A2780 and A2780-CP cell lines are shown for the range of 1900-700 cm-1 which can be related to β-sheet secondary structure of amid I  contains the asymmetric and symmetric membrane lipids. CH2 symmetric and asymmetric stretching vibration bands are appeared at 2920 and 2852 cm-1  and CH3 stretching vibrations (at 2950 cm-1) are higher than resistant cell line. Our research is representing alterations in the lipophilicity of cell membrane between resistant and sensitive cells.The normalized FTIR spectra of A2780, A2780-CP cell lines in region 3300-2700 cm852 cm-1 . CH2 strData processing-1, while models 6 to 20 used four segmentations of FTIR wave number from 1000-1500 cm-1 , 1500-2000 cm-1, 2000-2500 cm-1 and 2500-3000 cm-1. The data were subjected to ANN, LDA and PCA analysis.The FTIR data of A2780 and A2780-cp cell lines were sorted randomly into 20 different data sets (numbered 1 to 20) each composed of 44 training variables and 16 testing variable. Models 1 to 5 used all FTIR wave number from 1000-3000 cmArtificial neural network -1 fragment was more accurate than the other ANN models for the discrimination of sensitive versus resistant cells  contains the asymmetric and symmetric membrane lipids (LDA was also used to analyze the same 20 data sets of FTIR spectra values. The results of these analyses are given in Table 2. Classification rates provided by the LDA models were about 85%. Comparison of the 20 LDA models indicates that using variables in segmentations of 2500-3000 cmA models . This mie lipids  have linComparison of LDA and ANN-1 is more correctly classified with ANN model while the data set between 2500-3000 cm-1 is a better candidate for LDA model. According to total data sets used, the ANN modeling performs better than LDA because of less variation. Our analyses demonstrate that it is possible to classify individual resistant cell lines from sensitive type based on the analysis FTIR spectra using multivariate ANN analysis.The comparison between LDA and ANN were done using paired student t-test. From the result of the t-test, it is obvious that the prediction accuracy in ANN models are different from the accuracy of LDA models with p-value ≤ 0.02. The data set between 1000-2000 cmPCA analysisPCA can be used to extract the most significant variations between groups of spectra of cells. Score plots in PCA model provide visualization of the data, whereby the loading of data is an indicator of biochemical similarity . PCA was-1. Moreover Figure 6 shows the loading plot of PC1 from four fragmented observation in these cell lines.The data of the resistant cells are in the central area of PCA projection. Based on this approach, the PCA correctly classified more than 95% of all spectra for representing the variety of cell line spectra. Thus PCA as unsupervised model provide a good separation for representing the variety of sensitive and resistant cell line spectrum between 1000-3000 cm-1 could be related to amid II (Analysis through direct observation of the spectra is not an easy task. Biochemical discriminatory spectra were calculated for the difference between the spectra of resistant and sensitive cells . Based o amid II .The pattResistance to Chemotherapy is a serious obstacle in treatment of cancer. In ovarian cancers, greater than 70% of patients initially respond to therapy with Cisplatin. However, this stabilized the five-year survival rate for ovarian carcinoma population less than 25% (-1 with less variability which addresses linear alteration in the CH stretching region of resistant and sensitive cells. We offer PCA among unsupervised modeling and ANN as supervised modeling for pattern recognition of sensitive and resistant type.Cluster separation of FTIR spectrum data in PCA, ANN and LDA was found in previous studies. Discrimination rates have been reported verify between 60 to 98% of data with multivariate analysis of FTIR data , 30. Thi


Inhibition of human islet amyloid polypeptide (hIAPP) fibrillisation by peptides incorporating a helicogenic amino acid, dehydrophenylalanine: implications for Type-2 diabetes. hIAPP fibrillization implicated in Type 2 diabetes pathology involves formation of oligomers toxic to insulin producing pancreatic β-cells. We report design, synthesis, 3D structure and functional characterization of dehydrophenylalanine (ΔF) containing peptides which inhibit hIAPP fibrillization. The inhibitor protects β-cells from hIAPP induced toxicity. A large amount of evidence favors its wide role in glucose metabolism.4 hIAPP is known to form amyloid fibrils with cross-beta structure,5 and amyloid deposits as the product of aggregation, but the process proceeds through oligomerization.7 It has been suggested that hIAPP oligomers of pore-like morphology are formed by association of helical monomers which then perform membrane fragmentation by pore formation.8 Thus, these prefibrillar oligomers are considered to be toxic and are implicated in β-cell dysfunction and death.9 Hence, the impairment of oligomerization of helices by using designed small molecule inhibitors such as short peptides is a therapeutically relevant strategy for the prevention of T2DM. In this report, we show that two pentapeptides related to one of the core fibrillization regions of hIAPP inhibit fibril formation of hIAPP. Crystal structure analysis revealed an anion receptor ‘nest’ motif in these inhibitors, which based on computational studies was shown to interact with helical monomers of hIAPP. We also propose a model for fibrillization inhibition by these peptides.Type 2 Diabetes Mellitus (T2DM) is one of the most prevalent endocrine disorders underlining the importance of developing molecular therapies to mitigate T2DM.10 hIAPP(22–27), i.e. NFGAIL, has been shown to form amyloid fibrils similar to those formed by the full-length polypeptide.11 Based on the motif hIAPP(22–27), we designed several peptides as possible inhibitors of hIAPP fibrillization by strategically incorporating a non-natural amino acid α,β-dehydrophenylalanine (ΔF). ΔF is an analogue of phenylalanine with a double bond between Cα and Cβ atoms and its presence induces β-turn in short peptides and helical secondary structures in longer peptides.12 Also, peptides containing ΔF resist enzymatic proteolysis,13 an added advantage for inhibitor design.Among the core fibrillization motifs/fragments of hIAPP,23 and I26, and their replacement with the helicogenic residue ΔF, individually or together, was a preferred choice for inhibitor design. I26 is an important residue; I26 → P mutation in full length hIAPP resulted in a hIAPP fibrillization inhibitor.14 Designed peptides  were positioned such that their main chain NH groups including free NH3+ formed a concave depression serving as an anion receptor, which interacted with terminal carboxyl oxygen from a symmetry related molecule. The ‘nest’ serves as the binding site of an ‘egg’ which is an atom or a group of atoms with full or partial negative charge.15 The peptide also showed a type-I β-turn formed by intramolecular N–H···O hydrogen bonding between L5i+3)( → G2i) with a shape complementarity value (Sc)18 of 0.83 indicating that hIAPP and the inhibitor have complementary binding surfaces. A helical wheel plot of hIAPP13–30  & L16V17 (hIAPP) and ΔF4 (peptide) & F23L27 (hIAPP).To investigate the possible modes of interaction of FGAΔFL with hIAPP, we performed molecular docking of FGAΔFL with the 3D structure of hIAPP (PDB: 2KB8)al mol–1 . In the APP13–30  shows thi.e. when the hIAPP was in prefibrillar form (3 h old hIAPP)  spectra of hIAPP in the presence and absence of inhibitors at two different stages of the hIAPP fibrillization process 4 In order to test whether the FGAΔFL would reduce the cytotoxity of hIAPP we carried out cell viability assay. Results of the MTT  assay revealed that cytotoxic effects of hIAPP on cultured pancreatic rat insulinoma cells (RIN 5fm) showed a noticeable reduction in the presence of FGAΔFL. The inhibitor on its own did not show any cytotoxic effects on the cell line.hIAPP is known to be highly cytotoxic to pancreatic cells.26 was replaced with a β branched amino acid, I, and determined its 3D structure through X-ray crystallography  , Govt. of India and ICGEB, VSC thanks Department of Biotechnology, Govt. of India and SR thanks DST for support. We thank the reviewers for their valuable comments.


The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies.We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics.This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question. A wide panel of intestinal cell lines is being used to study the biology of the intestine. All of these cell lines are either directly derived from primary colo-rectal cancers (CRCs) of different clinical stages and differentiation grades or from metastatic sites originated from a colon tumor. The major oncogenic pathways in colon cancer include loss of function mutations in APC, TP53 and SMAD4 (approximately 80–85% of sporadic tumors), or DNA mismatch repair genes, and activating mutations in beta-catenin . As a coWhile cancer drug discovery has mainly focused on targeting tumor cell proliferation, the outcome of a cancer depends largely on tumor invasion and dissemination . Recent The variety of available human cancer cell lines reflects the genomic heterogeneity across the human cancer population at least in part and has therefore regained attention notably to predict responsiveness of anticancer drugs ,16. StarIn this study we compare for the first time gene expression signatures relating to a wide panel of commonly known intestinal cell lines, primary cell cultures of human cancer-associated fibroblasts and laser-dissected human colonocytes, small intestinal enterocytes and tumor cells. We delineate selection criteria for CRC derived cell lines based on genomic expression patterns related to clinical parameters, migratory capacities and proliferative activities. While some cell lines are mainly being used to study mechanisms associated with tumor biology others serve as models for normal enterocytes studying drug absorption. Thus, we used the same strategy to assess the validity of models for oral drug absorption.Most dominant gene expression differences as revealed by principal component analysis (PCA) were observed between normal epithelium and all tumor-derived cell lines, and cells with epithelial versus mesenchymal properties.PCA was performed to assess the major gene expression differences between microdissected normal and tumor epithelium, primary cancer-associated fibroblast (CAF) cultures and the various CRC cell lines. PCA involves a mathematical procedure that transforms a number of possibly correlated variables into a smaller number of uncorrelated variables called principal components. Hence, the first principal component accounts for as much of the variability in the data as possible and each succeeding component accounts for as much of the remaining variability as possible. Two breast cancer cell lines with known pronounced MB231) or weak (MCF7) EMT traits  were inc or weak Next, the suitability of the various cell lines for tumor biology related studies was assessed. There are no standard models and selection criteria are not well defined. The activation of the Wnt pathway is a hallmark in colon carcinogenesis. Recent findings indicate that the Wnt activity defines CRC stem cells and is regulated by the microenvironment . Thus, tInvasive tumor cells acquire higher motility through EMT . Thus, wThe model of choice to evaluate drug absorption are Caco-2 cells as they undergo in vitro spontaneous enterocytic differentiation by developing an apical brush border membrane endowed with hydrolases such as sucrase-isomaltase and aminopeptidase N ,32-36. OConsequently, we made use of our objective approach to assess the suitability of the Caco-2 cells as model for oral drug absorption as compared to other CRC derived cell lines, taking inter-laboratory differences into account. As described above, PCA suggested that T84, HT29 and Caco-2 cells grown to confluency for 3 weeks were most similar to small intestinal enterocytes. Yet, when performing PCA filtering on solute carriers, transporters mostly responsible for active uptake, differentiated Caco-2 cells emerged as most akin to small intestinal enterocytes Figure . HoweverRecent reports suggest that chemotherapy may lead to a selection of tumor cells with EMT and stem cell properties and increased chemo-resistance . For exahttp://dtp.nci.nih.gov/docs/cancer/cancer_data.html).To extend this analysis we addressed more comprehensively to what extent EMT, HuISC and WNT signatures correlated with GI50 values of 50 commonly used chemotherapeutics in the NCI60 cell line panel . Clustering of the correlation coefficients revealed two distinct gene sets .In view of the heterogeneity of solid tumors ,43 and tScreening the literature with key words such as “absorption” and “transporters” revealed that, within the CRC cell line panel assessed herein, Caco-2 is the most widely used intestinal cell line for absorption-related studies -47. Comphttp://dtp.nci.nih.gov/docs/cancer/cancer_data.html). We therefore probed these data sets for a correlation between EMT and CSC signatures and chemoresistance. Using the EMT signature defined herein, we could indeed confirm such a correlation in the breast cancer data set of Gupta et al. l results,41,55-58Click here for fileFigure S4. Enrichment of small intestinal signature in Caco-2 cells upon differentiation. Heatmap representing enrichment scores obtained by gene set enrichment analysis of the selected panel of gene sets. Genes in the various cell lines were ranked according to their relative average expression (n = 2-3) in the given cell line compared to a panel of healthy epithelial tissues. Details see Additional file Click here for file


The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an ‘establishment’ phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research.Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma  Breast cancer is a leading cause of death worldwide and represents the primary cause of mortality among women in Brazil . Breast et al, ability to assemble colonies on agar plates and formation of tumors in immunodeficient mice  were evaluated using array comparative genomic hybridization (aCGH). These findings render MACL-1 and MGSO-3 the first characterized breast cancer cell lines to potentially be used for comparative research with other established breast cancer cell lines.et al were housed in filter-top cages, and sterile water and food were provided ad libitum. The manipulations were conducted aseptically inside a laminar flow hood. One million MACL-1 and MGSO-3 cells were diluted in phosphate buffer and injected subcutaneously between the scapulae of each animal, as described in our previous study . Subsequent to attaining confluence, the cells were fixed with buffered formalin for 1–2 min, washed with phosphate buffer, and stored in this solution until immunohistochemical staining was performed. This study was approved by the institutional Human Ethics Committee (ETIC 03120203000).Primary tumors were obtained from 2 breast cancer samples obtained from 2 patients (patients 1 and 2) who had presented at Santa Casa de Misericórdia Hospital in Belo Horizonte, Brazil. Samples were routinely processed, embedded in paraffin and 4-μm-thick sections were cut and stained with hematoxylin and eosin (H&E) to evaluate tumor morphology and grade. To evaluate tumor xenografts, the animals were sacrificed and the tumors were excised and fixed in 4% buffered formaldehyde for 24–48 h. Tumor fragments were then rinsed with phosphate buffer, dehydrated in a series of graded ethanol washes and embedded in paraffin. To compare the MACL-1 and MGSO-3 cells grown Immunohistochemical analysis was performed using the antibodies shown in 2 plates and incubated for 10 days. Colonies were stained using a mixture of 6.0% glutaraldehyde and 0.5% crystal violet, and then rinsed with water. Colonies with >50 cells were counted as survivors. Surviving fractions were normalized by the plating efficiency of MDA-MB-231 cells. Statistical analysis was carried out using GraphPad Prism 5 software  using one-way ANOVA and Duncan’s post-test. P<0.05 was considered to indicate a statistically significant difference.Cell survival was measured using clonogenic assay . Briefly2 ≤-0.13 and ≥+0.3 for the determination of a loss or gain region, respectively.Genomic DNA from MACL-1 and MGSO-3 cell lineages was obtained using SDS/proteinase K digestion, followed by phenol/chloroform extraction and ethanol precipitation  and treaThe breast tumor sample from patient 1 exhibited an invasive ductal carcinoma morphology that may be sub-classified as a luminal A subtype carcinoma (ER/PR-positive and HER2-negative) . Tumors in situ (DCIS) and presented with a HER2 subtype profile, given that the tumor was negative for ER/PR staining and showed strong HER2 staining (3+)  . Breast ing (3+) . Additio+/CD24− phenotype, as shown in the study by Al Hajj et al and the breast cancer stem cell markers  were absent in the primary tumor and cultured cells of patient 1, as well as the xenografts derived from the 2 cell lines .in vitro establishment of cells derived from primary tumors is a rare event, occurring in relatively few attempts  gene is located. The loss of this region could explain the lack of HER-2 expression in MGSO-3 cells and derived xenotransplants from this cell line in nude mice.The complexity of MACL-1 and MGSO-3 genomes has already been observed by our group when we attempted to explore the karyotype profile of these cells through G-banding or DAPI staining (data not shown). This impairment was promptly demonstrated through our aCGH data, which confirmed extremely complex alterations hindering chromosomal mapping through those techniques. MACL-1 and MGSO-3 cell lines displayed common alterations, such as loss of considerable portions of chromosomes 17, 19 and 22 . Losses et al showed tet al. AnotherFor chromosome 19 the affected regions were: 19q13.11-q13.32 in the MACL-1 and MGSO-3 cell lines and 19q13.33-q13.43 in the MGSO-3 cell line . Loss onOverall the biological processes involved in MACL-1 and MGSO-3 CNAs showed alterations in genes that are engaged in several activities including gene transcription and regulation, cell cycle, signal transduction and metabolic processes. As expected there does not appear to be a concise bias toward a particular biological process.In conclusion, MACL-1 and MGSO-3 cell lines changed their protein expression profile possibly due to a selection pressure for a more fitted phenotype on cell culture conditions. This phenotypic shift was conserved in tumor xenografts in immunodeficient mice. Despite carrying extensive chromosomal imbalances, these cells maintained a high proliferative ability. To the best of our knowledge, MACL-1 and MGSO-3 are the only Brazilian breast cancer cell lines that could be used for comparative studies with other known breast cancer cell lines.


There is an error in the second to last sentence of the "Introduction" section. The correct sentence for this section is:"Downregulation using siRNA and/or inhibition of EPS8 by mithramycin resulted in greater cell growth inhibition in non-EGFR mutant lung cancer cells and a bladder cell line following cisplatin treatment."