7 vs. 14.6 kPa, P>0.05). All these results might have caused some overlapping values www.selleckchem.com/products/17-AAG(Geldanamycin).html between patients with F3 and F4. The prediction of LRE development using LSM was imperfect. Although patients with baseline LSM values >19 kPa were at a significantly greater risk of LRE development (HR, 7.176) than were those with baseline LSM values ��19 kPa, 55.6% of patients with LSM values >19 kPa developed no LRE and 6.9% of those with LSM values ��19 kPa did. In the sub-group analysis, we found no significant predictor of discordant results regarding LRE development. Because this finding might be related to statistical error due to the short-term follow-up period and the low number of LREs, large-scale studies with long-term follow-up are needed to elucidate a novel serological predictor of LRE development.

When we increased the sensitivity of cutoff LSM value up to 89.5%, 9.1 kPa was selected. Using this cutoff value, we can clinically identify the sub-group of patients at low risk of LRE development (1.6%), such that these patients can be reassured. LSM has been known to predict fibrosis regression in response to long-term antiviral treatment [46]. Thus, we further analyzed the role of LSM as a dynamic indicator of LRE development using cutoff LSM values of 19 kPa or relative change in LSM values from baseline, but the results were negative. However, small sample size of some groups (n=11 and 12, respectively) might be related to type II error. Furthermore, because the study by Jung et al.

[14] revealed that LSM change, similar to baseline LSM value, can influence HCC development, large cohorts with sufficient events will likely be required to investigate the usefulness of serial LSM value follow-up in patients with chronic viral hepatitis. We included only patients with histologically advanced liver fibrosis (��F3). However, because histologic evaluation grades liver fibrosis categorically, it cannot exactly represent the continuous spectrum of liver fibrosis, especially between adjacent fibrosis grades. Furthermore, because histologic evaluation of liver fibrosis can be influenced by intra- and interobserver variability, the over- or underestimation of liver fibrosis inevitably occurs. Considering these limitations of histologic evaluation, some patients who were excluded due to F0�CF2 liver fibrosis may represent underestimations of F3 or F4 fibrosis, which might have resulted in selection bias, a major limitation of this study.

Thus, further validation of LSM cutoff values for predicting advanced liver fibrosis regardless of histologic data should be performed. Furthermore, the significantly higher proportion of patients with F4 fibrosis may be another bias of this study. Batimastat In conclusion, our data suggest that LSM can be a useful predictor of LRE development in CHB patients showing histologically advanced liver fibrosis.

The observed bone loss in patients with IBD is from either osteopenia or osteoporosis. It is estimated click here that 31�C59% of adult patients with IBD are classified as osteopenic, whereas 18�C24% are diagnosed with osteoporosis (1, 4, 13). The pathogenesis of IBDs is not fully understood, but the cytokine profiles of the patients hint at the possibility of cytokines playing a prominent role since these patients have an imbalance of proinflammatory cytokines (12, 26). Patients with IBD have increased levels in number of cytokines including IL-1, IL-6, interferon (IFN-��), and tumor necrosis factor-�� (TNF-��) (17). TNF-�� has been named the mastermind of the inflammatory response since treatment of anti-TNF-�� antibodies controls the progression of IBD and also increases bone mineral density (6, 16).

Phosphate is an important element for the body because it is essential for ATP synthesis, acid/base regulation, and the formation of nucleotides. Most importantly, it is a key component of bone. In fact, the bone matrix stores 85% of the body phosphate. Mammalian phosphate homeostasis is tightly regulated by controlling intestinal and renal epithelial transport mechanisms. In the renal proximal tubules, phosphate reabsorption is mediated by type IIa sodium-phosphate cotransporters (NaPi-IIa). Intestinal absorption of dietary phosphate is mediated by another subtype of type II NaPi cotransporters expressed on the apical membranes of enterocytes named NaPi-IIb (14, 19, 37). Enterocytes also express another NaPi cotransport protein on the basolateral membranes called NaPi-III, and this isoform most likely plays a role in cellular phosphate homeostasis (3, 9).

For intestinal phosphate absorption, however, NaPi-IIb is the most important of these cotransporters to study. Phosphate transport in the intestine has already been shown to be regulated by age (2, 36, 38), vitamin D3 (8, 11, 36), and hormones such as glucocorticoid (2) and estrogen (42). Since metabolic bone diseases like osteopenia and osteoporosis are associated with IBD, it is necessary to understand the molecular mechanisms of aberrant phosphate homeostasis in patients with IBD. Although TNF-�� has been found to affect bone density by inducing osteoclasts to erode the bone and by inhibiting osteoblasts to lay new bone matrix (35), how the state of inflammation affects the intestinal phosphate absorption is unknown.

Because of the fact that phosphate absorption is segment specific (ileum in mouse and jejuna in rat), we chose both of the animals as our in vivo model in our present studies. We aim to study the effects of inflammation on intestinal phosphate transport and ultimately bone health. MATERIALS AND METHODS Animals. Carfilzomib Six-week-old male Balb/C mice or 3-wk-old male Sprague Dawley rats were administered trinitrobenzene sulphonic acid (TNBS) in 50% ethanol (2 mg/mouse or 1 mg/rat) by an enema into the colonic lumen.

The expression of genes involved in selleck chemicals cell proliferation and cell cycle regulation (such as overexpressed CDKN3, BTG2, TGFB1, CNOT8, KAT2B, RARRES3, CDKN2C, RARRES1, MAGED1, PPAP2A, MXD4, TENC1, SESN1, and downregulated CDCA4, VEGFA), intracellular signal transduction, transcription regulation, metabolic and transport processes and apoptosis (overexpressed CDKN2C, BIK, CASP6, TIA, DAPK3, and dowregulated ANXA1, CEBPB, CBX4) are mainly changed under NS398 treatment. However, the function of several differentially expressed transcripts is not known yet. The functional classification of genes is represented in Figure 1. Figure 1 Functional classification of differentially expressed genes in HT29 cells under NS398 treatment. (A) Distribution of differentially expressed transcripts in the main cell functional groups.

(B) Distribution of downregulated transcripts in the main cell … In correlation with the mRNA expression findings, significant dose-dependent cell proliferation inhibition was measured using MTT assay, which was carried out to optimise the treatment concentration of NS398 COX2 inhibitor. Changes in colorectal adenoma and cancer-related mRNA expression patterns under NS398 treatment Seventeen of these 20 genes changed in a reverse manner in HT29 colon adenocarcinoma cells under NS398 COX2 inhibitor treatment, 14 of them (including upregulated somatostatin, claudin 8, peptide YY, and downregulated cadherin 3, KIAA1199) at a significance of P<0.05 (Figure 2A). The expression of 12 of the 38 CRC-related markers (such as carbonic anhydrase 7, interleukin 8, melanoma cell adhesion molecule) was changed in a reverse manner under NS398 treatment (Figure 2B).

Figure 2 Changes in colorectal adenoma and cancer-related mRNA expression patterns under NS398 treatment. (A) Expression of adenoma vs normal discriminatory genes in biopsy samples and in HT29 colon adenocarcinoma cells under NS398 treatment (B). Expression of … HT29 immunocytochemistry and western blot results Dose-dependent inhibition of COX2 protein expression was observed under NS398 treatment. COX2-positive cell/total cell ratio was 80.5% in untreated control samples, whereas it decreased to 77.0% under 10��M, to 61.2% under 25��M NS398 treatment. Further elevation of the NS398 dose (100��M) caused a significant decrease in the positive cell ratio (53.1%).

Strong granular and/or diffuse cytoplasmatic immunostaining was detected in COX2-positive AV-951 cells (Figure 3A and B). Western blot results showed correlation to the immunocytochemistry findings (Figure 3C). More considerable reduction in COX2 protein expression was detected after 96h of NS398 treatment at 50 and 100��M concentrations. Figure 3 The decrease in COX2 protein expression under NS398 treatment. Dose-dependent inhibition of COX2 protein expression was observed under NS398 treatment. Strong granular and/or diffuse cytoplasmatic immunostaining was detected in COX2-positive cells. ( …

Altered expression of ApoA-1 and Ig �� chain c-region containing fraction ApoA-1 and Ig �� chain c-region were identified in SEB8 fraction. Although a major proportion of breast cancer and benign breast disease no patients exhibited up-regulation of this protein fraction, breast cancer patients cannot be discriminated from benign breast disease patients on the basis of the expression of these proteins (P = 0.099; Table 2). Expression pattern of this protein fraction seems to be influenced by the status of chemotherapeutic treatment and HCV infection (P = 0.032). Moreover, it is interesting to note that non-chemotherapeutically treated patients were found to have predominant up-regulation (65.63%) whereas a higher proportion of chemotherapeutically treated and HCV positive breast cancer patients showed down-regulation of this protein fraction (Table 3).

Similarly, the apoA-1 and Ig �� chain c-region containing protein fraction was significantly up-regulated in infiltrating/invasive ductal carcinoma patients and patients whose carcinoma type was not identified, but predominantly down-regulated in in-situ duct carcinoma patients (Table 3). X2 analysis has revealed a 23.9% chance that the deviation from normal is due to chance only (P = 0.239). Hence, type of breast cancer did not contribute differentially towards the altered expression of this protein fraction (Table 3). Effect of chemotherapy on the expression of various proteins A small fraction of the BC patients (n = 8) investigated in the present study had undergone 2�C6 cycles of chemotherapy.

In these patients, all identified protein fractions were significantly down-regulated with the exception of SEB3 and SEB4. SEB3 (modified TTR) was expressed in a small fraction of chemotherapeutically treated patients whereas there was no change in the expression level of the SEB4 fraction as compared to the normal controls (Table 3). Combined effect of HBV/HCV infection and BC The current study included two lobular carcinoma patients and one spindle cell neoplasm patient. One patient was found to be HBV-positive and had also been treated chemotherapeutically. Batimastat SEB1, 5 and 7 were up-regulated while SEB2 and 6 were down-regulated in the patient suffering from spindle cell neoplasm. Lobular carcinoma patients exhibited down-regulation of SEB6 and SEB7, while out of 2 lobular carcinoma patients, one exhibited up- and other down-regulation of SEB1, 2 and 5. In HBV-positive and chemotherapeutically-treated BC patient, SEB1, 2, 5 and 7 were down-regulated but SEB6 was up-regulated. Modified TTR (SEB4) expression was documented in one lobular carcinoma patient as well as in the patient suffering from spindle cell neoplasm.

TNF stimulated increased COX-2 levels by 3 h, and expression peaked at ~16 h in these cells (Fig. 2C); 100 ng/ml TNF most strongly induced COX-2 expression (Fig. 2D). These selleck chem inhibitor data confirm that the concentration of TNF that exhibits a strong cytotoxic effect in COX-2?/? cells is the strongest inducer of COX-2 protein expression and that TNF induction of COX-2 expression is similar in colon and gastric epithelial cells. Fig. 2. TNF or EGF stimulates COX-2 expression in YAMC and ImSt cells. YAMC (A) or ImSt (C) cells were treated with TNF (100 ng/ml) or EGF (10 ng/ml) for 0�C24 h. YAMC (B) or IMST (D) cells were treated with TNF (0�C1,000 ng/ml) or EGF (10 ng/ml) … TNF signals through TNFR1 to induce COX-2 expression.

To determine which of the two TNFRs is required for TNF-induced COX-2 expression, we used conditionally immortalized TNFR1?/? or TNFR2?/? MCE to generate TNFR add-back cell lines. TNFR1?/? MCE cells were infected with recombinant retrovirus to introduce a Vec, a ��DD TNFR1 mutant, or WT TNFR1 (Fig. 3A). TNFR2?/? MCE cells were also infected to introduce Vec or WT TNFR2 (Fig. 3B). TNF did not stimulate COX-2 expression in TNFR1?/? Vec or ��DD cells (Fig. 3C); however, TNF induction of COX-2 was rescued in TNFR1 WT cells. TNF and EGF induced expression of COX-2 in TNFR2?/? Vec cells (Fig. 3D). Interestingly, the basal and TNF- and EGF-stimulated COX-2 expression levels were lower in TNFR2 WT cells. The same pattern of COX-2 expression occurs in WT, TNFR1?/?, and TNFR2?/? ImSt cells (Fig. 3E). Thus, TNF induces COX-2 expression through TNFR1 in GI epithelial cells.

Fig. 3. TNF signals through TNF receptor (TNFR) 1 to induce COX-2 expression. Expression of hemagglutinin (HA)-tagged death domain deletion (��DD) mutant TNFR1 and wild-type (WT) TNFR1 in TNFR1?/? MCE cells (A) and WT TNFR2 in TNFR2?/? … TNF stimulation of COX-2 expression requires EGFR kinase activity. We determined whether EGFR has a role in TNF-induced COX-2 expression by assessing COX-2 expression in the presence of a pharmacological EGFR inhibitor or in the absence of EGFR expression. YAMC cells were treated with TNF in the presence or absence of AG-1478, a small-molecule inhibitor of EGFR kinase activity (Fig. 4A). To quantify the effect of AG-1478 on TNF-stimulated COX-2 expression, we performed a load-response Western blot analysis of the cell lysates (Fig. 4B).

GSK-3 AG-1478 reduced TNF-stimulated COX-2 expression to near-basal levels (Fig. 4C). As expected, the inhibitor also blocked EGF-stimulated COX-2 protein expression. In contrast, AG-1478 did not affect basal COX-2 expression. Interestingly, the concentration of TNF that maximally stimulates COX-2 in YAMC and ImSt cells also maximally stimulated transactivation of EGFR in ImSt (Fig. 4D) and YAMC (76) cells.

Figure 3 Effect of different prostaglandin receptor inhibitors in MH1C1 cells. A) The EP4 inhibitor L-161982 (10��M) was added 30min prior to stimulation with PGE2 (100��M) for 5min. B) The selleck chemical EP1 inhibitor SC51322 … Evidence of a role for Ca2+, but not PKC, in the PGE2-induced transactivation of EGFR We next tried to determine which pathways downstream of PLC�� are mediating the PGE2-induced transactivation of EGFR. InsP3 and DAG stimulate cytosolic Ca2+ release and protein kinase C (PKC) activity, respectively. Pretreatment of the cells with the PKC inhibitor GF109203X did not prevent the effects of PGE2 on the phosphorylation of the EGFR, ERK, or Akt in the MH1C1 cells (Figure4A).

Furthermore, the data in Figure4B, comparing PGE2 and the direct PKC activator tetradecanoylphorbol acetate (TPA), showed that TPA did not mimic the effect of PGE2 on Akt, and its stimulation of ERK, unlike the effect of PGE2, was blocked by GF109203X. Interestingly, pretreatment of the cells with GF109203X consistently increased basal and PGE2-induced Akt phosphorylation in the cells. This might result from a reduced feedback inhibition by PKC [47]. In contrast to TPA, thapsigargin, which increases the intracellular Ca2+ level by inhibiting the ��sarco/endoplasmic reticulum Ca2+-ATPase�� (SERCA) pump [48], induced gefitinib-sensitive phosphorylation of EGFR, ERK, and Akt (Figure4C). Taken together, these data suggest that Ca2+ rather than PKC mediates the PGE2-induced transactivation of the EGFR in these cells. Figure 4 Role of Ca2+ and PKC in responses to PGE2 in MH1C1 cells.

A) MH1C1 cells were pretreated for 30min with the PKC inhibitor GF109203X (3.5��M) before stimulation with PGE2 (100��M) for 5min. B) MH1C1 cells … Role of Src and metalloproteinases in the transactivation of the EGFR To further elucidate mechanisms involved in transactivation of the EGFR, we investigated the effects of Src inhibitors. As shown in Figure5A, pretreatment of the cells with the Src inhibitor CGP77675 almost completely abolished the PGE2-induced phosphorylation of EGFR and the activation of ERK and Akt, but, in contrast, had little or no effect on the phosphorylation of these proteins elicited by EGF. The Src inhibitor PP2 similarly prevented the phosphorylation of ERK in response to PGE2, while the response to EGF was not significantly affected (Figure5B).

These results suggest an involvement of a Src family kinase in the PGE2-induced transactivation of EGFR in MH1C1 cells. Figure 5 Effect of Src and MMP inhibitors on phosphorylation of EGFR and downstream targets. A) MH1C1 cells were pretreated for 90min with the Src inhibitor CGP 77675 (10��M). Cells were then stimulated with either PGE2 (100��M) … Previous evidence has implicated proteinases of the ��a-disintegrin-and-metalloproteinase�� (ADAM) family in EGFR transactivation by GPCRs GSK-3 in various cells [2,49,50].

A healthy control sample yielded no PCR product, the 17-DMAG cost wild-type allele being too large (~27kb) to be amplified. Sequencing the PCR product revealed a simple deletion of 24720bp (Supplementary Figure 1). This deletion was also detected in three other affected individuals with available DNA samples (Figure 2: individuals II:4, II:7, and II:10 of family 1). Figure 4 Illustration of two possible mechanisms underlying the complex rearrangement detected in patient BEA. (a) In the SRS model,42, 43 the newly synthesized leading strand (C) misaligned with the lagging template strand (B) through inverted short repeats (the … Discussion In this study, we showed for the first time that a significant subset of patients with symptomatic cholestasis/cholelithiasis has underlying ABCB4 deletions.

Partial or complete heterozygous ABCB4 deletions were found in 7% of the patients with LPAC and in ~2% of the patients with CIC. A large family of 12 affected patients with severe LPAC and cholecystectomy (family 1) was notably reported (Table 1; Figure 2). Recent gene dosage methodologies have allowed identification of these ABCB4 deletions. Our observations urge to systematically test the patients with LPAC for the presence of ABCB4 deletions. MLPA, a sensitive, rapid, and cost-effective approach seems particularly adapted to routine diagnosis in molecular genetics. We developed a molecular algorithm tailored to ABCB4 routine analysis that includes ABCB4 gene dosage by MLPA, in case of ABCB4 negative sequencing in patients with suggestive phenotype.

MLPA allows a fast and inexpensive first-line screening for both the partial and complete ABCB4 deletions, complementary to a high-resolution technique such as array-CGH that can be used to characterize larger deletions. Real-time PCR-based gene dosage is useful for deletion’s confirmation, particularly Drug_discovery for the samples with ratio profiles considered as doubtful, that is, between 0.6 and 0.85 (patient ROC; Figure 1). The two intragenic deletion breakpoints were cloned at the nucleotide level and no recurrent breakpoints were found. That no significant sequence similarity was found between the centromeric and telomeric breakpoints of both the deletions, effectively excluded homologous recombination as the underlying mutational mechanisms in both the cases. However, the presence of microhomology in each of the aberrant junctions is consistent with both the microhomology-dependent replication-based recombination (MMRDR) and non-homologous end-joining (NHEJ) mechanisms.

Treatment of HBV involves finite administration of pegylated or unpegylated interferon alfa, or indefinite administration of anti-HBV nucleoside/nucleotide analogues. Five such sellckchem analogues are currently available. Lamivudine, a deoxycytidine analogue, was the first nucleoside approved for use in HBV and lamivudine monotherapy remains common despite high rates of treatment-emergent drug resistance [2]. Entecavir is a deoxyguanosine analogue with a high genetic barrier to resistance in treatment-naive patients [3]. However, lamivudine resistance predisposes HBV to subsequent entecavir resistance [4]. Telbivudine is an L-deoxythymidine analogue with superior efficacy to lamivudine [5] but a similar resistance profile [6].

Finally, the nucleotides adefovir and tenofovir are both acyclic mimetics of deoxyadenosine monophosphate which retain activity against lamivudine- and telbivudine-resistant HBV [6]. However, adefovir is associated with dose-dependent nephrotoxicity which restricts its dosing to 10 mg daily [7], at which dose it demonstrates inferior virologic efficacy to the other agents [8]�C[10]. There are also concerns about the long-term safety of tenofovir, which is associated with significant loss of renal function in HIV treatment [11]. HBV viral replication is a key driver for disease progression and is associated with the development of cirrhosis and HCC [12]. The initial goal of treatment is to suppress viral replication; thereafter, sustained (on-treatment) or maintained (off-treatment) suppression of circulating HBV DNA is associated with improved serological responses and long-term outcomes [13], [14].

The emergence of drug-resistant HBV results in breakthrough viremia leading to hepatitis and liver disease progression. To ensure good long-term outcomes, the conservation of HBV DNA suppression is essential. Early virologic response, particularly at Week 24, is associated with better long-term outcomes in chronic HBV, while detectable HBV DNA at Week 24 is associated with a higher incidence of on-therapy drug resistance [14], [15]. This predictive association has lead an international group of experts to propose the so-called ��Roadmap�� concept �C a therapeutic algorithm for the conditional intensification of nucleoside monotherapy based on early virologic response [16]. In the Roadmap, monotherapy is continued if plasma virus is undetectable (HBV DNA <300 copies/mL) at Week 24; while for those with detectable HBV DNA defined Batimastat options exist for either intensification or continued monotherapy. The Roadmap principle is widely accepted in clinical practice [17], but has yet to be prospectively evaluated.

.. Budding cells have been credited with the properties of malignant stem cells including the potential for redifferentiation both locally and at sites of distant metastasis and marking, what appears to be, the first histological event in tumor cell migration sellekchem and invasion. Supporting this hypothesis further is the presence of ��pseudopodia-like�� cytoplasmic protrusions in tumor buds which have been identified by both electron microscopy and recently by immunohistochemistry with pan-cytokeratins[17,20]. These podia appear to be in direct contact with the adjacent interstitial tissue suggesting their formation occurs during tumor cell migration. Moreover, Shinto et al[21] recently suggested that cytoplasmic pseudo-fragments could be used as a marker for an activated budding phenotype that is associated with cell motility and increased invasiveness independent of the extent of budding.

Not surprisingly, tumor buds have been shown to over-express proteins involved in extracellular matrix degradation and to under-express adhesion molecules. Previous studies on EMT and events occurring at the invasive tumor front implicate, in particular, the Wingless-INT (WNT) signalling pathway in the process of tumor budding evidenced by increased ��-catenin immunohistochemical staining in tumor buds, a concomitant loss of E-cadherin and over-expression of laminin5��2 along with activation of transcriptional repressors SLUG, and ZEB1[22,23].

Over-expression of urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase-7 and -9 (MMP7, MMP9), matrilysin, CD44, Ep-CAM, and extensive staining of ��(III)-tubulin, a major constituent Anacetrapib of microtubules, have all been reported[20,23-30] suggestive of the invasion and migration potential of tumor buds. Tumor buds seem to over-express CXCL12, a stromal cell-derived factor whose receptor CXC4 is involved in chemotaxis and angiogenesis[31]. In addition, we recently documented the over-expression of the putative colorectal CSC marker ABCG5 within tumor buds leading to a poorer outcome of patients including those with node-negative disease (Hostettler, World Journal of Gastroenterology, in press). Whether a sub-population of tumor buds may in fact represent malignant stem cells is still an open question which necessitates further investigation. Prognostic impact of tumor budding Since tumor budding appears to play a critical role in the initiation of metastasis, several authors have investigated the potential of this feature to predict dissemination of tumor cells to regional lymph nodes. A significant association between tumor budding and lymph node positivity has been consistently demonstrated correlating with tumor aggressiveness and more advanced TNM stage[32-43].

An alternative approach could be to selleck kinase inhibitor use licensed iron chelators such as deferoxamine (DFO) or deferasirox, which have a proven safety profile (Merlot et al., 2012). Both in vitro and in vivo data highlight their potential as possible anti-cancer agents (Richardson, 2002; Whitnall et al., 2006; Yu et al., 2006; Merlot et al., 2012). The iron chelator, DFO, has shown promise as an anti-tumour agent in human clinical trials involving neuroblastoma and leukaemia (Estrov et al., 1987; Donfrancesco et al., 1990; 1992; 1995). However, the utility of iron chelators in treating patients with oesophageal cancer has not yet been addressed. Therefore, the aims of this investigation were to assess whether DFO and deferasirox could inhibit iron-mediated tumour-promoting effects observed in oesophageal models and whether they possess anti-oesophageal cancer activity in vivo.

These studies could provide a sound rationale for evaluating the usefulness of these agents in human clinical trials as treatments for oesophageal cancer and also as potential chemo-sensitizers. The latter is particularly relevant in oesophageal cancer, as the majority of patients either receive neo-adjuvant or palliative chemotherapy. Methods Iron chelators and chemotherapeutic drugs DFO was purchased from Sigma-Aldrich (St. Louis, MO) and was used throughout this study at its IC50 value (10 ��M). Deferasirox (a kind gift from Novartis, Basel, Switzerland) was used at concentrations of 0�C40 ��M. The ligand, Dp44mT, was synthesized as previously described and used at 1 ��M, where it shows high anti-tumour activity (Yuan et al.

, 2004). Epirubicin (Mayne Pharma Plc, Warwickshire, UK), cisplatin (TEVA UK, Eastbourne, UK) and fluorouracil (5-FU; Mayne Pharma Plc) were used at concentrations between 0 and 32 ��M (IC50 values: 1, 8 and 8 ��M respectively). The IC50 values above were determined over a 48 h period using the OE33, OE19 and OE21 oesophageal cell lines. Cell culture The oesophageal adenocarcinoma lines, OE19 and OE33, and oesophageal squamous cell carcinoma lines OE21 and TE4, were routinely cultured in DMEM with 10% FCS (Invitrogen, Mulgrave, VIC, Australia) (Rockett et al., 1997; Takashima et al., 2011). In addition, the oesophageal squamous cell line, TE4 (kind gift from Prof W Dinjens, University of Rotterdam), previously reported to be cisplatin-resistant, was cultured with and without cisplatin (2 ��M) to maintain resistance (Takashima et al.

, 2011). Assessment of cellular iron uptake and efflux Inhibition of cellular 59Fe uptake by iron chelators Cells (1 �� 105 mL?1) were plated in triplicate to achieve 70% confluence Drug_discovery in culture dishes (35 �� 10 mm). Cell monolayers were then incubated for 3 h/37��C with media (1 mL) containing 59Fe-transferrin (59Fe-Tf; 60 ��g?mL?1) and the iron chelators (1�C20 ��M) (Richardson et al., 1995).