Non-neoplastic Cell Line Research Articles

Abstract 1841: Selective inhibitors of nuclear export (SINE) activate multiple tumor suppressor pathways and kill prostate cancer cells across multiple genotypes in vitro and in vivo

Abstract CRM1 (Xpo1) is a key nuclear export protein which controls the nuclear export of multiple tumor suppressor proteins (TSP) and cell proliferation pathways including p53, p21, FOXO, PI3K/AKT, Wnt/ß-catenin and NF-κB. Mislocalization of TSP can abrogate their functions as well as render chemotherapies ineffective. Induction of nuclear expression of TSP and chemotherapy targets, by CRM1 inhibition can restore their tumor suppressor functions and increase drug sensitivity. We have developed orally active, small molecule SINE that irreversibly and potently inhibit CRM1 mediated nuclear export of multiple TSP and other cargoes (IC50 < 100 nM). Here, we describe in vitro results using SINE compounds, KPT-185 in vitro and KPT-251 in vitro and in vivo on seven prostate cancer (PrCa) cell lines representing distinct differentiation/progression states of disease and genotypes: LAPC-4 (Androgen receptor [AR] positive, androgen dependent with low Akt/mTOR activities, p53 wt); LnCaP (AR positive, androgen dependent with high Akt/mTOR activities, p53 wt); LnCaP-C81 and LnCaP-C4-2B (AR positive, androgen independent with high Akt/mTOR activities, p53 wt); 22rv1 (AR positive, androgen independent with low Akt/mTOR activities, p53 wt); PC3 (AR negative, with high Akt/mTOR activites and no p53 function (p53 del) and DU145 (AR negative, with low Akt/mTOR activites and mutant p53). Benign prostatic hyperplasia line (BPH1) and Prostatic epithelial line (EPN) were used as non-neoplastic controls. We show that SINE block CRM1 mediated nuclear export of FOXO and p53 with a IC50 values <100 nM in both PrCa and non-neoplastic cell lines. KPT compounds are selectively cytotoxic to PrCa lines with EC50s between 10 and 1000 nM, and show limited cytotoxicity on the non-neoplastic EPN and BPH1 lines (EC50 >5-20 μM). SINE cytotoxic effects are independent of p53 status, and induce caspase-3 activation. SINE display synergistic effects in combination with cisplatin and docetaxel in vitro with combination indices between 0.3 and 0.8. In vivo results indicate that KPT-251 show a dose-dependent inhibition of tumor growth. KPT251 was also synergistic when administered at 30mg/kg/5 days/week per os with docetaxel and cisplatin by using the aggressive p53 wt 22rv1 xenografts. Taken together, CRM1 inhibition represents a completely novel, neoplasia-selective and well- tolerated target for use as single agent or in combination with chemotherapy for PrCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1841. doi:1538-7445.AM2012-1841

Abstract 113: Effect of targeting Aurora kinase A in human oral squamous cell carcinoma cells

Abstract Oncogene addiction has provided therapeutic opportunities in many human malignancies, but molecular targeted therapy for oral squamous cell carcinoma (OSCC) is not available. In this study, we identified an appropriate molecule for treatment of patients with OSCC. We determined the gene expression profiles in 10 human OSCC cell lines and a non-neoplastic keratinocyte cell line by microarray analysis, and then identified Aurora kinase A (AURKA) as a cancer-related gene which was commonly overexpressed in OSCC cells. We also confirmed the overexpression of AURKA mRNA in OSCC tissues and compared to normal oral mucosa tissues by quantitative RT-PCR. AURKA plays an essential role in centrosome duplication, mitoic entry, and spindle assembly. Overexpression of AURKA in human malignancies is associated with gene amplification, genetic instability, poor histological differentiation, and poor prognosis. To determine if the AURKA is a plausible therapeutic target, we investigated the effect of small interfering RNAs specific for AURKA(siAURKA) and an AURKA selective inhibitor MLN8237 on the growth of human OSCC cells in vitro and in vivo. Knockdown of AURKA by RNA interference (RNAi) markedly inhibited the growth of OSCC cells and observed the change of colony formation and cell size looked like cell cycle arrest. We examined the RNAi effect of siAURKA in OSCC cells by Western blotting. Compared with non-target siRNA, the protein expression level of AURKA was almost completely suppressed. MLN8237 also reduced the cell growth, but showed relatively resistant to cells expressing AURKA at high levels. Transfection of siAURKA inhibited the growth of OSCC cells more potent than treatment with MLN8237 in vitro. Furthermore, we confirmed the effect of siAURKA and MLN8237 on the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice. Atelocollagen-siAURKA (8 nmol) complexes were injected into the tail vein every 3 days and MLN8237 (30 mg/kg) were received orally for 14 consecutive days. We found that siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSSC tumors. Interestingly, the combination of siAURKA and MLN8237 induced remarkable anti-tumor activity compared with single agent groups. These results suggest that AURKA functions as a critical gene for supporting the growth of human OSCC cells and targeting AURKA may be a useful therapeutic strategy for OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 113. doi:1538-7445.AM2012-113

The selective cytotoxic activity in breast cancer cells by an anthranilic alcohol-derived acyclic 5-fluorouracil O,N-acetal is mediated by endoplasmic reticulum stress-induced apoptosis

Advance in the knowledge of molecular biology has thrown light on many aspects of apoptosis regulation mechanisms. This has allowed a change in anti-cancer therapy trends, from classic cytotoxic strategies to the development of new non-harmful therapies which target the apoptosis response selectively only in tumour cells. We have selected an anthranilic alcohol-derived acyclic 5-fluorouracil O,N-acetal (5) to carry out the anti-cancer studies. This compound shows activity as a potent growth inhibitor of the tumour cell line MCF-7 at a very low concentration. Moreover, when this compound was administered to the non-neoplastic cell line, MCF-10A displayed less toxicity resulting in lower rates of apoptosis. Further studies by microarray hybridization, real-time PCR and western blot showed that when administered to human breast cancer cells, MCF-7, 5 had no activity against classic pro-apoptotic genes such as p53, and even induced the down-regulation of anti-apoptotic genes such as Bcl-2. In contrast, several pro-apoptotic genes related with the endoplasmic reticulum (ER)-stress-induced apoptosis, such as BBC3 and Noxa, appeared up-regulated. These results seem to show that the mechanism of action and selectivity of 5 was via the activation of the ER stress-induced apoptosis. The selective activity of this compound against tumour cells via the ER stress-induced apoptosis supposes a great advantage for future therapeutic use.

P1-03-04: In-Vitro Characterization of a Breast Cancer Microenvironment with Epithelial-to-Mesenchymal Transition (EMT) Characteristics.

Abstract Non-neoplastic tissues adjacent to tumor is known to influence the progression of the disease. We and others found that there are two types of microenvironment, one of these highly expresses genes associated with epithelial-to-mesenchymal transition (EMT). Tumor adjacent microenvironment (TAME) with wound responses have been observed in >90% of tumor-adjacent tissues, but EMT- characteristics seems to be less common and are observed in less than half of all patients. Our data suggest that the patients with EMT-TAME have worse breast cancer outcome, particularly among estrogen receptor positive patients. Thus, understanding the molecular cross talk between tumor cells and adjacent cells located in the TAME with EMT properties may identify targetable pathways that play a important role in breast cancer progression. Toward this end, we developed an in-vitro model system that can be used to probe paracrine signaling between cancer cells and adjacent cells undergoing EMT. We hypothesized that the presence of EMT signals in the TAME will alter the gene expression and phenotype of epithelial cells. We further hypothesized that the interactions between cancer cells and their TAME would be subtype dependent. Using cell line models of luminal and basal-like breast cancer, we performed cocultures with cancer cells and cell line models of EMT. The latter cell lines were HMLE cells transduced with either Snail or Twist, representing an intermediate state between an epithelial and a fully mesenchymal phenotype. This coculture system allows the study of heterotypic interactions between cancers and TAME cells with EMT characteristics. Gene expression, proliferation, migration, anchorage independent growth and cytokine production of different breast cancer and non-neoplastic cell lines were evaluated. We observed that coculture with models of EMT microenvironments altered the behavior of epithelial cells and that the gene expression changes were relevant to in-vivo in public datasets, showing correlations with both subtype and outcome. Cocultured cells acquired more migratory behavior and reduced expression of cellular adhesion genes, while simultaneously increasing anchorage independent growth and production of cytokines such as IL-6 and IL-8. Furthermore, we observed different response by luminal and basal-like breast cancer cells to the coculture condition, suggesting that the tumor-TAME interactions occur in a tumor subtype dependent fashion. These results reiterate the importance of the surrounding environment on cancer cell phenotypes and document that paracrine interactions with cells undergoing EMT may increase the aggressiveness of some cancers. Identification of specific signaling pathways that define interactions between specific breast cancer subtypes and their microenvironments will lead to new insights regarding tumor biology and identify potential pathway-specific biomarkers and/or targets for prognosis and treatment of breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-04.

Cytoplasmic Forkhead Box M1 (FoxM1) in Esophageal Squamous Cell Carcinoma Significantly Correlates with Pathological Disease Stage

Esophageal cancer is a deadly cancer with esophageal squamous cell carcinoma (ESCC) as the major type. Until now there has been a lack of reliable prognostic markers for this malignancy. This study aims to investigate the clinical correlation between Forkhead box M1 (FoxM1) and patients’ parameters in ESCC.MethodsImmunohistochemistry was performed to investigate the expression and localization of FoxM1 in 64 ESCC tissues and 10 nontumor esophageal tissues randomly selected from 64 patients before these data were used for clinical correlations.ResultsCytoplasmic and nuclear expressions of FoxM1 were found in 63 and 16 of the 64 ESCC tissues, respectively. Low cytoplasmic expression of FoxM1 was correlated with early pathological stage in ESCC (P = 0.018), while patients with nuclear FoxM1 were younger in age than those without nuclear expression (P < 0.001). Upregulation of FoxM1 mRNA was found in five ESCC cell lines (HKESC-1, HKESC-2, HKESC-3, HKESC-4, and SLMT-1) when compared to non-neoplastic esophageal squamous cell line NE-1 using quantitative polymerase chain reaction (qPCR). Except for HKESC-3, all studied ESCC cell lines demonstrated a high expression of FoxM1 protein using immunoblot. A high mRNA level of FoxM1 was observed in all of the ESCC tissues examined when compared to their adjacent nontumor tissues using qPCR.ConclusionCytoplasmic FoxM1 was correlated with pathological stage and might be a biomarker for advanced ESCC.

Barrett's esophagus: genetic and cell changes

The following includes commentaries on how genetic code of Barrett's esophagus (BE) patients, the mechanisms for GERD-induced esophageal expression of caudal homeobox, and the development of Barrett's metaplasia are increasingly better known, including the role of stromal genes in oncogenesis. Additional lessons have been learned in vitro models in nonneoplastic cell lines, yet there are limitations to what can be expected from BE-derived cell lines. Other topics discussed include clonal diversity in Barrett's esophagus; the application of peptide arrays to clinical samples of metaplastic mucosa; proliferation and apoptosis of Barrett's cell lines; tissue biomarkers for neoplasia; and transcription factors associated with BE.

Differences in Eotaxin-3 Expression After Stimulation With IL-13 and IL-4 in Esophageal Squamous Cell Lines From Patients With Eosinophilic Esophagitis and GERD.

Introduction: In eosinophilic esophagitis (EoE), food and other allergens appear to activate Th2 cells to release IL-13 and IL-4, cytokines that can induce esophageal squamous epithelial cells to produce the potent eosinophil chemoattractant eotaxin-3. GERD also can cause an eosinophilic infiltration of the esophagus, usually milder than that seen in EoE, through mechanisms that are not clear. In earlier studies, we showed that IL-13 and IL-4 caused a marked increase in eotaxin-3 protein secretion in an esophageal squamous cell line derived from a patient with EoE, but only a minimal increase in eotaxin-3 protein secretion in esophageal squamous cell lines derived from GERD patients. To explore this mechanism further, we studied the effects of IL-13 and IL-4 on transcriptional activation of eotaxin-3 in esophageal squamous cell lines from patients with EoE and GERD. Methods: Two telomerase-immortalized, non-neoplastic esophageal squamous cell lines derived from patients with GERD (NES-B10T & NES-G4T), and one derived from a patient with EoE (EoE1-T) were stimulated with IL-13 (100 ng/ml) or IL-4 (1 ng/ml) for times ranging from 24-48 hours. Eotaxin-3 mRNA expression levels were determined by RT-PCR, and an eotaxin-3 promoter (-800bp) construct attached to a luciferase reporter was used to measure transcriptional activity. Results: In the GERD cells, IL-13 and IL-4 induced only slight increases in eotaxin-3 mRNA expression levels at 24 and 48 hours. In contrast, both cytokines caused a marked increase in eotaxin-3 mRNA expression levels at 24 and 48 hours in the EoE cells. Both IL-13 and IL-4 significantly increased eotaxin-3 promoter activity in all 3 cell lines. There was no significant difference in the degree of eotaxin-3 promoter induction by IL-13 and IL-4 between the two GERD cell lines (NES-G4T cells IL-13: 431 ± 42.5 SEM RLUs % control, IL-4: 279± 38; NES-B10T cells IL-13: 554 ± 109, IL-4: 463 ± 54). In contrast, the cytokine-stimulated increase in eotaxin-3 promoter activity in the EoE1 cells (IL-13: 1522 ± 225, p<0.01; IL-4: 1714 ± 385, p<0.01) was significantly greater than that in the two GERD cell lines. Conclusions: In an EoE esophageal squamous cell line, stimulation with IL-13 and IL-4 caused a far greater increase in eotaxin-3 mRNA expression and promoter transcriptional activity than in esophageal squamous cell lines from GERD patients. These observations suggests that the esophagus of patients with EoE produces more eotaxin3 in response to Th2 cytokine stimulation than the esophagus of patients with GERD, and that differences among patients in the degree of cytokine-stimulated production of eotaxin3 by esophageal squamous cells might underlie the degree of esophageal infiltration by activated eosinophils.

Markers of Tyrosine Kinase Activity for Diagnosis and Treatment Response in Eosinophilic Esophagitis: A Pilot Study of pERK 1/2 and pSTAT5

Introduction: In eosinophilic esophagitis (EoE), food and other allergens appear to activate Th2 cells to release IL-13 and IL-4, cytokines that can induce esophageal squamous epithelial cells to produce the potent eosinophil chemoattractant eotaxin-3. GERD also can cause an eosinophilic infiltration of the esophagus, usually milder than that seen in EoE, through mechanisms that are not clear. In earlier studies, we showed that IL-13 and IL-4 caused a marked increase in eotaxin-3 protein secretion in an esophageal squamous cell line derived from a patient with EoE, but only a minimal increase in eotaxin-3 protein secretion in esophageal squamous cell lines derived from GERD patients. To explore this mechanism further, we studied the effects of IL-13 and IL-4 on transcriptional activation of eotaxin-3 in esophageal squamous cell lines from patients with EoE and GERD. Methods: Two telomerase-immortalized, non-neoplastic esophageal squamous cell lines derived from patients with GERD (NES-B10T & NES-G4T), and one derived from a patient with EoE (EoE1-T) were stimulated with IL-13 (100 ng/ml) or IL-4 (1 ng/ml) for times ranging from 24-48 hours. Eotaxin-3 mRNA expression levels were determined by RT-PCR, and an eotaxin-3 promoter (-800bp) construct attached to a luciferase reporter was used to measure transcriptional activity. Results: In the GERD cells, IL-13 and IL-4 induced only slight increases in eotaxin-3 mRNA expression levels at 24 and 48 hours. In contrast, both cytokines caused a marked increase in eotaxin-3 mRNA expression levels at 24 and 48 hours in the EoE cells. Both IL-13 and IL-4 significantly increased eotaxin-3 promoter activity in all 3 cell lines. There was no significant difference in the degree of eotaxin-3 promoter induction by IL-13 and IL-4 between the two GERD cell lines (NES-G4T cells IL-13: 431 ± 42.5 SEM RLUs % control, IL-4: 279± 38; NES-B10T cells IL-13: 554 ± 109, IL-4: 463 ± 54). In contrast, the cytokine-stimulated increase in eotaxin-3 promoter activity in the EoE1 cells (IL-13: 1522 ± 225, p<0.01; IL-4: 1714 ± 385, p<0.01) was significantly greater than that in the two GERD cell lines. Conclusions: In an EoE esophageal squamous cell line, stimulation with IL-13 and IL-4 caused a far greater increase in eotaxin-3 mRNA expression and promoter transcriptional activity than in esophageal squamous cell lines from GERD patients. These observations suggests that the esophagus of patients with EoE produces more eotaxin3 in response to Th2 cytokine stimulation than the esophagus of patients with GERD, and that differences among patients in the degree of cytokine-stimulated production of eotaxin3 by esophageal squamous cells might underlie the degree of esophageal infiltration by activated eosinophils.

Absence of the JAZF1/SUZ12 chimeric transcript in the immortalized non-neoplastic endometrial stromal cell line T HESCs

Endometrial stromal sarcomas are rare malignancies, accounting for less than 10% of uterine sarcomas. The most characteristic chromosomal aberration of this tumor type is the translocation t(7;17)(p15-p21;q12-q21) leading to the fusion of two zinc finger genes, JAZF1 and SUZ12. Recently, the presence of the neoplastic JAZF1/SUZ12 fusion transcript was reported in normal cells of human endometrium. One of the positive samples for the JAZF1/SUZ12 transcript was the immortalized T HESCs cell line. This cell line was derived from the stromal cells obtained from an adult female with myomas and immortalized by transfection of a human telomerase gene. Since T HESCs has a normal karyotype and no fusion of the two genes occurs at the genomic level, the JAZF1/SUZ12 transcript was proposed to be generated by regulated trans-splicing between precursor RNAs for JAZF1 and SUZ12. However, no confirmatory reports currently exist. To determine whether the results could be reproduced, the T HESCs cell line was subjected to three different RT-PCR amplifications for the JAZF1/SUZ12 fusion transcript. RT-PCR assays did not amplify JUZF1/SUZ12 cDNA fragments in the T HESCs cell line, whereas the same assays easily generated JUZF1/SUZ12-amplified transcripts in an endometrial stromal cell sarcoma carrying the t(7;17) chromosomal aberration. Thus, the presence, if any, of a JUZF1/SUZ12 chimeric transcript in the immortalized normal T HESCs is not a constant, reproducible result.

The role of intrinsic epithelial activation in the pathogenesis of Sjögren’s syndrome

Sjögren’s syndrome (SS) is a chronic autoimmune disorder that is characterized by dysfunction and destruction of the exocrine glands. Exocrinopathy is associated with periductal mononuclear cell infiltrates in the affected exocrine glands and B-cell hyperreactivity. Epithelial cells are thought to play an important pathogenetic role, as suggested by the occurrence of infiltrating lesions in various epithelial tissues (described as autoimmune epithelitis) as well as the increased epithelial expression of several inflammatory proteins in the histopathologic lesions of patients. The application of long-term cultured non-neoplastic salivary gland epithelial cell (SGEC) lines has permitted the more explicit investigation of the role of these cells in the pathophysiology of SS. These studies have revealed the inherent capacity of SGEC to induce and promote chronic inflammatory reactions, as corroborated by the constitutive or inducible expression of various molecules implicated in innate and acquired immune responses. Furthermore, significantly increased constitutive expression of several molecules has been observed in SGEC lines derived from SS patients, as compared to those obtained from disease control patients. This fact strongly indicates the operation of intrinsic activation mechanisms in the epithelia of SS patients and further supports the active participation of these cells in the pathogenesis of the disorder.

DNA Methylation Inhibites ANXA1 Gene Expression in Nasopharyngeal Carcinoma Cell Lines*

In order to investigate the messenger ribonucleic acid(mRNA) and protein expression status of ANXA1 gene in nasopharyngeal carcinoma cell lines, so as to explore the correlation between ANXA1 methylation status and gene expression, four nasopharyngeal carcinoma(NPC) cell lines, including CNE1, CNE2, 5-8F, 6-10B, and immortalized non-neoplastic human nasopharyngeal epithelial cell line NP69 were cultured in vitro for research.Methylation status of ANXA1 gene was detected by methylation specific polymerase chain reaction(MSP), while mRNA expression level were also evaluated by reverse transcriptional polymerase chain reaction(RT-PCR).Subsequently, different end concentration(0 μmol/L as control, 0.1 μmol/L, 1 μmol/L, 5 μmol/L,10 μmol/L) of 5-aza-2′-deoxycytidine(5-aza-2dC) were added into culture medium of NPC cell lines for 72 hours′ de-methylation treatment, then methylation status of ANXA1 gene were detected by MSP and mRNA expression were evaluated by RT-PCR.In addition, ANXA1 protein expression was detected by Western-blotting.Without de-methylation treatment, ANXA1 gene was methylated in all the four NPC cell lines but NP69, and the methylation extent is correlated with differentiation state and metastasis potential of the cells.mRNA expression was lower in all of the four NPC cell lines without de-methylation treatment compared with NP69, and the expression level was correlated with gene methylation level.5-aza-2dC de-methylation treatment reversed ANXA1 methylation status, and increased the expression levels of mRNA and protein in all the four NPC cell lines.In summary, the current research verified down-regulated ANXA1 gene expression in NPC cell lines from both mRNA and protein level, the expression down-regulation was mainly caused by gene methylation, and 5-aza-2dC de-methylation treatment restored the down-regulated expression of ANXA1 in NPC cell lines to the level in the non-neoplastic cell line NP69.

Cytotoxic effect of saffron stigma aqueous extract on human transitional cell carcinoma and mouse fibroblast.

Saffron has been suggested to have inhibitory effects on tumoral cells. We evaluated the cytotoxic effect of aqueous extract of saffron on human transitional cell carcinoma (TCC) and mouse non-neoplastic fibroblast cell lines. Human TCC 5637 cell line and mouse fibroblast cell line (L929) were cultivated and incubated with different concentrations of aqueous extract of saffron stigma (50 microg/mL to 4000 microg/mL). Cytotoxic effect of saffron was evaluated by morphologic observation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay after 24, 48, 72, and 120 hours in each cell line. After 24 hours, morphological observations showed growth inhibitory effects at saffron extract concentrations higher than 200 microg/mL for L929 cells and at concentrations of 50 microg/mL to 200 microg/mL for the TCC cells. These changes became more prominent after 48 hours. However, significant growth inhibitory effects of the extract were shown at concentrations of 400 microg/mL and 800 microg/mL. Higher concentrations of saffron correlated inversely with cell population of both cell lines. Significant reduction of the survived cells was seen at concentrations of 400 microg/mL and 2000 microg/mL for TCC and L929 cell lines, respectively. After 120 hours, decrease in the percentage of survived cells at higher concentrations of saffron extract was seen in both cell lines. At a concentration of 800 microg/mL, the survived L929 cells plummeted to less than 60% after 120 hours, while no TCC cells survived at this time. No L929 cells survived at 2000 microg/mL. Saffron aqueous extract has inhibitory effects on the growth of both TCC 5637 and normal L929 cell lines. This effect is dose dependent.

Role of cell cycle events and apoptosis in mediating the anti-cancer activity of a silver(I) complex of 4-hydroxy-3-nitro-coumarin-bis(phenanthroline) in human malignant cancer cells

The central objective of the current study was to investigate the potential in vitro anti-proliferative effect of 4-hydroxy-3-nitro-coumarin (hncH), and the mixed-ligand silver (I) complex of 4-oxy-3-nitro-coumarin-bis(phenanthroline), [Ag(hnc)(phen) 2] using four human-derived model cell lines. In addition, selected mechanistic studies were carried out using the most sensitive of the four cell lines. Results obtained show that the complex could decrease the proliferation of all four cell lines including neoplastic renal and hepatic, namely A-498 and HepG 2 cells, respectively, along with two non-neoplastic renal and hepatic cell lines, HK-2 and Chang, respectively. Furthermore, non-neoplastic hepatic cells (Chang) appeared to be less sensitive to the effect of the complex, but this effect was not replicated in the non-neoplastic renal (HK-2) cells. Based on IC 50 values [Ag(hnc)(phen) 2] was shown to be almost four times more potent than cisplatin, using HepG 2 cells. In addition, the observed anti-proliferative effect was shown to be both dose- and time-dependent. Furthermore, the complex was shown to decrease DNA synthesis, but did not intercalate with it. Moreover, there was no evidence that P-glycoprotein-mediated multi-drug resistance was likely to decrease anti-proliferative activity. Cytological stains, analysis of genomic DNA, and biochemical assays [caspase-3 and -9 and cleaved poly(ADP-ribose)-polymerase protein] showed that cell death appeared to result from apoptosis, with the possibility of secondary necrosis. Additionally, flow cytometric analysis showed that the complex functioned through an alteration in cell cycle progression. Taken together, [Ag(hnc)(phen) 2] has been shown to be a more potent anti-proliferative agent than cisplatin, capable of altering key biochemical events leading to cell death. Additional mechanistic studies are underway to probe more fully its mechanism of action.

Repeated exposure to acid and bile selectively induces colonic phenotype expression in a heterogeneous Barrett's epithelial cell line

Barrett's epithelium is a precancerous, specialized columnar metaplasia in the distal esophagus. We demonstrate the changes in cellular phenotype in a non-neoplastic Barrett's cell line (BAR-T), following exposure to acid and bile salt, the two important components of gastroesophageal refluxate. Cell phenotypes in BAR-T cell line were quantified by fluorescence-activated cell sorting (FACS) using monoclonal antibodies against markers: cytokeratin 8/18 (CK8/18) for columnar, CK4 for squamous, mAbDas-1 for colonic epithelial cell phenotype and p75NTR for esophageal progenitors. Cells were exposed for 5 min each day to 200 μM glycochenodeoxycholic acid at pH 4, pH 6 and pH 7.4 or only to acid (pH 4) for up to 6 weeks. The BAR-T cell line comprised 35±5.2% CK8/18, 32±3.5% mAbDas-1, 9.5±3% CK4 and 4±2.5% p75NTR-positive cells. Single exposure to acid and or bile did not change cell phenotypes. However, chronic treatment for at least 2 weeks significantly enhanced (P<0.05) the expression of colonic phenotype and CK8/18-positive cells, as evidenced by FACS analysis. Bile salt at pH 4 and bile salt followed by acid (pH 4) in succession were the strongest stimulators (P<0.01) for induction of colonic phenotype cells. Squamous (CK4+) phenotype did not change by the treatments. Cox-2 expression was induced after acute treatment and increased to twofold during chronic treatment, particularly in response to acidic pH. We conclude that BAR-T cells can be utilized as an ‘in vitro' model to study the effect of environmental factors and their influence on the cellular phenotype and molecular changes in the pathogenesis of esophageal cancer.

Preferential induction of cytochrome P450 1A1 over cytochrome P450 1B1 in human breast epithelial cells following exposure to quercetin

Estrogen metabolism is suggested to play an important role in estrogen-induced breast carcinogenesis. Epidemiologic studies suggest that diets rich in phytoestrogens are associated with a reduced risk of breast cancer. Phytoestrogens are biologically active plant compounds that structurally mimic 17β-estradiol (E 2). We hypothesize that phytoestrogens, may provide protection against breast carcinogenesis by altering the expression of estrogen-metabolizing enzymes cytochrome P450 1A1 (Cyp1A1) and 1B1 (Cyp1B1). Cyp1A1 and Cyp1B1 are responsible for the metabolism of E 2 to generate 2-hydroxyestradiol (2-OHE 2) and 4-hydroxyestradiol (4-OHE 2), respectively. Studies suggest that 2-OHE 2 and 2-methoxyestradiol may protect against breast carcinogenesis, while 4-OHE 2 is carcinogenic in rodent models. Thus, agents that increase the metabolism of E 2 by Cyp1A1 to produce 2-OHE 2 may have chemoprotective properties. The human immortalized non-neoplastic breast cell line MCF10F was treated with quercetin at 10 and 50 μM concentrations for time points ranging from 3 to 48 h. Total RNA and protein were isolated. Real-time PCR was used to measure the expression of Cyp1A1 and Cyp1B1 mRNA. Quercetin treatment produced differential regulation of Cyp1A1 and Cyp1B1 mRNA expression in a time- and dose-dependent manner. Treatment with 10 and 50 μM doses of quercetin produced 6- and 11-times greater inductions of Cyp1A1 mRNA over Cyp1B1 mRNA, respectively. Furthermore, quercetin dramatically increased Cyp1A1 protein levels and only slightly increased Cyp1B1 protein levels in MCF10F cells. Thus, our data suggest that phytoestrogens may provide protection against breast cancer by modulating expression of estrogen-metabolizing genes such that production of the highly carcinogenic estrogen metabolite 4-OHE 2 by Cyp1B1 is reduced and the production of the less genotoxic 2-OHE 2 by Cyp1A1 is increased.

Neoplastic transformation of rat liver epithelial cells is enhanced by non-transferrin-bound iron

BackgroundIron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. While most iron circulates in blood as transferrin-bound iron, non-transferrin-bound iron (NTBI) also becomes elevated and contributes to toxicity in the setting of iron overload. The mechanism for iron-related carcinogenesis is not well understood, in part due to a shortage of suitable experimental models. The primary aim of this study was to investigate NTBI-related hepatic carcinogenesis using T51B rat liver epithelial cells, a non-neoplastic cell line previously developed for carcinogenicity and tumor promotion studies.MethodsT51B cells were loaded with iron by repeated addition of ferric ammonium citrate (FAC) to the culture medium. Iron internalization was documented by chemical assay, ferritin induction, and loss of calcein fluorescence. Proliferative effects were determined by cell count, toxicity was determined by MTT assay, and neoplastic transformation was assessed by measuring colony formation in soft agar. Cyclin levels were measured by western blot.ResultsT51B cells readily internalized NTBI given as FAC. Within 1 week of treatment at 200 μM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01). FAC alone induced little or no colony formation in soft agar. In contrast, FAC addition to cells previously initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in a concentration dependent increase in colony formation. This was first detected at 12 weeks of FAC treatment and increased at longer times. At 16 weeks, colony formation increased more than 10 fold in cells treated with 200 μM FAC (p < 0.001). The iron chelator desferoxamine reduced both iron uptake and colony formation. Cells cultured with 200 μM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1.ConclusionThese results establish NTBI as a tumor promoter in T51B rat liver epithelial cells. Changes in cyclin proteins suggest cell cycle disregulation contributes to tumor promotion by NTBI in this liver cell model.

The Role of Epithelial Cells in the Pathogenesis of Sjögren’s Syndrome

Sjögren's syndrome (SS) is a chronic autoimmune exocrinopathy that is characterized by periductal mononuclear cell infiltrates in the affected exocrine glands. Epithelial cells are thought to play an important pathogenetic role, as suggested by the occurrence of infiltrating lesions in various epithelial tissues (described as autoimmune epithelitis) as well as the increased epithelial expression of several inflammatory proteins in the histopathologic lesions of patients. In the recent decade, the application of long-term cultured nonneoplastic salivary gland epithelial cell (SGEC) lines has permitted the more explicit analysis of the role of these cells in SS pathophysiology. In such studies, cultured SGEC have been demonstrated to express constitutively or inducibly various molecules that are implicated in innate and acquired immune responses, a fact that underscores the inherent capacity of these epithelial cells to induce and promote chronic inflammatory reactions. Furthermore, the parallel analysis of SGEC lines obtained from SS patients and disease controls has revealed the significantly increased constitutive expression of several molecules in cells derived from SS patients. This fact strongly suggests the operation of intrinsic activation mechanisms in the epithelia of patients and further supports the active participation of epithelia in SS pathogenesis.

Regulation of COX‐2 Expression in Canine Prostate Carcinoma: Increased COX‐2 Expression is Not Related to Inflammation

Background: Cyclooxygenase‐2 (COX‐2) expression has been documented in human and canine prostate carcinoma (PCA). Canine PCA is a histologically heterogeneous tumor, sometimes including inflammatory infiltrates. However, it is unknown whether COX‐2 expression in canine PCA is related to the histologic type of tumor, to the presence of inflammation, or to both. Moreover, little is known about the mechanisms regulating COX‐2 expression in neoplastic tissue. Hypothesis: COX‐2 expression is related to the presence of inflammation in canine PCA and correlates with the degree of tumor differentiation. Methods: The expression of COX‐2 was examined in 28 cases of canine PCA by immunohistochemistry. In addition, a neoplastic and a nonneoplastic canine prostatic cell line were used to investigate the effects of interleukin‐6 (IL‐6), tumor necrosis factor‐ç (TNF‐ç), phorbol 12‐myristate 13‐acetate (PMA), epithelial growth factor (EGF), and specific signal transduction pathway inhibitors on COX‐2 expression. Results: Twenty‐four of the 28 prostate tumors showed COX‐2 expression. The presence of inflammatory infiltrates in tumor tissue was associated with lower COX‐2 expression scores. In vitro, TNF‐ç, IL‐6, and EGF increased COX‐2 expression in nonneoplastic cells but not in PCA cells, where baseline expression was high. COX‐2 expression in PCA cells could be suppressed by means of specific phosphatidyl inositol‐3 kinase (PI3K), protein kinase C (PKC), or inhibitor of extracellular signal‐related kinase (ERK/MAPK) inhibitors. Conclusions and Clinical Importance: COX‐2 is expressed in canine PCA; however, expression is not related to the presence of inflammatory infiltrates. This conclusion is further supported by the finding that the cytokines TNF‐ç and IL‐6 and their involved signaling pathways do not stimulate COX‐2 expression in malignant canine prostate cells.

Neoplastic and Non-neoplastic Cell Lines from a Malignant Peripheral Nerve Sheath Tumour of the Cervix of a Rat

A homotransplantable tumour (LY) and cell lines (LY-PPB6 and LY-H12) were established from a spontaneous malignant peripheral nerve sheath tumour (PNST) of the uterine cervix of an F344 rat. Primary and LY tumours consisted of oval or spindle-shaped cells arranged in a flatfield or streaming fashion, and indistinct nuclear palisades were seen. Immunohistochemically, neoplastic cells reacted to vimentin, S-100 protein, neuron-specific enolase (NSE), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) in varying degrees, indicating neurogenic derivation. LY-PPB6-induced tumours in syngeneic rats developed cellular whorling patterns reacting particularly strongly to S-100 protein, NSE, MBP and GFAP. Nerve growth factor (NGF) mRNA expression was shown in LY-PPB6 cells by the reverse transcription-polymerase chain reaction (RT-PCR). By contrast, LY-H12 had a normal chromosomal number of 42, and did not produce tumours when injected into syngeneic rats. LY-H12 cells reacted to vimentin and alpha-smooth muscle actin (alpha-SMA), and the alpha-SMA-positive cell number was increased dose-dependently by the addition of transforming growth factor (TGF)-beta1, indicating a myofibroblastic nature. LY-PPB6 cells were neoplastic with properties of PNS cells, whereas LY-H12 cells were non-neoplastic stromal cells showing myofibroblastic differentiation. As TGF-beta1 mRNA expression was shown in both LY-PPB6 and LY-H12 cells by the RT-PCR, the myofibroblastic phenotype of LY-H12 cells may be mediated by paracrine or autocrine signalling in tumour tissues. LY-PPB6 and LY-H12 may prove useful for studies on the pathobiological nature of neoplastic cells and interactions between neoplastic and stromal cells in PNSTs.

The transcription factor ATF5 is widely expressed in carcinomas, and interference with its function selectively kills neoplastic, but not nontransformed, breast cell lines

ATF5, a transcription factor important in differentiation, proliferation and survival, has been found to be highly expressed in neural progenitor cells and in certain tumors including glioblastomas (GBMs), but its expression in other normal and neoplastic tissues has not been extensively investigated. A tissue microarray immunostained for ATF5 showed diffuse nuclear expression (as defined by the presence in greater than 25% of cells) in 63% (117/186) of neoplastic samples, when compared to only 32% (20/62) in nonneoplastic tissues. When analyzed by histologic subtype, a significantly greater proportion of adenocarcinomas, transitional cell carcinomas, squamous cell carcinomas and metastatic carcinomas of various tissue origins had nuclear staining when compared to nonneoplastic tissues. There was no significant difference in ATF5 expression in renal cell carcinomas, lymphomas and seminomas, when compared to nonneoplastic tissues. An expanded series of nonarray breast resection specimens revealed a significantly greater proportion of ATF5 positivity in ductal and lobular carcinomas, when compared to normal breast tissue. Past work found that loss of ATF5 function triggers death of GBM cells, but not of normal activated astrocytes. Here, we observed that loss of ATF5 function caused significant apoptotic death of neoplastic breast cell lines, but not of nonneoplastic breast cell lines. Our data demonstrate elevated ATF5 expression in a wide variety of neoplasms and that interference with ATF5 function selectively triggers death of breast carcinoma cells. Such findings may have potential therapeutic application.