Intestinal Tumor Cells Research Articles

Chemopreventive effects of berberine on intestinal tumor development in Apc min/+mice

BackgroundBerberine, an isoquinoline alkaloid, has shown inhibitory effects on growth of several tumor cell lines in vitro. The aim of this study was to investigate chemopreventive effects of berberine on intestinal tumor development in Apcmin/+ mice.MethodsFour-week old Apcmin/+ mice were treated with 0.05% or 0.1% berberine in drinking water for twelve weeks. The number and the size of tumors were measured to evaluate intestinal tumor development. Tissue sections were prepared for PCNA and Ki-67 immunostaining to detect cell proliferation, and TUNEL assay and cleaved caspase-3 immunostaining for apoptosis. Western blot analysis and immunostaining were performed to detect the activation of Wnt and epidermal growth factor receptor (EGFR) signaling pathways and COX-2 expression in the intestinal tumor cells. The prostaglandin E2 level in the small intestine was detected using ELISA.ResultsCompared with untreated Apcmin/+ mice, the total numbers of tumors in the small intestine and the colon were reduced by 39.6% and 62.5% in 0.05% and 0.1% berberine-treated mice, respectively. The numbers of tumors in proximal, middle, and distal segments of the small intestine in 0.1% berberine-treated mice were significantly reduced by 53.7%, 55.3%, and 76.5% respectively. Berberine treatment also decreased the numbers of all sizes of tumors (>2 mm, 1–2 mm, and <1 mm) in the small intestine. Berberine suppressed tumor cell proliferation and increased apoptosis. Furthermore, berberine decreased the activation levels of Wnt and EGFR signaling pathways, and down-regulated COX-2 expression in intestinal tumor cells and prostaglandin E2 production in the small intestine.ConclusionsBerberine inhibits intestinal tumor development, which is correlated with its activity to suppress tumor cell proliferation and increase apoptosis in Apcmin/+ mice. Down-regulation of Wnt and EGFR signaling pathways and COX-2 expression by berberine may be involved in its anti-tumorigenic effects.

Breast Cancer Expression of DAP12 is Associated With Skeletal and Liver Metastases and Poor Survival

BackgroundThe transmembrane adapter protein, DAP12, transduces activation signals for several arrays of receptors, including human signal-regulatory protein, DAP12-associating lectin-1, triggering receptor expressed on myeloid cells-1, -2, and -3, in natural killer cells, granulocytes, monocytes/macrophages, and dendritic cells. The macrophage-specific antigen, Cluster of Differentiation 163 (CD163), is expressed in breast and colorectal cancers and is associated with early cancer recurrence and poor prognosis. It was recently shown that fusion between intestinal tumor cells and macrophages results in nuclear reprogramming with hybrid transcripts from both cells of origin. The role of DAP12 in the fusion process is not known. This study investigates the expression of DAP12 in BRC cells, and its relation to other macrophage traits and to the clinical progression of disease. Materials and MethodsImmunostaining of DAP12 and CD163 was performed and evaluated in paraffin-embedded specimens from 132 patients with BRC. The outcomes were analyzed in relation to clinicopathological data. ResultsDAP12 expression in cancer cells was positive in 66 percent of the cancers and was associated with high tumor grade (P = .015), and with liver (P = .047) and skeletal (P = .067), but not with lung metastases (P = 1.00). Patients with BRC expressing DAP12 had poor prognosis, with higher recurrence rates of skeletal (P = .018) and liver metastases (P = .047), and shorter survival time (P = .0060). ConclusionWe suggest that macrophage traits in BRC cells facilitate the metastatic process and that DAP12 expression might promote metastatic homing to bone and liver tissues.

Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors

Well-differentiated small intestinal neuroendocrine tumors are rare malignancies. They arise from enterochromaffin cells and very little is known about differential microRNA (miRNA) expression. The aim of this study was to identify the miRNA profile of well-differentiated small intestinal neuroendocrine tumors, which may have a critical role in tumor development, progression and potentially develop miRNAs as novel clinical biomarkers. Specimens from two test groups, 24 small intestinal neuroendocrine tumor specimens at different stages of malignancy, are included in this study. Total RNA from the first test group, five primary tumors, five mesentery metastases and five liver metastases was hybridized onto the Affymetrix Genechip miRNA arrays to perform a genome-wide profile. The results were validated by using quantitative real-time PCR (QRT-PCR) and northern blot analyses. We then expanded the investigation to laser capture microdissected small intestinal neuroendocrine tumor cells and immuno-laser capture microdissected normal enterochromaffin cells of the first test group. Furthermore, a second test group, three primary tumors, three mesentery metastases and three liver metastases, was included in the study. Thus, two independent test groups validated the data by QRT-PCR. Moreover, we characterized nine miRNAs, five (miR-96, -182, -183, -196a and -200a), which are upregulated during tumor progression, whereas four (miR-31, -129-5p, -133a and -215) are downregulated. Several online software programs were used to predict potential miRNA target genes to map a number of putative target genes for the aberrantly regulated miRNAs, through an advanced and novel bioinformatics analysis. Our findings provide information about pivotal miRNAs, which may lead to further insights into tumorigenesis, progression mechanisms and novel therapeutic targets recognition.

Christia vespertilionis plant extracts as novel antiproliferative agent against human neuroendocrine tumor cells

Neuroendocrine tumors respond poorly to radiation and conventional chemotherapy, hence surgical removal of the neoplastic tissue is still the most effective way of treatment. In an attempt to find new therapeutic plant extracts of Christia vespertilionis (CV) their antitumor potential in human medullary thyroid carcinoma (MTC) and human small intestinal neuroendocrine tumor (SI-NET) cell lines were tested. Proliferation and viability were analyzed using cell counting and WST-1 assay. Apoptosis was determined by microscopy, luminescence assays for caspases 3/7, and expression studies of apoptosis-related genes. CV extracts showed antiproliferative and proapoptotic effects in all MTC and SI-NET cell lines, whereby high growth inhibition was observed by treatment with the ethylacetate-extracts (CV-45) in tumor cell lines but not in normal human fibroblasts. Furthermore CV-45 treatment resulted in alterations of gene expression of PDCD5, MTDH and TNFRSF10b in MTC as well as in SI-NET cells. The results indicate that Christia vespertilionis could serve as an anticancer therapeutic for treatment of neuroendocrine tumors.

Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target

Human SMC2 is part of the condensin complex, which is responsible for tightly packaging replicated genomic DNA prior to segregation into daughter cells. Engagement of the WNT signaling pathway is known to have a mitogenic effect on cells, but relatively little is known about WNT interaction with mitotic structural organizer proteins. In this work, we described the novel transcriptional regulation of SMC2 protein by direct binding of the β-catenin·TCF4 transcription factor to the SMC2 promoter. Furthermore, we identified the precise region in the SMC2 promoter that is required for β-catenin-mediated promoter activation. Finally, we explored the functional significance of down-regulating SMC2 protein in vivo. Treatment of WNT-activated intestinal tumor cells with SMC2 siRNA significantly reduced cell proliferation in nude mice, compared with untreated controls (p = 0.02). Therefore, we propose that WNT signaling can directly activate SMC2 transcription as a key player in the mitotic cell division machinery. Furthermore, SMC2 represents a new target for oncological therapeutic intervention.

Expression of Cocaine- and Amphetamine-Regulated Transcript Is Associated with Worse Survival in Small Bowel Carcinoid Tumors

Cocaine- and amphetamine-regulated transcript (CART) peptide exerts several regulatory functions acting both as neurotransmitter and hormone. We recently showed that CART is expressed in various neuroendocrine tumors, including small bowel carcinoids. The main objective of the present study was to examine whether CART expression is associated with survival in patients with small bowel carcinoid. Secondary aims were to assess whether CART expression is associated with other tumor characteristics or clinical symptoms. Specimens from 97 patients with small bowel carcinoids were examined for CART expression using immunohistochemistry. A CART score was introduced on the basis of the proportion of CART immunoreactive cells. On inclusion, specimens were examined by routine histopathologic methods and detailed clinical patient data were retrieved. The effect of CART on cell viability was assessed in vitro using two intestinal tumor cell lines. Expression of CART (P = 0.011) and increasing CART score (P = 0.033) were associated with worse disease-specific survival. Adjusting for age, disease stage, and tumor grade in multivariable analysis, CART expression was still associated with worse survival [Low CART HR, 5.47; 95% confidence interval (CI), 0.71-42.46; and High CART HR, 9.44; 95% CI, 1.14-78.14]. CART expression was not associated with patient age, disease stage, tumor grade, or any presenting symptom. Supporting our clinical data, we found that CART promoted tumor cell viability in vitro in two different tumor cell lines. Expression of CART in small bowel carcinoid tumors is associated with worse survival.

Microencapsulation of small intestinal neuroendocrine neoplasm cells for tumor model studies

Basic cancer research is dependent on reliable in vitro and in vivo tumor models. The serotonin (5-HT) producing small intestinal neuroendocrine tumor cell line KRJ-1 has been used in in vitro proliferation and secretion studies, but its use in in vivo models has been hampered by problems related to the xeno-barrier and tumor formation. This may be overcome by the encapsulation of tumor cells into alginate microspheres, which can function as bioreactors and protect against the host immune system. We used alginate encapsulation of KRJ-1 cells to achieve long-term functionality, growth and survival. Different conditions, including capsule size, variations in M/G content, gelling ions (Ca(2+) /Ba(2+)) and microcapsule core properties, and variations in KRJ-1 cell condition (single cells/spheroids) were tested. Viability and cell growth was evaluated with MTT, and confocal laser scanner microscopy combined with LIVE/DEAD viability stains. 5-HT secretion was measured to determine functionality. Under all conditions, single cell encapsulation proved unfavorable due to gradual cell death, while encapsulation of aggregates/spheroids resulted in surviving, functional bioreactors. The most ideal spheroids for encapsulation were 200-350 μm. Long-term survival (>30 days) was seen with solid Ca(2+) /Ba(2+) microbeads and hollow microcapsules. Basal 5-HT secretion was increased (sixfold) after hollow microcapsule encapsulation, while Ca(2+) /Ba(2+) microbeads was associated with normal basal secretion and responsiveness to cAMP/PKA activation. In conclusion, encapsulation of KRJ-1 cells into hollow microcapsules produces a bioreactor with a high constitutively activate basal 5-HT secretion, while Ca(2+) /Ba(2+) microbeads provide a more stable bioreactor similar to non-encapsulated cells. Alginate microspheres technology can thus be used to tailor different functional bioreactors for both in vitro and in vivo studies.

Abstract 5223: The role of bone-marrow derived cells in the intestinal tumor microenvironment of apcmin/+ mouse on tumor response to inhibitors of thymidylate synthase

Abstract The vast majority of chemotherapeutic agents have been developed to specifically target actively dividing neoplastic cells. However, tumors are not exclusively composed of a heterogeneous population of neoplastic cells. They also include a heterogeneous collection of host derived infiltrating non-neoplastic cells that together with the extracellular matrix and secreted molecules compose the tumor stroma or microenvironment. Multiple interactions between stromal and tumor cells play a critical role in tumor growth and progression. Therefore, tumor response to chemotherapy and recurrence post-therapy must also be influenced by these interactions. For many decades, inhibitors of the enzyme thymidylate synthase (TS), such as fluoropyrimidines, have been used in the clinical management of various cancers; however, their clinical efficacy continues to be limited by their toxicity to normal cells and acquired resistance by tumor cells. Therapeutic efficacy is often measured by its impact on tumor burden, however, the impact of TS inhibitors on tumor stromal cells and their role on tumor response to therapy is largely unknown. The goal of this project is to determine the role of bone marrow derived tumor stromal cells (BMDCs) in mediating tumor response to systemic therapy with TS inhibitors. We hypothesize that, in addition to targeting tumor cells, TS inhibitors also directly target stromal cells and that the sensitivity of stromal cells to the therapy determines tumor response. Using the ApcMin/+ mouse as a model of intestinal tumorigenesis, we identified and characterized BMDCs in the intestinal tumor stroma. These included Macrophages, Fibroblasts, Pan T cells, Helper T cells, Leukocytes, Mast cells, T-regulatory cells (T-reg) and Myeloid derived suppressor cells (MDSCs), among others. We generated chimeric mice wherein the chemosensitivity of intestinal tumor cells to TS inhibitors was distinct from that of the BMDCs by transplanting eGFP positive bone marrow cells that were genetically modified to express a drug resistant variant of TS. We examined the effect of 5-FU on MDSCs, mast cells, and T-regs, that have previously been shown to promote intestinal tumor development in ApcMin/+ mice. The results showed that MDSCs were specifically sensitive to 5-FU due to its apoptotic effect on these cells. Current studies are aimed at identifying the combination of stromal cells that mediate tumor response to TS inhibitors in order to identify cellular targets for combined therapy that would enhance reduction of tumor burden or delay tumor recurrence. 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 5223. doi:1538-7445.AM2012-5223

β-catenin/TCF4 complex induces the epithelial-to-mesenchymal transition (EMT)-activator ZEB1 to regulate tumor invasiveness

In most carcinomas, invasion of malignant cells into surrounding tissues involves their molecular reprogramming as part of an epithelial-to-mesenchymal transition (EMT). Mutation of the APC gene in most colorectal carcinomas (CRCs) contributes to the nuclear translocation of the oncoprotein β-catenin that upon binding to T-cell and lymphoid enhancer (TCF-LEF) factors triggers an EMT and a proinvasive gene expression profile. A key inducer of EMT is the ZEB1 transcription factor whose expression promotes tumorigenesis and metastasis in carcinomas. As inhibitor of the epithelial phenotype, ZEB1 is never present in the epithelium of normal colon or the tumor center of CRCs where β-catenin remains membranous. We show here that ZEB1 is expressed by epithelial cells in intestinal tumors from human patients (familial adenomatous polyposis) and mouse models (APC(Min/+)) with germline mutations of APC that result in nuclear accumulation of β-catenin. However, ZEB1 is not expressed in the epithelium of hereditary forms of CRCs that carry wild-type APC and where β-catenin is excluded from the nucleus (Lynch syndrome). We found that β-catenin/TCF4 binds directly to the ZEB1 promoter and activates its transcription. Knockdown of β-catenin and TCF4 in APC-mutated CRC cells inhibited endogenous ZEB1, whereas forced translocation of β-catenin to the nucleus in APC-wild-type CRC cells induced de novo expression of ZEB1. Upregulation of MT1-MMP and LAMC2 by β-catenin/TCF4 has been linked to invasiveness in CRCs, and we show here that both proteins are activated by ZEB1 coexpressing with it in primary colorectal tumors with mutated APC. These results set ZEB1 as an effector of β-catenin/TCF4 signaling in EMT and tumor progression.

A water soluble tri-cationic porphyrin–EDTA conjugate induces apoptosis in human neuroendocrine tumor cell lines

In this study, a completely water soluble tri-cationic porphyrin–EDTA conjugate was synthesized. We present data demonstrating the tumoristatic effects of the novel fully water soluble cationic porphyrin TMPy3PhenEDTA-P-Cl4 in the dark, in the medullary thyroid carcinoma cell lines MTC-SK and SHER-I and weaker effects in the small intestinal neuroendocrine tumor cell line KRJ-I. In addition, cytotoxic effects were also studied in normal human fibroblasts that represent normal tissue and the results are compared to the tumor cell lines.

AP1B plays an important role in intestinal tumorigenesis with the truncating mutation of an APC gene

Recent evidence has suggested that carcinoma is accompanied by the loss of cell polarity. An epithelial cell-specific form of the AP-1 clathrin adaptor complex, AP1B, is involved in the polarized transport of membrane proteins to the basolateral surface of epithelial cells. In our study, we investigated whether AP1B is involved in intestinal tumorigenesis. The cellular polarity of intestinal tumor cells was examined using APC(Min/+) mice as an in vivo model and SW480 cells with a truncating mutation in the adenomatous polyposis coli (APC) gene as an in vitro model by confocal microscopy. Next, the expression of AP1B in intestinal tumor cells was examined by real-time polymerase chain reaction (PCR) and Western blotting. The localization of β-catenin and the expression of AP1B in the tumor tissue of patients with colorectal cancer were evaluated by confocal microscopy and real-time PCR, respectively, and the relationships among cell polarity, AP1B expression and intestinal tumorigenesis were examined. Cellular polarity was lost in intestinal tumor cells, and the expression of AP1B was downregulated. In addition, the reduction in the expression level of AP1B correlated with the nuclear localization of β-catenin in human colorectal cancer. Our study indicates the close associations between AP1B, intestinal tumorigenesis and mutations in the APC gene. This is the first report to reveal the relationships among AP1B, cellular polarity and intestinal tumorigenesis, and achieving a detailed understanding of AP1B will hopefully lead to discovery of therapeutic targets and novel biomarkers for intestinal cancer.

Abstract 2408: Generation of an inducible Wnt5a transgenic mouse model to study the contribution of increased Wnt5a expression to intestinal tumor growth

Abstract Introduction: The importance of the interaction between tumor cells and stromal tissue for tumor invasion and metastasis is becoming well-recognized lately. Noncanonical Wnt5a ligand is expressed at increased levels by stromal cells in intestinal tumors. Despite well known importance of Wnt signaling in colorectal cancer, Wnt5a is one out of only two Wnt ligands described to be upregulated in gastrointestinal tumors. In vitro data suggest involvement of Wnt5a in tumor cell migration, angiogenesis and epithelial to mesenchymal transition. At present, it is unclear however to which extent and through which mechanisms Wnt5a contributes to the malignant behavior of colorectal tumors in vivo. Therefore, we have generated a mouse model in which expression of Wnt5a can be controlled in a tissue-specific and temporal fashion. Methods: We generated a transgenic mouse line carrying the Wnt5a gene driven by a doxycycline (dox)-responsive promoter, which was crossed with hnRNP-rtTA animals expressing the rtTA2 transcription factor in all their tissues. In this way a model was generated in which Wnt5a expression can be induced by dox administration in double transgenic animals. Subsequently, double transgenics were crossed with Apc1638N mice that develop gastrointestinal tumors spontaneously. These mice were provided with dox in their drinking water from the age of 3-8 months, where after they were analyzed for the development of intestinal tumors. Results: We identified transgenic lines showing a tightly controllable dox-induced expression of Wnt5a in vitro and in vivo. Whereas induced Wnt5a expression appears well-tolerated in adult mice, the induction during embryonic development results in a severe embryonic phenotype. Induction of transgenic Wnt5a expression in the developing gastrointestinal tumors is successful. Despite this, the enhanced Wnt5a expression appears not to affect tumor number, tumor size or degree of malignancy. We observe a minor shift in the distribution pattern of the tumors towards a more distal intestinal location. Analysis of tumor cell proliferation and stromal content of the tumors shows no alteration upon Wnt5a induction. Conclusions: We have successfully generated a transgenic model in which robust and tightly controllable Wnt5a expression can be induced upon dox administration. Although induced Wnt5a expression appears to be well-tolerated in adult animals, embryonic development is severely affected. Additionally we have successfully induced Wnt5a expression within the intestinal tumors developing in an Apc mutant model. Our results suggest that increased Wnt5a expression has no major effect on intestinal tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2408. doi:10.1158/1538-7445.AM2011-2408

Abstract 2396: Function of HLTF in the development of colon cancer and in the maintenance of genomic stability

Abstract HLTF (Helicase like Transcription Factor) is a DNA helicase protein which is homologous to SNF/SWI family. It has also been shown to be a functional homolog of yeast rad5, required for the maintenance of genomic stability through the ubiquitination of proliferating cell nuclear antigen (PCNA). Although the physiologic role of HLTF is largely unknown, inactivation of HLTF by promoter methylation has been demonstrated in more than 40% human colon cancers. In this study, we have applied mouse transgenic approaches to determine whether loss of HLTF function could be important for colorectal carcinogenesis. HLTF knockout mice were created by the deletion of first 5 exons of HLTF genome. The complete loss of HLTF expression in these mice was confirmed by Northern blot and RT-PCR assays. HLTF knockout mice did not show any developmental defects within a 2-year observation, indicating that HLTF is dispensable for normal development, and loss of HLTF function alone is not sufficient to drive colon carcinogenesis. To determine whether loss of HLTF function could cooperate with other tumor suppressors in the formation of colon cancers, we have bred HLTF knockout mice with the mutant mice for p53 and Apc (adenomtous polyposis coli). In HLTF−/−/Apc+/− mice, the significantly increased formation of intestinal adenocarcinoma and colon cancers were observed. Although very few HLTF−/−/p53−/− mice developed colon cancers, these mice had increased incidence of lymphoma formation. This finding suggests that loss of HLTF function could not only promote colon cancer formation, but also for other types of tumor. Cytogenetic analysis of colon cancer cells derived from HLTF−/−Apc+/− mice demonstrated a high incidence of gross chromosomal instabilities, including Robertsonian fusions, fragments and aneuploidy. All these genetic alterations were not observed in the intestinal tumor cells from Apc+/−, implicating that loss of HLTF function could induce genomic instability which contributes to colorectal carcinogenesis. To further establish the role of HLTF in the maintenance of genomic stability, we have also derived HLTF deficient mouse embryonic stem (ES) cells, and demonstrated that HLTF−/− ES cells exhibited similar, but a milder genetic alterations as observed in HLTF deficient colon cancer cells. Taken together, our work provides direct genetic evidence that loss of HLTF function as observed in a majority of human colon cancers is able to promote colorectal carcinogenesis. This function could be mediated by the induced genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2396. doi:10.1158/1538-7445.AM2011-2396

JNK Signaling Promotes Intestinal Tumorigenesis Through Activation of mTOR Complex 1 in ApcΔ716 Mice

Signaling by the mammalian target of rapamycin complex 1 (mTORC1) has been implicated in various human cancers. mTORC1 signaling is activated in intestinal tumors of adenomatous polyposis coli (Apc(Δ716)) mice, a model of familial adenomatous polyposis; in these mice, the mTORC1 inhibitor RAD001 can block tumor formation. However, the precise mechanism of mTORC1 signaling in intestinal tumors is not clear. We investigated whether c-Jun-NH(2) terminal kinase (JNK) is involved in the mTORC1 activation. We investigated the effects of an inhibitor and an activator of JNK, as well as small interfering RNA against JNK, on mTORC1 in Apc(Δ716) mice and colon cancer cell lines. We also determined the role of JNK in mTORC1 signaling using in vitro kinase assays. JNK was activated in intestinal polyps of Apc((Δ716) mice); the JNK inhibitor SP600125 significantly suppressed tumor formation. In colorectal cancer cell lines, the JNK activator anisomycin activated mTORC1, whereas SP600125 or small interfering RNAs against JNK suppressed signaling. Importantly, JNK stimulated the mTORC1 kinase activity in vitro, through direct phosphorylation of Raptor at serine 863. JNK is required for activation of mTORC1 in intestinal tumor cells. JNK inhibitors might be developed as therapeutics or to prevent development of intestinal tumors.

S1680 Mechanism of Expression Alterations of Keratins and Vimentin in Intestinal Tumor Cell Lines During Epithelial-Mesenchymal Transition

S1680 Mechanism of Expression Alterations of Keratins and Vimentin in Intestinal Tumor Cell Lines During Epithelial-Mesenchymal Transition

The integrated stress response-associated signals modulates intestinal tumor cell growth by NSAID-activated gene 1 (NAG-1/MIC-1/PTGF- )

Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2alpha) is a critical convergence point of the integrated stress response (ISR), which supports eukaryotic cellular adaptation to diverse stressful conditions, including the endoplasmic reticulum (ER) stress by global protein translational arrest and induction of numerous stress-triggered cytoprotective genes. Challenge with non-steroidal anti-inflammatory drug (NSAID) leads to ER perturbation that may sensitize cancer cells to drug-induced apoptosis. Here, we examined the ER stress signals in the context of NSAID exposure and the induction of the critical tumor suppressor, NSAID-activated gene 1 (NAG-1), in the epithelial cancer cells. Sulindac sulfide, the active sulindac metabolite, was shown to trigger the ISRs via eIF2alpha kinase such as RNA-dependent protein kinase-related endoplasmic reticulum kinase (PERK) and RNA-dependent protein kinase (PKR). ER stress markers such as glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and activating transcription factor (ATF)-3 were enhanced by sulindac sulfide in colon cancer cells. In these cells, the PERK-activated ATF3-CHOP signaling pathway mediated the gene expression of pro-apoptotic NAG-1- and NSAID-induced apoptosis. In contrast, PKR protein was not involved in the signaling cascade for the gene expression of CHOP-linked NAG-1. Instead, PKR mediated activation of pro-survival extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, which was enhanced by NAG-1 suppression in response to cytotoxic sulindac sulfide exposure. PKR-ERK1/2 activation may thus contribute to the defensive cellular response to cytotoxic NSAIDs while drug-mediated ER stress triggers the pro-apoptotic NAG-1 production in human colon cancer cells.

The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

BackgroundIgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium.Methodology/Principal FindingsFcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains.Conclusions/SignificanceOur data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested.

Asymmetrically substituted cationic indole- and fluorene porphyrins inhibit tumor proliferation in small intestinal neuroendocrine tumors and medullary thyroid carcinomas

In this study we demonstrate anticancer activity of novel fully water soluble cationic porphyrins. The two cationic porphyrins 5,10,15-tris(N-methylpyridinium-4-yl)-20-[1-phenyl-4-(3-N-phenylsulfonylindolyl)]-21H,23H-porphyrin chloride (TMPy3PhenIndolprot1P–Cl3) and 5-{5-[2-(9,9-Dimethyl)fluorenyl]-N-methylpyridinium-3-yl}-10,15,20-tris(N-methyl-pyridinium-4-yl)-21H,23H-porphyrin chloride (TMPy3PyFluorenyl1P–Cl4) were prepared and their antiproliferative effects were studied in two human tumor cell lines and a normal human fibroblast cell line. Effects of the novel porphyrin compounds were evaluated in the small intestinal neuroendocrine tumor cell line KRJ-I, the medullary thyroid carcinoma cell line MTC-SK and the normal human fibroblast cell line HF-SAR by cell counting, cell proliferation assays and cell cytotoxicity analyses. TMPy3PhenIndolprot1P–Cl3 and TMPy3PyFluorenyl1P–Cl4 showed antiproliferative effects in the tumor cell lines MTC-SK and KRJ-I; cell viability was decreased and cytotoxic effects were quantified, while no significant alterations of the human fibroblasts were noted. With the advantage of full water solubility and antiproliferative effects in tumor cell lines, the novel porphyrin compounds TMPy3PhenIndolprot1P–Cl3 and TMPy3PyFluorenyl1P–Cl4 could be a new therapeutic option in anticancer treatment.

Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells

Casein phosphopeptides (CPPs), originating by in vitro and/or in vivo casein digestion, are characterized by the ability to complex and solubilize calcium ions preventing their precipitation. Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines, are able to re-mineralize carious lesions in a dental enamel, and, as components of a diet, affect bone weight and calcium content in rats. The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization. Primary human osteoblast-like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery. Commercial mixtures of bovine casein phosphopeptides were used. The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2-fluorescence assays. Results show that CPPs: (i) stimulate calcium uptake by primary human osteoblast-like cells; (ii) increase the expression and activity of alkaline phosphatase, a marker of human osteoblast differentiation; (iii) affect the cell proliferation rate and the apoptotic level; (iv) enhance nodule formation by human SaOS-2. Taken together these results confirm the possibility that CPPs play a role as modulator of bone cell activity, probably sustained by their ability as calcium carriers. Although the exact mechanism by which CPPs act remains not completely clarified, they can be considered as potential anabolic factors for bone tissue engineering.

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.