Angela Maria Acquaviva

IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells

Nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer and this effect is mediated in part through inhibition of type 2 prostaglandin endoperoxide synthase/cyclo-oxygenase (COX-2). In the present study, we demonstrate that COX-2 expression and PGE2 synthesis are up-regulated by an IGF-II/IGF-I receptor autocrine pathway in Caco-2 colon carcinoma cells. COX-2 mRNA and PGE2 levels are higher in proliferating cells compared with post-confluent differentiated cells and in cells that constitutively overexpress IGF-II. Up-regulation of COX-2 expression by IGF-II is mediated through activation of IGF-I receptor because: (i) treatment of Caco-2 cells with a blocking antibody to the IGF-I receptor inhibits COX-2 mRNA expression; (ii) transfection of Caco-2 cells with a dominant negative IGF-I receptor reduces COX-2 expression and activity. Also, the blockade of the PI3-kinase, that mediates the proliferative effect of IGF-I receptor in Caco-2 cells, inhibits IGF-II-dependent COX-2 up-regulation and PGE2 synthesis. Moreover, COX-2 expression and activity inversely correlate with the increase of apoptosis in parental, IGF-II and dominant-negative IGF-I receptor transfected cells. This study suggests that induction of proliferation and tumor progression of colon cancer cells by the IGF-II/IGF-I receptor pathway may depend on the activation of COX-2-related events.

Inhibitory effects of anti-CXCR4 antibodies on human colon cancer cells.

Background: CXCR4, the chemokine receptor for CXCL12, has recently been involved in the metastatic process of several neoplasms. Materials and methods: The expression of CXCR4 was evaluated by immunohistochemistry of colorectal tissue samples and by flow cytometry on Caco2, GEO, SW480, SW48, Lovo and SW620 human colon carcinoma cell lines. Correlations with pathological characteristics of the specimens were analysed with chi-square test. To verify the functional status of CXCR4, cell lines were tested in adhesion, migration, and proliferation assays. Results: We studied the expression of CXCR4 in 88 human colorectal tissues and we found that CXCR4 was expressed in >10% of epithelial cells in 50% of normal mucosae (7/14), in 55% of polyps (29/53), in all of carcinomas (16/16) and hepatic metastasis (5/5). Notably, CXCR4 was significantly over-expressed in cancerous lesions (carcinomas and metastasis) compared to non-cancerous lesions (normal mucosa and polyps) (P=0.003) and in adenomatous polyps versus hyperplastic polyps (P=0.009). The diameter of a polyp was also significantly associated with CXCR4 expression (P=0.031). SW480, SW48 and SW620 cell lines showed the highest levels of CXCR4 (60–80% of positive cells). Adhesion, migration, and proliferation increased in response to the CXCL12 chemokine. These effects were abrogated by the addition of anti-CXCR4 antibodies. Further, CXCL12 activated ERK1/2 in SW480 cells. Conclusions: These data suggest that CXCR4 might play a role in colon cancer cell properties and that anti-CXCR4 antibodies could have therapeutic effects against colorectal cancer.

Effect of aspirin on cell proliferation and differentiation of colon adenocarcinoma Caco‐2 cells

Several lines of evidence suggest that long‐term treatment with non‐steroidal anti‐inflammatory drugs may reduce the risk of colon cancer and the size and number of colonic polyps in patients with familial adenomatous polyposis. Aspirin has also been shown to inhibit cell proliferation in human tumor cell lines and to induce apoptosis in colonic mucosa of familial polyposis patients. To elucidate the molecular mechanisms of the antiproliferative action of aspirin, we studied the effects of aspirin on cell growth and differentiation of the human colon carcinoma Caco‐2 cell line. These cells represent a useful tool for studying the mechanisms involved in the regulation of cell growth and differentiation of intestinal epithelial cells since they spontaneously differentiate into polarized cells, expressing brush border enzymes. We show in this study that aspirin (0.1–10 mM) induces a profound inhibition of cell replication as assessed either by cell counts or thymidine incorporation. Moreover, aspirin concentrations of 5 and 10 mM induce apoptosis, whereas concentrations of 1 and 2 mM do not. The inhibition of growth is associated with a dose‐dependent reduction in insulin‐like growth factor II mRNA expression and with an increase in sucrase activity (a brush border enzyme) and apolipoprotein A‐I mRNA expression, 2 specific markers of the differentiative status of this cell line. Our data thus show that aspirin‐dependent inhibition of cell growth is associated with the enterocyte‐like differentiation of Caco‐2 cells. Int. J. Cancer 73:880–884, 1997.

Extra-mitochondrial localisation of frataxin and its association with IscU1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2.

Friedreichs ataxia is a recessive neurodegenerative disease due to insufficient expression of the mitochondrial protein frataxin. Although it has been shown that frataxin is involved in the control of intracellular iron metabolism, by interfering with the mitochondrial biosynthesis of proteins with iron/sulphur (Fe/S) clusters its role has not been well established. We studied frataxin protein and mRNA expression and localisation during cellular differentiation. We used the human colon adenocarcinoma cell line Caco-2, as it is considered a good model for intestinal epithelial differentiation and the study of intestinal iron metabolism. Here we report that the protein, but not the mRNA frataxin levels, increase during the enterocyte-like differentiation of Caco-2 cells, as well as in in-vivo-differentiated enterocytes at the upper half of the crypt-villus axis. Furthermore, subcellular fractionation and double immunostaining, followed by confocal analysis, reveal that frataxin localisation changes during Caco-2 cell differentiation. In particular, we found an extramitochondrial localisation of frataxin in differentiated cells. Finally, we demonstrate a physical interaction between extramitochondrial frataxin and IscU1, a cytoplasmic isoform of the human Fe/S cluster assembly machinery. Based on our data, we postulate that frataxin could be involved in the biosynthesis of iron-sulphur proteins not only within the mitochondria, but also in the extramitochondrial compartment. These findings might be of relevance for the understanding of both the pathogenesis of Friedreichs ataxia and the basic mechanism of Fe/S cluster biosynthesis.

Anti-proliferative effect of rhein, an anthraquinone isolated from Cassia species, on Caco-2 human adenocarcinoma cells

In recent years, the use of anthraquinone laxatives, in particular senna, has been associated with damage to the intestinal epithelial layer and an increased risk of developing colorectal cancer. In this study, we evaluated the cytotoxicity of rhein, the active metabolite of senna, on human colon adenocarcinoma cells (Caco‐2) and its effect on cell proliferation. Cytotoxicity studies were performed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), neutral red (NR) and trans‐epithelial electrical resistance (TEER) assays whereas 3H‐thymidine incorporation and Western blot analysis were used to evaluate the effect of rhein on cell proliferation. Moreover, for genoprotection studies Comet assay and oxidative biomarkers measurement (malondialdehyde and reactive oxygen species) were used. Rhein (0.1–10 μg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco‐2 cells. Rhein (0.1 and 1 μg/ml) significantly reduced cell proliferation as well as mitogen‐activated protein (MAP) kinase activation; by contrast, at high concentration (10 μg/ml) rhein significantly increased cell proliferation and extracellular‐signal‐related kinase (ERK) phosphorylation. Moreover, rhein (0.1–10 μg/ml): (i) did not adversely affect the integrity of tight junctions and hence epithelial barrier function; (ii) did not induce DNA damage, rather it was able to reduce H2O2‐induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and reactive oxygen species (ROS) levels induced by H2O2/Fe2+. Rhein was devoid of cytotoxic and genotoxic effects in colon adenocarcinoma cells. Moreover, at concentrations present in the colon after a human therapeutic dosage of senna, rhein inhibited cell proliferation via a mechanism that seems to involve directly the MAP kinase pathway. Finally, rhein prevents the DNA damage probably via an anti‐oxidant mechanism.

Cell-Free Synthesis of Rat Insulin-like Growth Factor II

Total RNA extracted from a rat liver cell line (BRL 3A) that synthesizes rat insulin-like growth factor II (IGF-II) was translated in a reticulocyte lysate cell-free system. Incubation of the translation products with antisera to mature rat IGF-II (Mr 8700) selectively immunoprecipitated a Mr 21,600 protein. We propose that this protein represents pre-pro-IGF-II.

Up-regulation of prostaglandin biosynthesis by leukotriene C4 in elicited mice peritoneal macrophages activated with lipopolysaccharide/interferon-γ

Leukotrienes (LT) and prostaglandins (PG) are proinflammatory mediators generated by the conversion of arachidonic acid via 5‐lipoxygenase (5‐LO) and cyclooxygenase (COX) pathways. It has long been proposed that the inhibition of the 5‐LO could enhance the COX pathway leading to an increased PG generation. We have found that in in vitro models of inflammation, such as mice‐elicited peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon‐γ (IFN‐γ), the deletion of the gene encoding for 5‐LO or the enzyme activity inhibition corresponded to a negative modulation of the COX pathway. Moreover, exogenously added LTC4, but not LTD4, LTE4, and LTB4, was able to increase PG production in stimulated cells from 5‐LO wild‐type and knockout mice. LTC4 was not able to induce COX‐2 expression by itself but rather potentiated the action of LPS/IFN‐γ through the extracellular signal‐regulated kinase‐1/2 activation, as demonstrated by the use of a specific mitogen‐activated protein kinase (MAPK) kinase inhibitor. The LT‐induced increase in PG generation, as well as MAPK activation, was dependent by a specific ligand‐receptor interaction, as demonstrated by the use of a cys‐LT1 receptor antagonist, although also a direct action of the antagonist used, on PG generation, cannot be excluded. Thus, the balance between COX and 5‐LO metabolites could be of great importance in controlling macrophage functions and consequently, inflammation and tumor promotion.

Effect of non-steroidal anti-inflammatory drugs on colon carcinoma Caco-2 cell responsiveness to topoisomerase inhibitor drugs

Numerous studies demonstrate that the chemopreventive effect of non-steroidal anti-inflammatory drugs on colon cancer is mediated through inhibition of cell growth and induction of apoptosis. For these effects non-steroidal anti-inflammatory drugs have been recently employed as sensitising agents in chemotherapy. We have shown previously that treatments with aspirin and NS-398, a cyclo-oxygenase-2 selective inhibitor, affect proliferation, differentiation and apoptosis of the human colon adenocarcinoma Caco-2 cells. In the present study, we have evaluated the effects of aspirin and NS-398 non-steroidal anti-inflammatory drugs on sensitivity of Caco-2 cells to irinotecan (CPT 11) and etoposide (Vp-16) topoisomerase poisons. We find that aspirin co-treatment is able to prevent anticancer drug-induced toxicity, whereas NS-398 co-treatment poorly affects anticancer drug-induced apoptosis. These effects correlate with the different ability of aspirin and NS-398 to interfere with cell cycle during anticancer drug co-treatment. Furthermore, aspirin treatment is associated with an increase in bcl-2 expression, which persists in the presence of the anticancer drugs. Our data indicate that aspirin, but not NS-398, determines a cell cycle arrest associated with death suppression. This provides a plausible mechanism for the inhibition of apoptosis and increase in survival observed in anticancer drug and aspirin co-treatment.

Cell cycle block at G1-S or G2-M phase correlates with differentiation of Caco-2 cells: Effect of constitutive insulin-like growth factor II expression

BACKGROUND & AIMS We have previously shown that autocrine insulin-like growth factor (IGF)-II synthesis through IGF-I receptor stimulates proliferation and inhibits differentiation of Caco-2 cells. To demonstrate whether differentiation of Caco-2 cells is dependent on cell growth status, we analyzed the effect of cell cycle arrest on differentiation of wild-type and IGF-II-overexpressing cells. METHODS Cells were treated with drugs that inhibit the progression either to S phase (l-b-D-arabinofuranosylcytosine or M phase (nocodazole). Cell differentiation was analyzed by assessing apolipoprotein A-1 and sucrase-isomaltase expression. Cell proliferation and DNA content were assessed by thymidine incorporation and fluorescence-activated cell sorter analysis, respectively. Cell cycle regulatory molecules were analyzed by assessing p21 and retinoplasma protein (pRb) expression and pRb phosphorylation. RESULTS Cell cycle block at G1-S phase was associated with increased expression of differentiation markers in both parental and IGF-II-transfected cells. On the contrary, cell cycle arrest at G2-M phase correlated with the expression of differentiation markers in parental but not in IGF-II-transfected cells. Constitutive IGF-II-expressing cells actively incorporated thymidine and showed an increase in the proportion of cells with >4N DNA ploidy in the presence of nocodazole. Nocodazole treatment of constitutive IGF-II-expressing cells stimulated p21 expression in the presence of hyperphosphorylated pRb. CONCLUSIONS The data show that cell cycle arrest increases differentiation of Caco-2 cells. IGF-II-mediated proliferation may prevent cell differentiation through effects on control cell checkpoint proteins.

HLA class II molecules transduce accesory signals affecting the CD3 but not the interleukin-2 activation pathway in T blasts

MHC class II molecules play a central role in the control of the immune response, but their biologic function and mechanism of action on the surface of activated human T lymphocytes are not entirely understood. In our study, the functional role of HLA class II molecules in T-blast proliferation was investigated by analyzing in parallel the IL-2- and CD3-driven activation pathways. The results indicate that the cross-linking of class II and CD3 molecules significantly increased the CD3-mediated T-blast proliferation, while no effect was observed on the IL-2-driven cell activation. This phenomenon was not confined to either CD4+ or CD8+ subsets nor was specifically affected by CD45 triggering. Biochemical studies showed that signaling via MHC class II molecules in T blasts led to PKC membrane translocation and IP accumulation. The simultaneous triggering of CD3 and HLA class II molecules led to a synergistic effect on IP accumulation but did not increase the CD3-mediated PKC membrane translocation. Our data suggest that HLA class II molecules are involved in T-cell-T-cell interactions and can mediate accessory signals, affecting the T-lymphocyte activation state.