Aluminum Enters Mammalian Cells and Destabilizes Chromosome Structure and Number.

Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum-in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues-incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.

Genomic Instability Is an Early Event in Aluminium-Induced Tumorigenesis.

Genomic instability is generally considered as a hallmark of tumorigenesis and a prerequisite condition for malignant transformation. Aluminium salts are suspected environmental carcinogens that transform mammary epithelial cells in vitro through unknown mechanisms. We report here that long-term culture in the presence of aluminium chloride (AlCl3) enables HC11 normal mouse mammary epithelial cells to form tumours and metastases when injected into the syngeneic and immunocompetent BALB/cByJ strain. We demonstrate that AlCl3 rapidly increases chromosomal structural abnormalities in mammary epithelial cells, while we failed to detect direct modulation of specific mRNA pathways. Our observations provide evidence that clastogenic activity-a well-recognized inducer of genomic instability-might account in part for the transforming abilities of aluminium in mammary epithelial cells.

Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells.

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti-acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long-term exposure to concentrations of aluminium-in the form of aluminium chloride (AlCl3 )-in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl3 form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl3 . Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well-established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens.

Cooperative expression of junctional adhesion molecule-C and -B supports growth and invasion of glioma.

Brain invasion is a biological hallmark of glioma that contributes to its aggressiveness and limits the potential of surgery and irradiation. Deregulated expression of adhesion molecules on glioma cells is thought to contribute to this process. Junctional adhesion molecules (JAMs) include several IgSF members involved in leukocyte trafficking, angiogenesis, and cell polarity. They are expressed mainly by endothelial cells, white blood cells, and platelets. Here, we report JAM-C expression by human gliomas, but not by their normal cellular counterpart. This expression correlates with the expression of genes involved in cytoskeleton remodeling and cell migration. These genes, identified by a transcriptomic approach, include poliovirus receptor and cystein-rich 61, both known to promote glioma invasion, as well as actin filament associated protein, a c-Src binding partner. Gliomas also aberrantly express JAM-B, a high affinity JAM-C ligand. Their interaction activates the c-Src proto-oncogene, a central upstream molecule in the pathways regulating cell migration and invasion. In the tumor microenvironment, this co-expression may thus promote glioma invasion through paracrine stimuli from both tumor cells and endothelial cells. Accordingly, JAM-C/B blocking antibodies impair in vivo glioma growth and invasion, highlighting the potential of JAM-C and JAM-B as new targets for the treatment of human gliomas.

Arachidonylethanolamide induces apoptosis of human glioma cells through vanilloid receptor-1.

The anti-tumor properties of cannabinoids have recently been evidenced, mainly with delta9-tetrahydrocannabinol (THC). However, the clinical application of this drug is limited by possible undesirable side effects due to a broad expression of cannabinoid receptors (CB1 and CB2). An attractive field of research therefore is to identify molecules with more selective tumor targeting. This is particularly important for malignant gliomas, considering their poor prognosis and their location in the brain. Here we investigated whether the most potent endogenous cannabinoid, arachidonylethanolamide (AEA), could be a candidate. We observed that AEA induced apoptosis in long-term and recently established glioma cell lines via aberrantly expressed vanilloid receptor-1 (VR1). In contrast with their role in THC-mediated death, both CB1 and CB2 partially protected glioma against AEA-induced apoptosis. These data show that the selective targeting of VR1 by AEA or more stable analogues is an attractive research area for the treatment of glioma.

Arachidonyl ethanolamide induces apoptosis of uterine cervix cancer cells via aberrantly expressed vanilloid receptor-1.

OBJECTIVES: Delta(9)-Tetrahydrocannabinol, the active agent of Cannabis sativa, exhibits well-documented antitumor properties, but little is known about the possible effects mediated by endogenous cannabinoids on human tumors. In the present study, we analyzed the effect of arachidonyl ethanolamide (AEA) on cervical carcinoma (CxCa) cell lines. METHODS: To assess the sensitivity of CxCa cells to AEA, we selected three cell lines that were exposed to increasing doses of AEA with or without antagonists to receptors to AEA. DNA fragmentation and caspase-7 activity were used as apoptosis markers. The expression of receptors to AEA were analyzed in CxCa cell lines as well as CxCa biopsies. RESULTS: The major finding was that AEA induced apoptosis of CxCa cell lines via aberrantly expressed vanilloid receptor-1, whereas AEA binding to the classical CB1 and CB2 cannabinoid receptors mediated a protective effect. Furthermore, unexpectedly, a strong expression of the three forms of AEA receptors was observed in ex vivo CxCa biopsies. CONCLUSIONS: Overall, these data suggest that the specific targeting of VR1 by endogenous cannabinoids or synthetic molecules offers attractive opportunities for the development of novel potent anticancer drugs.

Anoxia induces macrophage inhibitory cytokine-1 (MIC-1) in glioblastoma cells independently of p53 and HIF-1.

Human astrocytic brain tumors select for mutations in the p53 tumor suppressor gene early in malignant progression. p53 is activated upon various kinds of cellular stress leading to apoptosis or cell cycle arrest, but is also implicated in complex biological processes such as inhibition of angiogenesis and metastasis. In an effort to shed light on consequences mediated by p53 inactivation in gliomas, we established the Tet-On system for p53 in the LN-Z308 glioblastoma cell line. The macrophage inhibitory cytokine-1 (MIC-1) gene was identified as a most prominent p53 target gene upon gene expression profiling. Oxygen deprivation, an important cellular stress, revealed MIC-1 as an anoxia responsive gene in glioblastoma cell lines. MIC-1 up-regulation by anoxia is mediated through an alternative, p53 and hypoxia inducible factor 1 (HIF-1) independent pathway. Furthermore, ectopic expression of MIC-1 in LN-Z308 cell line completely abolished its inherent tumorigenicity in nude mice, while proliferation in vitro was not affected. In the present experimental model MIC-1 may exert its anti-tumorigenic properties via a paracrine mechanism mediated by host cells in vivo. Taken together, these data suggest that MIC-1 is an important downstream mediator of p53 function, while acting itself as an intercessor of cellular stress signaling and exerting anti-tumorigenic activities.

Overexpression of thrombospondin-1 reduces growth and vascular index but not perfusion in glioblastoma.

Little is known about the effects of antiangiogenic therapy on perfusion of human tumors and the mechanisms by which tumors can adapt to these treatments and recur. Here, we examined the effects of serial passaging of LN-229 human glioma xenografts overexpressing thrombospondin (TSP)-1 on tumor growth, vascularity, and perfusion. Persistence of TSP-1 overexpression was confirmed after three serial s.c. passages of small xenografted tumor blocks of cells stably transfected with TSP-1 cDNA (clones C9 and E7) or vector controls (pooled clones A7-A9) in immunodeficient nu/nu mice. The tumor vascularity was estimated by noninvasive near infrared spectroscopy measuring blood volume at 800 +/- 10 nm and by histological vessel scores in CD31-immunostained cryosections. The tumor perfusion was assessed by noninvasive laser Doppler flowmetry. Overexpression of TSP-1 significantly inhibited tumor growth. In size-matched tumors (approximately 300 mm(3)), the blood volume and the histological vessel scores were lower in the TSP-1-transfected tumors than in controls, and this effect was more pronounced in tumors derived from the clone with the highest TSP-1 expression (clone E9). Despite this clear reduction in tumor vascularity, the tumor perfusion was the same in TSP-1-transfected tumors and controls. This study shows that TSP-1 overexpression slows glioma growth in vivo but does not prevent it from reaching a large size (300 mm(3)). Whereas a clear reduction in blood volume during tumor growth and a reduced vascular index at sacrifice are observed in TSP-1-transfected tumors, this did not affect perfusion when size-matched comparisons were performed. Given the increased time needed to reach equal size, it indicates that a fixed rate of perfusion must be maintained in the tumor to allow for growth. Elucidation of the mechanisms that allow this to happen has important consequences for the understanding of tumor recurrence after antiangiogenic therapy.