Sheri F T Fong

We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with beta-aminopropionitrile (betaAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of betaAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with betaAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on betaAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer.

Lysyl oxidase-like 2 (LOXL2) belongs to an amine oxidase family whose members have been implicated in crosslink formation in stromal collagens and elastin, cell motility, and tumor development and progression. We previously demonstrated the association between increased LOXL2 expression and invasive/metastatic behavior in human breast cancer cells and mouse squamous and spindle cell carcinomas, interaction between LOXL2 and SNAIL in epithelial-mesenchymal transition, and localization of the LOXL2 gene to 8p21.2-21.3, within a minimally deleted region in several cancers, including colon and esophagus. In the present study, we analyzed LOXL2 expression in colon and esophageal tumors, and explored methylation as a regulator of LOXL2 expression. Immunohistochemistry using normal tissues demonstrated intracellular localization of LOXL2 in colonic enteroendocrine cells and esophageal squamous cells at the luminal surface, but not in mitotically active cells. Tissue array analysis of 52 colon adenocarcinomas and 50 esophageal squamous cell carcinomas revealed presence of LOXL2 expression in 83 and 92% of the samples, respectively, and a significant association between increased number of LOXL2-expressing cells and less-differentiated colon carcinomas. We determined that the methylation status of the 1150 bp 5' CpG island may contribute to the regulation of the gene. Loss of heterozygosity studies, using a microsatellite within intron 4 of the LOXL2 gene, revealed that loss of LOXL2 was unlikely to play a major role in either colon or esophageal tumors. These results suggest that increased LOXL2 expression in colon and esophageal cancer may contribute to tumor progression.

Two known glycosides and a novel trisaccharide fatty acid ester were isolated from the n-butanol-soluble fraction of the fruits of Morinda citrifolia (noni). Structure determination was carried out by spectral techniques such as MS, IR, NMR, and 2D-NMR. The novel trisaccharide fatty acid ester was elucidated as 2,6-di-O-(β-d-glucopyranosyl)-1-O-octanoyl-β-d-glucopyranose. The known compounds were identified as rutin and asperulosidic acid. Keywords: Noni; Morinda citrifolia; trisaccharide fatty acid ester; 2,6-di-O-(β-d-glucopyranosyl-1-O-octanoyl-β-d-glucopyranose; rutin; asperulosidic acid

Lysyl oxidase (LOX), a copper-dependent amine oxidase, has been implicated in tumor suppression and cell growth regulation. The chromosomal locus of LOX, 5q23, is affected by loss of heterozygosity (LOH) in colon cancer, suggesting that the LOX gene could be affected by LOH and consequently, loss or reduction of LOX function contribute to the tumorigenic process. Identification of microsatellite markers within the LOX locus has allowed us to map the LOX gene within the 5q23.1 region. Analysis of this locus and flanking loci in matched tumor and blood DNA samples from a panel of colorectal cancer patients, demonstrated that 38% (16/42) of informative samples were affected by LOH or allelic imbalance. Furthermore, 75% (6/8) of these tumor samples were shown to have significantly reduced LOX mRNA levels. Similar reduction in LOX levels were detected in a panel of matched normal colon and colon tumor samples. Tumor samples demonstrating LOH by RFLP, were subject to mutational analysis, including RT-PCR, exonic deletion detection by PCR, cDNA and genomic DNA sequencing, and were found to have a spectrum of alterations and mutations affecting the LOX gene. These results confirm that loss or reduction of LOX function during tumor development is a direct consequence of somatic mutations and is associated with colon tumor pathogenesis.