Comparative studies of TIMP-1 immunohistochemistry, TIMP-1 FISH analysis and plasma TIMP-1 in glioblastoma patients

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been associated with poor prognosis and resistance towards chemotherapy in several cancer forms. In a previous study we found an association between a low TIMP-1 tumor immunoreactivity and increased survival for glioblastoma patients, when compared to moderate and high TIMP-1 tumor immunoreactivity. The aim of the present study was to further evaluate TIMP-1 as a biomarker in gliomas by studying TIMP-1 gene copy numbers by fluorescence in situ hybridization (FISH) on 33 glioblastoma biopsies and by measuring levels of TIMP-1 in plasma obtained pre-operatively from 43 patients (31 gliomas including 21 glioblastomas) by enzyme-linked immunosorbent assay (ELISA). The results showed TIMP-1 gene copy numbers per cell ranging from 1 to 5 and the TIMP-1/CEN-X ratio ranging between 0.7 and 1.09, suggesting neither amplification nor loss of the TIMP-1 gene. The TIMP-1 protein levels measured in plasma were not significantly higher than TIMP-1 levels measured in healthy subjects. No correlation was identified between TIMP-1 tumor cell immunoreactivities and the TIMP-1 gene copy numbers or the plasma TIMP-1 levels. In conclusion, high immunohistochemical TIMP-1 protein levels in glioblastomas were not caused by TIMP-1 gene amplification and TIMP-1 in plasma was low and not directly related to tumor TIMP-1 immunoreactivity. The study suggests that TIMP-1 immunohistochemistry is the method of choice for future clinical studies evaluating TIMP-1 as a biomarker in glioblastomas. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11060-016-2252-4) contains supplementary material, which is available to authorized users

The glutamate transport inhibitor DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) differentially affects SN38- and oxaliplatin-induced death of drug-resistant colorectal cancer cells

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer death globally and new biomarkers and treatments are severely needed. METHODS: Here, we employed HCT116 and LoVo human CRC cells made resistant to either SN38 or oxaliplatin, to investigate whether altered expression of the high affinity glutamate transporters Solute Carrier (SLC)-1A1 and -1A3 (EAAT3, EAAT1) is associated with the resistant phenotypes. Analyses included real-time quantitative PCR, immunoblotting and immunofluorescence analyses, radioactive tracer flux measurements, and biochemical analyses of cell viability and glutathione content. Results were evaluated using one- and two-way ANOVA and Students two-tailed t-test, as relevant. RESULTS: In SN38-resistant HCT116 and LoVo cells, SLC1A1 expression was down-regulated ~60 % and up-regulated ~4-fold, respectively, at both mRNA and protein level, whereas SLC1A3 protein was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-β-Benzyloxyaspartic acid (TBOA)-sensitive, UCPH101-insensitive [(3)H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration-dependently augmented loss of cell viability induced by SN38, while strongly counteracting that induced by oxaliplatin, in both HCT116 and LoVo cells. This reflected neither altered expression of the oxaliplatin transporter Cu(2+)-transporter-1 (CTR1), nor changes in cellular reduced glutathione (GSH), although HCT116 cell resistance per se correlated with increased cellular GSH. DL-TBOA did not significantly alter cellular levels of p21, cleaved PARP-1, or phospho-Retinoblastoma protein, yet altered SLC1A1 subcellular localization, and reduced chemotherapy-induced p53 induction. CONCLUSIONS: SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while attenuating that induced by oxaliplatin. These findings may point to novel treatment options in treatment-resistant CRC. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1405-8) contains supplementary material, which is available to authorized users

Mechanisms of topoisomerase I (<em>TOP1</em>) gene copy number increase in a stage III colorectal cancer patient cohort

BACKGROUND: Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH). METHODS: Nine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN-20) or chromosome 2 (CEN-2). Tissue sections from 154 chemonaive stage III CRC patients, previously studied with TOP1/CEN-20, were analyzed with TOP1/CEN-2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and time to local recurrence (LR; rectal cancer only) were determined. RESULTS: TOP1 aberrations were observed in four cell line metaphases. In all cell lines CEN-2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN-2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN-2≥1.5). One hundred and three patients (68.2%) had TOP1 gain, of which 15 patients (14.6%) harbored an amplification (TOP1/CEN-20≥2.0). TOP1 gene gain did not have any association with clinical endpoints, whereas TOP1 amplification showed a non-significant trend towards longer TTR (multivariate HR: 0.50, p = 0.08). Once amplified cases were segregated from other cases of gene gain, non-amplified gene increases (TOP1/CEN-2≥1.5 and TOP1/CEN-20<2.0) showed a trend towards shorter TTR (univariate HR: 1.57, p = 0.07). CONCLUSIONS: TOP1 gene copy number increase occurs frequently in stage III CRC in a mechanism that often includes CEN-20. Using CEN-2 as a measurement for tumor ploidy levels, we were able to discriminate between different mechanisms of gene gain, which appeared to differ in prognostic impact. TOP1 FISH guidelines have been updated