Francisco Javier Rodríguez-Berrocal

Cytotoxicity of selected imidazolium-derived ionic liquids in the human Caco-2 cell line. Sub-structural toxicological interpretation through a QSAR study

Room-temperature ionic liquids (ILs) are considered green chemicals that may replace volatile organic solvents currently used by industry. However, toxicological effects of ILs are not well known. In this study, we describe the cytotoxicity of selected imidazolium-derived ILs in Caco-2 cells, prototypical human epithelial cells. The most toxic IL was 1-decyl-3-methylimidazolium chloride ([C10mim][Cl]), whereas the least toxic was 1,3-dimethylimidazolium methyl sulfate ([C1mim][MSO4]). Using the toxicological experimental data obtained we developed a Quantitative Structure–Activity Relationship (QSAR) study using the Topological Sub-Structural Molecular Design (TOPS-MODE) approach. The model found showed excellent statistical parameters and from their interpretation, we arrived to some important conclusions such as: For 1-alkyl-3-methylimidazolium chloride derivatives, a correlation between the R2 alkyl chain length and toxicity was observable. A positive contribution of p-chloro substituent in benzyl ring is detected among 1-methylarylimidazolium chloride derivatives. Regarding the contribution of the anion, anion chloride presents a positive contribution to toxicity whereas for compounds [C1mim][MSO4] and [C1eim][ESO4], a negative fragment contribution can be detected in anion methyl or ethyl sulfate. The EC50 values determined for the ILs analysed in this study in Caco-2 cells are lower than the data reported in the literature on the toxicity of classical solvents assessed with cell lines. In conclusion, our results indicate that the possible cytotoxic effect of ILs should be considered in the design and overall evaluation of these compounds. Nevertheless, any toxicity evaluation of ILs should take their bioavailability into account, which will be affected by their low volatility, compared to conventional solvents.

Preoperative serum CD26 levels: diagnostic efficiency and predictive value for colorectal cancer.

CD26 is an ectoenzyme with dipeptidyl peptidase IV activity expressed on a variety of cell types. Although the function of the high concentration of serum-soluble CD26 (sCD26) is unknown, it may be related to the cleavage of biologically active polypeptides. As CD26 or enzymatic activity levels were previously associated with cancer, we examined the potential diagnostic and prognostic value of preoperative sCD26 measurements by ELISA in colorectal carcinoma patients. We found a highly significant difference between sCD26 levels in healthy donors (mean 559.7 ± 125.5 μg l−1) and cancer patients (mean 261.7 ± 138.1 μg l−1) (P < 0.001). A cut-off at 410 μg l−1 gave 90% sensitivity with 90% specificity which means that the diagnostic efficiency of sCD26 is higher than that shown by other markers, particularly in patients at early stages. Moreover, sCD26 as a variable is not related with Dukes’ stage classification, age, gender, tumour location or degree of differentiation. With a follow-up of 2 years until recurrence, preliminary data show that sCD26 can be managed as a prognostic variable of early carcinoma patients. In addition, the origin of sCD26 is discussed.

Proteomics for discovery of candidate colorectal cancer biomarkers

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in Europe and other Western countries, mainly due to the lack of well-validated clinically useful biomarkers with enough sensitivity and specificity to detect this disease at early stages. Although it is well known that the pathogenesis of CRC is a progressive accumulation of mutations in multiple genes, much less is known at the proteome level. Therefore, in the last years many proteomic studies have been conducted to find new candidate protein biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of colorectal carcinogenesis. An important advantage of the proteomic approaches is the capacity to look for multiple differentially expressed proteins in a single study. This review provides an overview of the recent reports describing the different proteomic tools used for the discovery of new protein markers for CRC such as two-dimensional electrophoresis methods, quantitative mass spectrometry-based techniques or protein microarrays. Additionally, we will also focus on the diverse biological samples used for CRC biomarker discovery such as tissue, serum and faeces, besides cell lines and murine models, discussing their advantages and disadvantages, and summarize the most frequently identified candidate CRC markers.

On the identification of biomarkers for non-small cell lung cancer in serum and pleural effusion

The current imperative need for new biomarkers of non-small cell lung cancer (NSCLC) prompted us to compare the proteome of serum and pleural effusion samples from cancer patients with those with benign lung diseases as pneumonia or tuberculosis. Samples were prefractionated through affinity chromatography prior to 2D-DIGE to detect proteins with altered expression in cancer patients. Overall, we identified more potential biomarkers in pleural effusion, which is closer to the affected organ, than in serum. Nevertheless, in both cases principal component analysis demonstrated that the pattern of significantly altered proteins discriminates between disease groups. The biomarker candidates comprise proteins increased in malignant pleural effusions as gelsolin and the metalloproteinase inhibitor 2, and others with lower levels as S100-A8 and S100-A9. The most interesting protein was the pigment epithelium-derived factor (PEDF), which is related to angiogenesis inhibition, and was significantly overexpressed both in serum and pleural effusion from NSCLC patients. More than 12 PEDF isoforms were specifically immunodetected in both fluids in 2-D blots, most of them overexpressed in NSCLC. Thus, further validation would be ideally directed to quantify individual PEDF isoforms, as it may be only one or some of them the ones altered in the cancer process.

Differential Expression of Serum Clusterin Isoforms in Colorectal Cancer

Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.

Value of the Serum Alpha-L-Fucosidase Activity in the Diagnosis of Colorectal Cancer

Objectives: The purpose of this study was to assess the value of the serum levels of α-L-fucosidase activity in the diagnosis of patients with colorectal cancer. Methods: Using a fluorometric method we analyzed the α-L-fucosidase activity in preoperative sera from 137 colorectal cancer patients and in sera from 232 donors. Results: The enzymatic activity of α-L-fucosidase was significantly lower (p < 0.001) in patients (4.8 ± 3.09 U/ml) than in donors (10.5 ± 5.46 U/ml). Using the ROC curve, the ideal cut-off for the diagnostic value of α-L-fucosidase activity was determined to be 5.6 U/ml. The diagnostic efficiency for colorectal cancer of α-L-fucosidase activity was higher than that observed for carcinoembryonic antigen (cut-off 5.0 ng/ml), especially for tumors at an early stage. Conclusions: Our results suggest that preoperative serum α-L-fucosidase activity may be used as a cheap and easy complementary test, in addition to standard clinical procedures routinely used for the diagnosis of colorectal cancer.

Absence of Activating Mutations in the EGFR Kinase Domain in Spanish Head and Neck Cancer Patients

The discovery of kinase domain mutations in the epidermal growth factor receptor gene (EGFR) in never-smoker patients, associated with an increased sensitivity to tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib, has been one of the most relevant findings ever in non-small cell lung carcinomas (NSCLC). Since treatment with TKIs has furthermore shown a clinical benefit in head and neck squamous cell carcinoma (HNSCC) patients, we hypothesized that these mutations could also be present in this neoplasia. Current studies looking for EGFR mutations in HNSCC are limited and results are still controversial. In this work, we screened for EGFR tyrosine kinase mutations in tumour DNA obtained from 31 Spanish patients with HNSCC by PCR-single-strand conformational polymorphism analysis. None of the patients displayed a somatic EGFR mutation, previously described in NSCLC, but other DNA sequence variations were found in 9 of 31 HNSCC patients. Accordingly, activating EGFR mutations in HNSCC patients seem to be a rare event in Spanish patients, suggesting that there is little room for the administration of TKIs in HNSCC based on the presence of these mutations. Additional investigations about EGFR amplification are indicated to establish a potential relationship between EGFR overexpression and the response to anti-EGFR therapies.

Cholinesterases are down-expressed in human colorectal carcinoma

Abstract.The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G2A) and monomers (G1A) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A12, G4H and PRiMA-containing G4A AChE forms, besides G4H, G4A and G1H BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and overstimulating muscarinic receptors.

Immunohistochemical Analysis of Sialic Acid and Fucose Composition in Human Colorectal Adenocarcinoma

The expression of different sialoglycoconjugates and fucoglycoconjugates in normal mucosa and adenocarcinoma samples from 43 colorectal cancer patients was investigated by using specific lectins and applying a semiquantitative analysis. A pronounced decrease in the intracellular binding of the Maackia amurensis lectin, which recognizes α(2,3)-linked sialic acid residues, was found in the tumoral tissue. In contrast, a significant increase in the staining with the Sambucus nigra lectin (SNA I), which binds to α(2,6)-linked sialic acid residues, was detected in the epithelial cells as well as in the mucins from tumors. No significant differences in the reactivity with the Aleuria aurantia lectin, which recognizes the sequence Fuc(α1,6)GlcNAc, between normal and malignant colorectal tissues were detected. Furthermore, the correlation between lectin-binding profiles and the prognosis of colorectal cancer patients was examined. After an average postoperative follow-up period of 31 months, patients with tumors showing a strong SNA I staining presented a greater probability of disease recurrence. This result suggests that the intensity of staining with SNA I could be a valid parameter for predicting recurrence in colorectal cancer.

Serum CD26 is related to histopathological polyp traits and behaves as a marker for colorectal cancer and advanced adenomas

BackgroundSerum CD26 (sCD26) levels were previously found diminished in colorectal cancer (CRC) patients compared to healthy donors, suggesting its potential utility for early diagnosis. Therefore we aimed to estimate the utility of the sCD26 as a biomarker for CRC and advanced adenomas in a high-risk group of patients. The relationship of this molecule with polyp characteristics was also addressed.MethodssCD26 levels were measured by ELISA in 299 symptomatic and asymptomatic patients who had undergone a colonoscopy. Patients were diagnosed as having no colorectal pathology, non-inflammatory or inflammatory bowel disease, polyps (hyperplastic, non-advanced and advanced adenomas) or CRC.ResultsAt a 460 ng/mL cut-off, the sCD26 has a sensitivity and specificity of 81.8% (95% CI, 64.5-93.0%) and 72.3% (95% CI, 65.0-77.2%) for CRC regarding no or benign colorectal pathology. Clinicopathological analysis of polyps showed a relationship between the sCD26 and the grade of dysplasia and the presence of advanced adenomas. Hence, a 58.0% (95% CI, 46.5-68.9%) sensitivity detecting CRC and advanced adenomas was obtained, with a specificity of 75.5% (95% CI, 68.5-81.0%).ConclusionsOur preliminary results show that measurement of the sCD26 is a non-invasive and reasonably sensitive assay, which could be combined with others such as the faecal occult blood test for the early diagnosis and screening of CRC and advanced adenomas. Additional comparative studies in average-risk populations are necessary.