Eloah Rabello Suarez

Heparan sulfate mediates trastuzumab effect in breast cancer cells

BackgroundTrastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components—heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)—in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab.MethodsThe cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). The profile of sulfated glycosaminoglycans was also investigated by [35S]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate.ResultsBreast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. The addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. In addition, HS shedding was increased significantly in these resistant cells.ConclusionTrastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. In addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.

Glycosaminoglycan synthesis and shedding induced by growth factors are cell and compound specific.

The interactions between growth factors and sulphated glycosaminoglycans (GAG) have been extensively studied. The aim of this study is to investigate if growth factors would show specificity of action on the synthesis and shedding of sulphated GAG, using two different cell lines: endothelial and smooth muscle cells. The cells were grown in the presence or absence of growth factors: EGF, FGF2, VEGF121, VEGF165. Transfection assays were also performed using recombinant pcDNA3.1, containing VEGF165 cDNA. In order to analyse the different types of GAG the cells were metabolically labelled with [35S]-sulphate. At low doses, VEGF121 was the only growth factor able to increase both the synthesis and secretion of heparan sulphate (HS) in endothelial cells. Over expression of VEGF165 stimulated HS synthesis in both cells. The combined results showed that growth factors affect GAG synthesis in a cell specific and dose dependent manner.

Heparanase expression and glycosaminoglycans profile in renal cell carcinoma

A better understanding of the molecular mechanisms of renal cell carcinogenesis could contribute to a decrease in the mortality rate of this disease. The aim of this study was to evaluate the glycosaminoglycans profile and heparanase expression in renal cell carcinoma. The study included 24 patients submitted to nephrectomy with confirmed pathological diagnosis of renal cell carcinoma. The majority of the samples (87.5%) were classified in the initial stage of renal cell carcinoma (clinical stages I and II). Heparanase messenger ribonucleic acid expression was evaluated by quantitative real‐time reverse transcription polymerase chain reaction, and sulfated glycosaminoglycans were identified and quantified by agarose gel electrophoresis of renal cell carcinoma samples or non‐neoplastic tissues obtained from the same patients (control group). The sulfated glycosaminoglycans and hyaluronic acid were analyzed in urine samples of the patients before and after surgery. The data showed a significant statistical increase in chondroitin sulfate, and a decrease in heparan sulfate and dermatan sulfate present in neoplastic tissues compared with non‐neoplastic tissues. Higher heparanase messenger ribonucleic acid expression in the neoplastic tissues was also shown, compared with the non‐neoplastic tissues. The urine glycosaminoglycans profile showed no significant difference between renal cell carcinoma and control samples. Extracellular matrix changes observed in the present study clarify that heparanase is possibly involved with heparan sulfate turnover, and that heparanase and the glycosaminoglycans can modulate initial events of renal cell carcinoma development.

The Profile of Heparanase Expression Distinguishes Differentiated Thyroid Carcinoma from Benign Neoplasms

Introduction The search for a specific marker that could help to distinguish between differentiated thyroid carcinoma and benign lesions remains elusive in clinical practice. Heparanase (HPSE) is an endo-beta-glucoronidase implicated in the process of tumor invasion, and the heparanase-2 (HPSE2) modulates HPSE activity. The aim of this study was to evaluate the role of heparanases in the development and differential diagnosis of follicular pattern thyroid lesions. Methods HPSE and HPSE2 expression by qRT-PCR, immunohistochemistry evaluation, western blot analysis and HPSE enzymatic activity were evaluated. Results The expression of heparanases by qRT-PCR showed an increase of HPSE2 in thyroid carcinoma (P = 0.001). HPSE activity was found to be higher in the malignant neoplasms than in the benign tumors (P<0.0001). On Western blot analysis, HPSE2 isoforms were detected only in malignant tumors. The immunohistochemical assay allowed us to establish a distinct pattern for malignant and benign tumors. Carcinomas showed a typical combination of positive labeling for neoplastic cells and negative immunostaining in colloid, when compared to benign tumors (P<0.0001). The proposed diagnostic test presents sensitivity and negative predictive value of around 100%, showing itself to be an accurate test for distinguishing between malignant and benign lesions. Conclusions This study shows, for the first time, a distinct profile of HPSE expression in thyroid carcinoma suggesting its role in carcinogenesis.

Glycosaminoglycans affect heparanase location in CHO cell lines.

Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-β-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

A phase I study of gemcitabine combined with vinblastine in patients with solid tumors.

Aims and background Gemcitabine and vinblastine are chemotherapeutic drugs with a wide spectrum of antitumor activity. We conducted a phase I trial to define the maximal tolerated dose of this combination. Methods Twenty-nine patients with a variety of solid tumors with measurable disease were included. Twenty-seven of these patients had been previously treated with chemotherapy. The use of hematological growth factors was not allowed. The maximal tolerated dose was defined as the level in which WHO grades 3 or 4 non-hematological toxicity would occur in at least a third of the patients. Results The maximal tolerated dose was reached at level 5, which consisted of gemcitabine (1000 mg/m2) with vinblastine (3 mg/m2) given intravenously on days 1, 8 and 15 every 28 days. At this level, one patient had WHO grade 3 cerebellar toxicity and another WHO grade 4 anorexia. Furthermore, patients were able to receive all 3 weekly doses of chemotherapy in only 2 out of 10 cycles of treatment given at this level. We also observed 5 episodes of WHO grades 1 and 2 neuropathy, most of which (4/5) occurred in patients previously exposed to neurotoxic chemotherapy. We recorded 2 partial responses (one at level 3 and the other at level 4) and 8 instances of stable disease after 3 scheduled cycles of the combination. Conclusions We therefore recommend gemcitabine (1100 mg/m2) plus vinblastine (5.5 mg/m2) given on days 1 and 8 every 21 days (level 4) for previously treated patients with solid tumors, with special caution in patients previously exposed to neurotoxic chemotherapy.

From Combinatorial Display Techniques to Microarray Technology: New Approaches to the Development and Toxicological Profiling of Targeted Nanomedicines

Advances in genome scale analysis provide an alternative tool to combine large-scale screening of chemicals with detailed mechanistic studies, in which expression signatures for specific factors can be used as predictors of response or serve as new therapeutic targets in vitro and in vivo. Recently, the possibility of analyzing the effects of nanomaterials on a large number of genes has led to toxicogenomic studies in nanotechnology. Furthermore, expression signatures are providing potential targets for the design of ligand-mediated tumor targeting. In this respect, the integration of phage display technique with nanotechnology has been explored as a new approach to generate cancer-targeted nanomedicines. In this chapter we outline advances in microarray technology and in the selection of peptides and antibodies by display techniques. Furthermore, we add insights into their application in the field of toxicogenomics and in the development of targeted nanomedicines.