Acquisition of anoikis resistance up-regulates syndecan-4 expression in endothelial cells.

Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonization of distant organs. Cell adhesion plays an important role in neoplastic transformation. Tumors produce several molecules that facilitate their proliferation, invasion and maintenance, especially proteoglycans. The syndecan-4, a heparan sulfate proteoglycan, can act as a co-receptor of growth factors and proteins of the extracellular matrix by increasing the affinity of adhesion molecules to their specific receptors. It participates together with integrins in cell adhesion at focal contacts connecting the extracellular matrix to the cytoskeleton. Changes in the expression of syndecan-4 have been observed in tumor cells, indicating its involvement in cancer. This study investigates the role of syndecan-4 in the process of anoikis and cell transformation. Endothelial cells were submitted to sequential cycles of forced anchorage impediment and distinct lineages were obtained. Anoikis-resistant endothelial cells display morphological alterations, high rate of proliferation, poor adhesion to fibronectin, laminin and collagen IV and deregulation of the cell cycle, becoming less serum-dependent. Furthermore, anoikis-resistant cell lines display a high invasive potential and a low rate of apoptosis. This is accompanied by an increase in the levels of heparan sulfate and chondroitin sulfate as well as by changes in the expression of syndecan-4 and heparanase. These results indicate that syndecan-4 plays a important role in acquisition of anoikis resistance and that the conferral of anoikis resistance may suffice to transform endothelial cells.

A clinical trial testing the efficacy of PDT in preventing amputation in diabetic patients.

The feet of diabetic patients continue to be an unsolved problem in medicine. Uncontrolled neuropathy, ulceration and infection usually lead to amputation and presently there is no effective and reliable method that can be used to provide an efficient cure. Overall improvement in the salvage strategies, based on comprehensive pre-clinical evaluation, debridement, antibiotic therapy and follow up, has shown improvements in certain hospital settings, but the general picture for patients with diabetic foot is to have some sort of amputation, especially in underserved populations. It is clearly necessary to develop novel treatment strategies for this worldwide health problem. Photodynamic therapy (PDT) is a treatment modality that uses light to generate in situ reactive oxygen species, which can cause cell death. PDT can be used to treat several diseases, including foot infections that do not respond well to antibiotic therapy. There are several characteristics of PDT that make it potentially ideal to treat diabetic feet: the photosensitizer is non-toxic in the dark, but after illumination it becomes a very efficient antimicrobial agent with topical use, and it can regenerate small bones, such as the phalanges. However, PDT is still not used in clinical practice to treat diabetic feet. Therefore, we decided to perform a clinical study to prove that PDT is an effective method to avoid amputation of infected diabetic feet. An inexpensive PDT protocol was developed and applied to 18 patients with osteomyelitis, classified as Grade 3 on the Wagner scale. Only one of these patients suffered amputation. At least two of them were cured from resistant bacteria strains without intravenous antibiotic therapy. In the control group of 16 patients, all of them ended up suffering amputation. The rate of amputation in the PDT group was 0.029 times the rate in the control group and the difference is clearly statistically significant (p=0.002).

Heparanase-2, syndecan-1, and extracellular matrix remodeling in colorectal carcinoma.

AIM: To propose a quantitative method to detect heparanase-2 (HPA2) and syndecan-1 (Syn-1) using immunohistochemistry in colorectal (colon and rectal) carcinomas compared with nonneoplastic tissues and evaluate the possible role of these molecules in tumor development and extracellular remodeling. METHODS: Cytoplasmic staining of HPA2 and Syn-1 was obtained by standard immunohistochemical reactions in 50 colorectal carcinoma and 20 nonneoplastic large bowels tissues. An image system was used to quantify the immunoexpression by digital computer-assisted method (Matos et al. 2006). The cutoff point for the immunohistochemistry variable was defined by sensibility and specificity curves. Statistical analysis was performed using SPSS version 13.0. RESULTS: HPA2 was over-expressed in colorectal cancer (131.1+/-24.9 o.u./microm) when compared with nonneoplastic tissues (27.9+/-12.2 o.u./microm) (P<0.0001). However, an opposite correlation was observed between Syn-1 and tumor presence, where colorectal tissues expressed lower Syn-1 proteoglycan compared with nonneoplastic tissues, respectively (39.2+/-17.8 o.u./microm) and (102.2+/-25.2 o.u./microm) (P<0.0001). CONCLUSION: A methodology with high sensitivity and specificity is proposed with a cutoff value for HPA2 and Syn-1 in the immunohistochemistry assay to define the presence of tumor. It was demonstrated for the first time in the literature that HPA2 is over-expressed in colorectal carcinoma tissues compared with nonneoplastic tissues. HPA2 over-expression could be possibly related to Syn-1 shedding despite the fact that HPA2 does not present enzymatic activity as HPA1.

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 [(35)S]-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.

Peripheral blood c-erbB-2 expression by reverse transcriptase-polymerase chain reaction in breast cancer patients receiving chemotherapy.

Minimal residual disease (MRD) evaluation in breast cancer patients is a promising tool to improve current staging procedures. In a previous work employing a CK-19-based reverse transcriptase-polymerase chain reaction (RT-PCR) technique for MRD detection, we identified a group of women who exhibited persistent negativity for this assay and for whom this technique was considered noninformative. In order to improve the yield of MRD detection in these patients, we evaluated the usefulness of RT-PCR detection of c-erbB-2 expression. We were able to detect up to 1 MCF-7 cell (positive for c-erbB-2 expression) in a mixture of 1,000,000 CCRF-CEM cells (negative for c-erbB-2 expression). We evaluated the specificity of this technique in the peripheral-blood mononuclear cells (PBMCs) of 20 healthy women and found that 2 of these women were positive for c-erbB-2 expression. In the PBMCs of a group of 16 women with breast cancer, 25% of the samples were positive for c-erbB-2 expression before chemotherapy. Except for race (P = 0.017), no other significant correlations were found, including c-erbB-2 expression in the primary tumor by immunoperoxidase. Interestingly, in the subgroup of 6 patients for whom this technique was informative, we found that 80% of the samples obtained while on chemotherapy were negative compared to only 10% obtained off treatment (P = 0.017). Additionally, 2 patients for whom CK-19 expression was noninformative had at least 1 c-erbB-2-positive sample. We conclude that this technique might be useful for MRD detection in breast cancer patients, but further studies are necessary to confirm our findings.