Colorectal Cancer Cell Invasion and Functional Properties Depend on Peri-Tumoral Extracellular Matrix.

We investigated how the extracellular matrix (ECM) affects LoVo colorectal cancer cells behavior during a spatiotemporal invasion. Epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and morphological phenotypes expressed by LoVo-S (doxorubicin-sensitive) and higher aggressive LoVo-R (doxorubicin-resistant) were evaluated in cells cultured for 3 and 24 h on Millipore filters covered by Matrigel, mimicking the basement membrane, or type I Collagen reproducing a desmoplastic lamina propria. EMT and invasiveness were investigated with RT-qPCR, Western blot, and scanning electron microscopy. As time went by, most gene expressions decreased, but in type I Collagen samples, a strong reduction and high increase in MMP-2 expression in LoVo-S and -R cells occurred, respectively. These data were confirmed by the development of an epithelial morphological phenotype in LoVo-S and invading phenotypes with invadopodia in LoVo-R cells as well as by protein-level analysis. We suggest that the duration of culturing and type of substrate influence the morphological phenotype and aggressiveness of both these cell types differently. In particular, the type I collagen meshwork, consisting of large fibrils confining inter fibrillar micropores, affects the two cell types differently. It attenuates drug-sensitive LoVo-S cell aggressiveness but improves a proteolytic invasion in drug-resistant LoVo-R cells as time goes by. Experimental studies on CRC cells should examine the peri-tumoral ECM components, as well as the dynamic physical conditions of TME, which affect the behavior and aggressiveness of both drug-sensitive and drug-resistant LoVo cells differently.

Protein extracts from regenerating lizard tail show an inhibitory effect on human cancer cells cultivated in-vitro.

BACKGROUND: accumulating evidence indicates that during tail regeneration in lizards the initial stage of regenerative blastema is a tumor-like proliferative outgrowth that rapidly elongates into a new tail composed of fully differentiated tissues. Both oncogenes and tumor-suppressors are expressed during regeneration, and it has been hypothesized that an efficient control of cell proliferation avoids that the blastema is turned into a tumor outgrowth. METHODS: in order to determine whether functional tumor-suppressors are present in the growing blastema we have utilized protein extracts collected from early regenerating tails of 3-5 mm that have been tested for a potential anti-tumor effect on in-vitro culture by using cancer cell lines from human mammary gland (MDA-MB-231) and prostate cancer (DU145). RESULTS: at specific dilutions, the extract determines a reduction of viability in cancer cells after 2-4 days of culture, as supported by statistical and morphological analyses. While control cells appear viable, treated cells result damaged and produce an intense cytoplasmic granulation and degeneration. CONCLUSIONS: this negative effect on cell viability and proliferation is absent using tissues from the original tail supporting the hypothesis that only regenerating tissues synthesize tumor-suppressor molecules. The study suggests that the regenerating tail of lizard at the stages here selected contains some molecules that determine inhibition of cell viability on the cancer cells analyzed.

Substrate Type and Concentration Differently Affect Colon Cancer Cells Ultrastructural Morphology, EMT Markers, and Matrix Degrading Enzymes.

Aim of the study was to understand the behavior of colon cancer LoVo-R cells (doxorubicin-resistant) vs. LoVo-S (doxorubicin sensitive) in the initial steps of extracellular matrix (ECM) invasion. We investigated how the matrix substrates Matrigel and type I collagen-mimicking the basement membrane (BM) and the normal or desmoplastic lamina propria, respectively-could affect the expression of epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and phenotypes. Gene expression with RT-qPCR, E-cadherin protein expression using Western blot, and phenotypes using scanning electron microscopy (SEM) were analyzed. The type and different concentrations of matrix substrates differently affected colon cancer cells. In LoVo-S cells, the higher concentrated collagen, mimicking the desmoplastic lamina propria, strongly induced EMT, as also confirmed by the expression of Snail, metalloproteases (MMPs)-2, -9, -14 and heparanase (HPSE), as well as mesenchymal phenotypes. Stimulation in E-cadherin expression in LoVo-S groups suggests that these cells develop a hybrid EMT phenotype. Differently, LoVo-R cells did not increase their aggressiveness: no changes in EMT markers, matrix effectors, and phenotypes were evident. The low influence of ECM components in LoVo-R cells might be related to their intrinsic aggressiveness related to chemoresistance. These results improve understanding of the critical role of tumor microenvironment in colon cancer cell invasion, driving the development of new therapeutic approaches.

The role of SPATA2 in TNF signaling, cancer, and spermatogenesis.

The activation of TNF receptors can lead to cell death with a mechanism of cell necrosis regulated genetically and distinct from apoptosis which is defined as necroptosis. Necroptosis has been one of the most studied emerging cell death/signaling pathways in recent years, especially in light of the role of this process in human disease. However, not all regulatory components of TNF signaling have been identified in relation to both physiological and pathological conditions. In 2008, Spata2 (Spermatogenesis-associated protein 2) was identified as one of the seven fundamental genes for the cellular signaling network that regulates necroptosis and apoptosis. This gene had been cloned by our group and named Spata2 as its expression was found to be elevated in the testis compared to other tissues, localized at the Sertoli cell level and FSH-dependent. More recently, it has been demonstrated that deletion of Spata2 gene causes increased inhibin α expression and attenuated fertility in male mice. However, more importantly, five recently published reports have highlighted that SPATA2 is crucial for recruiting CYLD to the TNFR1 signaling complex thus promoting its activation leading to TNF-induced cell death. Loss of SPATA2 increases transcriptional activation of NF-kB and limits TNF-induced necroptosis. Here we will discuss these important findings regarding SPATA2 and, in particular, focus attention on the evidence that suggests a role for this protein in the TNF signaling pathway.

Heparanase regulates EMT and cancer stem cell properties in prostate tumors.

Prostate cancer displays a certain phenotypic plasticity that allows for the transition of cells from the epithelial to the mesenchymal state. This process, known as epithelial-mesenchymal transition (EMT), is one of the factors that give the tumor cells greater invasive and migratory capacity with subsequent formation of metastases. In addition, many cancers, including prostate cancer, are derived from a cell population that shows the properties of stem cells. These cells, called cancer stem cells (CSCs) or tumor-initiating cells, not only initiate the tumor process and growth but are also able to mediate metastasis and drug resistance. However, the impact of EMT and CSCs in prostate cancer progression and patient survival is still far from fully understood. Heparanase (HPSE), the sole mammalian endoglycosidase capable of degrading heparan sulfate (HS), is also involved in prostate cancer progression. We had previously proved that HPSE regulates EMT in non-cancerous pathologies. Two prostate cancer cell lines (DU145 and PC3) were silenced and overexpressed for HPSE. Expression of EMT and stemness markers was evaluated. Results showed that the expression of several EMT markers are modified by HPSE expression in both the prostate cancer cell lines analyzed. In the same way, the stemness markers and features are also modulated by HPSE expression. Taken together, the present findings seem to prove a new mechanism of action of HPSE in sustaining prostate cancer growth and diffusion. As for other tumors, these results highlight the importance of HPSE as a potential pharmacological target in prostate cancer treatment.

Fibrosis of Peritoneal Membrane as Target of New Therapies in Peritoneal Dialysis.

Peritoneal dialysis (PD) is an efficient renal replacement therapy for patients with end-stage renal disease. Even if it ensures an outcome equivalent to hemodialysis and a better quality of life, in the long-term, PD is associated with the development of peritoneal fibrosis and the consequents patient morbidity and PD technique failure. This unfavorable effect is mostly due to the bio-incompatibility of PD solution (mainly based on high glucose concentration). In the present review, we described the mechanisms and the signaling pathway that governs peritoneal fibrosis, epithelial to mesenchymal transition of mesothelial cells, and angiogenesis. Lastly, we summarize the present and future strategies for developing more biocompatible PD solutions.

Heparanase as active player in endothelial glycocalyx remodeling.

The surface of all animal cells is coated with a layer of carbohydrates linked in various ways to the outer side of the plasma membrane. These carbohydrates are mainly bound to proteins in the form of glycoproteins and proteoglycans and together with the glycolipids constitute the so-called glycocalyx. In particular, the endothelial glycocalyx that covers the luminal layer of the endothelium is composed of glycosaminoglycans (heparan sulphate -HS and hyaluronic acid -HA), proteoglycans (syndecans and glypicans) and adsorbed plasma proteins. Thanks to its ability to absorb water, this structure contributes to making the surface of the vessels slippery but at the same time acts by modulating the mechano-transduction of the vessels, the vascular permeability and the adhesion of leukocytes in thus regulating several physiological and pathological events. Among the various enzymes involved in the degradation of the glycocalyx, heparanase (HPSE) has been shown to be particularly involved. This enzyme is responsible for the cutting of heparan sulfate (HS) chains at the level of the proteoglycans of the endothelial glycocalyx whose dysfunction appears to have a role in organ fibrosis, sepsis and viral infection. In this mini-review, we describe the mechanisms by which HPSE contributes to glycocalyx remodeling and then examine the role of glycocalyx degradation in the development of pathological conditions and pharmacological strategies to preserve glycocalyx during disease pathogenesis.

Renal effect of severe hypoxia evaluated By NGAL measurements: An in vivo and in vitro study.

PURPOSE: To investigate possible renal damage in healthy men exposed to extreme hypobaric hypoxia, using urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL) concentration as biomarker. The value of NGAL as a biomarker of proximal tubular cell damage under hypoxic conditions was also tested in vitro experiments. METHODS: NGAL was assayed in a cohort of air cadets (n = 16) exposed to hypobaric hypoxia in a hypobaric chamber during their training program. In all subjects, urine creatinine (Cr) and urinary NGAL levels were measured immediately before, 3, and 24 h after hypobaric environment exposure. Three in vitro experiments using proximal tubular cell cultures were also performed to measure NGAL gene expression, NGAL secretion in the culture medium and to evaluate apoptosis under two cycles of hypoxia and reoxygenation. RESULTS: In the in vivo study, geometric means of urinary NGAL/Cr ratio measured 24 h after hypobaric hypoxia in the hypobaric chamber were significantly lower than baseline values (13.4 vs 25.9 ng/mg, p = 0.01). In cell cultures, hypoxia down-regulated NGAL gene expression without significantly changing NGAL secretion in the culture medium. Hypoxia significantly increased the percentage of apoptotic/necrotic cells, especially after the second hypoxia-reoxygenation cycle. CONCLUSIONS: Exposure to hypobaric-hypoxic environments does not cause significant and irreversible renal tubular injury in vivo and in vitro, except than in a late stage. The hypoxic insult does not seem to be mirrored by an increase of urinary NGAL in healthy men nor of NGAL gene expression in HK-2 cell culture or secretion in the culture medium in the in vitro conditions reported in the present study.

Biological Effects of XyloCore, a Glucose Sparing PD Solution, on Mesothelial Cells: Focus on Mesothelial-Mesenchymal Transition, Inflammation and Angiogenesis.

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.

Endothelial Glycocalyx as a Regulator of Fibrotic Processes.

The endothelial glycocalyx, the gel layer covering the endothelium, is composed of glycosaminoglycans, proteoglycans, and adsorbed plasma proteins. This structure modulates vessels' mechanotransduction, vascular permeability, and leukocyte adhesion. Thus, it regulates several physiological and pathological events. In the present review, we described the mechanisms that disturb glycocalyx stability such as reactive oxygen species, matrix metalloproteinases, and heparanase. We then focused our attention on the role of glycocalyx degradation in the induction of profibrotic events and on the possible pharmacological strategies to preserve this delicate structure.