Impact of carbamylation and glycation of collagen type I on migration of HT1080 human fibrosarcoma cells.

Collagen type I is an abundant component of the extracellular matrix and due to its longevity, constitutes a prominent target of non-enzymatic post-translational in vivo modifications such as carbamylation and glycation. These protein modifications involved in aging, renal diseases and diabetes, are linked to elevated cancer risk. In this in vitro study, we investigated the impact of carbamylated and glycated collagen type I on the migratory behavior of the highly invasive HT1080 human fibrosarcoma cells. The proliferation of HT1080 on modified collagens did not differ from that on native form. The glycated collagen delayed the cell adhesion time whereas the carbamylated one had no effect. The migration ability of HT1080 was studied by quantifying single cell speed using videomicroscopy. Glycation strongly inhibited mean cell speed by 47% whereas carbamylation moderately affected it by 12%. In addition, the influence of these collagen modifications on actin and vinculin organization was studied. On the glycated collagen, 63% of cells revealed a dramatic loss of actin stress fibers vs. 28% on the carbamylated one. In these cells, disorganized F-actin was accompanied with a disturbance of vinculin and both proteins were localized at the rim of the cells. Concerning the focal adhesion kinase (FAK) expression, glycated collagen only induced a significant inhibition. Whereas, both collagen modifications provoked a differential inhibition of FAK phosphorylation state by 25% for carbamylation and 60% for glycation. In conclusion, our data suggest that, in vivo, collagen glycation and carbamylation may affect tumor cell metastasis. This suggestion is supported by clinical studies reporting less aggressive tumors in diabetic or uremic patients. Consequently, the impact of such post-translational modifications has to be taken into account in order to better understand the link between aging, diabetes or uremia and cancer progression.

3D collagen type I matrix inhibits the antimigratory effect of doxorubicin.

BACKGROUND: The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D) context which simulates a natural microenvironment. METHODS: To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK) and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation. RESULTS: We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D) culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected beta1 integrin expression nor the state of phosphorylation of FAK and RhoA. CONCLUSION: This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.

Extracellular matrix proteins protect human HT1080 cells against the antimigratory effect of doxorubicin.

In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. They reveal cell locomotion as a key factor of microenvironment-mediated drug resistance. This new concept needs to be exploited in in vitro models to optimize the screening of new antimigratory drugs.

Temporal changes in free iron levels after brain ischemia Relevance to the timing of iron chelation therapy in stroke.

Whereas iron chelators have been proposed as therapeutic agents in stroke, changes in free iron levels have never been explored after focal brain ischemia. Therefore, free and total iron levels in cortical tissue and free iron levels in plasma were measured before and after (1, 4 and 24h) photothrombotic occlusion of cortical vessels in rats. Brain ferritin expression and localization were also investigated before and after (24, 72 and 192 h) occlusion. The results showed that free iron remained below detectable levels in plasma and that the lesion exhibited high levels of free and total iron. As compared to contralateral values, free iron levels in ischemic core and penumbra increased (+50%) at 1h and returned to control values at 4h post-occlusion. In contrast, the increase in total iron levels (+20-30%) was long-lasting, but confined to the ischemic core. A time-dependent increase in the expression of both chains of ferritin was detected in regions that previously exhibited free iron accumulation. Finally, ischemic damage was reduced by the liposoluble iron chelator 2,2'-dipyridyl (20 mg/kg, i.p.) when injected 15 min or 1 h post-occlusion, yet not later (4 h). In conclusion, our results show that focal brain ischemia results in an early and transient elevation in free iron levels in the ischemic tissue and suggest that free iron excess does not originate in blood. They also highlight the importance of starting iron chelation therapy as soon as possible after stroke.