Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization

Abstract

Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

“…4, A, B, E, and F). Since pThr 18 /Ser 19 -MRLC localized with stress fibers and in the nucleus, we investigated whether pThr 18 -MRLC and pSer 19 -MRLC had differential localizations using phosphorylation site-specific antibodies. pThr 18 -MRLC extensively localized with stress fibers; however, pSer 19 -MRLC localized in the nucleus of the cells (Fig.…”

“…Mammalian protein extraction reagent (M-PER) and the bicinchoninic acid protein assay reagent kit were purchased from Pierce (Rockford, IL). Antibodies for pMRLC (Thr 18 /Ser 19 , Thr 18 , and Ser 19 ) and total MRLC were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Rac1 and anti-focal adhesion kinase (FAK) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY).…”

“…Attachment and spreading assay. The attachment and spreading assay was performed as previously described (19). Cells were harvested by trypsinization, counted by a Beckman Coulter counter, and resuspended in serum-free DMEM.…”

“…The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr 18 /Ser 19 -MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr 18 -and pSer 19 -MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr 18 -MRLC without any effect on nuclear pSer 19 -MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells.…”

“…Myosin II plays important roles in many contractile-like cell functions including cell migration, adhesion, and retraction (36,42). Myosin II is activated by myosin regulatory light chain (MRLC) phosphorylation on Ser 19 (and Thr 18 ), whereas MRLC dephosphorylation leads to myosin II inactivation (4,5). It has been suggested that phosphorylated (p)MRLC might stabilize actomyosin, thus inhibiting the turnover of stress fibers (21,43).…”