Plasminogen/plasmin affects expression of glycolysis regulator TIGAR and induces autophagy in lung adenocarcinoma A549 cells.

Pericellular plasmin generation triggers apoptosis/anoikis in normal adherent cells. However, cancer cells are notoriously resistant to anoikis, enabling metastasis and new tumor growth beyond their original environment. Autophagy can be a major contributor to anoikis resistance in cancer. AIM: To investigate if protective autophagy can be induced in lung adenocarcinoma cells in response to plasminogen treatment. MATERIALS AND METHODS: Human lung adenocarcinoma A549 cells were incubated with Glu-plasminogen (0.1-1.0 µM) for 24 h. Pericellular plasmin activity was monitored spectrophotometrically by a cleavage of the specific chromogenic- substrate S-2251. Cell survival was assessed by 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)-test. Degradation of fibronectin, levels of autophagy markers (beclin-1 and light chain 3 (LC3)) and glycolysis regulator (TIGAR) were evaluated by western blot. Intracellular localization of LC-3 was visualized by immunocytochemistry. RESULTS: It was shown that plasminogen is converted into plasmin on the surface of adenocarcinoma cells in a dose-dependent manner. Plasmin disrupted cellular adhesive contacts resulting in cell detachment. A549 cells did not loss their viability after plasminogen treatment for 24 h, while 1.0 µM plasminogen was cytotoxic for non-transformed fibroblasts. Plasminogen 0.1, 0.5, and 1.0 µM induced 7.08-, 5.18-, and 3.78-fold elevation of TIGAR expression (p < 0.05), respectively. Enhanced TIGAR expression indicates switch on pentose phosphate pathway, protection against oxidative stress to prevent apoptosis, facilitation of DNA repair and the degradation of their own organelles (autophagy). Exposure of adenocarcinoma cells to plasminogen in concentrations of 0.1 and 0.5 µM caused 1.74- and 2.19-fold elevation of beclin-1 expression vs untreated cells (p < 0.05), respectively. Unlike K1-3 fragment, plasminogen treatment (0.1-0.5 µM) resulted in increased expression of LC3-I and stimulated rapid conversion of LC3-I to LC3-II. Up-regulation of beclin-1 levels and enhanced LC3-I/II conversion in plasminogen-treated A549 cells are the hallmarks of autophagy induction. According to immunocytochemistry data, increased LC3 puncta and autophagosome formation after exposure to plasminogen could reflect autophagy activation. CONCLUSIONS: Therefore, we showed stimulation of prosurvival signals and induction of autophagy in plasminogen-treated adenocarcinoma cells rendering them resistant to apoptosis/anoikis. Based on the obtained data, autophagy has a great potential for novel targets that affect cancer cell death, in addition to the current cytotoxic agents.

Effect of nicotinamide on amino acids content in bone collagen depending on biological availability of vitamins in diabetic rats.

Connective tissue is highly susceptible to imbalances induced by diabetes. Diabetes-related osteopenia, decreased bone strength etc. may be associated with altered metabolism of various collagens: Although it is assumed that alterations in collagen amino acids (AA) may strongly affect protein properties andphysiological functions, however, very limited evidences are present at the moment regarding AA composition of bone type I collagen and its relevance to abnormal availability of vitamins which are necessary for collagen synthesis in diabetes. We have tested whether nicotinamide (NAm) can influence type Icollagen formation and AA composition as well as vitamins availability in diabetes. After 4 weeks of STZ-induced diabetes (60 mg/ kg) male Wistar rats were injected for 2 weeks with/without NAm (200 mg/kg b. w). Acid extraction of type I collagen from the bones was performed with following stepwise salting out. The content of type I collagen after its acid extraction from the bones was estimated by the amounts of hydroxyproline. Amino acids were assayed by cation exchange chromatography Diabetes-associated changes in AA composition of type I collagen mainly affect those amino acids which are known to be involved in helix formation and cross-linking of the molecules. Diabetes was found to significantly reduce bone collagen contents of o-Pro, Gly, Ala, o-Lys and Pro, whereas Lys, His, Arg, Glu, Thr, Leu, Phe contents were elevated (P < 0.05). NAm treatment was able to partially normalise AA contents. In diabetes, blood serum and hepatic vitamin C and B3 contents were shown to be significantly lowered, whereas a-tocopherol was slightly increased compared with control (P < 0.05). Restoration of circulatory and liver vitamin C and B3 was observed. The data demonstrate the close relationship between the diabetes-associated decrease in type I collagen deposition, altered amino acids metabolism and impaired availability of vitamins, which are necessary for collagen synthesis. Thus, NAm might be a useful agent for treatment of bone failures related to diabetes.