The inhibitory effects of calpain inhibitors on epithelial-mesenchymal transition of MCF-10A mammary epithelial cells induced by fibronectin / 药学学报

The aim of this research is to investigate the inhibitory effects of calpain inhibitors (ALLN and calpain inhibitor IV) on mammary epithelial-mesenchymal transition (EMT) of MCF-10A cells induced by fibronectin (FN). After FN treatment of MCF-10A cells for 48 h, cell migration and invasion were determined by scratch repair assay and matrigel coated transwell assay, respectively. Vimentin, E-cadherin, snail and calpain-2 protein expression were measured by Western blot. The results suggest that FN induced morphological changes in MCF-10A cells, significantly increased the migration and invasion abilities of MCF-10A cells, upregulated the expression of calpain-2, vimentin and snail, and downregulated the expression of E-cadherin. All such changes by FN were reversed with ALLN and calpain inhibitor IV. In conclusion, the upregulation of calpain-2 was involved in FN-induced EMT of MCF-10A mammary epithelial cells, which may be inhibited by ALLN and calpain inhibitor IV.

Rescuing axons from degeneration does not affect retinal ganglion cell death

After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.

Effects of Calpain inhibitor Ⅰ on glucocorticoid receptor expression and its transcript activation ability / 第三军医大学学报

Objective To investigate the effects of Calpain inhibitor Ⅰ on glucocorticoid receptor and its transcript activation ability. Methods Raw-264.7 cells were treated respectively with Calpain inhibitor Ⅰ and dexamethasone or both for 24 h. The changes of glucocorticoid receptor were observed. COS-7 cells were co-transfected with PRsh-GR? and pMAMneo-CAT vectors, and then the effects of Calpain inhibitor Ⅰ on glucocorticoid receptor and its transcript activation ability were detected. Results The glucocorticoid receptors was decreased after Raw-264.7 cells were treated with dexamethasone for 24 h. Calpain inhibitor Ⅰ could inhibit this effect to some extent. Co-transfection experiment revealed that Calpain inhibitor Ⅰ could also promote glucocorticoid receptor transcript activation ability. Conclusion Calpain inhibitor Ⅰ can inhibit the down-regulation of dexamethasone on glucocorticoid receptor, but promote glucocorticoid receptor transcript activation ability.

Systemic Administration of a Novel Calpain Inhibitor Attenuates Subarachnoid Hemorrhage Induced Vasospasm

The calcium-sensitive neutral protease, calpain, is activated in the basilar artery after subarachnoid hemorrhage. Pathological activation of this proteolytic enzyme has been suggested to contribute to cerebral vasospasm after subarachnoid hemorrhage. The present study was undertaken to evaluate the effects of a newly developed calpain inhibitor, CX-287 on vasospasm. A blind, randomized trial was utilized in which CX-287 was injected intravenously into subarachnoid hemorrhage rabbits. Two days after subarachnoid hemorrhage, animals were sacrificed by perfusion-fixation and cross-sectional areas of the basilar arteries were measured using histological techniques. Expressing the cross-sectional area in the untreated SAH animals as a percentage of control value, it was 38.4+/-5.7%. Basilar artery area of the treatment groups with 1.5mg/kg CX-287(b.i.d. or t.i.d) showed no statistical differences from subarachnoid only group(b.i.d.: 34.7%, t.i.d.: 49.0%). However, the treatment group with 3mg/kg CX-287 showed significant reversal of the subarachnoid hemorrhage-induced constriction(b.i.d.: 73.4%, p<0.0003, t.i.d.: 58.7%, p<0.05). These findings support the important role of calcium activated proteolysis by calpain in the pathophysiology of vasospasm after subarachnoid hemorrhage. Furthermore, these results provide the first demonstration that a calpain inhibitor can inhibit cerebral vasospasm.

Calpain inhibitors reduce the cornified cell envelope formation by inhibiting proteolytic processing of transglutaminase 1

Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.