Calpain Expression Research Articles

Suppression of calpain expression by NSAIDs is associated with inhibition of cell migration in rat duodenum.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the alleviation of pain and inflammation, but these drugs are also associated with a suite of negative side effects. Gastrointestinal (GI) toxicity is particularly concerning since it affects an estimated 70% of individuals taking NSAIDs routinely, and evidence suggests the majority of toxicity is occurring in the small intestine. Traditionally, NSAID-induced GI toxicity has been associated with indiscriminate inhibition of cyclooxygenase isoforms, but other mechanisms, including inhibition of cell migration, intestinal restitution, and wound healing, are likely to contribute to toxicity. Previous efforts demonstrated that treatment of cultured intestinal epithelial cells (IEC) with NSAIDs inhibits expression and activity of calpain proteases, but the effects of specific inhibition of calpain expression in vitro or the effects of NSAIDs on intestinal cell migration in vivo remain to be determined. Accordingly, we examined the effect of suppression of calpain protease expression with siRNA on cell migration in cultured IECs and evaluated the effects of NSAID treatment on epithelial cell migration and calpain protease expression in rat duodenum. Our results show that calpain siRNA inhibits protease expression and slows migration in cultured IECs. Additionally, NSAID treatment of rats slowed migration up the villus axis and suppressed calpain expression in duodenal epithelial cells. Our results are supportive of the hypothesis that suppression of calpain expression leading to slowing of cell migration is a potential mechanism through which NSAIDs cause GI toxicity.

Cyclosporine-A induces endoplasmic reticulum stress and influences pro-apoptotic factors in human gingival fibroblasts.

Cyclosporine-A (CsA) induces gingival overgrowth. Cyclosporine's anti-apoptotic activity in human gingival fibroblast is due to desensitization of mitochondrial permeability transition pore (MPTP) and augmentation of anti-apoptotic, Bcl2. Alternative mechanisms of apoptosis exist involving enzymes like calcium-dependent Calpain and signaling events related to apoptosis, like Glycogen synthase kinase 3β (GSK3β) and protein kinase A (PKA). Cyclosporine-A in renal tubular cells induces endoplasmic reticulum stress (ER stress) which has not been explored in gingival overgrowth. Hence, this study was carried out to assess the influence of Cyclosporine-on ER stress and on the alternate anti-apoptotic mechanisms. Human gingival fibroblasts were treated with CsA, and expression of ER stress markers, such as binding immunoglobulin protein and CCAAT/enhancer-binding protein homologous protein (CHOP), MPTP, and expression of Calpain & GSK3β /PKA were estimated. The results showed CsA-added fibroblast significantly increasing the expression of Endoplasmic reticulum stress markers. Contrary to usual ER stress outcome of apoptosis, we observed Cyclosporine's anti-apoptotic action in spite of augmented ER stress markers. We conclude that CsA's independent action on different organelles may alter the conventional outcome of ER stress.

The role of calpains in ventilator-induced diaphragm atrophy

BackgroundControlled mechanical ventilation (CMV) is associated with diaphragm dysfunction. Dysfunction results from muscle atrophy and injury of diaphragm muscle fibers. Enhanced proteolysis and reduced protein synthesis play an important role in the development of atrophy. The current study is to evaluate the effects of the calpains inhibitor calpeptin on the development of diaphragm atrophy and activation of key enzymes of the ubiquitin-proteasome pathway in rats under CMV.MethodsThree groups of rats were studied: control animals (CON, n = 8), rats subjected to 24 h of MV (CMV, n = 8), and rats subjected to 24 h of MV after administration of the calpain inhibitor calpeptin (CMVC, n = 8). The diaphragm was analyzed for calpain activity, myosin heavy chain (MHC) content, and cross-sectional area (CSA) of diaphragmatic muscle fibers as a marker for muscle atrophy. In addition, key enzymes of the ubiquitin-proteasome pathway (MAFbx and MuRF1) were also studied.ResultsCMV resulted in loss of both MHCfast and MHCslow. Furthermore, the CSA of diaphragmatic muscle fibers was significantly decreased after 24 h of CMV. However, calpain inhibitor calpeptin prevented loss of MHC and CSA after CMV. In addition, calpeptin prevented the increase in protein expression of calpain1 and calpain2 and reduced calpain activity as indicated by reduced generation of the calpain cleavage product αII-spectrin in the diaphragm. CMV-induced upregulation of both MAFbx and MuRF1 protein levels was attenuated by treatment with calpeptin.ConclusionsThe calpain inhibitor calpeptin prevents MV-induced muscle atrophy. In addition, calpeptin attenuated the expression of key proteolytic enzymes known to be involved in ventilator-induced diaphragm atrophy, including MAFbx and MuRF1.

CaSR and calpain contribute to the ischemia reperfusion injury of spinal cord

Spinal cord ischemia reperfusion injury (SCIRI) can cause spinal cord dysfunction and even devastating paraplegia. Calcium-sensing receptor (CaSR) and calpain are two calcium related molecules which have been reported to be involved in the ischemia reperfusion injury of cardiomyocytes and the subsequent apoptosis. Here, we studied the expression of CaSR and calpain in spinal cord neurons and tissues, followed by the further investigation of the role of CaSR/calpain axis in the cellular apoptosis process during SCIRI. The results of in vitro and in vivo studies showed that the expression of CaSR and calpain in spinal cord neurons increased during SCIRI. Moreover, the CaSR agonist GdCl3 and antagonist NPS-2390 enhanced or decreased the expression of CaSR and calpain respectively. The expressions of CaSR and calpain were also consistent with the cellular apoptosis in spinal cord. Taken together, CaSR-calpain contributes to the SCIRI apoptosis, and CaSR antagonist might be a helpful drug for alleviating SCIRI.

N-Butylphthalide Improves Cognitive Function in Rats after Carbon Monoxide Poisoning.

Cognitive impairment is the most common neurologic sequelae after carbon monoxide (CO) poisoning, and the previous investigations have demonstrated that N-Butylphthalide (NBP) could exert a broad spectrum of neuroprotective properties. The current study aimed to investigate the effect of NBP on cognitive dysfunction in rats after acute severe CO poisoning. Rats were randomly divided into a normal control group, a CO poisoning group and a CO+NBP group. The animal model of CO poisoning was established by exposure to CO in a chamber, and then all rats received hyperbaric oxygen therapy once daily, while rats in CO+NBP group were administered orally NBP (6 mg/ 100g) by gavage twice a day additionally. The results indicated that CO poisoning could induce cognitive impairment. The ultrastructure of hippocampus was seriously damaged under transmission electron microscopy, and the expressions of calpain 1 and CaMK II proteins were significantly elevated after CO exposure according to the analysis of immunofluorescence staining and western blot. NBP treatment could evidently improve cognitive function, and maintain ultrastructure integrity of hippocampus. The expression levels of both calpain 1 and CaMK II proteins in CO+NBP group were considerably lower than that of CO poisoning group (P < 0.05). Taken together, this study highlights the molecular mechanism of cognitive dysfunction in rats after CO exposure via the upregulation of both calpain 1 and CaMK II proteins. The administration of NBP could balance the expressions of calpain 1 and CaMK II proteins and improve cognitive function through maintaining ultrastructural integrity of hippocampus, and thus may play a neuroprotective role in brain tissue in rats with CO poisoning.

Decreased calpain 6 expression is associated with tumorigenesis and poor prognosis in HNSCC.

Calpains are a family of intracellular cysteine proteases involved in various biological processes. Previously, the family was identified to have abnormal expression in several types of malignant tumor. Calpain 6 was less well known; however, it was recently identified to be involved in the carcinogenesis of certain types of malignant tumor. However, the expression of calpain 6 in head and neck squamous cell carcinoma (HNSCC) remains unclear. A total of six datasets from the Gene Expression Omnibus (GEO) was analyzed and an association between calpain 6 expression levels and HNSCC was identified, with the expression of calpain 6 observed to be significantly decreased in HNSCC (P<0.01). However, the expression of calpain 6 may vary between distinct tumor stages of HNSCC. Furthermore, calpain 6 expression was positively associated with the survival rate in patients with HNSCC (P<0.05), with increased expression of calpain 6 associated with an improved survival outcome. Calpain 6 expression was analyzed using an HNSCC tissue microarray and these results were consistent with the statistical analysis of the bioinformatics data from the GEO, indicating that calpain 6 may be a tumor suppressor protein in HNSCC.

Negative feedback regulation between microRNA let-7g and LOX-1 mediated hypoxia-induced PASMCs proliferation

BackgroundPulmonary hypertension (PH) is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Our lately study demonstrated that let-7g inhibited hypoxia-induced proliferation of PASMCs via repressing c-myc-Bmi-1-p16 signaling pathway. However, the upstream of let-7g has not yet been fully defined. Previous studies have shown that LOX-1, a target of let-7g, could also regulate the expression of let-7g in human aortic endothelial cells. In this present study, we aimed to investigate whether there is a negative feedback regulation between microRNA let-7g and LOX-1 in hypoxia-induced proliferation of PASMCs. MethodsSD Rats were exposed to hypoxia (10% O2, 3 weeks) to induce PH. HE staining was used to evaluate pulmonary artery remodeling. in situ hybridization and immunohistochemistry were performed to assess the expression and distribution of let-7g and LOX-1, respectively. MTS, EDU and flow cytometry were performed to evaluate PASMCs proliferation. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were conducted to assess the expression of let-7g, LOX-1, calpain-1,-2,-4, and OCT-1. ResultsThe expression of let-7g was significantly down-regulated in pulmonary arteries of hypoxia-induced PH rats accompanied by pulmonary vascular remodeling, whereas let-7g mimic inhibited hypoxia-induced proliferation of PASMCs and up-regulation of LOX-1 expression. LOX-1 blocking reversed hypoxia-induced down-regulation of let-7g expression. Calpains, protein kinase C and OCT-1 were involved in negative feedback regulation between let-7g and LOX-1. ConclusionNegative feedback regulation between let-7g and LOX-1 mediated hypoxia-induced proliferation of in PASMCs.

Calpain-1 Expression in Triple-Negative Breast Cancer: A Potential Prognostic Factor Independent of the Proliferative/Apoptotic Index.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer in which calpain system plays an important role in its cellular processes including apoptosis and proliferation. Although such roles have been assessed in tumor pathogenesis, the correlation of its expression to the proliferating/apoptotic index has not been studied yet. Immunohistochemical staining of calpain-1 was performed on paraffin-embedded tissues to correlate its expression with clinicopathological variables and outcome. The proliferation activity was determined by calculating the percentage of cells expressing the Ki-67 antigen. The apoptotic index was assessed morphologically and biochemically using Haematoxylin & Eosin method and Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, respectively. Calpain-1 was significantly expressed in TNBC tissues varying from low to high with a significant correlation to lymph node status but not with the other clinicopathological variables, suggesting its role as a prognostic factor. In addition, a positive correlation was found between both apoptotic counts assays (P < 0.001, r = 0.547) as well as with proliferation (P = 0.045). Calpain-1 expression had no significant correlation with either proliferation (P = 0.29) or apoptotic indices (P = 0.071 and P = 0.100). Determining calpain-1 expression may provide relevant prognostic value for TNBC cancer patients.

Gonadal steroids block the calpain-1-dependent intrinsic pathway of apoptosis in an experimental rat stroke model

Objectives: Apoptosis plays an important role in the progression of the ischemic penumbra after reperfusion. Estrogen and progesterone have neuroprotective effects against ischemic brain damage, however the exact mechanisms of neuroprotection and signaling pathways is not completely understood. In this study, we investigated the possible regulatory effects of a combined steroid treatment on extrinsic and intrinsic apoptotic signaling pathways after cerebral ischemia.Methods: Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (tMCAO) using an intraluminal filament technique for 1 h followed by 23 h reperfusion. Estrogen and progesterone were immediately injected after tMCAO subcutaneously. Sensorimotor functional tests and the infarct volume were evaluated 24 h after ischemia. Protein expression of calpain-1 and Fas receptor (FasR), key members of intrinsic and extrinsic apoptosis, were determined in the penumbra region of the ischemic brain using western blot analysis, immunohistochemistry, and TUNEL staining.Results: Neurological deficits and infarct volume were significantly reduced following hormone therapy. Calpain-1 up-regulation and caspase-3 activation were apparent 24 h after ischemia in the peri-infarct area of the cerebral cortex. Steroid hormone treatment reduced infarct pathology and attenuated the induction of both proteases. FasR protein levels were not affected by ischemia and hormone application.Conclusion: We conclude that a combined steroid treatment inhibits ischemia-induced neuronal apoptosis through the regulation of intrinsic pathways.

A novel benzofuran derivative, ACDB, induces apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress.

Chondrosarcoma is one of the bone tumor with high mortality in respond to poor radiation and chemotherapy treatment. Here, we analyze the antitumor activity of a novel benzofuran derivative, 2-amino-3-(2-chlorophenyl)-6-(4-dimethylaminophenyl)benzofuran-4-yl acetate (ACDB), in human chondrosarcoma cells. ACDB increased the cell apoptosis of human chondrosarcomas without harm in chondrocytes. ACDB also enhanced endoplasmic reticulum (ER) stress, which was characterized by varieties in the cytosolic calcium levels and induced the expression of glucose-regulated protein (GRP) and calpain. Furthermore, the ACDB-induced chondrosarcoma apoptosis was associated with the upregulation of the B cell lymphoma-2 (Bcl-2) family members including pro- and anti-apoptotic proteins, downregulation of dysfunctional mitochondria that released cytochrome C, and subsequent activation of caspases-3. In addition, the ACDB-mediated cellular apoptosis was suppressed by transfecting cells with glucose-regulated protein (GRP) and calpain siRNA or treating cells with ER stress chelators and caspase inhibitors. Interestingly, animal experiments illustrated a reduction in the tumor volume following ACDB treatment. Together, these results suggest that ACDB may be a novel tumor suppressor of chondrosarcoma, and this study demonstrates that the novel antitumor agent, ACDB, induced apoptosis by mitochondrial dysfunction and ER stress in human chondrosarcoma cells in vitro and in vivo.

Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival

Impaired apoptosis is one of the hallmarks of cancer. Caspase-3 and -8 are key regulators of the apoptotic response and have been shown to interact with the calpain family, a group of cysteine proteases, during tumorigenesis. The current study sought to investigate the prognostic potential of caspase-3 and -8 in breast cancer, as well as the prognostic value of combinatorial caspase and calpain expression. A large cohort (n = 1902) of early stage invasive breast cancer patients was used to explore the expression of caspase-3 and -8. Protein expression was examined using standard immunohistochemistry on tissue microarrays. High caspase-3 expression, but not caspase-8, is significantly associated with adverse breast cancer-specific survival (P = 0.008 and P = 0.056, respectively). Multivariate analysis showed that caspase-3 remained an independent factor when confounding factors were included (hazard ratio (HR) 1.347, 95% confidence interval (CI) 1.086–1.670; P = 0.007). The analyses in individual subgroups demonstrated the significance of caspase-3 expression in clinical outcomes in receptor positive (ER, PR or HER2) subgroups (P = 0.001) and in non-basal like subgroup (P = 0.029). Calpain expression had been previously assessed. Significant association was also found between high caspase-3/high calpain-1 and breast cancer-specific survival in the total patient cohort (P = 0.005) and basal-like subgroup (P = 0.034), as indicated by Kaplan–Meier analysis. Caspase-3 expression is associated with adverse breast cancer-specific survival in breast cancer patients, and provides additional prognostic values in distinct phenotypes. Combinatorial caspase and calpain expression can predict worse prognosis, especially in basal-like phenotypes. The findings warrant further validation studies in independent multi-centre patient cohorts.

Inhibition of calpain on oxygen glucose deprivation-induced RGC-5 necroptosis.

The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.

Calpain inhibitor calpeptin suppresses pancreatic cancer by disrupting cancer-stromal interactions in a mouse xenograft model.

Desmoplasia contributes to the aggressive behavior of pancreatic cancer. However, recent clinical trials testing several antifibrotic agents on pancreatic cancer have not shown clear efficacy. Therefore, further investigation of desmoplasia‐targeting antifibrotic agents by another mechanism is needed. Calpeptin, an inhibitor of calpains, suppressed fibroblast function and inhibited fibrosis. In this study, we investigated the anticancer effects of calpeptin on pancreatic cancer. We investigated whether calpeptin inhibited tumor progression using a mouse xenograft model. We used quantitative RT‐PCR to evaluate the expression of calpain‐1 and calpain‐2 mRNA in pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs). We also undertook functional assays, including proliferation, migration, and invasion, to evaluate the inhibitory effects of calpeptin on PCCs and PSCs. Quantitative RT‐PCR indicated that PCCs and PSCs expressed calpain‐2 mRNA. Calpeptin reduced tumor volume (P = 0.0473) and tumor weight (P = 0.0471) and inhibited the tumor desmoplastic reaction (P < 0.001) in xenograft tumors in nude mice. Calpeptin also inhibited the biologic functions of PCCs and PSCs including proliferation (P = 0.017), migration (P = 0.027), and invasion (P = 0.035) in vitro. Furthermore, calpeptin reduced the migration of PCCs and PSCs by disrupting the cancer–stromal interaction (P = 0.0002). Our findings indicate that calpeptin is a promising antitumor agent for pancreatic cancer, due not only to its suppressive effect on PCCs and PSCs but also its disruption of the cancer–stromal interaction.

Deciphering the role of ferulic acid against streptozotocin-induced cellular stress in the cardiac tissue of diabetic rats

The cardiomyocytes are one of the major sources of hyperglycemia induced ROS generation. The present study focuses on the ameliorative role of ferulic acid in combating cardiac complications in diabetic rats. Induction of diabetes by STZ in male Wistar rats (at a dose of 50 mg kg−1 body wt, i.p.) reduced body weight and plasma insulin level, enhanced blood glucose, disturbed the intra-cellular antioxidant machineries and disintegrated the normal radiation pattern of cardiac muscle fibers. Induction of ER stress (up-regulation in the levels of CHOP, GRP78, eIF2α signaling, increased calpain-1 expression), caspase-3 activation, PARP cleavage and DNA fragmentation were evidenced from immunoblot analyses and DNA fragmentation assay. However, ferulic acid administration, (at a dose of 50 mg kg−1 body wt, orally for eight weeks) in post-hyperglycemia could reverse such adverse effects. Also, the molecule increased GLUT-4 translocation to the cardiac membrane by enhanced phosphorylation of PI3Kinase, AKT and inactivation of GSK-3β thereby altering the hyperglycemic condition in the cardiac tissue of diabetic rats. Therefore, as a potential therapeutic, ferulic acid, exhibiting antioxidant and hypoglycemic effects, may hold promise in circumventing stress mediated diabetic cardiomyopathy in rats.

Effect of 8-hydroxyquinoline and derivatives on human neuroblastoma SH-SY5Y cells under high glucose

8-Hydroxyquinoline and derivatives exhibit multifunctional properties, including antioxidant, antineurodegenerative, anticancer, anti-inflammatory and antidiabetic activities. In biological systems, elevation of intracellular calcium can cause calpain activation, leading to cell death. Here, the effect of 8-hydroxyquinoline and derivatives (5-chloro-7-iodo-8-hydroxyquinoline or clioquinol and 8-hydroxy-5-nitroquinoline or nitroxoline) on calpain-dependent (calpain-calpastatin) pathways in human neuroblastoma (SH-SY5Y) cells was investigated. 8-Hydroxyquinoline and derivatives ameliorated high glucose toxicity in SH-SY5Y cells. The investigated compounds, particularly clioquinol, attenuated the increased expression of calpain, even under high-glucose conditions. 8-Hydroxyquinoline and derivatives thus adversely affected the promotion of neuronal cell death by high glucose via the calpain-calpastatin signaling pathways. These findings support the beneficial effects of 8-hydroxyquinolines for further therapeutic development.

Simvastatin inhibited oxLDL-induced proatherogenic effects through calpain-1-PPARγ-CD36 pathway.

We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, inhibits atherosclerosis in rats. The present study was designed to investigate the effect of simvastatin on mouse peritoneal macrophage foam cell formation, the early feature of atherosclerosis, and explore its mechanisms. The results showed that simvastatin decreased cholesterol content and DiI-oxLDL (1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate - oxidized low-density lipoprotein) uptake, reduced the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the medium, down-regulated the mRNA and protein expression of CD36 (a fatty acid receptor), and reduced the mRNA expressions of peroxisome proliferator-activated receptor gamma (PPARγ), TNF-α, and IL-6 in macrophages treated with oxLDL. However, PPARγ agonist troglitazone partly abolished the effects of simvastatin on foam cells. In addition, simvastatin reduced the protein expression of calpain-1, a Ca2+-sensitive cysteine protease, in oxLDL-treated macrophages. Furthermore, PD150606, a specific calpain inhibitor, reduced mRNA expressions of PPARγ and CD36 in macrophages treated with oxLDL. Combination of simvastatin and PD150606 had no further effect on mRNA expression of PPARγ and CD36 compared with either alone. However, over-expression of calpain-1 in macrophages partly reversed the simvastatin effects, including cell cholesterol content, mRNA expressions of PPARγ, and CD36. The results suggested that simvastatin inhibits foam cell formation of oxLDL-treated macrophages through a calpain-1-PPARγ-CD36 pathway.

Molecular and biochemical evidence on the protective effects of quercetin in isoproterenol-induced acute myocardial injury in rats.

Cardioprotection represents one of the most important and realistic aspects of preventive therapy today. Quercetin, a naturally occurring dietary flavone, has been studied extensively for its antioxidant properties. The objective of present study is to find out the cardioprotective activity and to explore the underlying mechanisms of quercetin pretreatment (50 mg/kg body weight, orally) for 14 days against isoproterenol (ISO; 100 mg/kg body weight, subcutaneously) induced myocardial infarction in Wistar rats. Cardiac diagnostic markers, oxidative stress, inflammatory cytokines, histopathology along with gene expression analysis of calpain 1 and 2 were carried out in experimental rats. Quercetin pretreatment showed protective effects on heart by significantly attenuating the ISO-induced oxidative stress, inflammation, protecting heart architecture, and by downregulation of the expression of calpain. Overall, these findings revealed the cardio-protective potential of quercetin and its mechanism of action against ISO-induced MI in rats.

Astaxanthin intake attenuates muscle atrophy caused by immobilization in rats

Astaxanthin is a carotenoid pigment and has been shown to be an effective inhibitor of oxidative damage. We tested the hypothesis that astaxanthin intake would attenuate immobilization‐induced muscle atrophy in rats. Male Wistar rats (14‐week old) were fed for 24 days with either astaxanthin or placebo diet. After 14 days of each experimental diet intake, the hindlimb muscles of one leg were immobilized in plantar flexion position using a plaster cast. Following 10 days of immobilization, both the atrophic and the contralateral plantaris muscles were removed and analyzed to determine the level of muscle atrophy along with measurement of the protein levels of CuZn‐superoxide dismutase (CuZn‐SOD) and selected proteases. Compared with placebo diet animals, the degree of muscle atrophy in response to immobilization was significantly reduced in astaxanthin diet animals. Further, astaxanthin supplementation significantly prevented the immobilization‐induced increase in the expression of CuZn‐SOD, cathepsin L, calpain, and ubiquitin in the atrophied muscle. These results support the postulate that dietary astaxanthin intake attenuates the rate of disuse muscle atrophy by inhibiting oxidative stress and proteolysis via three major proteolytic pathways.

Calpain Genetic Disruption and HSP90 Inhibition Combine To Attenuate Mammary Tumorigenesis

Calpain is an intracellular Ca(2+)-regulated protease system whose substrates include proteins involved in proliferation, survival, migration, invasion, and sensitivity to therapeutic drugs. Genetic disruption of calpain attenuated the tumorigenic potential of breast cancer cells and hypersensitized cells to 17AAG, an inhibitor of the molecular chaperone HSP90. Calpain-1 or -2 overexpression rendered cells resistant to 17AAG, whereas downregulation or inhibition of calpain-1/2 led to increased cell death in multiple breast cancer cell lines, including models of HER2(+) (SKBR3) and triple-negative basal-cell-like (MDA-MB-231) breast cancer. In an MDA-MB-231 orthotopic xenograft model, calpain knockdown or 17AAG treatment independently attenuated tumor growth and metastasis, while the combination was most effective. Calpain knockdown was associated with increased 17AAG-induced degradation of the HSP90 clients cyclin D1 and AKT and multidrug resistance protein 2, which correlated with increased expression of antimitogenic p27(KIP1) and proapoptotic BIM proteins. Like other therapeutics, 17AAG can be effluxed by specific ABC transporters. Calpain expression positively correlated with the expression of P glycoprotein in mouse embryonic fibroblasts. Importantly, we show that calpain affects ABC transporter function and efflux of clinically relevant doxorubicin. These observations provide a compelling rationale for exploring the combination of calpain inhibition with new or existing cancer therapeutics.

The inhibition of calpains ameliorates vascular restenosis through MMP2/TGF-β1 pathway.

Restenosis limits the efficacy of vascular percutaneous intervention, in which vascular smooth muscle cell (VSMC) proliferation and activation of inflammation are two primary causal factors. Calpains influence VSMC proliferation and collagen synthesis. However, the roles of calpastatin and calpains in vascular restenosis remain unclear. Here, restenosis was induced by ligating the left carotid artery, and VSMCs were pretreated with platelet-derived growth factor (PDGF)-BB. Adenovirus vector carrying MMP2 sequence and specific small interfering RNA against calpain-1/2 were introduced. Finally, restenosis enhanced the expression of calpain-1/2, but reduced calpastatin content. In calpastatin transgenic mice, lumen narrowing was attenuated gradually and peaked on days 14–21. Cell proliferation and migration as well as collagen synthesis were inhibited in transgenic mice, and expression of calpain-1/2 and MMP2/transforming growth factor-β1 (TGF-β1). Consistently, in VSMCs pretreated with PDGF-BB, calpastatin induction and calpains inhibition suppressed the proliferation and migration of VSMCs and collagen synthesis, and reduced expression of calpain-1/2 and MMP2/TGF-β1. Moreover, simvastatin improved restenosis indicators by suppressing the HIF-1α/calpains/MMP2/TGF-β1 pathway. However, MMP2 supplementation eliminated the vascular protection of calpastatin induction and simvastatin. Collectively, calpains inhibition plays crucial roles in vascular restenosis by preventing neointimal hyperplasia at the early stage via suppression of the MMP2/TGF-β1 pathway.