Astragaloside IV mitigates hypoxia-induced cardiac hypertrophy through calpain-1-mediated mTOR activation.

BACKGROUND: Astragaloside IV (AsIV), a key functioning element of Astragalus membranaceus, has been recognized for its potential cardiovascular protective properties. However, there is a need to elucidate the impacts of AsIV on myocardial hypertrophy under hypoxia conditions and its root mechanisms. PURPOSE: This study scrutinized the influence of AsIV on cardiac injury under hypoxia, with particular emphasis on the role of calpain-1 (CAPN1) in mediating mTOR pathways. METHODS: Hypoxia-triggered cardiac hypertrophy was examined in vivo with CAPN1 knockout and wild-type C57BL/6 mice and in vitro with H9C2 cells. The impacts of AsIV, 3-methyladenine, and CAPN1 inhibition on hypertrophy, autophagy, apoptosis, [Ca2+]i, and CAPN1 and mTOR levels in cardiac tissues and H9C2 cells were investigated. RESULTS: Both AsIV treatment and CAPN1 knockout mitigated hypoxia-induced cardiac hypertrophy, autophagy, and apoptosis in mice and H9C2 cells. Moreover, AsIV, 3-methyladenine, and CAPN1 inhibition augmented p-mTOR level but reduced [Ca2+]i and CAPN1 level. Additionally, lentivirus-mediated CAPN1 overexpression in H9C2 cells exacerbated myocardial hypertrophy, apoptosis, and p-mTOR inhibition under hypoxia. Specifically, AsIV treatment reversed the impacts of increased CAPN1 expression on cardiac injury and the inhibition of p-mTOR. CONCLUSION: These findings suggest that AsIV may alleviate cardiac hypertrophy under hypoxia by attenuating apoptosis and autophagy through CAPN1-mediated mTOR activation.

Calpain and Cardiometabolic Diseases.

Calpain is defined as a member of the superfamily of cysteine proteases possessing the CysPC motif within the gene. Calpain-1 and -2, which are categorized as conventional isozymes, execute limited proteolysis in a calcium-dependent fashion. Accordingly, the calpain system participates in physiological and pathological phenomena, including cell migration, apoptosis, and synaptic plasticity. Recent investigations have unveiled the contributions of both conventional and unconventional calpains to the pathogenesis of cardiometabolic disorders. In the context of atherosclerosis, overactivation of conventional calpain attenuates the barrier function of vascular endothelial cells and decreases the immunosuppressive effects attributed to lymphatic endothelial cells. In addition, calpain-6 induces aberrant mRNA splicing in macrophages, conferring atheroprone properties. In terms of diabetes, polymorphisms of the calpain-10 gene can modify insulin secretion and glucose disposal. Moreover, conventional calpain reportedly participates in amino acid production from vascular endothelial cells to induce alteration of amino acid composition in the liver microenvironment, thereby facilitating steatohepatitis. Such multifaceted functionality of calpain underscores its potential as a promising candidate for pharmaceutical targets for the treatment of cardiometabolic diseases. Consequently, the present review highlights the pivotal role of calpains in the complications of cardiometabolic diseases and embarks upon a characterization of calpains as molecular targets.

Calpain inhibition decreases oxidative stress via mitochondrial regulation in a swine model of chronic myocardial ischemia.

INTRODUCTION: Calpain overexpression is implicated in mitochondrial damage leading to tissue oxidative stress and myocardial ischemic injury. The aim of this study was to determine the effects of calpain inhibition (CI) on mitochondrial impairment and oxidative stress in a swine model of chronic myocardial ischemia and metabolic syndrome. METHODS: Yorkshire swine were fed a high-fat diet for 4 weeks to induce metabolic syndrome then underwent placement of an ameroid constrictor to the left circumflex artery. Three weeks later, animals received: no drug (control, "CON"; n= 7); a low-dose calpain inhibitor (0.12 mg/kg; "LCI", n= 7); or high-dose calpain inhibitor (0.25 mg/kg; "HCI", n=7). Treatment continued for 5 weeks, followed by tissue harvest. Cardiac tissue was assayed for protein carbonyl content, as well as antioxidant and mitochondrial protein expression. Reactive oxygen species (ROS) and mitochondrial respiration was measured in H9c2 cells following exposure to normoxia or hypoxia (1%) for 24 h with or without CI. RESULTS: In ischemic myocardial tissue, CI was associated with decreased total oxidative stress compared to control. CI was also associated with increased expression of mitochondrial proteins superoxide dismutase 1, SDHA, and pyruvate dehydrogenase compared to control. 100 nM of calpain inhibitor decreased ROS levels and respiration in both normoxic and hypoxic H9c2 cardiomyoblasts. CONCLUSIONS: In the setting of metabolic syndrome, CI improves oxidative stress in chronically ischemic myocardial tissue. Decreased oxidative stress may be via modulation of mitochondrial proteins involved in free radical scavenging and production.

Implication of Vegetable Oil-Derived Hydroxynonenal in the Lysosomal Cell Death for Lifestyle-Related Diseases.

Lysosomes are membrane-bound vesicular structures that mediate degradation and recycling of damaged macromolecules and organelles within the cell. For ensuring the place of degradation within the acidic organelle, the integrity of the lysosomal-limiting membrane is critical in order to not injure the cell. As lysosomes fade away in response to acute intense insults or long-term mild insults, dissolving lysosomes are hardly detected during the phase of cell degeneration. If observed at the right time, however, lysosomal membrane rupture/permeabilization can be detected using an electron microscope. In both the experimental and clinical materials, here the author reviewed electron microphotographs showing disintegrity of the lysosomal-limiting membrane. Regardless of insults, cell types, organs, diseases, or species, leakage of lysosomal content occurred either by the apparent disruption of the lysosomal membrane (rupture) and/or through the ultrastructurally blurred membrane (permeabilization). Since lysosomal rupture occurs in the early phase of necrotic cell death, it is difficult to find vivid lysosomes after the cell death or disease are completed. A lipid peroxidation product, 4-hydroxy-2-nonenal (hydroxynonenal), is incorporated into the serum by the intake of ω-6 polyunsaturated fatty acid-rich vegetable oils (exogenous), and/or is generated by the peroxidation of membrane lipids due to the oxidative stress (intrinsic). Exogenous and intrinsic hydroxynonenal may synergically oxidize the representative cell stress protein Hsp70.1, which has dual functions as a 'chaperone protein' and 'lysosomal stabilizer'. Hydroxynonenal-mediated carbonylation of Hsp70.1 facilitates calpain-mediated cleavage to induce lysosomal membrane rupture and the resultant cell death. Currently, vegetable oils such as soybean and canola oils are the most widely consumed cooking oils at home and in restaurants worldwide. Accordingly, high linoleic acid content may be a major health concern, because cells can become damaged by its major end product, hydroxynonenal. By focusing on dynamic changes of the lysosomal membrane integrity at the ultrastructural level, implications of its rupture/permeabilization on cell death/degeneration were discussed as an etiology of lifestyle-related diseases.

A Nanocurcumin and Pyrroloquinoline Quinone Formulation Prevents Hypobaric Hypoxia-Induced Skeletal Muscle Atrophy by Modulating NF-κB Signaling Pathway.

Kushwaha, Asha D., and Deepika Saraswat. A nanocurcumin and pyrroloquinoline quinone formulation prevents hypobaric hypoxia-induced skeletal muscle atrophy by modulating NF-κB signaling pathway. High Alt Med Biol. 23:249-263, 2022. Background: Hypobaric hypoxia (HH)-induced deleterious skeletal muscle damage depends on exposure time and availability of oxygen at cellular level, which eventually can limit human work performance at high altitude (HA). Despite the advancements made in pharmacological (performance enhancer, antioxidants) and nonpharmacological therapeutics (acclimatization strategies), only partial success has been achieved in improving physical performance at HA. A distinctive combination of nanocurcumin (NC) and pyrroloquinoline quinone (PQQ) has been formulated (named NCF [nanocurcumin formulation], Indian patent No. 302877) in our laboratory, and has proven very promising in improving cardiomyocyte adaptation to chronic HH. We hypothesized that NCF might improve skeletal muscle adaptation and could be a performance enhancer at HA. Material and Methods: Adult Sprague-Dawley rats (220 ± 10 g) were divided into five groups (n = 6/group): normoxia vehicle control, hypoxia vehicle control, hypoxia NCF, hypoxia NC, and hypoxia PQQ. All the animals (except those in normoxia) were exposed to simulated HH in a chamber at temperature 22°C ± 2°C, humidity 50% ± 5%, altitude 25,000 ft for 1, 3, or 7 days. After completion of the stipulated exposure time, gastrocnemius and soleus muscles were excised from animals for further analysis. Results: Greater lengths of hypoxic exposure caused progressively increased muscle ring finger-1 (MuRF-1; p < 0.01) expression and calpain activation (0.56 ± 0.05 vs. 0.13 ± 0.02 and 0.44 ± 0.03 vs. 0.12 ± 0.021) by day 7, respectively in the gastrocnemius and soleus muscles. Myosin heavy chain type I (slow oxidative) fibers significantly (p > 0.01) decreased in gastrocnemius (>50%) and soleus (>46%) muscles by the seventh day of exposure. NCF supplementation showed (p ≤ 0.05) tremendous improvement in skeletal muscle acclimatization through effective alleviation of oxidative damage, and changes in calpain activity and atrophic markers at HA compared with hypoxia control or treatment alone with NC/PQQ. Conclusion: Thus, NCF-mediated anti-oxidative, anti-inflammatory effects lead to decreased proteolysis resulting in mitigated skeletal muscle atrophy under HH.

Calpain inhibition decreases myocardial fibrosis in chronically ischemic hypercholesterolemic swine.

OBJECTIVES: Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium. METHODS: Yorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and CI were continued for 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content. RESULTS: In the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters. CONCLUSIONS: Calpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities.

Effect of chronic alcohol consumption on myocardial apoptosis in the rat model of isoproterenol-induced myocardial injury and investigation on the cardioprotective role of calpain inhibitor 1.

We investigated the presence of myocardial apoptosis on isoproterenol (ISO)-induced myocardial injury (MI) after long-term high dose alcohol consumption and examined the antiapoptotic role of calpain inhibitor 1. Male Wistar Albino rats (n = 108) were divided into six groups: Control, alcohol (ethanol was given during 30 days for chronic alcohol consumption), MI (150 mg/kg ISO injection at last two days of alcohol consumption), alcohol + MI, alcohol + MI + calpain inhibitor 1 (10 mg/kg inhibitor was injected at 15 min before ISO injections) and Dimethyl Sulfoxide (DMSO) groups. Biochemical, histological, and morphometric methods determined apoptosis levels in the heart tissue of rats. Cytochrome c, caspase 3, and calpain levels were significantly high in alcohol, MI, and alcohol + MI groups. In contrast, mitochondrial cardiolipin content was found to be low in alcohol, MI, and alcohol + MI groups. These parameters were close to the control group in the therapy group. Histological and morphometric data have supported biochemical results. As a result of our biochemical data, myocardial apoptosis was seen in the alcohol, MI, and especially alcohol after MI groups. Calpain inhibitor 1 reduced apoptotic cell death and prevented myocardial tissue injury in these groups. The efficiency of calpain inhibitor was very marked in MI after long-term high dose alcohol consumption.

Protective Effects of Glutamine and Leucine Supplementation on Sepsis-Induced Skeletal Muscle Injuries.

This study investigated the effects of l-glutamine (Gln) and/or l-leucine (Leu) administration on sepsis-induced skeletal muscle injuries. C57BL/6J mice were subjected to cecal ligation and puncture to induce polymicrobial sepsis and then given an intraperitoneal injection of Gln, Leu, or Gln plus Leu beginning at 1 h after the operation with re-injections every 24 h. All mice were sacrificed on either day 1 or day 4 after the operation. Blood and muscles were collected for analysis of inflammation and oxidative damage-related biomolecules. Results indicated that both Gln and Leu supplementation alleviated sepsis-induced skeletal muscle damage by reducing monocyte infiltration, calpain activity, and mRNA expression levels of inflammatory cytokines and hypoxia-inducible factor-1α. Furthermore, septic mice treated with Gln had higher percentages of blood anti-inflammatory monocytes and muscle M2 macrophages, whereas Leu treatment enhanced the muscle expressions of mitochondrion-related genes. However, there were no synergistic effects when Gln and Leu were simultaneously administered. These findings suggest that both Gln and Leu had prominent abilities to attenuate inflammation and degradation of skeletal muscles in the early and/or late phases of sepsis. Moreover, Gln promoted the switch of leukocytes toward an anti-inflammatory phenotype, while Leu treatment maintained muscle bioenergetic function.

Inhibition of Calpain Alleviates Apoptosis in Coxsackievirus B3-induced Acute Virus Myocarditis Through Suppressing Endoplasmic Reticulum Stress.

Virus myocarditis (VMC) is a common cardiovascular disease and a major cause of sudden death in young adults. However, there is still a lack of effective treatments. Our previous studies found that calpain activation was involved in VMC pathogenesis. This study aims to explore the underlying mechanisms further. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin (Tg-CAST), the endogenous calpain inhibitor, were used to establish VMC model. Hematoxylin and eosin and Masson staining revealed inflammatory cell infiltration and fibrosis. An ELISA array detected myocardial injury. Cardiac function was measured using echocardiography. CVB3 replication was assessed by capsid protein VP1. Apoptosis was measured by TUNEL staining, flow cytometry, and western blot. The endoplasmic reticulum (ER) stress-related proteins were detected by western blot. Our data showed that CVB3 infection resulted in cardiac injury, as evidenced by increased inflammatory responses and fibrosis, which induced myocardial apoptosis. Inhibiting calpain, both by PD150606 and calpastatin overexpression, could attenuate these effects. Furthermore, ER stress was activated during CVB3 infection. However, calpain inhibition could downregulate some ER stress-associated protein levels such as GRP78, pancreatic ER kinase-like ER kinase (PERK), and inositol-requiring enzyme-1α (IRE-1α), and ER stress-related apoptotic factors, during CVB3 infection. In conclusion, calpain inhibition attenuated CVB3-induced myocarditis by suppressing ER stress, thereby inhibiting cardiomyocyte apoptosis.

Dopamine receptor D1 agonist inhibits glioblastoma via calpain­mediated ER stress and mitochondrial dysfunction.

Recent studies have reported the important roles of dopamine receptors in the early development and progression of glioblastoma (GBM). The present research aimed to explore the antineoplastic effect and intrinsic pathways of action of dopamine receptor D1 agonist SKF83959 on GBM cells. Flow cytometric analysis revealed a significant level of apoptotic cell death under SKF83959 treatment. SKF83959 administration increased intracellular calcium levels and oxidative stress through the phospholipase C/inositol trisphosphate pathway. The downstream calpains were activated and dysregulated by the increased calcium levels. The mitochondrial membrane potential­dependent staining assay revealed decreased mitochondrial transmembrane potential in GBM cells under SKF83959 treatment. The mitochondrial/cytosolic fraction and western blotting further demonstrated mitochondrial dysfunction and endoplasmic reticulum stress, followed by apoptosis. The calpain inhibitor, calpastatin, significantly reversed the increase in mitochondrial injury and endoplasmic reticulum stress and eventually ameliorated GBM cell apoptosis during SKF83959 treatment. Finally, the in vivo inhibitory efficacy of SKF83959 was verified in GBM xenograft models. In addition, immunohistochemistry and western blotting both revealed increased expression of calpains in xenograft GBM tissues. These results suggested a potential therapeutic target for human GBM treatment regarding calpain expression and activity regulation.

Calpain-Associated Proteolytic Regulation of the Stromal Microenvironment in Cancer.

BACKGROUND: Normalization of the stromal microenvironment is a promising strategy for cancer control. Cancer-associated fibroblasts, tumor-associated macrophages, and mesenchymal stromal cells have a central role in stromal functions. Accordingly, understanding these stromal cells is indispensable for the development of next-generation cancer therapies. Growing evidence suggests that calpain-induced intracellular proteolysis is responsible for cancer growth and stromal regulation. Calpain is a family of stress-responsive intracellular proteases and is inducible in cancer and stromal cells during carcinogenesis. OBJECTIVE: Here, we shed light on the recent advances that have been made in understanding how calpain contributes to stromal regulation in cancer. CONCLUSION: Calpains are activated in stromal cells, including pancreatic stellate cells and mesenchymal cells. They induce fibrogenic responses in cancer stroma. Moreover, these molecules contribute to epithelial-mesenchymal transition and endothelial-mesenchymal transition to provide mesenchymal stromal cells in the microenvironment and concomitantly participate in cancer angiogenesis. In addition to the conventional calpains, the unconventional calpain-9 is associated with epithelial-mesenchymal transition. Animal experiments showed that targeting calpain systems antagonizes cancer development; thus, this approach is promising for cancer control.

Relationship between beta-adrenergic agonists, calpain system activity and beef texture: A systematic review.

The objective was to conduct a systematic review to evaluate the effects of dietary supplementation with beta-adrenergic agonists on calpains and calpastatin activity in bovine muscle and changes in meat tenderness. A survey was conducted in June 2019 on Science Direct, Web of Science, Scopus, PubMed and Capes Periodicals, using four keyword combinations: agonist and calpain and cattle; agonist and calpain and bovine; agonist and calpain and heifers; agonist and calpain and steers. Thirteen studies were selected, 54% concluded that supplementation with beta-adrenergic agonists increases calpastatin activity, 23% observed increase in their gene expression and 23% reported no effect on activity or expression of this enzyme. Nine studies evaluated the influence of beta-adrenergic agonists supplementation on meat texture and all found an increase in shear force values. There is strong evidence that beta-adrenergic agonists may increase calpastatin activity in the muscle, causing damage to meat tenderness.

Pseudoginsenoside-F11 attenuates cognitive dysfunction and tau phosphorylation in sporadic Alzheimer's disease rat model.

We previously reported that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, significantly ameliorated Alzheimer's disease (AD)-associated cognitive defects in APP/PS1 and SAMP8 mice by inhibiting Aß aggregation and tau hyperphosphorylation, suggesting a potential therapeutic effect of PF11 in the treatment of AD. In the present study we further evaluated the therapeutic effects of PF11 on relieving cognitive impairment in a rat model of sporadic AD (SAD). SAD was induced in rats by bilateral icv infusion of streptozotocin (STZ, 3 mg/kg). The rats were treated with PF11 (2, 4, 8 mg·kg-1·d-1, ig) or a positive control drug donepezil (5 mg·kg-1·d-1, ig) for 4 weeks. Their cognitive function was assessed in the nest building, Y-maze, and Morris water maze tests. We showed that STZ icv infusion significantly affected the cognitive function, tau phosphorylation, and insulin signaling pathway in the hippocampus. Furthermore, STZ icv infusion resulted in significant upregulation of the calpain I/cyclin-dependent protein kinase 5 (CDK5) signaling pathway in the hippocampus. Oral administration of PF11 dose-dependently ameliorated STZ-induced learning and memory defects. In addition, PF11 treatment markedly reduced the neuronal loss, protected the synapse structure, and modulated STZ-induced expression of tau phosphorylation by regulating the insulin signaling pathway and calpain I/CDK5 signaling pathway in the hippocampus. Donepezil treatment exerted similar beneficial effects in STZ-infused rats as the high dose of PF11 did. This study highlights the excellent therapeutic potential of PF11 in managing AD.

Magnesium deficiency with high calcium-to-magnesium ratio promotes a metastatic phenotype in the CT26 colon cancer cell line.

Magnesium (Mg) plays important roles in maintaining genomic stability and cellular redox. Mg also serves as nature's physiological calcium (Ca) channel antagonist, controlling intracellular Ca entry. Because Ca is the most important second messenger, its intracellular concentration is tightly regulated. Excess intracellular Ca can activate aberrant signaling pathways leading to the acquisition of pathological characteristics and cell injury. Several epidemiological studies have linked Mg deficiency (MgD) and increased Ca:Mg ratios with higher incidences of colon cancer and increased mortality. While it is estimated that less than 50% of the US population consumes the recommended daily allowance for Mg, Ca supplementation is widespread. Therefore, we studied the effect of MgD, with variable Ca:Mg ratios on cellular oxidative stress, cell migration, calpain activity, and associated signaling pathways using the CT26 colon cancer cell line. MgD (with Ca:Mg ratios >1) elevated intracellular Ca levels, calpain activity and TRPM7 expression, as well as oxidative stress and cell migration, consistent with observed degradation of full-length E-cadherin, ß-catenin, and N-terminal FAK. MgD was accompanied by enhanced degradation of IκBα and the transactivation domain containing the C-terminus of NF-κB p65 (RelA). MgD-exposed CT26 cells exhibited increased p53 degradation and aneuploidy, markers of genomic instability. By contrast, these pathological changes were not observed when CT26 were cultured under MgD conditions where the Ca:Mg ratio was kept at 1. Together, these data support that exposure of colon cancer cells to MgD with physiological Ca concentrations (or increasing Ca:Mg ratios) leads to the acquisition of a more aggressive, metastatic phenotype.

Electroacupuncture Pretreatment Elicits Tolerance to Cerebral Ischemia/Reperfusion through Inhibition of the GluN2B/m-Calpain/p38 MAPK Proapoptotic Pathway.

Background: As one of the first steps in the pathology of cerebral ischemia, glutamate-induced excitotoxicity progresses too fast to be the target of postischemic intervention. However, ischemic preconditioning including electroacupuncture (EA) might elicit cerebral ischemic tolerance through ameliorating excitotoxicity. Objective: To investigate whether EA pretreatment based on TCM theory could elicit cerebral tolerance against ischemia/reperfusion (I/R) injury, and explore its potential excitotoxicity inhibition mechanism from regulating proapoptotic pathway of the NMDA subtype of glutamate receptor (GluN2B). Methods: The experimental procedure included 5 consecutive days of pretreatment stage and the subsequent modeling stage for one day. All rats were evenly randomized into three groups: sham MCAO/R, MCAO/R, and EA+MCAO/R. During pretreatment procedure, only rats in the EA+MCAO/R group received EA intervention on GV20, SP6, and PC6 once a day for 5 days. Model preparation for MCAO/R or sham MCAO/R started 2 hours after the last pretreatment. 24 hours after model preparation, the Garcia neurobehavioral scoring criteria was used for the evaluation of neurological deficits, TTC for the measurement of infarct volume, TUNEL staining for determination of neural cell apoptosis at hippocampal CA1 area, and WB and double immunofluorescence staining for expression and the cellular localization of GluN2B and m-calpain and p38 MAPK. Results: This EA pretreatment regime could improve neurofunction, decrease cerebral infarction volume, and reduce neuronal apoptosis 24 hours after cerebral I/R injury. And EA pretreatment might inhibit the excessive activation of GluN2B receptor, the GluN2B downstream proapoptotic mediator m-calpain, and the phosphorylation of its transcription factor p38 MAPK in the hippocampal neurons after cerebral I/R injury. Conclusion: The EA regime might induce tolerance against I/R injury partially through the regulation of the proapoptotic GluN2B/m-calpain/p38 MAPK pathway of glutamate.

Calpain-2 participates in the process of calpain-1 inactivation.

Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at specific sites, thus modifying their properties such as function or activity. These isoforms have long been considered to function in a redundant or complementary manner, as they are both ubiquitously expressed and activated in a Ca2+- dependent manner. However, studies using isoform-specific knockout and knockdown strategies revealed that each calpain species carries out specific functions in vivo. To understand the mechanisms that differentiate calpain-1 and calpain-2, we focused on the efficiency and longevity of each calpain species after activation. Using an in vitro proteolysis assay of troponin T in combination with mass spectrometry, we revealed distinctive aspects of each isoform. Proteolysis mediated by calpain-1 was more sustained, lasting as long as several hours, whereas proteolysis mediated by calpain-2 was quickly blunted. Calpain-1 and calpain-2 also differed from each other in their patterns of autolysis. Calpain-2-specific autolysis sites in its PC1 domain are not cleaved by calpain-1, but calpain-2 cuts calpain-1 at the corresponding position. Moreover, at least in vitro, calpain-1 and calpain-2 do not perform substrate proteolysis in a synergistic manner. On the contrary, calpain-1 activity is suppressed in the presence of calpain-2, possibly because it is cleaved by the latter protein. These results suggest that calpain-2 functions as a down-regulation of calpain-1, a mechanism that may be applicable to other calpain species as well.

Ursolic acid protects against cisplatin­induced ototoxicity by inhibiting oxidative stress and TRPV1­mediated Ca2+­signaling.

Cisplatin (CDDP) is widely used in clinical settings for the treatment of various cancers. However, ototoxicity is a major side effect of CDDP, and there is an associated risk of irreversible hearing loss. We previously demonstrated that CDDP could induce ototoxicity via activation of the transient receptor potential vanilloid receptor 1 (TRPV1) pathway and subsequent induction of oxidative stress. The present study investigated whether ursolic acid (UA) treatment could protect against CDDP­induced ototoxicity. UA is a triterpenoid with strong antioxidant activity widely used in China for the treatment of liver diseases. This traditional Chinese medicine is mainly isolated from bearberry, a Chinese herb. The present results showed that CDDP increased auditory brainstem response threshold shifts in frequencies associated with observed damage to the outer hair cells. Moreover, CDDP increased the expression of TRPV1, calpain 2 and caspase­3 in the cochlea, and the levels of Ca2+ and 4­hydroxynonenal. UA co­treatment significantly attenuated CDDP­induced hearing loss and inhibited TRPV1 pathway activation. In addition, UA enhanced CDDP­induced growth inhibition in the human ovarian cancer cell line SKOV3, suggesting that UA synergizes with CDDP in vitro. Collectively, the present data suggested that UA could effectively attenuate CDDP­induced hearing loss by inhibiting the TRPV1/Ca²+/calpain­oxidative stress pathway without impairing the antitumor effects of CDDP.

Cryptotanshinone-Induced p53-Dependent Sensitization of Colon Cancer Cells to Apoptotic Drive by Regulation of Calpain and Calcium Homeostasis.

Over-expression of calpains in tumor tissues can be associated with cancer progression. Thus, inhibition of calpain activity using specific inhibitors has become a novel approach to control tumor growth. In this study, the anticancer potential of cryptotanshinone in combination with calpain inhibitor had been investigated in colon cancer cells and tumor xenograft. Cryptotanshinone elicited an initial endoplasmic reticular (ER) stress response, whereas prolonged stress would result in the promotion of apoptosis. It was then discovered that cryptotanshinone could cause rapid and sustained increase in cytosolic calcium in colon cancer cells accompanied by early GRP78 overexpression, which could be attenuated by pre-treatment of the calcium chelator BAPTA-AM. Cryptotanshinone also facilitated an early increase in calpain activity, which could be blocked by BAPTA-AM or the calpain inhibitor PD150606. A dynamic interaction between GRP78 and calpain during the action of cryptotanshinone was unveiled. This together with the altered NF-[Formula: see text]B signaling could be abolished by calpain inhibitor. GRP78 knockdown increased the sensitivity of cancer cells to cryptotanshinone-evoked apoptosis and reduction of cancer cell colony formation. Such sensitization of drug action had been confirmed to be p53-dependent by using p53-mutated (HT-29) and p53-deficient (HCT116 p53-∕-) cells. The synergistic antitumor effect of cryptotanshinone and calpain inhibitor was further exhibited in vivo. Taken together, findings in this study exemplify a new chemotherapeutic regimen comprising cryptotanshinone and calpain inhibitor by regulation of calpain and calcium homeostasis. This has provided us with new insights in the search of a potential target-specific neoadjuvant therapy against colon cancer.

Acid-sensing ion channel 1a mediates acid-induced pyroptosis through calpain-2/calcineurin pathway in rat articular chondrocytes.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1ß level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.

Prognostic and predictive value of a five-molecule panel in resected pancreatic ductal adenocarcinoma: A multicentre study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis. The performance of clinicopathologic parameters and molecules as prognostic factors remains limited and inconsistent. The present study aimed to construct a multi-molecule biomarker panel to more accurately predict post-resectional prognosis of PDAC patients. METHODS: Firstly, a novel computational strategy integrating prognostic evidence from omics and literature on the basis of bioinformatics prediction (CIPHER) to generate the network, was designed to systematically identify potential high-confidence PDAC-related prognostic candidates. After specimens from 605 resected PDAC patients were retrospectively collected, 23 candidates were detected immunohistochemically in tissue-microarrays for the development cohort to construct a multi-molecule panel. Lastly, the panel was validated in two independent cohorts. FINDINGS: According to the constructed five-molecule panel, disease-specific survival (DSS) was significantly poorer in high-risk patients than in low-risk ones in development cohort (HR 2.15, 95%CI 1.51-3.05, P<0.0001; AUC 0.67). In two validation cohorts, similar significant differences between the two groups were also observed (HR 3.18 and 3.31, 95%CI 1.89-5.37 and 1.78-6.16, All P<0.0001; AUC 0.72 and 0.73). In multivariate analyses, this panel was the sole prognosticator that was significant in each cohort. Furthermore, its predictive power for long-term survival, higher than its individual constituents, could be largely enhanced by combination with traditional clinicopathological variables. Finally, adjuvant chemotherapy (ACT) correlated with better DSS only in high-risk patients, uni- and multi-variately, in all the cohorts. INTERPRETATION: The novel prognostic panel developed by a systematically network-based strategy presents strong ability in prediction of post-resectional survival of PDAC patients. Furthermore, panel-defined high-risk patients might benefit more from ACT.