P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells

Aim： To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. Methods： The viability of oridonin- treated MCF-7 cells was measured by MTT （thiazole blue） assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein （Hsp）90, p53, p-p53, p21, Poly （ADP-ribose） polymerase （PARP）, and the inhibitor of caspase-activated DNase （ICAD） protein expressions were detected by Western blot analysis. Results： Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of △ψmit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3. Condusion： DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.     

Inhibition of mitochondrial and cytosolic calpain attenuates atrophy in myotubes co-cultured with colon carcinoma cells

Cancer cachexia is a life-threatening syndrome characterized by muscle atrophy. Cancer cachectic muscle atrophy (CCMA) is associated with mitochondrial injury. Mitochondrial calpains have been reported to induce mitochondrial injury in mouse cardiomyocytes and pulmonary smooth muscle. In the present study, the presence of calpain in the mitochondria of skeletal muscle and its potential role in CCMA were investigated. Transwell plates were used to develop a myotube-carcinoma cell co-culture model to simulate the cancer cachexia environment in vitro. The calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used to inhibit calpain activity in myotubes during co-culture. Calpain-1, calpain-2 and CAST were found to be present in mouse myotube mitochondria. Co-culture activated calpain in both cytoplasm and mitochondria, which caused myotube atrophy. CAST and CAPT treatment prevented calpain activation in both cytoplasm and mitochondria, which inhibited myotube atrophy during co-culture. Additionally, CAST and CAPT treatment increased mitochondrial complex I activity, decreased mitochondrial permeability transition pore opening and improved mitochondrial membrane potential in myotubes during co-culture. In addition, CAST and CAPT treatment increased AKT/mTOR activity, inhibited FoxO3a activity and decreased atrogin-1 content in myotubes during co-culture. The present findings provide new insights to understand the mechanism of CCMA and further help the development of focused approaches to treat CCMA by manipulating the mitochondrial and cytosolic calpain activity.     

L‐type calcium channel blocker ameliorates diabetic encephalopathy by modulating dysregulated calcium homeostasis

Diabetic encephalopathy is a complication of diabetes characterized by impaired cognitive functions. The objective of the present study was to examine the beneficial effect of the calcium channel blocker, nimodipine, on diabetes‐induced cognitive deficits and altered calcium homeostasis in the cerebral cortex. Diabetes was induced in mice by intraperitoneal injection of streptozotocin (40 mg/kg body wt) for 5 days. Nimodipine (10 mg/kg body weight) was administered intraperitoneally to the animals every 48 hr for 8 weeks. A significant impairment in spatial learning and memory was observed in diabetic animals, which was reversed by nimodipine treatment. Diabetic animals showed increased CaV1.2 mRNA and protein expression, which might be responsible for enhanced synaptosomal calcium uptake. Nimodipine treatment was found to lower CaV1.2 mRNA, protein expression, and calcium uptake. Mitochondrial Ca²⁺ uptake was reduced in diabetic brains, which was reversed with nimodipine treatment. Plasma membrane and sarcoplasmic reticulum Ca²⁺‐ATPase activity was found to be significantly decreased in diabetic animals, whereas nimodipine supplementation restored the activity of both Ca²⁺‐ATPases nearly to control values. Nimodipine treatment was shown to normalize intracellular free Ca²⁺ levels in diabetic animals. Nimodipine was shown to attenuate increased calpain activity measured in terms of hydrolysis of fluorogenic substrate and αII‐spectrin degradation. Nimodipine supplementation also reduced reactive oxygen species production and lipid peroxidation in diabetic animals. The data suggests that L‐type calcium channel blocker is beneficial in preventing cognitive deficits associated with diabetic encephalopathy through modulation of dysregulated calcium homeostasis. © 2014 Wiley Periodicals, Inc.     

Altered ubiquitin causes perturbed calcium homeostasis,hyperactivation of calpain,dysregulated differentiation,and cataract

Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca²⁺ elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca²⁺ elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.Many age-related diseases such as cataracts, macular degeneration, Alzheimer’s, Parkinson’s, Huntington’s, and several premature aging syndromes, appear to be causally associated with accumulation of abnormal proteins (1, 2). The accumulation of damaged proteins in many age-related diseases involves a vicious cycle of stress-induced postsynthetic modifications to bulk and catalytically critical molecules and limited capacity to remove the damaged proteins, thus accelerating accumulation of damaged proteins and protein precipitation (1–3). Clarity is essential for lens function. Age-related cataract is due to the aggregation and precipitation of proteins from the normally clear milieu and is the leading cause of adult blindness worldwide, affecting more than 18 million people (4). Congenital cataracts also involve protein precipitation (5).The lens is an excellent system to study specific relationships between proteolytic pathways, stress, and maintenance of protein quality because all of the cells are retained throughout life. The oldest lens tissue is found at the center or core of the lens. Crystallins, the major gene products of the lens, are very long-lived proteins, with half-lives of decades, and their aberrant synthesis or modification results in aggregation, insolubilization, and cataract (6). Common age-related stresses that confront proteins in the lens and other tissues during aging include oxidation, glycation, and methylation, as well as their sequels (3, 6–9). Effective stress-reducing systems including antioxidants, antioxidant and repair enzymes, and chaperone and proteolytic capacities help limit damage and maintain solubility and function in younger tissues (3).There are three general systems for intracellular proteolysis: lysosomal/autophagic mechanisms, calcium-activated proteases, or calpains, and the ubiquitin proteasome system (UPS). Because nuclei and lysosomes are removed from most of the lens cells in regions where most cataracts form, this leaves only cytoplasmic proteases, including the UPSs and calpains, to remove damaged proteins to retain lens function (3). There are two basic steps to the UPS: conjugation of substrates to multiple ubiquitins (Ubs) followed by degradation of the protein substrate. Ubiquitin is a highly conserved protein with seven lysines (10). The lysines are used, in the first step of the UPS, to form inter-Ub linkages that lead to Ub polymers that are conjugated to protein substrates. Commonly, proteins with K48-linked Ub oligomers attached are scheduled for degradation by the 26S proteasome. The UPS is also involved at multiple critical stages of proliferation, differentiation, and development in most tissues.It was recently noted that only a minute proportion of Ub conjugates use K6 on Ub (11). Thus, we were surprised to find that expression of higher levels of K6W-Ub in the lens produced cataracts (12). Other work showed that Ub mutations in which K6 is replaced by various amino acids, i.e., K6W-Ub, are conjugation competent but proteolytically incompetent. Thus, cells and tissues in which K6W-Ub is expressed accumulate Ub conjugates (13). Exchanging K for A or R has the same effects. Although structural information about Ub conjugates built using K6 is becoming available (14, 15), a complete understanding of features that render such conjugates biologically stable remain to be elucidated.Calpain is up-regulated by increased Ca²⁺. Gap junction proteins, or Connexins (Cxs), are required for maintaining Ca²⁺ homeostasis (16). UPS-dependent degradation of Cx has been observed in CHO and BWEM cells (17). The formation of cataracts in lenses that express K6W-Ub is compatible with novel functional connections between UPS activity, regulation of Cx and Ca²⁺, calpain activity, and lens clarity. To test the hypothesis, we targeted expression of K6W-Ub to the lens, and we monitored stability of multiple proteins, Cx function and ubiquitination, Ca²⁺, calpain activity, protein integrity, solubility, and localization, as well as lens clarity.     

Effect of diet on tissue protease activity

Rats 1,3,12, and 24 months old were fed diets low in protein (8% casein), and proteolytic activity in tissue from brain, liver, and lung was determined. After a low-protein diet was fed for 4 weeks to 1-month-old rats, there was a significant increase in cathepsin D activity in liver, and calpain activity was increased in lung. Little change was seen in proteolytic activity in brain. In 12-month-old rats, there was an increase in cathepsin D activity in brain and liver. In 24-month-old rats, cathepsin D activity in the liver and calpain activity in lung were increased. There was no change in proteolytic activity in the brain. When animals were fed diets supplemented with fatty acids or antioxidants for 2 months, in 3-month-old rats calpain activity was increased in brain but decreased in lung. Cathepsin D activity was significantly increased in young and adult animals in brain and in liver. These observations suggest that diet changes result in significant alteration in tissue calpain and cathepsin D levels, and possibly activity, in vivo. Generally, changes are greater for cathepsin D than for calpain, and are smaller in brain than in other tissues. © 1995 Wiley-Liss, Inc.     

Mechanism of apoptosis with the involvement of calpain and caspase cascades in human malignant neuroblastoma SH-SY5Y cells exposed to flavonoids

Neuroblastoma is the most common extracranial solid tumor in children causing death at pre-school age, as no cure has yet been developed. We investigated the proteolytic mechanisms for apoptosis in human malignant (N-type) neuroblastoma SH-SY5Y cells following exposure to flavonoids such as apigenin (APG), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST). We found decrease in viability of SH-SY5Y cells with an increase in dose of APG, EGC, EGCG and GST. Predominantly apoptosis occurred following exposure of SH-SY5Y cells to 50 microM APG, 50 microM EGC, 50 microM EGCG and 100 microM GST for 24 hr. Apoptosis was associated with increases in intracellular free [Ca(2+)] and Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and activation of caspase-9, calpain and caspase-3. Induction of apoptosis with APG and GST showed activation of caspase-12 as well. Activation of caspase-3 could cleave the inhibitor-of-caspase-activated DNase (ICAD) to release and translocate caspase-3-activated DNase (CAD) to the nucleus. Activation of caspase-8 cleaved Bid to truncated Bid (tBid) in cells treated with EGC and EGCG. EGC and EGCG induced apoptosis with caspase-8 activation and mitochondria-mediated pathway, whereas APG and GST caused apoptosis via an increase in intracellular free [Ca(2+)] with calpain activation and mitochondria-mediated pathway. Activation of different proteases for cell death was confirmed using caspase-8 inhibitor II, calpeptin (calpain inhibitor), caspase-9 inhibitor I and caspase-3 inhibitor IV. Thus, plant-derived flavonoids cause cell death with activation of proteolytic activities of calpain and caspases in SH-SY5Y cells, and therefore serve as potential therapeutic agents for controlling the growth of neuroblastoma.     

Utrophin is a calpain substrate in muscle cells

Calpains are Ca2+ -dependent cytosolic cysteine proteases that participate in the pathology of Duchenne muscular dystrophy (DMD). Utrophin is a functional homolog of dystrophin that partially compensates for dystrophin deficiency in myofibers of mdx mice. In this study, we investigated the susceptibility of utrophin to cleavage by calpain in vitro and in muscle cells. We found that utrophin is a direct in vitro substrate of purified calpain I and II. Cleavage of utrophin by calpain I or II generates specific degradation products that are also found in cultured control and DMD myotubes under conditions with elevated intracellular Ca2+ levels. In addition, we showed that activation of cellular calpains by Ca2+ ionophore treatment reduces utrophin protein levels in muscle cells and that calpain inhibition prevents this Ca2+ -induced reduction in utrophin levels. These observations suggest that, beside its known effect on general muscle protein degradation, calpain contributes to DMD pathology by specifically degrading the compensatory protein utrophin.     

急性离心运动后骨骼肌超微结构、钙依赖性蛋白酶和泛素的动态变化

目的:探讨1次离心运动后大鼠骨骼肌超微结构、Calpains和Ubiquitin的动态变化。方法:30只雄性SD大鼠随机分为对照组和1次离心运动后即刻组、24 h组和7天组。各运动组大鼠进行1次下坡跑运动,速度16 m/min,坡度?16°,运动100 min后休息10 min,再运动100 min,总时间200 min。分别在急性运动后即刻、24 h和第7天取材,测定血清LDH、CK活性和股四头肌超微结构及Calpain-1、Calpain-2和Ubiquitin含量。结果:①1次离心运动后即刻组大鼠股四头肌肌丝排列混杂、卷曲;运动后24 h出现轻度溶解、断裂,Z线不规则,局部Z线消失;运动后7天超微结构与对照组相比无显著变化。血清CK和LDH活性变化与股四头肌超微结构变化一致。②与对照组相比,运动后即刻组大鼠股四头肌Calpain-1、Calpain-2和Ubiquitin含量均有所降低,但均无显著性差异;运动后24 h组股四头肌Calpain-1、Calpain-2和Ubiquitin含量均显著高于对照组和运动后即刻组;运动后7天组与运动后24 h组相比均显著下降,而与对照组相比均无显著性差异。结论:①1次离心运动所致的骨骼肌损伤有一定的延迟性,骨骼肌损伤在运动后即刻从整体看并不严重,但在运动后24 h出现明显损伤,运动后7天基本恢复。②急性离心运动后骨骼肌Calpain和Ubiquitin含量变化与骨骼肌损伤的动态变化基本一致。     

丹曲林对大鼠缺血再灌注心肌嘌呤核苷酸代谢的影响

目的 探讨丹曲林对缺血再灌注心肌嘌呤核苷酸代谢的影响.方法 24只健康雄性Wistar大鼠(n=24)建立Langendorff离体心脏灌注模型,分为两组,对照组即缺血再灌注组和实验组即丹曲林处理组,应用高效液相色谱仪(HPLC)测定嘌呤核苷酸代谢产物腺苷,AMP,尿酸的含量变化,以揭示5'-核苷酸酶(5'-nu-cleotidase,5'-NT),腺苷脱氛酶(adenosine deaminase,ADA),黄嘌呤氧化酶(xanthine oxidase,XO)的活性.结果 丹曲林处理组与缺血再灌注组相比,5'-核苷酸酶,腺苷脱氛酶,黄嘌呤氧化酶活性均明显增加(P<0.05).结论 丹曲林通过抑制Ca2+超载,降低Ca2+活化的钙蛋白酶对细胞膜骨架蛋白和结构蛋白的降解,以及增强膜磷脂的稳定性,维持细胞完整性,保存细胞内的各种代谢酶,对缺血再灌注心肌起到保护作用.