Acid Cathepsin Research Articles

The role of lysosomal peptidases in glioma immune escape: underlying mechanisms and therapeutic strategies.

Glioblastoma is the most common primary malignant tumor of the central nervous system, which has the characteristics of strong invasion, frequent recurrence, and rapid progression. These characteristics are inseparable from the evasion of glioma cells from immune killing, which makes immune escape a great obstacle to the treatment of glioma, and studies have confirmed that glioma patients with immune escape tend to have poor prognosis. The lysosomal peptidase lysosome family plays an important role in the immune escape process of glioma, which mainly includes aspartic acid cathepsin, serine cathepsin, asparagine endopeptidases, and cysteine cathepsins. Among them, the cysteine cathepsin family plays a prominent role in the immune escape of glioma. Numerous studies have confirmed that glioma immune escape mediated by lysosomal peptidases has something to do with autophagy, cell signaling pathways, immune cells, cytokines, and other mechanisms, especially lysosome organization. The relationship between protease and autophagy is more complicated, and the current research is neither complete nor in-depth. Therefore, this article reviews how lysosomal peptidases mediate the immune escape of glioma through the above mechanisms and explores the possibility of lysosomal peptidases as a target of glioma immunotherapy.

Differentiation of Cells Isolated from Human Femoral Heads into Functional Osteoclasts.

Proper formation of the skeleton during development is crucial for the mobility of humans and the maintenance of essential organs. The production of bone is regulated by osteoblasts and osteoclasts. An imbalance of these cells can lead to a decrease in bone mineral density, which leads to fractures. While many studies are emerging to understand the role of osteoblasts, less studies are present about the role of osteoclasts. This present study utilized bone marrow cells isolated directly from the bone marrow of femoral heads obtained from osteoarthritic (OA) patients after undergoing hip replacement surgery. Here, we used tartrate resistant acid phosphatase (TRAP) staining, Cathepsin K, and nuclei to identity osteoclasts and their functionality after stimulation with macrophage-colony stimulation factor (M-CSF) and receptor activator of nuclear factor kappa-β ligand (RANKL). Our data demonstrated that isolated cells can be differentiated into functional osteoclasts, as indicated by the 92% and 83% of cells that stained positive for TRAP and Cathepsin K, respectively. Furthermore, isolated cells remain viable and terminally differentiate into osteoclasts when stimulated with RANKL. These data demonstrate that cells isolated from human femoral heads can be differentiated into osteoclasts to study bone disorders during development and adulthood.

Evaluating the activity of salivary enzymes as stress biomarkers under psychological stress and their relationship with rumination and personality traits

Background Salivary enzymes are used as non-invasive biomarkers to assess the activity of the sympathetic-adrenal-medullary system. The aim of this study was to evaluated levels of acid phosphatase, beta-glucuronidase and cathepsin salivary enzymes under psychological tension and their connection with rumination and personality traits. Methods A total of 60 medical students, who wanted to participate in the final exam, two months before the exam, the inventory emotional control questionnaire and the neo-short form were completed. Saliva samples were taken in both the basal conditions and under exam stress. Results A significant difference was found between the mean of level salivary enzymes in rest and under exam stress. Also, we found a positive and significant correlation between the activity of salivary enzymes and personality traits such as neuroticism, extraversion, agreeableness and rumination (p < .01, p < .05) level. Neuroticism, agreeableness and rumination predicted 45% of the variance of salivary acid phosphatase, neuroticism and rumination predicted 49% of the variance of salivary beta-glucuronidase and neuroticism, extraversion and rumination predicted 38% of the variance of salivary cathepsin under stress exam. Conclusion The results of this study show, levels of salivary enzymes may increase in individuals with traits of neuroticism, extraversion, agreeableness and rumination through response to psychological stressors.

Influence of Angptl1 on osteoclast formation and osteoblastic phenotype in mouse cells

BackgroundOsteoblasts and osteoclasts play important roles during the bone remodeling in the physiological and pathophysiological states. Although angiopoietin family Angiopoietin like proteins (Angptls), including Angptl1, have been reported to be involved in inflammation, lipid metabolism and angiogenesis, the roles of Angptl1 in bone have not been reported so far.MethodsWe examined the effects of Angptl1 on the osteoblast and osteoclast phenotypes using mouse cells.ResultsAngptl1 significantly inhibited the osteoclast formation and mRNA levels of tartrate-resistant acid phosphatase and cathepsin K enhanced by receptor activator of nuclear factor κB ligand in RAW 264.7 and mouse bone marrow cells. Moreover, Angptl1 overexpression significantly enhanced Osterix mRNA levels, alkaline phosphatase activity and mineralization induced by bone morphogenetic protein-2 in ST2 cells, although it did not affect the expression of osteogenic genes in MC3T3-E1 and mouse osteoblasts. On the other hand, Angptl1 overexpression significantly reduced the mRNA levels of peroxisome proliferator-activated receptor γ and adipocyte protein-2 as well as the lipid droplet formation induced by adipogenic medium in 3T3-L1 cells.ConclusionsThe present study first indicated that Angptl1 suppresses and enhances osteoclast formation and osteoblastic differentiation in mouse cells, respectively, although it inhibits adipogenic differentiation of 3T3-L1 cells. These data suggest the possibility that Angptl1 might be physiologically related to bone remodeling.

Docetaxel-Loaded Hyaluronic Acid-Cathepsin B-Cleavable-Peptide-Gold Nanoparticles for the Treatment of Cancer.

Gold nanoparticles are commonly used for medical applications such as drug delivery and as therapeutic and diagnostic materials because of their unique properties. In this study, we prepared docetaxel (DTX)-loaded hyaluronic acid-cleavable-peptide-gold nanoparticles for the treatment of cancer by selectively delivering DTX into the tumor and, thus, enhancing the therapeutic effect of DTX; further, we determined synergistic effects of the nanoparticles using laser treatment. The DTX-loaded hyaluronic acid-cleavable-peptide-gold nanoparticles prepared in this study had an average size of 75 nm and negative surface charge. The nanoparticles revealed greater cytotoxicity and higher tumor suppression efficacy in tumor models than free DTX under near-infrared laser irradiation. Therefore, the nanoparticle formulation prepared in this study could be utilized for targeted drug delivery and in combination with other cancer therapies.

Dynamics of activity of intracellular cysteine-dependent proteases and some peptidases in embryogenesis of Atlantic salmon Salmo salar L.

The dynamics of the activity of cysteine-dependent enzymes involved in intracellular proteolysis was studied in freshwater salmon Salmo salar L. during its early development: in the period from the blastodisc formation to hatching and in eggs before fertilization. The Ca2+-activated proteolytic activity of calpains, the activity of acidic lysosomal protease cathepsin B, and the activity of several peptidases that use the chromogenic component p-nitroaniline-containing peptides (L-Ala-pNA, L-Pro-pNA, L-Arg-pNA, N-Bzl-L-Arg-pNA, L-Phe-pNA, N-Glu-L-Phe-pNA, and Z-Gly-Pro-pNA) as substrates were analyzed. It is shown that the activity of the enzymes of interest increases at the stage of late blastula and early gastrula. The next increase in the activity of cathepsin B and calpain is observed at the stage of eye pigmentation, and the activity of peptidases and cathepsin B increases in eggs before hatching. The functional role of changes in intracellular proteolytic enzymes in the processes of protein metabolism associated with the Atlantic salmon embryo is discussed.

Enzyme-Cleavable Polymeric Micelles for the Intracellular Delivery of Proapoptotic Peptides.

Peptides derived from the third Bcl-2 homology domain (BH3) renormalize apoptotic signaling by antagonizing prosurvival Bcl-2 family members. These potential peptide drugs exhibit therapeutic activities but are limited by barriers including short circulation half-lives and poor penetration into cells. A diblock polymeric micelle carrier for the BIM BH3 peptide was recently described that demonstrated antitumor activity in a B-cell lymphoma xenograft model [Berguig et al., Mol. Ther. 2015, 23, 907-917]. However, the disulfide linkage used to conjugate the BIM peptide was shown to have nonoptimal blood stability. Here we describe a peptide macromonomer composed of BIM capped with a four amino acid cathepsin B substrate (FKFL) that possesses high blood stability and is cleaved to release the drug inside of target cells. Employing RAFT polymerization, the peptide macromonomer was directly integrated into a multifunctional diblock copolymer tailored for peptide delivery. The first polymer block was made as a macro-chain transfer agent (CTA) and composed of a pH-responsive endosomolytic formulation of N,N-diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA). The second polymer block was a copolymer of the peptide and polyethylene glycol methacrylate (PEGMA). PEGMA monomers of two sizes were investigated (300 Da and 950 Da). Protein gel analysis, high performance liquid chromatography, and coupled mass spectrometry (MS) showed that incubation with cathepsin B specifically cleaved the FKFL linker and released active BIM peptide with PEGMA300 but not with PEGMA950. MALDI-TOF MS showed that incubation of the peptide monomers alone in human serum resulted in partial cleavage at the FKFL linker after 12 h. However, formulation of the peptides into polymers protected against serum-mediated peptide degradation. Dynamic light scattering (DLS) demonstrated pH-dependent micelle disassembly (25 nm polymer micelles at pH 7.4 versus 6 nm unimers at pH 6.6), and a red blood cell lysis assay showed a corresponding increase in membrane destabilizing activity (<1% lysis at pH 7.4 versus 95% lysis at pH 6.6). The full carrier-drug system successfully induced apoptosis in SKOV3 ovarian cancer cells in a dose-dependent manner, in comparison to a control polymer containing a scrambled BIM peptide sequence. Mechanistic analysis verified target-dependent activation of caspase 3/7 activity (8.1-fold increase), and positive annexin V staining (72% increase). The increased blood stability of this enzyme-cleavable peptide polymer design, together with the direct polymerization approach that eliminated postsynthetic conjugation steps, suggests that this new carrier design could provide important benefits for intracellular peptide drug delivery.

Seropharmacological study on osteogenic effects of post-absorption ingredients of an osteoprotective herbal formula.

To further investigate the {ptin vitro} effects of an osteoprotective herbal formula "ELP" (Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae) using seropharmacological approach. Rats were fed with ELP or its individual component herbs for 2 days. The serum containing the postabsorbed ingredients of the herbal items were collected for cell culture using UMR106 cell, RAW264.7 cell and mesenchymal stem cell (MSC) isolated from the bone marrow of the rats. The effects of the herbal-containing serum on cell toxicity were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay; bromodeoxyuridine assay was conducted to measure the cell proliferation of UMR106 cell and MSC; cell activity was measured using colorimetric method, and mRNA expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteopontin (OPN) of UMR106 and MSC as well as matrix metalloproteinase 9 (MMP-9), tartrate-resistant acid phosphatase (TRAP) and cathepsin K of RAW264.7 were analyzed using real-time reverse-transcription polymerase chain reaction. ELP and its component serum exhibited no cytotoxic effects on the cells. The ELP-containing serum increased the proliferation of UMR106 cell and MSC by 25.7% and 14.4 %, respectively and the alkaline phosphatase activity of MSC was increased by 42.6%. On the contrary, it inhibited the RAW264.7 cell differentiation by 29.2 %. ELP serum upregulated the Runx2 expression of UMR and MSC by 1.18 fold and 1.27 fold, respectively. It also upregulated ALP and OPN expression in MSC by 1.69- and 2.12-fold, respectively. On the other hand, ELP serum down-regulated MMP-9 and cathepsin K expression of RAW264.7 cell by 0.46- and 0.36-fold, respectively. The serum of the animals fed with ELP contains active ingredients which are effective in promoting osteogenesis and inhibiting osteoclastogenesis.

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells.

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.

Determinants of chronic obstructive pulmonary disease in patients with intermediate levels of alpha-antitrypsin.

We examined elderly heterozygotes for alpha-antitrypsin deficiency (phenotype MZ) to determine which of several factors might be associated with the development of chronic obstructive pulmonary disease (COPD). Elderly heterozygotes who smoked had a very high incidence of COPD (70 per cent), whereas homozygotes (phenotype MM) who smoked had a significantly lower incidence of COPD (35 per cent). There was no difference in the incidence of COPD between subjects with MM and MZ phenotypes who did not smoke. To determine if leukocyte enzymes were related to the development of COPD in elderly heterozygotes, we measured the activities of monocytic acid cathepsin, neutrophil acid cathepsin, neutral cathepsin, and elastase. The activities of these enzymes were not associated with the development of COPD or with smoking history in the heterozygotes. In conclusion, cigarette smoking seemed to be a significant determinant of the development of COPD in heterozygotes for alpha-antitrypsin deficiency, but the leukocyte enzyme actitivty that we studied was unrelated.

Cloning and characterization of a basic cysteine-like protease (cathepsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae)

Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L’s, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in Rosetta™ Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His6 tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L1 from larval gut of D. abbreviatus.

Jagged1 promotes osteoclast differentiation and inhibits proliferation by activating of Notch signaling pathway

Objective To study the role of Jadded1 and Notch signaling pathway played in the differentiation and proliferation of osteoclasts derived from human circulating CD14 + monocytes.Methods Peripheral blood CD14 + monocytes were isolated from healthy adult by Ficoll density gradient centrifugation combined with magnetic beads separation.Recombinant protein Jagged1 and γ-Secretase Inhibitor (DAPT) with Jagged1 were added to the culture system of CD14 + monocytes respectively.The mRNA expression of osteoclast markers tartrate-resistant acid phosphatase (TRAP),cathepsin K (CK),calctionin receptor (CTR) and Notch key target genes hairy and enhancer of split-1 (HES-1) and HES-related with YRPW motif-1 (HEY-1) were measured by quantitative real-time quantitative polymerase chain reaction (Real-time PCR).The formation of osteoclast,bone resorption,Notch expression and proliferation of CD14 + monocytes were detected by TRAP staining,scanning electron microscope,and cell counting kit-8 (CCK-8).Results High expression of Jagged1 in bone metastases specimens was observed in contrast of no expression or low expression in adjacent tissues.TRAP,CK,CTR,HES-1 and HEY-1 mRNA expression were significantly higher than the control group and DAPT group in Jadded1 group (P ＜ 0.05).TRAP + cell count,osteolytic area was significantly increased in Jagged1 group when compared with control and DAPT group,and no significant difference observed between the last two groups.Cell proliferation was inhibited in Jagged1 group also (P ＜ 0.05).Conclusion Jagged1 promotes osteoclast differentiation and inhibits proliferation by activating Notch signaling pathway. Key words: Jagged1 ; Osteoclasts ; Notch ; Differentiation ; Proliferation

Peptide-Modulated Activity Enhancement of Acidic Protease Cathepsin E at Neutral pH

Enzymes are regulated by their activation and inhibition. Enzyme activators can often be effective tools for scientific and medical purposes, although they are more difficult to obtain than inhibitors. Here, using the paired peptide method, we report on protease-cathepsin-E-activating peptides that are obtained at neutral pH. These selected peptides also underwent molecular evolution, after which their cathepsin E activation capability improved. Thus, the activators we obtained could enhance cathepsin-E-induced cancer cell apoptosis, which indicated their potential as cancer drug precursors.

Mycoepoxydiene suppresses RANKL-induced osteoclast differentiation and reduces ovariectomy-induced bone loss in mice

Mycoepoxydiene (MED) is a compound isolated from the marine fungal Diaporthe sp. HLY-1 associated with mangroves. MED has various biological effects such as anti-microbial, anti-cancer, and anti-inflammatory activities. However, the effect of MED on the differentiation of osteoclasts, the multinucleated bone-resorbing cells which play a crucial role in bone remodeling, is still unknown. In this study, we showed that MED could inhibit receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and the expression of three well-known osteoclast markers such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K in bone marrow-derived macrophages. Furthermore, we found that MED inhibited the expression of nuclear factor of activated T cells c1, a key transcriptional factor in osteoclast differentiation, via inhibiting the phosphorylation of TAK1 and then blocking the activation of NF-κB and ERK1/2 pathways. Moreover, MED could prevent bone loss in ovariectomized mice. Taken together, we demonstrate for the first time that MED can suppress RANKL-induced osteoclast differentiation in vitro and ovariectomy-induced osteoporosis in vivo, suggesting that MED is a potential lead compound for the development of novel drugs for osteoporosis treatment.

Cysteine Cathepsins S and L Modulate Anti-angiogenic Activities of Human Endostatin

Human endostatin, a potent anti-angiogenic protein, is generated by release of the C terminus of collagen XVIII. Here, we propose that cysteine cathepsins are involved in both the liberation and activation of bioactive endostatin fragments, thus regulating their anti-angiogenic properties. Cathepsins B, S, and L efficiently cleaved in vitro FRET peptides that encompass the hinge region corresponding to the N terminus of endostatin. However, in human umbilical vein endothelial cell-based assays, silencing of cathepsins S and L, but not cathepsin B, impaired the generation of the ∼22-kDa endostatin species. Moreover, cathepsins L and S released two peptides from endostatin with increased angiostatic properties and both encompassing the NGR sequence, a vasculature homing motif. The G10T peptide (residues 1455-1464: collagen XVIII numbering) displayed compelling anti-proliferative (EC(50) = 0.23 nm) and proapoptotic properties. G10T inhibited aminopeptidase N (APN/CD13) and reduced tube formation of endothelial cells in a manner similar to bestatin. Combination of G10T with bestatin resulted in no further increase in anti-angiogenic activity. Taken together, these data suggest that endostatin-derived peptides may represent novel molecular links between cathepsins and APN/CD13 in the regulation of angiogenesis.

A Role for Serglycin Proteoglycan in Mast Cell Apoptosis Induced by a Secretory Granule-mediated Pathway

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.

Abstract P4-06-02: Systemic Deletion of c-Jun N-Terminal Kinase 1 or 2 Is Protective in Breast Cancer Bone Metastasis

Abstract Growth and survival of metastatic breast cancer cells in the bone is enhanced by the release of growth factors during osteoclast mediated bone resorption. In order to facilitate release of bone degradation products, breast cancer cells continually stimulate osteoblasts, which in turn differentiate osteoclast precursors. Mature osteoclasts are then activated to secrete factors such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K to degrade bone matrix. Identification of molecular targets involved in these signaling pathways may prove beneficial for treatment of aggressive tumor types. Results from our previous studies demonstrated that tumor cell expression of c-Jun N-Terminal Kinases (JNKs) mediate lung and bone metastasis. To determine if systemic JNKs play a role in breast cancer progression, jnk1-/- and jnk2-/- Balb/c mice were used to study host related contributions to bone metastasis. The highly metastatic 4T1.2 syngeneic mammary tumor cells were injected intra-cardiac into jnk1-/-, jnk2-/-, and wild-type mice. Both jnk1-/- and jnk2-/- mice survived significantly longer than wild-type controls, (p=0.0034 and p=0.0459, respectively). Wild-type mice succumbed to symptoms of morbidity as early as 10 days post-injection whereas; 60% of the jnk1-/- and 40% of the jnk2-/- mice remained asymptomatic at day 55 and day 70, respectively. Analysis of ribcage metastases post-mortem revealed a significant decrease in bone lesions in jnk1-/- and jnk2-/- mice compared to wild-type mice (P&amp;lt;0.0001 and p=0.0031, respectively). We then evaluated if jnk1 or 2 expression may inhibit osteoclast differentiation and slow tumor related osteolysis, Raw 264.7 osteoclast precursor cells were employed to knockdown Jnk1 or Jnk2 expression to study their proliferation and differentiation into osteoclasts. Proliferation of JNK1and JNK2 deficient osteoclast precursors (Raw shJNK1 and Raw shJNK2, respectively) was slightly decreased compared to wild-type Raw 264.7. In contrast, Receptor Activator for Nuclear Factor κB Ligand (RANKL) induced osteoclast differentiation was impaired in both Raw shJNK1 and Raw shJNK2 cells. Collectively, our results suggest the involvement of JNK1 and JNK2 in osteoclast differentiation, thus playing a vital role in breast cancer metastasis to the bone. These data suggest that therapeutic inhibition of JNKs may improve the morbidity and mortality associated with bone metastasis in breast cancer patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-02.

Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces fragments displaying in vitro ADP-ribosylating and apoptotic effects

To assess Pseudomonas exotoxin A (ETA) compartmentalization, processing and cytotoxicity in vivo, we have studied the fate of internalized ETA with the use of the in vivo rodent liver model following toxin administration, cell-free hepatic endosomes, and pure in vitro protease assays. ETA taken up into rat liver in vivo was rapidly associated with plasma membranes (5-30 min), internalized within endosomes (15-60 min), and later translocated into the cytosolic compartment (30-90 min). Coincident with endocytosis of intact ETA, in vivo association of the catalytic ETA-A subunit and low molecular mass ETA-A fragments was observed in the endosomal apparatus. After an in vitro proteolytic assay with an endosomal lysate and pure proteases, the ETA-degrading activity was attributed to the luminal species of endosomal acidic cathepsins B and D, with the major cleavages generated in vitro occurring mainly within domain III of ETA-A. Cell-free endosomes preloaded in vivo with ETA intraluminally processed and extraluminally released intact ETA and ETA-A in vitro in a pH-dependent and ATP-dependent manner. Rat hepatic cells underwent in vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by the mitochondrial release of cytochrome c, caspase-9 and caspase-3 activation, and DNA fragmentation. In an in vitro assay, intact ETA induced ADP-ribosylation of EF-2 and mitochondrial release of cytochrome c, with the former effect being efficiently increased by a cathepsin B/cathepsin D pretreatment. The data show a novel processing pathway for internalized ETA, involving cathepsins B and D, resulting in the production of ETA fragments that may participate in cytotoxicity and mitochondrial dysfunction.

Beneficial effects of omega-3 long-chain fatty acids in breast cancer and cardiovascular diseases: voltage-gated sodium channels as a common feature?

Cancers are among the leading causes of death worldwide. Voltage-gated sodium channels, among other ion channels, appear as new molecular players in epithelial cancers. Highly metastatic breast cancer cells express Na V1.5, the main isoform expressed in cardiac cells, where the current generated by the flux of sodium ions is responsible for the excitability. Breast cancer cells are not excitable and the protein activity regulates cell invasiveness, through the modulation of activity of acidic cathepsins, a characteristic involved in the metastatic phenotype. Interestingly, it is known that ω-3 LC-PUFA can exert beneficial effects by preventing post-myocardial infarction arrhythmias and by reducing the incidence of metastatic breast cancer. In this review, we compare the effects of some ω-3 LC-PUFA on Na V1.5 expressed in both cardiac and MDA-MB-231 breast cancer cells. We propose that some of the effects of ω-3 LC-PUFA act through common mechanisms involved in both diseases.

Cathepsin K/TRAP: Can they be used to induce osteogenesis?

Most of the strategies developed in bone tissue engineering in the past three decades have been aimed to repair/regenerate the tissue with forming elements such as osteoblasts and bone morphogenetic proteins. All these materials are selected as they are known to induce bone formation. Since it is known that bone turnover in basic multicellular units (BMUs) is at equilibrium, inducing an imbalance in this process via molecules known to resorb and transmit resorption signals and therefore initiate activation in bone forming cells from adjacent tissue may offer a radical approach to bone regeneration. Possible targets for such an approach may include resorbing molecules such as tartrate resistant acid phosphatase (TRAP) and cathepsin K. Delivering these enzymes (TRAP and cathepsin K) and/or other molecules involved in bone resorption into bone defects and thus obtaining a concentration difference in the levels of these materials may induce bone forming cells to balance bone turnover, therefore inducing bone regeneration.