Enzymatic Activity Of Cathepsin Research Articles

Design, synthesis, and biological evaluation of cathepsin B cleavage albumin-binding SN38 prodrug in breast cancer

Here, we introduce a novel and effective approach utilizing a cathepsin B cleavage albumin-binding SN38 prodrug specifically designed for the treatment of metastatic breast cancer. Termed Mal-va-mac-SN38, our prodrug exhibits a unique ability to rapidly and covalently bind with endogenous albumin, resulting in the formation of HSA-va-mac-SN38. This prodrug demonstrates exceptional stability in human plasma. Importantly, HSA-va-mac-SN38 showcases an impressive enhancement in cellular uptake by 4T1 breast cancer cells, primarily facilitated through caveolin-mediated endocytosis. Intriguingly, the release of the active SN38, is triggered by the enzymatic activity of cathepsin B within the lysosomal environment. In vivo studies employing a lung metastasis 4T1 breast cancer model underscore the potency of HSA-va-mac-SN38. Histological immunohistochemical analyses further illuminate the multifaceted impact of our prodrug, showcasing elevated levels of apoptosis, downregulated expression of matrix metalloproteinases, and inhibition of angiogenesis, all critical factors contributing to the anti-metastatic effect observed. Biodistribution studies elucidate the capacity of Mal-va-mac-SN38 to augment tumor accumulation through covalent binding to serum albumin, presenting a potential avenue for targeted therapeutic interventions. Collectively, our findings propose a promising therapeutic avenue for metastatic breast cancer, through the utilization of a cathepsin B-cleavable albumin-binding prodrug.

Effects of cold atmospheric plasma on endogenous enzyme activity and muscle protein oxidation in Trachinotus ovatus

In this study, we investigated the effects of cold atmospheric plasma (CAP) technology on endogenous enzyme characteristics and muscle protein properties of the golden pomfret (Trachinotus ovatus) under different treatment power and time conditions. Results showed that the enzymatic activity of cathepsin B, L, and calpain in crude protease extracts (CPE) decreased significantly as the treatment power and treatment time of CAP increased (p < 0.05). Oxidative degradation of the CPE after exposure to CAP resulted in significant changes in the structure, total sulfhydryl, and carbonyl content of the CPE (p < 0.05). CAP of an appropriate intensity resulted in significant improvements in the color parameters, hydration properties, and textural property parameters of muscle proteins (p < 0.05). These results suggest that CAP, as a non-thermophysical modification technique, can inhibit the activity of endogenous enzymes as well as alter the protein function in food.

Identification of a Novel Mutation in the Cathepsin K (CTSK) Gene in Iranian Patients with Pycnodysostosis

Background: Pycnodysostosis is a rare autosomal recessive skeletal dysplasia, which is characterized by various skeletal malformations and distinctive facial characteristics. The disorder is caused by mutations in CTSK gene at chromosomal region 1q21, which encodes a lysosomal cysteine protease, cathepsin K.&#x0D; Purpose: To describe the clinical, radiological, and molecular findings in a consanguineous Iranian family with 2 affected individuals demonstrating pycnodysostosis.&#x0D; Methods: Whole exome sequencing (WES) was utilized to detect disease-causing variants. To prove the impact of the identified variants, in silico analysis using SIFT and PROVEAN web servers were carried out. The effects of the novel mutation on the protein structure was evaluated by Chimera software using normal and mutated structures derived from SWISS model web server.&#x0D; Results: We identified the novel homozygous mutation, c.830C&gt;A, in exon 7 of the CTSK gene. This missense mutation leads to the substitution of the amino acid alanine at position 277 by glutamic acid (p.A277E) in cathepsin K protease. Sanger sequencing analysis confirmed this mutation in the affected individuals and revealed that the parents were heterozygous for this gene mutation. Bioinformatics databases and the variant’s protein structure proved the deleterious effect of this novel mutation on the enzymatic activity of cathepsin K protease.

Effect of dietary protein level and fasting length on enzymatic activity of cathepsin B in live muscle and in meat from heavy finishing pigs

To evaluate the effect of dietary protein level (13.0% and 9.5% CP) and duration of fasting on the enzymatic activity of cathepsin B in live muscle and pig, 44 fattening pigs weighing 138 ± 9 kg were reared to slaughter (171 ± 9 kg). The feed allowance was set at 3.2 kg/day. Two weeks prior to slaughter, subjects on each diet were randomly assigned to one of two different fasting treatments (no fasting or 48 h fasting). After this treatment, an individual longissimus dorsi(LD) muscle sample was taken from each pig by biopsy at the level of the third lumbar vertebra and frozen in liquid nitrogen until analysis. Then, the subjects within each diet × fasting interaction were assigned to different fasting times before slaughter (4 or 28 or 52 h). One day after slaughter, LD and semimembranous muscle (SM) samples were taken from each carcass, kept at 4 °C for 24 h and then frozen in liquid nitrogen. The activity of cathepsin B was measured in muscle homogenates by colorimetric method. Dietary CP did not affect cathepsin activity in LD obtained in vivo and daily live weight gain up to slaughter. Compared to the absence of fasting, a cathepsin activity greater than 18% in LD was observed after 48 h of fasting (P < 0.01). Cathepsin activity in LD and SM sampled after slaughter was not affected by dietary protein level and fasting 2 weeks prior to slaughter. Compared with 28 h fasting, feeding exclusion for 52 h prior to slaughter increased cathepsin activity in the LD and SM muscles by 18% (P < 0.05) and 12% (P = 0.07) respectively. The preslaughter fasting length had no effect on fresh meat quality parameters (pH at 1 and 24 h post mortem, colour, drip losses, cooking losses).

Expression of Cathepsins B, D, and G in Hypertrophic Port-wine Stain

Objectives: Cathepsins B, D, and G are expressed in vascular anomalies, fibroproliferative conditions, and malignancies. We investigated expression of these cathepsins and their localization to the embryonic stem cell (ESC)-like population in hypertrophic port-wine stain (hPWS). Methods: Immunohistochemical staining for cathepsins B, D, and G was performed on 15 hPWS tissue samples. Immunofluorescence dual staining investigated localization of the cathepsins with endothelial marker CD31, ESC markers OCT4 and SOX2, and mast cell markers chymase and tryptase on 2 hPWS tissue samples. Protein and transcript expression were investigated by western blotting and reverse-transcription quantitative polymerase chain reaction on 6 tissue samples and 3 hPWS-derived primary cell lines, respectively. Enzymatic activity assays of cathepsins B and D were performed on 6 tissue samples. Results: Immunohistochemical staining demonstrated expression of cathepsins B and D on the endothelium and media of lesional vessels and cells within the stroma. Cathepsin G was expressed in the stroma. Immunofluorescence staining showed localization of cathepsins B and D to the OCT4+/SOX2+ population, and cathepsin G to mast cells, in hPWS. Reverse-transcription quantitative polymerase chain reaction demonstrated transcript expression of all 3 cathepsins in hPWS tissues and cathepsins B and D in cell lines. Protein expression and enzymatic activity of cathepsins B and D was confirmed by western blotting and enzymatic activity assays, respectively. Conclusion: Cathepsins B and D are expressed by the ESC-like population on the endothelium and media of the lesional vessels and stroma, and cathepsin G is expressed by mast cells in hPWS. Functional investigations are needed to fully elucidate the functional role of these cathepsins in hPWS.

Role of Glycosaminoglycans in Procathepsin B Maturation: Molecular Mechanism Elucidated by a Computational Study.

Procathepsins are an inactive, immature form of cathepsins, predominantly cysteine proteases present in the extracellular matrix (ECM) and in lysosomes that play a key role in various biological processes such as bone resorption or intracellular proteolysis. The enzymatic activity of cathepsins can be mediated by glycosaminoglycans (GAGs), long unbranched periodic negatively charged polysaccharides found in ECM that take part in many biological processes such as anticoagulation, angiogenesis, and tissue regeneration. In addition to the known effects on mature cathepsins, GAGs can mediate the maturation process of procathepsins, in particular, procathepsin B. However, the detailed mechanism of this mediation at the molecular level is still unknown. In this study, for the first time, we aimed to unravel the role of GAGs in this process using computational approaches. We rigorously analyzed procathepsin B–GAG complexes in terms of their dynamics, energetics, and potential allosteric regulation. We revealed that GAGs can stabilize the conformation of the procathepsin B structure with the active site accessible for the substrate and concluded that GAGs most probably bind to procathepsin B once the zymogen adopts the enzymatically active conformation. Our data provided a novel mechanistic view of the maturation process of procathepsin B, while the approaches elaborated here might be useful to study other procathepsins. Furthermore, our data can serve as a rational guide for experimental work on procathepsin–GAG systems that are not characterized in vivo and in vitro yet.

The mRNA expression and enzymatic activity of three enzymes during embryonic development of the hard tick Haemaphysalis longicornis

BackgroundThree main enzymes including cathepsin B, cathepsin D and acid phosphatase are involved in vitellin degradation, which is a major biochemical event of the embryonic development and can provide nutrients and metabolites for tick embryos. In the present study, the mRNA expression profiles and enzymatic activity of cathepsin B, cathepsin D and acid phosphatase were investigated during embryonic development in the tick Haemaphysalis longicornis.ResultsThe results revealed that all three enzymes were expressed throughout embryonic development. Both cathepsin B and acid phosphatase transcripts were accumulated during the first four days. Cathepsin B reached its highest expression on day 5, whereas the peak expression of acid phosphatase and cathepsin D occurred on day 11. The highest activity of cathepsin B was observed on the first day of egg development, whereas cathepsin D reached its highest activity on day 13. Acid phosphatase activity increased gradually during the first five days and then remained stable until the end of egg development.ConclusionsThree enzymes were expressed and activated in eggs, and also presented different dynamic changes with the development of embryos. The profiles of both mRNA expression and enzymatic activity of these enzymes indicate that they are controlled orderly and play multiple roles during embryonic development in ticks.

High Dose Vitamin E Attenuates Diabetic Nephropathy via Alleviation of Autophagic Stress.

It has been reported that autophagic stress, which is involved in many diseases, plays a key role in the development of diabetic nephropathy (DN). In this study, we investigated the effects of high dose vitamin E on renal tubular epithelial cells and autophagic stress-related mechanisms in diabetes condition. In diabetic rats, high dose vitamin E treatment significantly decreased the serum creatinine, urea nitrogen, urinary albumin and urinary protein, reduced the levels of LCN2, HAVCR1, LDH and 8-OHdG in urine, and attenuated the cellular apoptosis and interstitial fibrosis in renal cortex. In vitro, vitamin E could reduce the release of LCN2 and HAVCR1 and the protein levels of caspase 3 and TGF-β1, as well as improve the growth inhibition in cultured HK-2 cells after exposure to advanced glycation end products (AGEs). Also, LC3-II and SQSTM1-positive dots were significantly increased in the renal tubular epithelial cells of DN patients and diabetic rats, and in HK-2 cells after exposure to AGEs, which were markedly declined by vitamin E. In addition, we found that the autophagosome formation was not affected by AGEs, as assessed by the mRNA levels of LC3B, Beclin-1, and ATG7. However, AGEs blocked the lysosomal degradation of autophagosome, which was characterized by a decrease in the enzymatic activity of cathepsin B/cathepsin L and DQ-ovalbumin degradation in HK-2 cells, indicating that AGEs-induced accumulation of autophagic vacuoles was a sign of autophagic stress. Interestingly, vitamin E exerted a protective effect on lysosomes to reduce the autophagic stress. Taken together, we conclude that autophagic stress may play an important part in the progression of DN, and alleviation of autophagic stress though improvement of lysosomal function provides a promising novel approach for treating DN.

Expression of cytotoxic T-lymphocyte antigen 2 alpha in mouse bronchiolar epithelial cells: anatomical and functional implications

The lung is one of the major sites of cathepsins (B, H, and L) expression where they are involved in several functions including degradation of extracellular matrix proteins, remodelling of the lung tissue and processing of the invariant chain of the MHC class II complex. Under pathological conditions, cathepsins have been observed in inflammation, tumour progression and metastasis. The enzymatic activity of cathepsins is regulated by changes in pH and their interaction with inhibitors. One of the potent and specific inhibitors of cathepsin L is the Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) which was originally expressed in mouse activated T-cells and mast cells. However, the cellular localization and distribution pattern of CTLA-2α in the lung is not known. In this study, CTLA-2α expression in the lung was evaluated by immunofluorescence. Strong immunoreactivity was specifically detected in the bronchiolar epithelial cells of primary, secondary and terminal segments; moderate in the lymphoid nodules and scanty in the lung parenchyma. The distribution pattern implicates an important role of this inhibitor protein in relation to matrix remodelling, immune response and their relationship to lung development and diseases.Keywords: CTLA-2α, immunofluorescence, mouse, lung

Graphene oxide quantum dots disrupt autophagic flux by inhibiting lysosome activity in GC-2 and TM4 cell lines.

Graphene oxide quantum dots (GOQDs) have broad application prospects in many areas including bioimaging, drug delivery, DNA cleavage system, sensors and photocatalyst. Recently, increasing concerns have been raised about their biocompatibility, but studies about the effects of GOQDs on male reproductive system are still lacking. In this work, we explored the effects and molecular mechanisms of GOQDs on GC-2 and TM4 cells. We found autophagosome accumulation in GC-2 and TM4 cells after GOQDs treatment. Both LC3-II/LC3-I ratio and p62 levels increased, and the chloroquine-induced accumulation of LC3-II didn't enhance in the presence of GOQDs, which indicated that GOQDs blocked autophagic flux. Further studies found that the fusion between autophagosome and lysosome was not inhibited by GOQDs, but the proteolytic capacity of lysosome was weakened and both the expression and activity of cathepsin B reduced. Taken together, these results suggested that GOQDs blocked autophagic flux by decreasing the amount and enzymatic activity of cathepsin B and inhibiting lysosome proteolytic capacity in GC-2 and TM4 cells, which might have a potential hazard to male reproduction.

Autophagy-Lysosome Pathway in Renal Tubular Epithelial Cells Is Disrupted by Advanced Glycation End Products in Diabetic Nephropathy

It has been suggested that autophagy protects renal tubular epithelial cells (TECs) from injury in diabetic nephropathy (DN). However, the manner in which the autophagy-lysosome pathway is changed in this state remains unclear. In this study of DN, we investigated the autophagic activity and lysosomal alterations in vivo and in vitro. We found that autophagic vacuoles and SQSTM1-positive proteins accumulated in TECs from patients with DN and in human renal tubular epithelial cell line (HK-2 cells) treated with advanced glycation end products (AGEs), the important factors that involved in the pathogenesis of DN. In HK-2 cells, exposure to AGEs caused a significant increase in autophagosomes but a marked decrease in autolysosomes, and the lysosomal turnover of LC3-II was not observed, although LC3-II puncta were co-localized with the irregular lysosomal-associated membrane protein1 granules after AGEs treatment. Furthermore, lysosomal membrane permeabilization was triggered by AGEs, which likely resulted in a decrease in the enzymatic activities of cathepsin B and cathepsin L, the defective acidification of lysosomes, and suppression of the lysosomal degradation of DQ-ovalbumin. Oxidative stress evoked by AGEs-receptor for AGE interaction likely played an important role in the lysosomal dysfunction. Additionally, ubiquitinated proteins were co-localized with SQSTM1-positive puncta and accumulated in HK-2 cells after exposure to AGEs, indicating blocked degradation of SQSTM1-positive and ubiquitinated aggregates. Taken together, the results show that lysosomal membrane permeabilization and lysosomal dysfunction are triggered by AGEs, which induce autophagic inactivation in TECs from patients with DN. Disruption of the autophagy-lysosome pathway should be focused when studying the mechanisms underlying DN.

BCM-95 and (2-hydroxypropyl)-β-cyclodextrin reverse autophagy dysfunction and deplete stored lipids in Sap C-deficient fibroblasts.

Saposin (Sap) C deficiency is a rare variant form of Gaucher disease caused by impaired Sap C expression or accelerated degradation, and associated with accumulation of glucosylceramide and other lipids in the endo/lysosomal compartment. No effective therapies are currently available for the treatment of Sap C deficiency. We previously reported that a reduced amount and enzymatic activity of cathepsin (Cath) B and Cath D, and defective autophagy occur in Sap C-deficient fibroblasts. Here, we explored the use of two compounds, BCM-95, a curcumin derivative, and (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), to improve lysosomal function of Sap C-deficient fibroblasts. Immunofluorescence and biochemical studies documented that each compound promotes an increase of the expression levels and activities of Cath B and Cath D, and efficient clearance of cholesterol (Chol) and ceramide (Cer) in lysosomes. We provide evidence that BCM-95 and HP-β-CD enhance lysosomal function promoting autophagic clearance capacity and lysosome reformation. Our findings suggest a novel pharmacological approach to Sap C deficiency directed to treat major secondary pathological aspects in this disorder.

Abstract 1868: Clinical significance of cathepsin L and B expression in pediatric acute myeloid leukemia

Abstract Introduction: Cathepsin L and B are commonly expressed cysteine proteinases that play a major role in lysosomal bulk proteolysis, protein processing, matrix degradation, and tissue remodelling. In tumors there is deregulation of these proteases that interferes with these normal biological processes. In a pilot study done in our lab expression of cathepsin L and B were found to be significantly elevated in peripheral blood mononuclear cells (PBMCs) of pediatric AML patients. However the expression of these proteases in bone marrow aspirate, which represents almost true population of blasts, has never been studied before. Methods: Enzymatic activities of cathepsin L and B were assayed in PBMCs and bone marrow mononuclear cells (BMMCs) of AML patients and healthy controls. Further expression of these proteases and its inhibitor were confirmed by western blotting and real time assay. Cell cycle analysis in PBMCs and BMMCs of these patients was performed by flow cytometry using propidium iodide. Results: In the PBMCs of pediatric AML patients activity of cathepsin L and B was found to be significantly elevated as compared to healthy controls. Similarly, increased activity of cathepsin L and B was also observed in the BMMCs when compared with PBMCs of these patients. Furthermore, protein and mRNA expression also confirmed the increased level of these proteases in pediatric AML. The protein and mRNA expression of their endogenous inhibitor cystatin-C was found to be decreased in PBMCs and BMMCs of AML patients. Moreover, expression of these proteases correlated positively with the percentage of blasts and percentage of cells in S-phase. Conclusion: This study for the first time demonstrates the increased expression of cathepsin L and B in the BMMCs and PBMCs of pediatric AML patients, along with a positive correlation with percentage of blasts, thereby suggesting a potential role of these proteases in the severity of this disease. Citation Format: Garima Pandey, Sameer Bakhshi, Ratnakar Singh, Shyam S. Chauhan. Clinical significance of cathepsin L and B expression in pediatric acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1868. doi:10.1158/1538-7445.AM2014-1868

Effect of iced-storage on the activity of cathepsin L and trypsin-like protease in carp dorsal muscle

Carps were stored in ice for 0, 2 and 4 days. The actomyosin was extracted from the ice-stored fish and the enzymatic activity of cathepsin L and trypsin-like protease in the extract was assayed. The two enzymes remained in the carp actomyosin despite intensive leaching during the extraction procedure. The activity of cathepsin L decreased slightly after 2 days of storage and remained relative constant thereafter, suggesting the enzyme was only slightly affected by the short term iced-storage. However the activity of the trypsin-like protease increased rapidly with iced-storage time, indicating that it might have been activated during the short term storage in ice.

Ginkgolide B prevents cathepsin-mediated cell death following cerebral ischemia/reperfusion injury

Ginkgolide B (GB) has been shown to exert neuroprotective effects against cerebral ischemia/reperfusion (I/R) injury. However, the underlying mechanism by which GB prevents ischemic cell death remains unclear. Lysosomal proteases, including cathepsins B and L, have been implicated in ischemic cell death following reperfusion. Therefore, in the present study, we investigated the role of GB with respect to cathepsin-mediated cell death following I/R. Both the expression and enzymatic activity of cathepsins B and L were significantly increased in the ischemic cortex following cerebral I/R injury. We found that GB treatment markedly decreased the activity and expression of cathepsins B and L following I/R. Moreover, GB reduced necrotic and apoptotic cell death following I/R. These data strongly suggest that GB prevents cathepsin-mediated cell death following focal cerebral I/R injury, and they might provide new insights into the mechanism of the neuroprotective effects of GB.

Differential pathways for interleukin-1β production activated by chromogranin A and amyloid β in microglia

Although chromogranin A (CGA) is frequently present in Alzheimer's disease (AD), senile plaques associated with microglial activation, little is known about basic difference between CGA and fibrillar amyloid-β (fAβ) as neuroinflammatory factors. Here we have compared the interleukin-1β (IL-1β) production pathways by CGA and fAβ in microglia. In cultured microglia, production of IL-1β was induced by CGA, but not by fAβ. CGA activated both nuclear factor–κB (NF-κB) and pro–caspase-1, whereas fAβ activated pro–caspase-1 only. For the activation of pro–caspase-1, both CGA and fAβ needed the enzymatic activity of cathepsin B (CatB), but only fAβ required cytosolic leakage of CatB and the NLRP3 inflammasome activation. In contrast, fAβ induced the IL-1β secretion from microglia isolated from the aged mouse brain. In AD brain, highly activated microglia, which showed intense immunoreactivity for CatB and IL-1β, surrounded CGA-positive plaques more frequently than Aβ-positive plaques. These observations indicate differential pathways for the microglial IL-1β production by CGA and fAβ, which may aid in better understanding of the pathological significance of neuroinflammation in AD.

Detection of pancreatic cancer tumours and precursor lesions by cathepsin E activity in mouse models

Background and AimsPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; however, only 20% of patients are...

Cystatin A protease inhibitor and cysteine proteases in human cervicovaginal fluid in term pregnancy and labor

The purpose of this study was to investigate the temporal changes in immunoreactive cystatin A and the enzymatic activity of cathepsins B, H, L, and S in human cervicovaginal fluid (CVF) in late pregnancy and spontaneous labor. CVF was collected weekly (n = 95 women) from 36 weeks gestation until spontaneous term labor. Cystatin A was quantified using enzyme-linked immunosorbent assay. The enzyme activity of cathepsins B, H, L, and S was measured with fluorometric enzyme assay kits. Cystatin A significantly decreased towards (P = .016, 2-way analysis of variance) and during labor (P < .001, 2-way analysis of variance). Enzymatic activity of cathepsins B, H, and S did not change with labor onset (P = .452, P = .703, P = .411, respectively, 2-way analysis of variance). In late gestation, CVF-decreased expression of the cysteine protease inhibitor, cystatin A, is associated with labor. Although the role and contribution of cystatin A to increased extracellular matrix remodeling has yet to be elucidated, the data that were obtained are consistent with this hypothesis.

Abstract 1570: Silibinin inhibits invasive properties of human breast cancer cell line MDA-MB-231 through suppression of cathepsin B and extracellular signal-regulated kinase-mediated induction of matrix metalloproteinase 2 and 9

Abstract The purpose of the current study is to evaluate the effects of silibinin on human breast adenocarcinoma MDA-MB-231 cells. BrdU cell proliferation assay, Cell-based extracellular signal-regulated kinase (ERK) 1/2 activation assay, cathepsin B activity assay, Gelatin zymography and quantitative real-time RT-PCR were employed to appraise the effects of silibinin on DNA synthesis, ERK 1/2 phosphorylation, cathepsin B activity and gelatinolytic activity in MDA-MB-231 cells. Silibinin inhibited cell proliferation, ERK 1/2 activation, cathepsin B enzymatic levels and gelatinase activity in a dose-dependent manner. In addition, an expressive decrease in mRNA levels of cathepsin B, hyaluronidase-2 (Hyal-2), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) coupled with a significant induction in transcriptional levels of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), Stefin A and Lipocalin 2 were observed. Altogether, these issues show for the first time that silibinin treatment could trammel cell growth and invasive features of a highly invasive human breast cancer cell line, MDA-MB-231, through suppression of ERK 1/2 both directly (through impeding ERK 1/2 phosphorylation) and indirectly (via transcriptional enhancement of Lipocalin 2). Furthermore, cramping enzymatic activity of cathepsin B by silibinin might cripple activation of gelatinases and thereby, reduces invasive potential in MDA-MB-231 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1570.

Silibinin inhibits invasive properties of human glioblastoma U87MG cells through suppression of cathepsin B and nuclear factor kappa B-mediated induction of matrix metalloproteinase 9

Glioblastoma multiforme remains one of the most devastating human malignancies because of its high infiltrative capacity. This study aimed to investigate the effects of silibinin on human glioblastoma U87MG cells. The microculture tetrazolium test, bromodeoxyuridine cell proliferation assay, cell-based nuclear factor kappa B (NF-[kappa]B) activation assessment, cathepsin B activity assay, gelatin zymography, and quantitative real-time reverse transcription-PCR were performed to appraise the effects of silibinin on the metabolic activity, DNA synthesis, NF-[kappa]B phosphorylation, cathepsin B activity, and gelatinolytic activity of U87 cells. Silibinin inhibited metabolic activity, cell proliferation, NF-[kappa]B activation, cathepsin B enzymatic levels, and gelatinase B activity in U87 cells. In addition, an expressive decrease in mRNA levels of matrix metalloproteinase-9, cathepsin B, urokinase plasminogen activator receptor, urokinase plasminogen activator, and intercellular adhesion molecule 1 coupled with a significant induction in transcriptional levels of stefin A was observed. Altogether, these issues show for the first time that silibinin treatment could trammel invasive features of a highly invasive human glioma cell line, U87, through suppression of NF-[kappa]B-mediated stimulation of matrix metalloproteinase-9. Furthermore, silibinin might cripple the activation of gelatinase B by cramping transcriptional and enzymatic activities of cathepsin B in U87 cells.