A bipartite molecular module controls cell death activation in the Basal cell lineage of plant embryos.

Plant zygote divides asymmetrically into an apical cell that develops into the embryo proper and a basal cell that generates the suspensor, a vital organ functioning as a conduit of nutrients and growth factors to the embryo proper. After the suspensor has fulfilled its function, it is removed by programmed cell death (PCD) at the late stages of embryogenesis. The molecular trigger of this PCD is unknown. Here we use tobacco (Nicotiana tabacum) embryogenesis as a model system to demonstrate that the mechanism triggering suspensor PCD is based on the antagonistic action of two proteins: a protease inhibitor, cystatin NtCYS, and its target, cathepsin H-like protease NtCP14. NtCYS is expressed in the basal cell of the proembryo, where encoded cystatin binds to and inhibits NtCP14, thereby preventing precocious onset of PCD. The anti-cell death effect of NtCYS is transcriptionally regulated and is repressed at the 32-celled embryo stage, leading to increased NtCP14 activity and initiation of PCD. Silencing of NtCYS or overexpression of NtCP14 induces precocious cell death in the basal cell lineage causing embryonic arrest and seed abortion. Conversely, overexpression of NtCYS or silencing of NtCP14 leads to profound delay of suspensor PCD. Our results demonstrate that NtCYS-mediated inhibition of NtCP14 protease acts as a bipartite molecular module to control initiation of PCD in the basal cell lineage of plant embryos.

A transgenic probiotic secreting a parasite immunomodulator for site-directed treatment of gut inflammation.

New treatment strategies for inflammatory bowel disease are needed and parasitic nematode infections or application of helminth components improve clinical and experimental gut inflammation. We genetically modified the probiotic bacterium Escherichia coli Nissle 1917 to secrete the powerful nematode immunomodulator cystatin in the gut. This treatment was tested in a murine colitis model and on post-weaning intestinal inflammation in pigs, an outbred model with a gastrointestinal system similar to humans. Application of the transgenic probiotic significantly decreased intestinal inflammation in murine acute colitis, associated with increased frequencies of Foxp3(+) Tregs, suppressed local interleukin (IL)-6 and IL-17A production, decreased macrophage inflammatory protein-1α/ß, monocyte chemoattractant protein -1/3, and regulated upon activation, normal T-cell expressed, and secreted expression and fewer inflammatory macrophages in the colon. High dosages of the transgenic probiotic were well tolerated by post-weaning piglets. Despite being recognized by T cells, secreted cystatin did not lead to changes in cytokine expression or macrophage activation in the colon. However, colon transepithelial resistance and barrier function were significantly improved in pigs receiving the transgenic probotic and post-weaning colon inflammation was reduced. Thus, the anti-inflammatory efficiency of a probiotic can be improved by a nematode-derived immunoregulatory transgene. This treatment regimen should be further investigated as a potential therapeutic option for inflammatory bowel disease.

Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin-containing SDS-PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E-64-sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity.

Differential fusion expression and purification of a cystatin in two different bacterial strains.

To date, the identification of the novel multifunctional properties of cysteine proteinase inhibitors "known as cystatins" is the great of interests for molecular biologists. The efficient production, purification and correctly folded form of these proteins are the most important requirements for their any basic research. To the best of our knowledge, maltose-binding protein (MBP) fusion tags are being used to overcome the impediment to their heterologous recombinant expression in Escherichia coli as insoluble and bio-inactive inclusion bodies. In the present work, to evaluate the expression efficiency of a cystatin molecule in E. coli cells by using MBP tags, the expression of Celosia cystatin was studied in two different strains of this bacterium. The quantitative analysis results based on the one-step purification yield of the fused product showed the excellency of the E. coli TB1 strain in comparison to E. coli DH5alpha for the high-level production of active product.

Recruitment of coregulator G9a by Runx2 for selective enhancement or suppression of transcription.

Runx2, best known for its role in regulating osteoblast-specific gene expression, also plays an increasingly recognized role in prostate and breast cancer metastasis. Using the C4-2B/Rx2(dox) prostate cancer cell line that conditionally expressed Runx2 in response to doxycycline treatment, we identified and characterized G9a, a histone methyltransferase, as a novel regulator for Runx2 activity. G9a function was locus-dependent. Whereas depletion of G9a reduced expression of many Runx2 target genes, including MMP9, CSF2, SDF1, and CST7, expression of others, such as MMP13 and PIP, was enhanced. Physical association between G9a and Runx2 was indicated by co-immunoprecipitation, GST-pulldown, immunofluorescence, and fluorescence recovery after photobleaching (FRAP) assays. Since G9a makes repressive histone methylation marks and is primarily known as a corepressor, we further investigated the mechanism by which G9a functioned as a positive regulator for Runx2 target genes. Transient reporter assays indicated that the histone methyltransferase activity of G9a was not required for transcriptional activation by Runx2. Chromatin immunoprecipitation assays for Runx2 and G9a showed that G9a was recruited to endogenous Runx2 binding sites. We conclude that a subset of cancer-related Runx2 target genes require recruitment of G9a for their expression, but do not depend on its histone methyltransferase activity.

Microglial cystatin F expression is a sensitive indicator for ongoing demyelination with concurrent remyelination.

Demyelination coincides with numerous changes of gene expression in the central nervous system (CNS). Cystatin F, which is a papain-like lysosomal cysteine proteinase inhibitor that is normally expressed by immune cells and not in the brain, is massively induced in the CNS during acute demyelination. We found that microglia, which are monocyte/macrophage-lineage cells in the CNS, express cystatin F only during demyelination. By using several demyelinating animal models and the spinal cord tissues from multiple sclerosis (MS) patients, we examined spatiotemporal expression pattern of cystatin F by in situ hybridization and immunohistochemistry. We found that the timing of cystatin F induction matches with ongoing demyelination, and the places with cystatin F expression overlapped with the remyelinating area. Most interestingly, cystatin F induction ceased in chronic demyelination, in which remyelinating ability was lost. These findings demonstrate that the expression of cystatin F indicates the occurrence of ongoing demyelination/remyelination and the absence of cystatin F expression indicates the cessation of remyelination in the demyelinating area.

Identification of cystatin B as a potential serum marker in hepatocellular carcinoma.

PURPOSE: The poor survival rate of hepatocellular carcinoma (HCC) is in part due to the inability to diagnose patients at an early stage. Therefore, the aim of this study was to search for candidate serum marker for HCC and to test their ability to distinguish a HCC from benign liver disease. EXPERIMENTAL DESIGN: Genome-wide analysis by a microarray in 40 HCC patients was done between HCC and paired nontumor liver tissues. Expression of cystatin B (CSTB) was examined by mRNA expression analysis and immunohistochemistry. The serum CSTB levels were measured using a sandwich ELISA method in four groups, including normal healthy subjects (group 1, n = 52) and patients with noncirrhotic chronic hepatitis (group 2, n = 53), cirrhosis (group 3, n = 43), and HCC (group 4, n = 62). RESULTS: Microarray and statistical analyses identified 248 genes that were expressed differently between HCC and nontumor liver tissues. One of them, CSTB, was expressed preferentially in the HCCs compared with the nontumor tissues, 36 of 45 specimens (80%) by Northern blot and semiquantitative reverse transcription-PCR analyses. The serum CSTB level was much higher in HCC patients than in those with nonmalignant chronic liver disease (groups 2 and 3; P < 0.0001). The receiver operating characteristic curve indicated 5.34 ng/mL to be the optimal value for CSTB, and the sensitivity and specificity for this CSTB value were 85.5% (95% confidence interval, 74.2-93.1%) and 53.1% (95% confidence interval, 42.7-63.4%), respectively, in distinguishing between patients with HCC and those with nonmalignant chronic liver disease. CONCLUSION: CSTB is specifically overexpressed in most HCCs and is also elevated in the serum of a large proportion of HCC patients. CSTB or the combination of CSTB and alpha-fetoprotein may be a useful marker for diagnosing patients with HCC with a high sensitivity.

Over-expression of cysteine proteinase inhibitor cystatin 6 promotes pancreatic cancer growth.

Pancreatic ductal adenocarcinoma (PDAC) shows the worst mortality among the common malignancies and development of novel therapies for PDAC through identification of good molecular targets is an urgent issue. Among dozens of over-expressing genes identified through our gene-expression profile analysis of PDAC cells, we here report CST6 (Cystatin 6 or E/M) as a candidate of molecular targets for PDAC treatment. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis confirmed over-expression of CST6 in PDAC cells, but no or limited expression of CST6 was observed in normal pancreas and other vital organs. Knock-down of endogenous CST6 expression by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in maintaining viability of PDAC cells. Concordantly, constitutive expression of CST6 in CST6-null cells promoted their growth in vitro and in vivo. Furthermore, the addition of mature recombinant CST6 in culture medium also promoted cell proliferation in a dose-dependent manner, whereas recombinant CST6 lacking its proteinase-inhibitor domain and its non-glycosylated form did not. Over-expression of CST6 inhibited the intracellular activity of cathepsin B, which is one of the putative substrates of CST6 proteinase inhibitor and can intracellularly function as a pro-apoptotic factor. These findings imply that CST6 is likely to involve in the proliferation and survival of pancreatic cancer probably through its proteinase inhibitory activity, and it is a promising molecular target for development of new therapeutic strategies for PDAC.

Cathepsin B expression in prostate cancer of native Japanese and Japanese-American patients: an immunohistochemical study.

BACKGROUND: Japanese-American (J-A) men who have immigrated to the U.S.A. and acquired the Western lifestyle usually have more invasive prostate cancer (PCa) than native Japanese (NJ) living in Japan. The specific reasons for these differences remain unknown. The objective of this study was to examine immunostainings of cathepsin B (CB) and its endogenous inhibitor stefin A (SA) in tissue microarray (TMA) and radical prostatectomy (RP) tissue sections in the hope of obtaining insights into the invasiveness of PCa in Japanese patients. PATIENTS AND METHODS: TMA and RP sections were evaluated in 50 men (25 NJ and 25 J-A) for CB and SA reaction products. The CB and SA immunostainings were imaged directly from microscope slides to a computer using a high performance charge coupled device (CCD) digital camera, quantified using Metamorph software, analyzed using the two-sample t-test, and confirmed by multiple regression analysis. RESULTS: The CB and SA proteins were localized in the carcinomatous glands and isolated cancer cells in the TMA and RP sections. The Gleason scores and pre-surgery serum total prostate-specific antigen (PSA) levels did not differ significantly in the NJ and J-A patients (p = 0.14, p = 0.16, respectively). The Chi-square analysis of clinical stage versus place of birth showed that the NJ patients had significantly more T2a and T2b clinical stages than the J-A patients who had more advanced T2c and T3a stages (p = 0.003). The CB and SA immunostainings and their ratios in Gleason score 6 tumors did not show any difference, but the CB:SA ratios in score > or = 7 tumors approached significance levels. CONCLUSION: The overall matching of specimens according to the Gleason grade/score, pre-RP serum total PSA levels, clinical stage and age prior to evaluation of immunostainings greatly minimizes subjectivity associated with the evaluation of markers in this ethnic sub-population of PCa patients. CB and SA immunostaining is similar in Japanese patients who have organ-confined and moderately-differentiated PCa. Analysis of the reaction product data provides indirect evidence that invasiveness of PCa is similar in the two Japanese patient populations.

Cloning, characterization and identification of Rcet1-v1 and Rcet1-v2, two novel splice variants of mouse Rcet1 related to Cres subgroup of family 2 cystatins.

Cystatins are physiological cysteine proteinase inhibitors. We used digital differential display (DDD) to clone two novel splice variants Rcet1-v1 and Rcet1-v2 which were isolated from adult mouse testis cDNA library. Sequence analysis revealed that Rcet1-v1 and Rcet1-v2 cDNAs are 454 and 610 bp in length, respectively, and each has four exons, but the lengths of their second and third exons are different, with the results that these cDNAs encoded two different putative proteins. The deduced proteins were 88 amino acid residues (RCET1-v1) and 140 residues (RCET1-v2) in length and have one potential signal peptide and one cystatin domain, respectively, but lack part critical consensus sites important for cysteine protease inhibition. These characteristics are seen in CRES subgroup, which related to the family 2 cystatins and primarily expressed in reproductive tract. RT-PCR analysis showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse testis, epididymis and cerebrum, but higher in testis than in epididymis and cerebrum. RT-PCR analysis also showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse pituitary and spermatogonium, but not expressed in spermatozoa. Results of in situ hybridization showed that Rcet1 gene expressed abundantly in mouse spermatogonium, spermatocytes and round spermatids; did not expressed in spermatozoa. At mouse testis different development stages, Rcet1-v1 and Rcet1-v2 were expressed very low from postnatal 1 day to postnatal 3 weeks; after postnatal 4 weeks, expressed steadily increased from postnatal 4 to 7 weeks, highest in postnatal 7 to 8 weeks, then keeping on the expressing level of postnatal 6 weeks in postnatal 13-57 weeks. All these indicated that Rcet1-v1 and Rcet1-v2 primarily expressed in mouse male reproductive tract and may play important roles in mouse spermatocytes and round spermatid development. Rcet1-v1 and Rcet1-v2 may be new members of Cres subgroup of the family 2 cystatins.

Differential expression of cathepsins and cystatin C in ovine uteroplacental tissues.

Cathepsins (CTSs) are peptidases that have biological roles in degrading extracellular matrix, catabolism of intracellular proteins, and processing of pro-hormones. Cystatin C (CST3) is a secreted inhibitor of lysosomal cysteine proteases cathepsin B (CTSB) and CTSL. Our working hypothesis is that cathepsins and cystatins play important roles in implantation and placentation in sheep. Expression of CTSB, CTSD, CTSH, CTSK, CTSL, CTSS, CTSZ, and CST3 mRNAs was detected in ovine uteroplacental tissues with distinct temporal and/or spatial expression patterns between Days 40 and 120 of pregnancy. Of particular note, CTSB, CTSD, and CTSZ mRNAs were predominantly detected in the chorion of the placenta and were more abundant in the placentomes than the intercaruncular endometria. CTSL and CST3 mRNAs were abundant in the endometrial epithelia and chorion, whereas CTSK, CTSS and CTSH mRNAs were most abundant in the stratum compactum stroma of the intercaruncular endometrium. Consistent with localisation of mRNAs, immunoreactive CTSL and CST3 proteins were mainly observed in the intercaruncular endometrial glands and intercotyledonary placenta during later pregnancy. These results support the working hypothesis that CTS and CST3 in uteroplacental tissues are involved in endometrial remodelling and placentation in sheep.

The upregulation of cystatin C in oral submucous fibrosis.

Cystatin C is a 13kDa non-glycosylated basic protein belonging to cystatin family. It is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare cystatin C expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induction of cystatin C expression. Twenty-five OSF specimens and six of normal buccal mucosa were examined by immunohistochemistry. The activity of cystatin C from fibroblasts cultured from OSF and normal buccal mucosa were evaluated by using reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Furthermore, the effect of arecoline, the major areca nut alkaloid, was explored. Cystatin C expression was significantly higher in OSF specimens (p<0.05) and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. OSF demonstrated significantly higher cystatin C expression than normal buccal mucosa fibroblasts both in mRNA and protein levels (p<0.05). In addition, arecoline was also found to elevate cystatin C mRNA and protein expression in a dose-dependent manner (p<0.05). Taken together, the data demonstrate that cystatin C expression is significantly upregulated in OSF from areca quid chewers and arecoline may be responsible for the enhanced cystatin C expression in vivo.

Cathepsin B expression is similar in African-American and Caucasian prostate cancer patients.

BACKGROUND: Increased incidence and mortality of prostate cancer (PCa) suggest that U.S. African-American men have more invasive cancer than Caucasian men. Invasive PCa requires several proteases, including the cysteine protease cathepsin B (CB), for degradation of basement membrane and extracellular matrix proteins prior to cancer cell migration across biological compartments. Our objective was to determine whether CB immunostaining patterns, in relation to clinical data, could distinguish invasive PCa in African-American and Caucasian patients. PATIENTS AND METHODS: Fifty Gleason score 6/7 PCa cases were selected for similar clinical data with benign prostatic hyperplasia (BPH) samples as controls. Immunostainings were imaged directly from microscope slides to a computer using a digital camera. Data were quantified using Metamorph software, analyzed using the two-sample t-test and confirmed by multiple regression. RESULTS: Ratios of CB to its endogenous inhibitor stefin A (SA) immunostainings were greater in PCa than BPH, but were not significantly different in PCa of either race. The African-American patients did not show increased CB immunostaining, indicating that the contribution of this protease to invasiveness was similar in both races. CONCLUSION: When veterans received equal medical care at the Minneapolis Veterans Affairs Medical Center, African-American patients did not show increased PCa invasiveness. Our conclusion is supported by analysis of post-surgery serum total PSA levels and cancer cell invasion to margins/capsules, seminal vesicles and/or lymph nodes. Invasiveness of PCa does not appear to be race-dependent. The previous conclusion of race-based differences in PCa requires re-evaluation with respect to the role of proteases (such as CB, matrix metalloproteinase) in invasion and metastasis of cancer cells.

Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host.

Tumor cell invasion and metastasis are associated with degradation of components of the extracellular matrix by different proteinases. Among those, papain-like cysteine proteases, such as cathepsin B, seem to play an important role, as they are associated with poor clinical outcome in different cancers. In this study, we tested whether cystatin C, a natural extracellular inhibitor of papain-like cysteine proteases, can inhibit metastasis when overexpressed at the tumor-host interface. Local overexpression of cystatin C in liver and lungs of CD1 nu/nu mice was achieved by gene transfer with a novel adenoviral construct, which also led to the presence of 60 ng/mL of cystatin C in the serum. Three days after gene transfer, these mice were challenged by i.v. inoculation of lacZ-tagged human fibrosarcoma cells (HT1080lacZ-K15), leading to the formation of experimental lung and liver metastases. In this model, formation of experimental metastatic foci correlated with expression of cathepsin B in lungs, whereas there was no correlation with metastasis to the liver. In mice overexpressing cystatin C, the number of lung metastases was significantly reduced by 92%, as compared with mice receiving control adenovirus. The efficacy of extravasation of HT1080lacZ-K15 cells into the liver was not affected, indicating the independence of this process from the activity of cysteine-cathepsins. The present report is the first evidence of successful reduction of metastasis by inhibition of cysteine-cathepsins by cystatin C overexpression in the host microenvironment. Furthermore, organ-specific protease expression during tumor-host cell interactions could affect the success of antiproteolytic intervention against metastasis.

The role of cystatin C in cerebral amyloid angiopathy and stroke: cell biology and animal models.

A variant of the cysteine protease inhibitor, cystatin C, forms amyloid deposited in the cerebral vasculature of patients with hereditary cerebral hemorrhage with amyloidosis, Icelandic type (HCHWA-I), leading to cerebral hemorrhages early in life. However, cystatin C is also implicated in neuronal degenerative diseases in which it does not form the amyloid protein, such as Alzheimer disease (AD). Accumulating data suggest involvement of cystatin C in the pathogenic processes leading to amyloid deposition in cerebral vasculature and most significantly to cerebral hemorrhage in patients with cerebral amyloid angiopathy (CAA). This review focuses on cell culture and animal models used to study the role of cystatin C in these processes.

Cystatin C expression is increased in the hippocampus following photothrombotic stroke in rat.

Stroke is a major cause of epilepsy, but the molecular mechanisms underlying post-stroke epileptogenesis are unknown. The expression of cystatin C, a cysteine protease inhibitor, is increased in the hippocampus during status epilepticus (SE)-induced epileptogenesis, and regulates both cell death and birth. To test the hypothesis that increased cystatin C expression represents a common molecular alteration induced by epileptogenic brain insults, we investigated the time course, cellular localization, and association of cystatin C expression with neuronal damage during post-stroke epileptogenesis. Stroke was induced with photothrombosis, which leads to epilepsy in approximately 20-30% of rats. Cystatin C expression was increased in the CA1 area of the hippocampus 4 days after photothrombosis, when the diameter of the lesion was the largest. Double-labeling and confocal analysis indicated that cystatin C was expressed in astrocytes and microglia. Unlike after SE, cystatin C expression did not change in the dentate gyrus. Also, increased cystatin C expression was not associated with neurodegeneration, which was demonstrated as an absence of Fluoro Jade B-positive cells in adjacent sections. The present study provides evidence that cystatin C may be involved in cellular alterations that occur after an epileptogenic insult, not only after SE but also after photothrombotic stroke.

Cystatins in non-small cell lung cancer: tissue levels, localization and relation to prognosis.

Cystatins regulate tumour-associated cysteine proteases, however, their role in tumour progression is not clear yet. To assess their relevance in the progression of non-small cell lung cancer (NSCLC) the protein level, cysteine protease activity (CPI) and localization of type I (stefins A and B) and type II (C, E/M and F) cystatins were defined in tumours and control lung counterparts from 165 patients. The medians of CPI activity, stefins A and B were significantly greater in tumour than in lung tissue (2.1-fold, 1.7-fold, 1.2-fold, respectively, all p<0.001). The median levels of cystatin C and cystatin E/M were lower in tumour tissue (0.9-fold, p=0.06; 0.6-fold, p<0.01). In all the samples the levels of cystatin F were below the detection limit. Immunohistochemical analysis revealed the presence of all cystatins in tumour cells and infiltrated inflammatory cells such as macrophages and neutrophils. In univariate survival analysis patients with high levels of stefin A, stefin B and CPI activity exhibited a better survival probability (p=0.05, p=0.05, p<0.01, respectively). In contrast, cystatins C and E/M provided no prognostic information. In multivariate analysis the most powerful predictor of survival was the pTNM stage (p<0.0001; RR 3.5), followed by stefin A, stefin B and CPI activity (all p=0.03; RR 1.5). Our results suggest that only stefins A and B, i.e. type I cystatins, are up-regulated in lung tumours and thus able to counteract harmful tumour-associated proteolytic activity. As biological markers they may add independent prognostic information for better assessment of low- and high-risk patients with NSCLC.

Enhanced cystatin C and lysosomal protease expression following 6-hydroxydopamine exposure.

6-Hydroxydopamine (6-OHDA) is a selective neurotoxin used to induce apoptosis in catecholamine-containing neurons. Although biochemical products and reactive oxygen species (ROS) of 6-OHDA have been well documented, the activation of cellular pathways following exposure are not well understood. Apoptosis in PC12 (Pheochromocytoma) cells was induced by 6-OHDA in a dose (10-150 microM) and time-dependent (24-72 h) manner compared to experimental controls (no treatment). PC 12 cells exposed to 50 microM 6-OHDA demonstrated the involvement of caspase 3 and lysosomal protease alterations. Following 6-OHDA exposure, the caspase 3-like inhibitor Ac-DEVD-CHO significantly decreased 6-OHDA induced cell death. In addition, alterations in expression of the lysosomal cysteine and aspartic proteases, cathepsin B (CB) and cathepsin D (CD) and the endogenous cysteine protease inhibitor cystatin C were observed utilizing immunocytochemical analysis at 24, 48, and 72 h following 6-OHDA exposure. Furthermore, CB and CD and cystatin C immuno-like reactivity was more pronounced in TUNEL positive cells. Moreover, Western blot analysis confirmed a significant increase in protein expression for CB and CD at 72 h and a temporal and concentration dependent increase in cystatin C in response to 6-OHDA. Cells treated with pepstatin A, an inhibitor for CD, showed a significant decrease in cell death, however, CA-074ME, a specific inhibitor for CB, failed to protect cells from 6-OHDA induced cell death. Thus, these results suggest that apoptosis induced by 6-OHDA exposure is mediated in part through caspase 3 activation and lysosomal protease CD.

Cystatin m: a novel candidate tumor suppressor gene for breast cancer.

The contribution of pericellular proteolysis to tumor progression is well documented. To better understand protease biology and facilitate clinical translation, specific proteolytic systems need to be better defined. In particular, the precise role of endogenous protease inhibitors still needs to be deciphered. We reported previously that cystatin M, a potent endogenous inhibitor of lysosomal cysteine proteases, significantly suppressed in vitro cell proliferation, migration, and Matrigel invasion. Here, we show that scid mice orthotopically implanted with breast cancer cells expressing cystatin M show significantly delayed primary tumor growth and lower metastatic burden in the lungs and liver when compared with mice implanted with mock controls. The incidence of metastasis, however, appeared to be unaltered between the cystatin M group and the control group. Experimental metastasis assays suggest that cystatin M suppressed tumor cell proliferation at the secondary site. By using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we found consistent expression of cystatin M in normal human breast epithelial cells, whereas expression was decreased by 86% in invasive ductal carcinoma (IDC) cells of stage I to IV patients. Complete loss of expression of cystatin M was observed in two of three IDCs from stage IV patients. Immunohistochemical studies confirmed that expression of cystatin M in IDCs was partially or completely lost. We propose cystatin M as a novel candidate tumor suppressor gene for breast cancer.