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Some secreted cysteine protease inhibitors of the cystatin family appear to affect intracellular proteolysis and growth of human cells, as a result of internalization. Here, we studied the effects of external addition of the most abundant human cystatin, cystatin C, on viability and proliferation of cancer cells in culture. A dose‐dependent decrease in viable cells was seen for A375 melanoma, MCF‐7 breast cancer, and PC‐3 prostate cancer cells cultured in 1–5 µm cystatin C after 24 h. Real‐time assessment of growth rates in A375 cell cultures for 48 h by digital holographic microscopy showed an increased doubling time for cells cultured in the presence of 5 µm cystatin C (20.1 h) compared with control cells (14.7 h). A prolonged doubling time was already observed during the first 12 h, indicating a rapid general decrease in cell proliferation at the population level. Tracking of individual cells in phase holographic images showed that dividing cells incubated with 5 µm cystatin C underwent fewer mitoses during 48 h than control cells. In addition, the time between cell divisions was longer, especially for the first cell cycle. Incubation with the variant W106F‐cystatin C (with high cellular uptake rate) resulted in a lower number of viable cells and a prolonged doubling time than when cells were incubated with wild‐type cystatin C, but no effect was observed for (R24A,R25A)‐cystatin C (low cellular uptake). Thus, cystatin C causes prolonged cell division leading to decreased proliferation of melanoma cells, and internalization seems to be a prerequisite for this effect. Addition of cystatin C, a secreted cysteine protease inhibitor, to A375 melanoma cell cultures resulted in rapid decrease in cell numbers. Tracking of individual cells in phase holographic microscopy images demonstrated that cystatin C causes a prolonged cell cycle rather than increased cell death. Cystatin C variants with altered uptake properties indicated that internalization is needed for the proliferation effect.

The secreted cysteine protease inhibitors, cystatin C and cystatin D, were added externally to leukemic U937, Jurkat, and HL‐60 cell cultures. The cystatins were internalized into endo‐lysosomal vesicles resulting in augmented hydrogen peroxide‐induced apoptosis, as well as decreased proliferation, both in apoptotic and nonapoptotic cells. Cysteine proteases are implicated in proteolysis events favoring cancer cell growth, spread, and death by apoptosis. Herein, we have studied whether the net growth and survival of the leukemic cell lines Jurkat, U937, and HL‐60 are affected by external addition of five proteins acting as natural cysteine protease inhibitors. None of the cystatins examined (A, C, D, and E/M) or chagasin showed consistent effects on Fas‐induced apoptosis when evaluated at 1 µm. In contrast, when the intrinsic apoptosis pathway was activated by hydrogen peroxide, addition of cystatin D augmented caspase‐3‐like activity within all three cell lines. Flow cytometric analysis of U937 cells also showed increased numbers of annexin V‐positive cells when hydrogen peroxide was used to initiate apoptosis and cells were cultured in the presence of cystatin D or C. Moreover, stimulation of hydrogen peroxide‐induced apoptotic U937 cells with either cystatin C or D resulted in a dose‐dependent decrease in the number of cells. Cell viability was also decreased when U937 cells were cultured in the presence of cystatin C or D (1–9 µm) only, demonstrating that these cystatins can reduce cell proliferation by themselves in addition to enhancing apoptosis induced by oxidative stress. These effects on U937 cells were paralleled by internalization of cystatins C and D, indicating these effects are caused by downregulation of intracellular proteolysis. External addition of cystatins C and D to HL‐60 and Jurkat cells demonstrated similar degrees of cystatin D uptake and decreased viability as for U937 cells, indicating that these effects are general for leukemic cells.

Cystatin C is believed to prevent tumor progression by inhibiting the activities of a family of lysosomal cysteine proteases. However, little is known about the precise mechanism of cystatin C function in prostate cancer. In the present study, we examined the expression of cystatin C and its association with matrix metalloproteinases 2 (MMP2) and androgen receptor (AR) in a tissue microarray comparing benign and malignant specimens from 448 patients who underwent radical prostatectomy for localized prostate cancer. Cystatin C expression was significantly lower in cancer specimens than in benign tissues (p<0.001) and there was a statistically significant inverse correlation between expression of cystatin C and MMP2 (r(s) (2) = -0.056, p = 0.05). There was a clear trend that patients with decreased level of cystatin C had lower overall survival. Targeted inhibition of cystatin C using specific siRNA resulted in an increased invasiveness of PC3 cells, whereas induction of cystatin C overexpression greatly reduced invasion rate of PC3 in vitro. The effect of cystatin C on modulating the PC3 cell invasion was provoked by Erk2 inhibitor that specifically inhibited MAPK/Erk2 activity. This suggests that cystatin C may mediate tumor cell invasion by modulating the activity of MAPK/Erk cascades. Consistent with our immunohistochemical findings that patients with low expression of cystatin C and high expression of androgen receptor (AR) tend to have worse overall survival than patients with high expression of cystatin C and high AR expression, induced overexpression of AR in PC3 cells expressing cystatin C siRNA greatly enhanced the invasiveness of PC3 cells. This suggests that there may be a crosstalk between cystatin C and AR-mediated pathways. Our study uncovers a novel role for cystatin C and its associated cellular pathways in prostate cancer invasion and metastasis.

Background High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied. Methods A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay. Results Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay. Conclusions These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.

ObjectivesTo explore the relationship between creatinine and cystatin C based estimated glomerular filtration rate (eGFR) in actively working sugarcane cutters.MethodsThis cohort study included 458 sugarcane cutters from Nicaragua and El Salvador. Serum samples were taken before and at end of harvest seasons and analysed for creatinine and cystatin C. Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formulas were used to calculate eGFRs based on creatinine (eGFRcr), cystatin C (eGFRcys) and both creatinine and cystatin C (eGFRcrcys) at each time point. Bland-Altman plots and paired t-tests were used to compare the difference between eGFRcr and eGFRcys, and the difference in eGFRs between before and at end of the harvest seasons.ResultsThe mean eGFRcr was higher than eGFRcys in both cohorts; absolute difference 22 mL/min/1.73 m2 (95% CI 21 to 23) in Nicaragua and 13 mL/min/1.73 m2 (95% CI 11 to 15) in El Salvador. Correlations between eGFRcr and eGFRcys were high, with r=0.69, 0.77 and 0.67 in Nicaragua at pre-harvest, end-harvest and cross-harvest, and r=0.89, 0.89 and 0.49 in El Salvador.ConclusionsCreatinine increases among heat-stressed workers reflect reduced glomerular filtration as estimated using eGFRcys, a marker independent of muscle mass and metabolism. The discrepancy between eGFRcr and eGFRcys may indicate reduced glomerular filtration of larger molecules and/or systemic bias in CKD-EPI performance in this population.

As a secreted protein, cystatin C is assumed to play its role in the extracellular compartment, where it can inhibit virtually all cysteine proteases of families C1 (cathepsin B, L, S) and C13 (mammalian legumain-related proteases). Since many of its potential target enzymes in the eye reside in intracellular compartments, we sought evidence for a cellular uptake of the inhibitor in ocular tissues. Fluorescence-labeled human cystatin C was injected intravitreally into normal rat eyes. Ocular tissues were subsequently examined using ELISA, fluorescence microscopy, and immunohistochemistry. Cystatin C uptake was additionally studied in an in vitro retina model. Cystatin C administered intravitreally in vivo is taken up into cells of the corneal endothelium and epithelium, the epithelial cells lining the ciliary processes, and into cells in the neuroretina (mostly ganglion cells) and the retinal pigment epithelium. The uptake is demonstrable also in vitro and was, in the neuroretina, found to be a high-affinity system, inhibited by cooling the specimens or by adding the microfilament polymerization inhibitor, cytochalasin D, to the medium. There is an active, temperature-dependent uptake system for cystatin C into several cell types in the cornea, ciliary body, and retina. The cell types that take up cystatin C are generally the same that contain endogenous cystatin C, suggesting that much or all cystatin C seen intracellularly in the normal eye may have been taken up from the surrounding extracellular space. The uptake indicates that the inhibitor may exert biological functions in intracellular compartments. It is also possible that this uptake system may regulate the extracellular levels of cystatin C in the eye.

Cysteine proteinases are important not only in the intracellular catabolism of peptides and proteins and in the processing of prohormones and proenzymes, but also in the penetration of normal human tissue by malignant cells and possibly microorganisms, including viruses. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. We have synthesized peptide derivatives mimicking the proposed proteinase-binding centre of cystatin C and find that they irreversibly inhibit cysteine proteinases. Several bacteria produce proteinases, so we tested a tripeptide derivative (Z-LVG-CHN2) for in vitro anti-bacterial activity against a large number of bacterial strains belonging to thirteen different species. It was found to inhibit specifically the growth of all strains of group A streptococci. The susceptibility of these human pathogens to the peptide was compared with that to well-established anti-streptococcal antibiotics such as tetracycline and bacitracin. Moreover, the peptide was active in vivo against group A streptococci: mice injected with lethal doses of these bacteria were cured by a single injection of Z-LVG-CHN2. The cysteine proteinase produced by group A streptococci was isolated and found to be inhibited by Z-LVG-CHN2; moreover, excess proteinase relieved the growth inhibition caused by the peptide derivative, suggesting that the antibacterial activity of Z-LVG-CHN2 is due to inhibition of this cysteine proteinase. This strategy of blocking proteinases with peptide derivatives that mimic naturally occurring inhibitors could be useful in the construction of new agents against other microorganisms, including viruses.

Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.

Hereditary cystatin C amyloid angiopathy is a dominantly inherited disorder, characterized by dementia, paralysis, and death from cerebral hemorrhage in early adult life. A variant of the cysteine proteinase inhibitor, cystatin C, is deposited as amyloid in the tissues of the patients and their spinal-fluid level of cystatin C is abnormally low. The disease-associated Leu-68 → Gln mutant (L68Q) cystatin C has been produced in an Escherichia coli expression system and isolated by use of denaturing buffers, immunosorption, and gel filtration. Parallel physicochemical and functional investigations of L68Q-cystatin C and wild-type cystatin C revealed that both proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation, 0.4 and 0.5 nM, respectively) but differ considerably in their tendency to dimerize and form aggregates. While wild-type cystatin C is monomeric and functionally active even after prolonged storage at elevated temperatures, L68Q-cystatin C starts to dimerize and lose biological activity immediately after it is transferred to a nondenaturing buffer. The dimerization of L68Q-cystatin C is highly temperature-dependent, with a rise in incubation temperature from 37 to 40⚬C resulting in a 150% increase in dimerization rate. The aggregation at physiological concentrations is likewise increased at 40 compared to 37⚬C, by ≈60%. These properties of L68Q-cystatin C have bearing upon our understanding of the pathophysiological process of hereditary cystatin C amyloid angiopathy. They might also be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates.