Cellular Effects of Cystatins

The exact cellular functions of cystatins are still unknown, and the molecular mechanism leading to their internalization into cells is not yet fully understood. Commonly the cystatins are known to inhibit human lysosomal cysteine proteases (cysteine cathepsins) and it is supposed that this is their primary physiological role. The cystatins are traditionally believed to mediate regulation of cysteine cathepsins sometimes leaking out from damaged lysosomes of apoptotic cells and, also, to defend against invading microbial pathogens utilizing cysteine proteases. The balance between cysteine cathepsins and their inhibitors of the cystatin family is apparently pivotal in maintaining a healthy state (cell homeostasis). In several diseases such as atherosclerosis, rheumatoid arthritis, Alzheimer’s and Parkinson’s diseases and neoplasia this balance is perturbed, with either higher or lower expression levels of the inhibitors or increased or decreased activity of the enzymes. Historically, the concept of cellular internalization of cystatins was indicated from a few studies using chicken egg-white cystatin, which seemed to have intracellular activity when externally added to cell cultures; it inhibited the replication of polio virus. Later, a biologically active human cystatin C was produced in Escherichia coli, which was shown to inhibit the replication of herpes simplex- and corona-viruses following its external addition to trans-infected cells. Furthermore, when human recombinant cystatin C was injected intraperitoneally into cystatin C-deficient mice, it was detected within cells in many tissues and organs containing endogenous cystatin C in normal mice.In this thesis project, we aimed to study the cellular effects of secreted cystatins on cancer cell growth and death, and whether their function to inhibit cysteine cathepsins is associated with these effects. Our studies were focused on the cellular effects of externally added cystatins C and D on viable cell number, cell death, and proliferation under several oxidative stress conditions. A dose-dependent decrease in the viable cell number was observed in leukemic (Jurkat, U937, and HL-60) and epithelial (A375, MCF-7, and PC-3) cancer cells cultured with cystatin C or D. Internalized cystatin D augmented caspase-3-like activity in U937 cells when the intrinsic pathway of the apoptotic cascade was induced by hydrogen peroxide (H2O2). In contrast, none of the cystatins showed consistent effects on the extrinsic apoptotic pathway induced by anti-Fas in these cells. Cathepsin C activity was high in U937 cells cultured under normoxia and hypoxia and was significantly inhibited by internalized cystatin D. Induction of lysosomal damage using the cathepsin C substrate, Gly-Phe-β-naphtylamide (GPN), leads to galectin puncta formation, which was used to provide an evidence for the inhibition of cathepsin C by internalized cystatin D within lysosomes. A partial co-localization of cystatin D and galectin was seen using confocal laser scanning microscopy. Inhibition of cathepsin C activity was seen in lysates of U937 cells cultured in the presence of cystatin D or C, which paralleled an observed decrease in cell numbers. This provided complimentary, although circumstantial, evidence that internalized cystatins decrease cell proliferation by a mechanism involving cathepsin C inhibition. To be able to study the effects of cystatins on the proliferation of individual cells over time, digital holographic microscopy was used for live time-lapse assessment of growth rate and doubling time in A375 melanoma cell cultures. A prolonged doubling time for cells cultured with cystatin C compared to untreated control cells was observed. Tracking of individual cells in holographic images showed that cells incubated with cystatin C underwent fewer mitoses than control cells, which was observable already the first 12 h. The results from incubation of the cells with the two inhibitor variants W106F- and (R24A,R25A)-cystatin C with altered internalization properties supported that internalization is a prerequisite for the cellular effects of cystatins. Preliminary findings showed that externally added cystatin D is as effective as cystatin C in downregulating the growth of A375 melanoma cells.In all, the results of this thesis demonstrate that 1) Cystatins C and D can reduce the proliferation of cultured cancer cell lines by themselves, in addition to enhancing apoptosis induced by oxidative stress, indicating downregulation of intracellular cysteine proteases; 2) In leukemic cells (U937), internalized cystatin D co-localizes with and targets lysosomal cathepsin C activity, implicating that cathepsin C may be a key enzyme that is involved in the mechanism(s) leading to cystatins’ anti-proliferative effects on cancer cells and, possibly also, promoting cell death; 3) Externally added cystatin C and D could prolong the cell cycle and division time of melanoma cells leading to reduced proliferation. These effects on cancer cells were paralleled by the cellular uptake of cystatins, indicating that internalization is a prerequisite for all

Externally added cystatin C reduces growth of A375 melanoma cells by increasing cell cycle time

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

Externally added cystatin C reduces growth of A375 melanoma cells by increasing cell cycle time

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

Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic 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

Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic 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