Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic cells

Abstract

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.

Keywords: caspase-3; cystatin C; cystatin D; cysteine peptidase; cysteine protease; protease inhibitor.

Fig. 1

Effects of cystatins A, C, D, E/M, and chagasin on caspase‐3‐like activity in cells following activation of the extrinsic apoptosis pathway. An initial number of 500 000 Jurkat (A) or U937 (B) cells were seeded in 12‐well plates. The cells were incubated for 12 (Jurkat) or 15 (U937) h with 0.2 µg·mL⁻¹ anti‐Fas as well as cystatins A, C, D, E/M, or chagasin at a final concentration of 1 µm. Control cells were incubated in standard medium. Caspase‐3‐like activity in cell homogenates was monitored by cleavage of the fluorescent substrate Z‐DEVD‐NHMec. Raw assay data from 3 to 4 independent cell experiments were grouped and are all shown, with red bars indicating median values for each group. Groups of data (8–16 data points, each being the mean result of three assay measurements) were compared and analyzed statistically by Mann–Whitney test. Significant differences between groups are indicated (***P < 0.001).

Fig. 2

Effects of cystatins A, C, D, E/M, and chagasin on caspase‐3‐like activity in cells following activation of the intrinsic apoptosis pathway. A number of 500 000 Jurkat (A), U937 (B), or HL‐60 (C) cells were seeded into 12‐well plates. The cells were incubated for 12 (Jurkat) or 15 (U937 and HL‐60) h with 40 µm H₂O₂ and 1 µm cystatins A, C, D, E/M, or chagasin. Control cells were incubated in standard medium. Caspase‐3‐like activity in homogenates of the cells was monitored by cleavage of the fluorescent substrate Z‐DEVD‐NHMec. Raw assay data from 3 to 4 independent cell experiments were grouped and are all shown, with red bars indicating median values for each group. Groups of data (8–16 data points, each being the mean result of three assay measurements) were compared and analyzed statistically by Mann–Whitney test. Significant differences between groups are indicated (***P < 0.001; **P < 0.01; *P < 0.05).

Fig. 3

Effects of cystatins A, C, D, E/M, and chagasin to induce or inhibit caspase‐3‐like activity in (A) cells grown without apoptosis induction or (B) a homogenate of apoptosis‐induced cells in vitro. (A) U937 cells were incubated with medium containing 1 µm of cystatins A, C, D, E/M, or chagasin for 15 h, without simultaneous apoptosis induction. Control cells were incubated in standard medium. Caspase‐3‐like activity in homogenates of the cells was monitored by cleavage of the fluorescent substrate Z‐DEVD‐NHMec. (B) Cystatins A, C, D, E/M, or chagasin was added to a final 1 µm concentration, to a lysate of U937 cells in which apoptosis had been induced by incubation with an antibody against Fas for 15 h, prior to assay with the fluorescent substrate Z‐DEVD‐NHMec. Raw assay data from two independent cell experiments were grouped and are all shown, with red bars indicating median values for each group. Groups of data (4–8 data points, each being the mean result of three assay measurements) were compared and analyzed statistically by Mann–Whitney test (**P < 0.01).

Fig. 4

Augmented apoptosis by cystatins C and D seen by annexin V staining. U937 cells were incubated for 24 h with 40 µm H₂O₂ and 1 or 5 µm cystatin C (A) or cystatin D (B). Apoptosis was determined by flow cytometry using annexin V‐APC staining. Flow cytometry measurements were made in triplicate wells in three independent experiments. The bars show mean results, with error bars representing SD. Statistics were calculated by Mann–Whitney test (*P < 0.05; ***P < 0.001).

Fig. 5

Cystatins C and D decrease U937 cell viability, both after apoptosis induction and in normal cells. U937 cells were incubated for 48 h with 0, 1, 3, or 9 µm cystatin C or cystatin D, and (A) with 40 µm H₂O₂ for apoptosis induction or (B) without apoptosis induction. For analysis of viability effects over time, U937 cells were incubated with 0–9 µm (C) cystatin C or (D) cystatin D without apoptosis induction for 24–72 h. After incubations with cystatins, MTT was added to the cell cultures for 4 h. Crystals formed in living cells were dissolved by DMSO, and the absorbance was measured at 540 nm. The results are expressed in relation to those for the control cells (0 µm cystatin). The mean ± SD for the mean results from three independent experiments are shown by bars in (A), (B). Statistics (9 data points/group) were calculated by Mann–Whitney test (***P < 0.001; **P < 0.01; *P < 0.05).) Errors bars for the SD of the mean values after 24, 48, or 72 h cultures shown in (C), (D) have been omitted as they overlap, indicating no significant difference in the relative viability of the cystatin treated cells at the different time points.

Fig. 6

Uptake of cystatins in U937 cells. U937 cells were incubated for 5 h with 1 µm wild‐type cystatin C, (R24A,R25A)‐cystatin C, W106F‐cystatin C, or wild‐type cystatin D. Control cells were incubated in standard medium. The intracellular cystatin content was measured in lysates of the cells by ELISA assays specific for either cystatin C (A) or cystatin D (B). The measured values were correlated to the protein content of each lysate. Bars represent mean values of two independent experiments with four culture wells each. Error bars represent SD. Statistics were calculated by Mann–Whitney test (***P < 0.001). Cystatin D values for control cells were below the detection limit of the ELISA (< 0.26 ng·mg⁻¹ protein).

Fig. 7

Intracellular localization of internalized cystatin C and cystatin D. U937 cells were incubated in medium containing both 3 µm Alexa Fluor 568‐cystatin C and Alexa Fluor 488‐cystatin D for 5 h. CLSM was used to visualize internalized cystatins in vesicular compartments. (A) cystatin C (red), (B) cystatin D (green) and (C) merge of (A) and (B) (yellow, colocalization). Nuclei were stained with DAPI (blue). Scale bar: 10 µm.

Fig. 8

Effect of cystatin C variants on U937 cell viability. U937 cells were incubated with 3 µm wild‐type cystatin C, or any of the cystatin C variants (R24A,R25A)‐ and W106F‐cystatin C for 48 h. After incubations with cystatins, MTT was added to the cell cultures for 4 h. Crystals formed in living cells were then dissolved by DMSO, and the absorbance was measured at 540 nm. Measurements were made in two independent experiments, six wells each time, and results were related to the control cells. The bars show mean results, with error bars denoting SD. Statistics were calculated by Mann–Whitney test (*P < 0.05; ***P < 0.001).

Fig. 9

Effect of cystatin C and D on HL‐60, Jurkat, and U937 cell viability. HL‐60, Jurkat, and U937 cells were cultured under normal conditions or in medium containing 40 µm H₂O₂ to induce apoptosis. Cystatin C or D (3 µm) was externally added to the cell cultures 48 h before MTT assay. Experiments were made on three independent occasions, and 16–24 individual wells were analyzed for each condition, each time. The mean result of the control cells in each experiment was set to 100%, and the rest of the values were correlated with that. Bars illustrate mean values of all three experiments, and symbols represent mean results for each experiment (triangles, experiment 1; squares, experiment 2; circles, experiment 3). Statistics were calculated by Mann–Whitney test on groups of raw data (**P < 0.01; ***P < 0.001).

Fig. 10

Internalization of cystatin D in leukemic cells. HL‐60, Jurkat, and U937 cells were incubated with 5 µm cystatin D for 5 h before fixation and immunostaining with a polyclonal antiserum directed against human cystatin D. CLSM was used to visualize the internalized cystatin D (red). Nuclei were stained with DAPI (blue). White arrows highlight staining in vesicular compartments. Scale bars: 50 µm (40× images) or 20 µm (100× images).