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.

中文翻译：

---

分泌的胱抑素可减少人类白血病细胞的增殖并增强其凋亡。

半胱氨酸蛋白酶与促进癌细胞生长、扩散和细胞凋亡死亡的蛋白水解事件有关。在此，我们研究了白血病细胞系 Jurkat、U937 和 HL-60 的净生长和存活是否受到外部添加五种充当天然半胱氨酸蛋白酶抑制剂的蛋白质的影响。当在 1 µm下评估时，所检查的半胱氨酸蛋白酶抑制剂（A、C、D 和 E/M）或 chagasin 均未显示出对 Fas 诱导的细胞凋亡的一致影响. 相反，当过氧化氢激活内在凋亡途径时，添加胱抑素 D 可增强所有三种细胞系中的 caspase-3 样活性。U937 细胞的流式细胞术分析还显示，当使用过氧化氢启动细胞凋亡并在胱抑素 D 或 C 存在下培养细胞时，膜联蛋白 V 阳性细胞的数量增加。此外，用过氧化氢诱导的凋亡 U937 细胞胱抑素 C 或 D 导致细胞数量呈剂量依赖性减少。当 U937 细胞在胱抑素 C 或 D（1-9 µm) 仅，证明这些胱抑素除了增强氧化应激诱导的细胞凋亡外，还可以通过自身减少细胞增殖。这些对 U937 细胞的影响与胱抑素 C 和 D 的内化平行，表明这些影响是由细胞内蛋白水解的下调引起的。向 HL-60 和 Jurkat 细胞外部添加胱抑素 C 和 D 显示出与 U937 细胞相似程度的胱抑素 D 摄取和活力降低，表明这些作用对于白血病细胞是普遍的。