Division of Clinical Chemistry & Pharmacology, Department of Laboratory Medicine, Lund University, Sweden.
FEBS Open Bio. 2021 Jun;11(6):1645-1658. doi: 10.1002/2211-5463.13162. Epub 2021 May 2.
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.
一些半胱氨酸蛋白酶抑制剂家族的分泌型胱抑素似乎通过内化作用影响人类细胞的细胞内蛋白水解和生长。在这里，我们研究了外源添加最丰富的人类胱抑素胱抑素 C 对培养的癌细胞活力和增殖的影响。在 24 小时后，用 1-5 µm 胱抑素 C 培养 A375 黑色素瘤、MCF-7 乳腺癌和 PC-3 前列腺癌细胞，可见活细胞数量呈剂量依赖性下降。通过数字全息显微镜对 A375 细胞培养物在 48 小时内的生长速率进行实时评估，结果显示在 5 µm 胱抑素 C 存在的情况下培养的细胞倍增时间（20.1 小时）比对照细胞（14.7 小时）延长。在最初的 12 小时内已经观察到延长的倍增时间，表明在群体水平上细胞增殖迅速普遍下降。在相位全息图像中跟踪单个细胞，结果显示与对照细胞相比，在 48 小时孵育期间，用 5 µm 胱抑素 C 孵育的分裂细胞经历的有丝分裂较少。此外，细胞分裂之间的时间间隔更长，尤其是在第一个细胞周期中。与野生型胱抑素 C 孵育相比，用变体 W106F-胱抑素 C（具有较高的细胞摄取率）孵育会导致活细胞数量减少和倍增时间延长，但对（R24A，R25A）-胱抑素 C（低细胞摄取率）没有影响。因此，胱抑素 C 导致细胞分裂延长，从而导致黑色素瘤细胞增殖减少，内化似乎是这种作用的先决条件。
