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Externally added cystatin C reduces growth of A375 melanoma cells by increasing cell cycle time.
FEBS Open Bio 2021 April 11
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 of 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 to control cells (14.7 h). A prolonged doubling time was already observed during the first 12 h, indicating a rapid general decrease of 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.
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