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

Abstract

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 M 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 M 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.

Chronic infrared‐A irradiation‐induced photoaging of human dermal fibroblasts from different donors at physiological temperature

“…After incubation for a pre‐determined time (24, 32, 48, 56, 72, 80, 96, 104, and 120 h), the cells were harvested and stained with trypan blue (0.4%) in PBS solution for cell counting on the hematocytometer using an inverted microscopy (Olympus, Tokyo, Japan). The doubling times of the CCD and NHDF cells were calculated based on the growth curve using the following equation: Doubling Time = duration of culture × log (2)/log [(Final Concentration) – log (Initial Concentration)] 18,19 …”

Molecularly Imprinted Polymers Exhibit Low Cytotoxic and Inflammatory Properties in Macrophages In Vitro

“…When the cells divided, the two daughter cells were selected for further analysis and connected to the mother cell. This enables the calculation of the time of each cell division for individual cells, [25,26]. Based on known time-points for cell divisions and from which cell the daughter cells originated, a cell family tree was drawn for each cell selected at time-point 0 h using RStudio software (v.2021.09.1, RStudio, Boston, MA, USA).…”

“…DHC is a non-phototoxic quantitative phase imaging technique that enables the monitoring of living cells [24]. The DHC technique allows for long time monitoring of the cells, acquiring high time resolution images, which can be used for longitudinal tracking of individual cells [25,26]. Tracking of individual cells using DHC demonstrated that the SA-MIPs had an impact on cell proliferation and cell division, but the reduction in cell numbers could not be attributed to SA-MIPs cytotoxicity.…”