Abstract
This study focused on the alterations that occur in larval molluscan cells after administration of apoptotic inducers and inhibitors used in mammalian cells in response to cold stress. This is the first report on apoptosis modulation in molluscan cells assessed by flow cytometry. Mitochondrial activity, general caspase activation, and membrane integrity of control molluscan cells were compared to those processes in frozen–thawed molluscan cells, primary mouse embryonic fibroblasts, and human colon tumor cells prior to treatment and after incubation with apoptotic inducers or inhibitors. We tested three apoptotic inducers (staurosporine, camptothecin, and mitomycin C, routinely used for the chemical induction of apoptosis in different mammalian cells) and found that only staurosporine resulted in an evident apoptotic increase in molluscan cell cultures: 9.06% early apoptotic cells in comparison with 5.63% in control frozen–thawed cells and 20.6% late apoptotic cells in comparison with 10.68% in controls. Camptothecin did not significantly induce molluscan cell apoptosis but did cause a slight increase in the number of active cells after thawing. Mitomycin C produced similar results, but its effect was less pronounced. In addition, we hypothesize that the use of the apoptotic inhibitors could reduce apoptosis, which is significant after cryopreservation in molluscan cells; however, our attempts failed. Development in this direction is important for understanding the mechanisms of marine organisms’ cold susceptibility.
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Acknowledgments
This study was supported in part by the Russian Science Foundation of Far Eastern Federal University (grant No 14-50-00034). Flow cytometric analysis was performed in the National Scientific Center of Marine Biology, FEB RAS (Vladivostok), and in the Laboratory of Marine Invertebrate Biology of the Far Eastern Federal University (Vladivostok, Russia). We would like to express special thanks to Dr. I.V. Kudryavtsev for his help in interpreting flow cytometric data and Dr. Mariia Miorova for technical assistance.
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Fig. S1
Results of flow cytometric detection of apoptotic and dead MEFs after a 24 h-treatment with inducers or inhibitors of apoptosis. Cells were stained with the green fluorescent stain, FLICA®, in conjunction with DAPI (a) or another green fluorescent stain, YO-PRO™-1, in conjunction with DAPI (b). The samples were analyzed with a CytoFLEX flow cytometer. Treatment key: control cells (C); cells undergoing STS-induced apoptosis (STS), cells undergoing CAM-induced apoptosis (CAM), cells undergoing MMC-induced apoptosis (MMC); cells cultivated with apoptotic inhibitors: cyclic pifithrin-α (Alpha), CHIR99021 (CHIR), Y-27632 (Y); cells undergoing oxidative stress (H2O2). The significance levels are *P < 0.05 and **P < 0.01 (PNG 89 kb)
Fig. S2
Results of flow cytometric detection of apoptotic and dead HCT 116 cells after a 24 h-treatment with inducers or inhibitors of apoptosis. Cells were stained with FLICA® in conjunction with DAPI. The samples were analyzed in a CytoFLEX flow cytometer. Treatment key: control cells (C); cells cultivated with apoptotic inducers: cells undergoing STS-induced apoptosis (STS); cells undergoing CAM-induced apoptosis (CAM); cells undergoing MMC-induced apoptosis (MMC); cells cultivated with apoptotic inhibitors: cyclic pifithrin-α (Alpha), CHIR99021 (CHIR), Y-27632 (Y); STS-treated cells incubated with apoptotic inhibitors: STS + Alpha, STS + CHIR, STS + Y; cells undergoing oxidative stress (H2O2). The significance levels are *P < 0.05 and **P < 0.01 (PNG 55 kb)
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Boroda, A.V., Kipryushina, Y.O. & Odintsova, N.A. Chemical modulation of apoptosis in molluscan cell cultures. Cell Stress and Chaperones 24, 905–916 (2019). https://doi.org/10.1007/s12192-019-01014-x
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DOI: https://doi.org/10.1007/s12192-019-01014-x