Chemical modulation of apoptosis in molluscan cell cultures

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.

Keywords: Apoptotic inducers; Apoptotic inhibitors; Cell death pathways; Flow cytometry; Mussel; Mytilus trossulus.

Fig. 1

A time course of alterations in mitochondrial activity of molluscan cells (active, non-active, and dead cells) after treatment with apoptotic inducers or inhibitors before and after a freeze–thaw cycle. Cells were cultivated for 6 h, 24 h, and 48 h and assessed by H₂DCFDA and DAPI staining. Treatment key: control unfrozen cells (C); unfrozen cells undergoing staurosporine-induced apoptosis (STS); unfrozen cells undergoing camptothecin-induced apoptosis (CAM); unfrozen cells undergoing mitomycin C-induced apoptosis (MMC); unfrozen cells cultivated with apoptotic inhibitors—cyclic pifithrin-α (Alpha), CHIR99021 (CHIR), Y-27632 (Y); unfrozen cells undergoing oxidative stress (H₂O₂); cells frozen with 5% Me₂SO (Fr). Standard deviations were less than 5.0%

Fig. 2

Two-dimensional (2D) plots from flow cytometric analysis of frozen–thawed cells cultivated for a 24-h recovery period. The samples were analyzed with a CytoFLEX flow cytometer. At least 20,000 events were evaluated for each sample. Cells were stained with the green fluorescent stains FLICA® or YO-PRO™-1 and the ultraviolet-fluorescent stain DAPI. The combination of FLICA® and DAPI was used to identify early apoptotic and dead cells, respectively (a). The combination of YO-PRO™-1 and DAPI was used to identify late apoptotic and dead cells, respectively (b). The most typical alterations in all Fr molluscan cells are presented. Of the apoptotic inhibitors, only the evident effect of CHIR99021 is presented which reduces a number both of early apoptotic and late apoptotic cells but shows an increased number of dead molluscan cells. Treatment key: frozen–thawed cells (Fr); frozen–thawed cells cultivated with apoptotic inducers: frozen–thawed cells undergoing 5 μM STS-induced apoptosis (Fr+STS), frozen–thawed cells undergoing 10 μM CAM-induced apoptosis (Fr+CAM), frozen–thawed cells incubated with 5 μM apoptotic inhibitor CHIR99021 (Fr+CHIR)

Fig. 3

Flow cytometric analysis of apoptosis- and necrosis-associated fluorescence in molluscan cells before and after a freeze–thaw cycle. Cells were cultivated for 24 h and 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). Treatment key: control unfrozen cells (C); cells frozen with 5% Me₂SO and then cultivated for 24 h (Fr); frozen–thawed cells undergoing STS-induced apoptosis (Fr+STS); frozen–thawed cells undergoing CAM-induced apoptosis (Fr+CAM); frozen–thawed cells undergoing MMC-induced apoptosis (Fr+MMC); frozen-thawed cells cultivated with apoptotic inhibitors: cyclic pifithrin-α (Fr+Alpha), CHIR99021 (Fr+CHIR), Y-27632 (Fr+Y). The significance levels are *P < 0.05 and **P < 0.01

Fig. 4

Morphologies of control non-frozen and frozen–thawed molluscan cells cultivated in different conditions for 24 h. Treatment key: control unfrozen cells (C); unfrozen cells undergoing STS-induced apoptosis (C+STS); frozen–thawed cells (Fr); frozen–thawed cells undergoing STS-induced apoptosis (Fr+STS); frozen–thawed cells undergoing CAM-induced apoptosis (Fr+CAM); frozen–thawed cells incubated with an apoptotic inhibitor, CHIR99021 (Fr+CHIR). Specimens were examined with a CKX41 inverted microscope (Olympus, Japan) equipped with phase-contrast optics and imaged with an Axiocam 105 color digital camera (Carl Zeiss, Germany). Scale bar, 25 μm