Induction of Apoptosis in MDA-231 Cells by Protein Synthesis Inhibitors Is Suppressed by Multiple Agents

Abstract

In the present study we investigated the ability of several diverse agents to inhibit MDA-231 cell death induced by two different protein synthesis inhibitors, cycloheximide (CHX) and ricin. Cell death was evaluated by several techniques: trypan blue staining, determination of the released lactic dehydrogenase, transmission electron microscopy, and DNA fragmentation. Results from DNA gel electrophoresis and electron microscopy suggest a mechanism of death by apoptosis which terminates in necrosis. Approximately 60% of cell death was induced either by a continuous exposure to 30 μg/ml CHX for 48 hr or by a 1-hr exposure to 250pg/ml ricin followed by a subsequent incubation of 48 hr in the absence of the drug. Cell survival, in the protein synthesis-inhibited cells, was enhanced by the following diverse agents: the growth factors EGF (20 ng/ml) and IGF-1 (20 ng/ml), the protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (5 ng/ml), the protein kinase A activator 8-bromoadenosine 3′:5′-cyclic monophosphate (650 μg/ml), the nuclease inhibitor aurintricarboxylic acid (100 μg/ml), and fetal bovine serum (5%). The survival agents that stimulated protein synthesis in the control untreated cells had no effect on the CHX—inhibited protein synthesis, which indicates that new protein synthesis is not required for cell survival. The same survival agents attenuated the continuous decrease in protein synthesis in the ricin-exposed cells; therefore, the involvement of new protein synthesis in the survival mechanism could not be excluded. The protein kinase C inhibitor staurosporine blocked, in a dose-dependent manner, the survival effect of 12-0-tetradecanoyl-phorbol-13-acetate and EGF, but not that of aurintricar-boxyclic acid or fetal bovine serum, in the protein synthesis-inhibited cells. These results provide evidence for several distinctive pathways, the activation of which inhibits MDA-231 cell death induced by protein synthesis inhibitors. Some of these pathways involved activation of protein kinases, probably protein kinase C.