Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide terminates in cell death. In the present study we investigated the effect of insulinlike growth factor-1, insulin, and epidermal growth factor on cell death induced by cycloheximide in the confluent MCF-7 cells, and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. After 48 h incubation, cycloheximide (10 to 50 micrograms/ml) was shown to induce cell death in a concentration-dependent manner. Insulinlike growth factor-1, at physiologic concentrations (0.2 to 5 ng/ml), reduced this cell death. Insulin at supraphysiologic concentrations (1 to 10 micrograms/ml) mimicked the effect of insulinlike growth factor-1, whereas epidermal growth factor (10 to 50 ng/ml) had no effect. More than 90% of protein synthesis measured by [3H]leucine incorporation was inhibited by 10 to 50 micrograms/ml cycloheximide. Insulinlike growth factor-1 and insulin at the concentrations that reduced cell death to control level, had no effect on the protein synthesis inhibition rate induced by cycloheximide. These results indicate that inhibition of cell death by insulinlike growth factor-1 does not depend on protein synthesis and may be mediated via a posttranslational modification effect.

Previously we have shown that IGF-I protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). In the present study we investigated the ability of protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the protein kinase A activator 8-bromoadenosine 3'5'-cyclic monophosphate (Br-cAMP), and the enzyme inhibitor aurintricarboxylic acid (ATA) to protect MCF-7 cells against death, due to a continuous presence of CHX. Cell death was evaluated after 48 h of incubation by several techniques (trypan blue staining, release of lactic dehydrogenase, cellular ATP content, transmission electron microscopy, and DNA fragmentation). Apoptosis which terminates in necrosis, characterized this mode of cell death. TPA and ATA at optimal concentrations of 40 ng/ml and 100 micrograms/ml, respectively, reduced cell death to the control level (without CHX), while Br-cAMP at an optimal concentration of 650 micrograms/ml reduced cell death only partially. IGF-1, TPA, and ATA, which stimulated protein synthesis in the control MCF-7 cells, had no effect on protein synthesis in the CHX-treated cells, indicating that the survival effect is not due to new protein synthesis. The protein kinase C inhibitor staurosporine blocked the survival effect of TPA and IGF-1 in a dose-dependent manner, however did not affect the survival effect of ATA. The tyrosine kinase inhibitor genistein blocked the survival effect of IGF-1, but not that of TPA and ATA. Our results provide evidence for several distinctive pathways, the activation of which protects MCF-7 cells against death, due to protein synthesis inhibition.

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The involvement of growth factors in cell survival in the presence of anticancer drugs was investigated. Cell death was induced in the human breast cancer cell line MCF-7, by the structurally and mechanistically unrelated chemotherapeutic drugs puromycin, actinomycin D, 5-fluorouracil, cisplatin, and adriamycin. The effect of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and insulin on cell death was evaluated by two different methods: (1) trypan blue dye exclusion test and (2) lactic dehydrogenase release into the culture medium. IGF-1 inhibited cell death induced by each of the diverse drugs in a concentration-dependent manner reaching a maximal effect at 40 ng/ml. Insulin mimicked the effect of IGF-1 only at supraphysiological concentration with an optimal effect at 10,000 ng/ml. EGF had no effect on cell death up to 100 ng/ml. Our finding that IGF-1 specifically enhanced MCF-7 cell survival in the presence of different anticancer drugs suggests the involvement of growth factors in the mechanism of drug resistance.