The combination of the antitumoural pyridyl cyanoguanidine CHS 828 and etoposide in vitro--from cytotoxic synergy to complete inhibition of apoptosis

Abstract

1. The present study was aimed at elucidating the apoptosis inhibitory properties of the cyanoguanidine CHS 828. CHS 828 exhibits impressive cytotoxic activity in vitro and in vivo. Apoptosis is not its main mode of cytotoxic effect, and we have previously proposed a dual mechanism, where CHS 828 inhibits its own cell death pathways. 2. Etoposide on the other hand, is a well-established anticancer agent with documented effect in a number of malignancies, induces apoptosis through extensively studied caspase dependent pathways. 3. Here we studied the combined effect of the two drugs in the human lymphoma cell line U-937 GTB. Cytotoxicity was evaluated as total viability measured by the fluorometric microculture cytotoxicity assay (FMCA). Caspase activity was assessed by colorimetric detection of specific cleavage products for caspases 3, 8 and 9, respectively. Morphology was evaluated in May-Grünwald/Giemsa stained preparations. Interaction analysis based on FMCA results of simple combination exposure revealed impressive synergistic effect on cell kill. 4. Detailed investigations of the kinetics involved showed that short pre-exposure (0-12 h) to CHS 828 enhanced caspase activation by etoposide, while longer pre-exposure (18-48 h) inhibited both caspase activation and apoptotic morphology otherwise induced by etoposide. The present results support the theory that CHS 828 block specific cell death pathways. 5. The synergistic results are promising for future combination trials in animals, however, different dosing schedules should be considered, in order to investigate whether the above findings translate into the in vivo setting.

Figure 1

Representative photos of May-Grünwald/Giemsa stained U-937 GTB cells. (A) Graphic representation of the proportion of apoptotic (black bar), necrotic (grey bar) and viable (open bar) cells in preparation (B–F). Cells were induced to undergo apoptosis by 4 h exposure to 25 μM etoposide (B). (C) 4 h co-exposure to 1.0 μM CHS 828 and 25 μM etoposide. (D) 4 h pre-exposure to CHS 828, before addition of 25 μM etoposide. (E) 24 h pre-exposure to 1.0 μM CHS 828, before addition of 25 μM etoposide. (F) control 24 h 1.0 μM CHS 828, without etoposide.

Figure 2

One representative MiCK experiment out of three. U-937 GTB response to 25 μM etoposide after indicated times of pre-exposure to 1.0 μM CHS 828.

Figure 3

Substrate specific activities of caspase 3 (A), caspase 8 (B) and caspase 9 (C) in U-937 GTB cells. Cultures were exposed as indicated and results are presented as means of three separate experiments±s.e. ^*P<0.05; ^**P<0.01, compared to 4 h etoposide exposure alone.