Ex vivo activity of cardiac glycosides in acute leukaemia

Abstract

Background: Despite years of interest in the anti-cancerous effects of cardiac glycosides (CGs), and numerous studies in vitro and in animals, it has not yet been possible to utilize this potential clinically. Reports have demonstrated promising in vitro effects on different targets as well as a possible therapeutic index/selectivity in vitro and in experimental animals. Recently, however, general inhibition of protein synthesis was suggested as the main mechanism of the anti-cancerous effects of CGs. In addition, evidence of species differences of a magnitude sufficient to explain the results of many studies called for reconsideration of earlier results.

Principal findings: In this report we identified primary B-precursor and T-ALL cells as being particularly susceptible to the cytotoxic effects of CGs. Digitoxin appeared most potent and IC(50) values for several patient samples were at concentrations that may be achieved in the clinic. Significant protein synthesis inhibition at concentrations corresponding to IC(50) was demonstrated in colorectal tumour cell lines moderately resistant to the cytotoxic effects of digoxin and digitoxin, but not in highly sensitive leukaemia cell lines.

Conclusion: It is suggested that further investigation regarding CGs may be focused on diagnoses like T- and B-precursor ALL.

Figure 1. Cytotoxic IC₅₀ values.

Cytotoxic IC₅₀ values (µM) for digitoxin (A) and oubain (B) in primary cultures of human leukaemia cells (T-ALL, B-precursor ALL, AML and CLL) and PBMCs.

Figure 2. Cytotoxic effects of digitoxin.

A: Cytotoxic effects of digitoxin (0.1 µM) expressed as Survival Index % (vs. untreated control cells) in the various leukaemia types (T-ALL, B precursor ALL, AML and CLL) and PBMC using the standard 72 h assay. B: Effects of therapeutically achievable digitoxin concentrations against leukaemic cells from patients and CCRF-CEM cell line over six days with daily medium change.

Figure 3. Effects of digitoxin on DNA synthesis in leukaemic cell lines.

Effects of digitoxin exposure on DNA synthesis (i.e. thymidine incorporation) in CCRF-CEM (A) and SUP-B15 (B) cells. The DNA polymerase inhibitor aphidicolin (15 µM) was used as a positive control. Effects of digitoxin exposure on protein synthesis (i.e. leucine incorporation) in CCRF-CEM (C) and SUP-B15 (D) cells. The ribosomal inhibitor cycloheximide (36 µM) was used as a positive control.

Figure 4. Comparison between colorectal and leukaemic cell lines.

For comparative purposes the effects of digitoxin on DNA and protein synthesis (A and B respectively) were also monitored in the colorectal adenocarcinoma cell line Hct116. Figure 4C shows differences in cytotoxic activity of digitoxin in the Hct116 cell line and the leukaemic cell lines (CCRF-CEM and SUP-B15) used in the present study. In Figure 4D the effects on survival (at 72 h, measured by FMCA) and protein synthesis at 24 hours at digitoxin concentrations slightly exceeding the IC₅₀ values in HT29 and SUP-B15 cells are shown.