Pancratistatin induces apoptosis in clinical leukemia samples with minimal effect on non-cancerous peripheral blood mononuclear cells

Abstract

Background: Pancratistatin, a natural compound extracted from Hymenocallis littoralis, can selectively induce apoptosis in several cancer cell lines. In this ex vivo study, we evaluated the effect of pancratistatin on peripheral blood mononuclear cells obtained from 15 leukemia patients prior to clinical intervention of newly diagnosed patients, as well as others of different ages in relapse and at various disease progression states.

Results: Mononuclear cells from healthy volunteers and leukemia patients were exposed to 1 microM pancratistatin for up to 48 h. Irrespective of leukemia type, pancratistatin induced apoptosis in the leukemic samples, with minimal effects on non-cancerous peripheral blood mononuclear control cells.

Conclusion: Our results show that pancratistatin is an effective and selective anti-cancer agent with potential for advancement to clinical trials.

Figure 1

Detection of apoptosis in cells treated for 24 h with paclitaxel or pancratistatin. Flow cytometry with Annexin-V AlexaFluor 488 was used to detect apoptosis in Jurkat cells (A) and patient-obtained leukemic PBMCs (B) that were untreated, treated with 500 nM paclitaxel or treated with 1 μM pancratistatin for 24 h. The leukemic PBMCs were obtained from a chemo-näive female patient (65 y) diagnosed with AML -- M1. Flipping of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, which binds to Annexin-V in the presence of calcium, is a characteristic feature of apoptosis. A minimum of 20 000 events were measured for each sample on a Beckman Coulter Cytomics FC500 flow cytometer.

Figure 2

Response of clinical leukemia and cultured Jurkat cells to treatment with pancratistatin. The apoptotic effect of 24 h exposure to 1 μM pancratistatin was observed by microscopy using cell-permeable Hoechst dye. Compared to the nuclear morphology of untreated cells (A, B), treatment with pancratistatin resulted in apoptosis characterized by condensed, brightly stained nuclei in Jurkat (E) and patient obtained leukemic PBMCs (F). Annexin-V binding, another characteristic feature of apoptosis, was minimal in untreated cells (C, D) in contrast to the binding observed after pancratistatin treatment (G, H). Hoechst and Annexin-V images are not of the same field.

Figure 3

Non-cancerous peripheral blood mono-nucleated cells (PBMCs) are relatively unaffected by pancratistatin. A) Flow cytometry analysis using Annexin-V AlexaFluor 488 was performed on PBMCs that were untreated or treated with 1 μM pancratistatin for 48 h. A minimum of 20 000 events were measured for each sample on a Beckman Coulter Cytomics FC500 flow cytometer. B) Hoechst staining and Annexin-V binding depicts the selective activity of pancratistatin; there is minimal difference in nuclear morphology (i, ii) and amount of externalized phosphatidylserine (iii, iv) between untreated and treated non-cancerous PBMCs.

Figure 4

Comparative effect of 48 h treatment with pancratistatin. Leukemia samples (n = 10) and non-cancerous PBMCs (n = 8) treated for 48 h with pancratistatin were stained with Hoechst dye and counted manually (a minimum of 5 fields with 100 cells per field); percent apoptosis was calculated per total cell number. Statistical analysis was performed (unpaired t-test, two-tailed p value); † represents p < 0.05 between untreated and pancratistatin treated leukemia samples.