Selective inhibition of human leukemia cell growth and induction of cell cycle arrest and apoptosis by pseudolaric acid B

Abstract

Purpose: The leukemias account for the largest number of cases of childhood cancer and remain the primary cause of cancer-related mortality among children in the United States. There is a need for novel antileukemia agents due to toxicity and resistant to existing chemotherapeutic agents. In this study, the effects of pseudolaric acid B (PAB) on three human leukemia cell lines, acute promyelocytic leukemia HL-60 cells, acute lymphoblastic leukemia CCRF-CEM cells, and human chronic myeloid leukemia blast-phase K562 cells were investigated in vitro, compared to normal human peripheral blood mononuclear cells (PBMC).

Methods: Cell viability was determined using CellTiter-Glo luminescent reagent. Colony formation was assessed by Microtitration cloning assay. Cell cycle analysis was carried out by flow cytometry. Tubulin polymerization was measured by recording the increase in absorbance. Inhibition of topoisomerase I (topo I) and topoisomerase II (topo II) enzyme activities was measured by DNA relaxation assay using topo I and II drug screening kit. Apoptosis was observed by DAPI staining assay and Caspase3/7 activities was measured using Caspase-Glo((R)) 3/7 assay kit.

Results: Pseudolaric acid B selectively inhibited the growth of human leukemia HL-60, CCRF-CEM and K562 cells, but not normal PBMC. PAB suppressed colony formation in HL-60 cells. Cell cycle analysis showed that PAB blocked the cell cycle at G(2)/M phase in HL-60 cells, suggesting that it suppresses mitosis. DNA topo I and topo II were not inhibited, but tubulin polymerization was inhibited. PAB-induced apoptosis and activated caspase-3/7 activity.

Conclusions: This study indicates that PAB has a potential for use against leukemia and its effects might be mediated by inhibiting tubulin polymerization, preventing cell division and activating caspase-3, which leads to apoptosis.

Fig. 1

The chemical structures of PAA and PAB

Fig. 2

Effects of PAB on the growths of human leukemia HL-60, CCRF-CEM and K562 cells, and normal PBMC. Cells were treated with 1 μM PAB or vehicle for 48 h and cell viability was determined by CellTiter-Glo^® luminescent cell viability assay, the results shown were mean ± SD (bars) of three experiments

Fig. 3

Effect of PAB on colony formation of HL-60 cells, after exposure for 2, 6 and 24 h, as determined by the microtitration cloning assay. Results are mean ± SD (bars) from three experiments

Fig. 4

Flow cytometry histograms of cell cycle distribution of HL-60 cells of untreated control and of PAB treated group at 1 μM. The time course of the cell cycle progress showed that HL-60 cells were treated with PAB for 6–48 h, progressive reduction of cells in G₀/G₁ phase occurred with a concomitant increase of cells arrested in the G₂/M phase and which were unable to proceed through mitosis to the next G₁ phase, instead accumulating in the G₂/M compartment. The proportion of cells in the S phase was comparatively unchanged by PAB treatment

Fig. 5

Inhibition of tubulin polymerization of bovine tubulin in vitro by PAB. In vitro polymerization of purified bovine brain tubulin in the presence of pseudolaric acid B was assessed as described in “Materials and methods”. Results are plotted as the extent of tubulin polymerization (A _340 nm) as function of time

Fig. 6

Induction of apoptosis by PAB in HL-60 cells. Cells were treated with vehicle (a) or with 1 μM PAB for 24 h (b). DAPI staining was conducted as described in “Materials and methods” and nuclear morphology was examined by using a fluorescent microscope. Exposure to 1 μM PAB for 24-h-induced apoptotic changes such as condensed chromatin, and fragmented nuclei (×1,000). Arrow indicates apoptotic cells with condensed and fragmented nuclei

Fig. 7

Time course of caspase-3 activation after treatment with PAB. HL-60 cells were treated with or without PAB (1 μM) for 2, 6, 12, 24, and 48 h. Enzymatic activity of caspase-3 were determined by adding 50 μL of caspase reagents. The activity of caspase-3 is expressed as the value relative to untreated cells. The results shown are mean ± SD (bars) of triplicate experiments. *P < 0.05; **P < 0.01