Induction of Apoptosis in Cancer Cells of pre-B ALL Patients after Exposure to Platelets, Platelet-Derived Microparticles and Soluble CD40 Ligand

Abstract

Objectives: The in vitro treatment of tumor cells with platelet (Plt) causes inhibition of tumor cell growth, although mechanism of this effect is not clear yet. Induction of apoptosis has been proposed as a mechanism of Plt effects on tumor cells. The purpose of this study was to clarify the role of Plts and Plt-derived components in the induction of apoptosis in the blood mononuclear cells of patients with leukemia.

Materials and methods: In this experimental study, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of five patients with childhood B-precursor acute lymphoblastic leukemia (pre-B ALL) and encountered with Plts, Plt-derived microparticles (Plt-MPs) as well as purified soluble CD40L (sCD40L). After 48 hours of co-culture, the anti-cancer activity of the aforementioned factors was surveyed using examination of apoptosis markers of the cells including active caspase-3 and CD95 using ELISA and flow cytometer techniques, respectively. Additionally, staining of the cells with 7-Aminoactinomycin D (7-AAD) was evaluated by flow cytometer technique. Trypan blue exclusion test and WST-1 method were also used to compare the death/survival status of the cells.

Results: Levels of CD95 and caspase-3 were significantly increased in the all treated groups (P<0.05). On the other hand, trypan blue, 7-AAD and WST-1 methods showed significantly lower number of the live cells in the treated groups (P<0.05).

Conclusions: This study can show the ability of Plts, Plt-MPs and sCD40L for the induction of apoptosis in PBMCs of pre-B-ALL patients. Further studies are necessary to elucidate the different effects of platelets on cancer cells in vitro and in vivo.

Keywords: Acute Lymphoblastic Leukemia; Apoptosis; CD40 Ligand; Microparticles; Platelet.

Fig.1

Flow cytometer plot and size distribution of platelet microparticles. Comparison of the size of fluorescent microspheres (1 µm microbeads) and unknown sized microparticles. Images represent, A. Gating of the microbeads (R1) and microparticles (R2), B. Diagram for the size range of microbeads, and C. Diagram for the size range of microparticles. The figure shows a smaller size and more heterogeneity in the size for platelet microparticles compared to 1 µm microbeads.

Fig.2

Soluble CD40L was purified from platelet concentrate by affinity chromatography using anti-human CD40L column. The specificity of CD40L was confirmed by western blotting using specific monoclonal antibody to CD40L. The results are represented for the purified samples. 1, 2, 3 and 4 referred to different lots of purified soluble CD40L.

Fig.3

Flow cytometer plot for 7-AAD staining. The gating and percentage of 7-AAD staining for peripheral blood mononuclear cells (PBMCs) of pre-B acute lymphoblastic leukemia patients after co-culture with: A. Plt, B. Thrombin-activated platelets (aPlt), C. Platelet-derived MPs, D. sCD40L, E. Control cells (PBMCs alone), and F. unstained PBMCs. *; Percentage of the dead cells after the co-culture time and MPs; Microparticles.

Fig.4

PBMCs of pre-B acute lymphoblastic leukemia patients were treated with each of the following factors: Plt, aPlt, platelet-derived MPs and sCD40L. Higher expression of CD95 was observed in all of the treated groups, compared to the control group. Data are presented as the mean ± SD of five independent experiments (*; P<0.05). PBMCs; Peripheral blood mononuclear cells, aPlt; Thrombinactivated platelets, and MPs; Microparticles.

Fig.5

Active caspase-3 levels in PBMCs of pre-B acute lymphoblastic leukemia patients after the co-culture time. Higher concentrations of caspase-3 are shown due to the vicinity of PBMCs with each of the following factors: Plt, aPlt, platelet-derived MPs and sCD40L. Higher expression of active caspase-3 was observed in all the treated groups, compared to the control group. Data are presented as the mean ± SD of five independent experiments (*; P<0.05). PBMCs; Peripheral blood mononuclear cells, aPlt; Thrombinactivated platelets, and MPs; Microparticles.

Fig.6

WST-1 results. Enzymatic cleavage of WST-1 to formazan by cellular mitochondrial dehydrogenases was measured by reading absorbance at the wavelength of 450 nm. Exposure to Plts, platelet-derived MPs or sCD40L caused lower viability and metabolic activity of the PBMCs of pre-B acute lymphoblastic leukemia patients. Lower survival was determined in all of the treated groups, compared to the control group. Data are presented as the mean ± SD of five independent experiments (*; P<0.05). PBMCs; Peripheral blood mononuclear cells, aPlt; Thrombin activated platelets, and MPs; Microparticles.