Influence of Human Bone Marrow Mesenchymal Stem Cells Secretome from Acute Myeloid Leukemia Patients on the Proliferation and Death of K562 and K562-Lucena Leukemia Cell Lineages

Abstract

Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.

Keywords: ABC transporters; bone marrow; cell death; cytokines; leukemia; mesenchymal stem cells; secretome.

Figure 1

Total cell ratio (A), viability (B), necrosis (C), apoptosis (D) and caspase 3/7 activity (E) in K562 and Lucena cells after transwell culture by 48 h with bone marrow-derived mesenchymal stem cells from patients with acute myeloid leukemia (MSC-AML) or from healthy donors (MSC-H). Data are expressed as mean ± SEM from at least three independent experiments in duplicate. (*) p values ≤ 0.05.

Figure 2

Cell cycle phases of K562 (A) and Lucena (B) cells after coculturing with mesenchymal stem cells from acute myeloid leukemia patients (MSC-AML) or mesenchymal stem cells from healthy donors (MSC-H) for 48 h. Data are expressed as mean ± SEM from 3 independent experiments in duplicate. Significant p values were considered as ≤0.050. Different symbols (*, # or $) in same cell cycle phases indicate statistical significancy.

Figure 3

Gene expression of cyclin D1 (CCND1) and cyclin D2 (CCND2) in K562 and Lucena cells after transwell culture by 48 h with bone marrow-derived mesenchymal stem cells from patients with acute myeloid leukemia (MSC-AML) or from healthy donors (MSC-H). (A) K562 gene expression of CCND1; (B) Lucena gene expression of CCND1; (C) K562 gene expression of CCND2; (D) Lucena gene expression of CCND2. Data are expressed as mean ± SEM from 3 independent experiments in duplicate. (*) p ≤ 0.05.

Figure 4

ABC transporters gene expression of Lucena cells transwell cultured with mesenchymal stem cells from healthy donors (MSC-H) compared with Lucena cells transwell cultured with mesenchymal stem cells from acute myeloid leukemia patients (MSC-AML). Results were obtained from a pool of cDNA samples from 3 independent experiments. Data are expressed as mean ± SEM. Dotted lines are the cutline to overexpression (RQ Log = 0.30) or inhibition (RQ Log = −0.30) of ABC transporter gene expressions. RQ: relative quantification. Dotted lines represent the significance limits of RQ (relative quantification) changed by at least two-fold: more than 2 (RQ log = 0.30) or less then 0,5 (RQ log = −0.30).