Human Mesenchymal Stem Cells-mediated Transcriptomic Regulation of Leukemic Cells in Delivering Anti-tumorigenic Effects

Abstract

Treatment of leukemia has become much difficult because of resistance to the existing anticancer therapies. This has thus expedited the search for alternativ therapies, and one of these is the exploitation of mesenchymal stem cells (MSCs) towards control of tumor cells. The present study investigated the effect of human umbilical cord-derived MSCs (UC-MSCs) on the proliferation of leukemic cells and gauged the transcriptomic modulation and the signaling pathways potentially affected by UC-MSCs. The inhibition of growth of leukemic tumor cell lines was assessed by proliferation assays, apoptosis and cell cycle analysis. BV173 and HL-60 cells were further analyzed using microarray gene expression profiling. The microarray results were validated by RT-qPCR and western blot assay for the corresponding expression of genes and proteins. The UC-MSCs attenuated leukemic cell viability and proliferation in a dose-dependent manner without inducing apoptosis. Cell cycle analysis revealed that the growth of tumor cells was arrested at the G₀/G₁ phase. The microarray results identified that HL-60 and BV173 share 35 differentially expressed genes (DEGs) (same expression direction) in the presence of UC-MSCs. In silico analysis of these selected DEGs indicated a significant influence in the cell cycle and cell cycle-related biological processes and signaling pathways. Among these, the expression of DBF4, MDM2, CCNE2, CDK6, CDKN1A, and CDKN2A was implicated in six different signaling pathways that play a pivotal role in the anti-tumorigenic activity exerted by UC-MSCs. The UC-MSCs perturbate the cell cycle process of leukemic cells via dysregulation of tumor suppressor and oncogene expression.

Keywords: cell cycle; gene expression profiling; leukemia; mesenchymal stem cells.

Figure 1.

Umbilical cord-derived MSCs inhibit the proliferation of leukemic tumor cells through cell-to-cell contact. Tumor cells at 0.5 × 10⁵ cells/well of 96-well plates were cultured in the presence or absence of UC-MSC at various ratios for 3 days. Cell viability and proliferation were assessed by MTS assay (Panel a) and ³H-TdR incorporation (Panel b) during the final 4 and 18 h of culture, respectively. The results represent the mean of at least four experiments ±SD. Panel (c) shows the tumor cells were cultured alone (Tumor cell), in direct contact with UC-MSC (Tumor+UC-MSCs), with UC-MSCs separated by Transwell (Transwell+Tumor) and in supernatant aspirated from UC-MSCs culture (SN+Tumor). Data show results from more than three independent experiments with mean ±SD. [*] statistically significant at p<0.05.

Figure 2.

Anti-proliferative effect of UC-MSCs surpasses apoptosis and prevent the tumor cells from entering into S or G₂/M phase of the cell cycle. One million leukemic tumor cells were co-cultured in presence or absence of UC-MSC at a 1:01 ratio for 24, 48 and 72 h. After incubation, cells were analyzed for apoptotic and necrotic markers, Annexin V and PI, respectively, using by flow cytometry. Data showed results from three different experiments ±SD (Panel a). For cell cycle analysis, 10⁶ cells/ml of leukemic tumor cells were co-cultured with or without of UC-MSC in 12-well plates. At day 3 the cells were collected and stained with PI and assessed by flow cytometry (Panel b).

Figure 3.

Protein expression validation of selected genes after co-culture with UC-MSCs. One million leukemic tumor cells were culture in presence or absence of UC-MSC at 1:01 ratio for 72 h. After 3 days cell were lysed and proteins were loaded onto SDS-PAGE gel and separating by electrophoresis. Then proteins were transferred onto PVDF membrane and incubated with primary antibodies and then secondary antibody for overnight and 1 h, respectively. All western blots were performed with two independent biological samples and repeated at least thrice. The representative data are shown.