Mesenchymal stem cells promote caspase-3 expression of SH-SY5Y neuroblastoma cells via reducing telomerase activity and telomere length

Abstract

Objectives: The use of mesenchymal stem cells in malignancies has attracted much attention due to their ability to deliver anticancer agents to tumors, including cytokines, chemokines, etc. This study aimed to investigate the effect of MSCs on the neuroblastoma SH-SY5Y cells through proliferation/apoptosis, senescence assessment, telomere length, and telomerase activity in vitro. BAX and BCL2 were also examined as potential signaling pathways in this process.

Materials and methods: For this reason, two cell populations (MSCs and SH-SY5Y cells) were co-cultured on trans-well plates for 7 days. In a subsequent step, SH-SY5Y cells were harvested from both control and experimental groups and subjected to flow cytometry, ELISA, real-time PCR, PCR-ELISA TRAP assay, and Western blotting assay for Ki67/Caspase3 investigation, β-Galactosidase assessment, telomere length, and telomerase activity assay. Also, expression of genes and proteins through real-time PCR and Western blotting demonstrated the involvement of the aforementioned signaling pathways in this process.

Results: It was found that MSCs contributed significantly to decrease and increase of Ki-67 and Caspase-3, respectively. Also, MSCs dramatically reduced the length of telomere and telomerase activity and increased the β-Galactosidase activity in a significant manner. In addition, significant increase and decrease were also seen in BAX and BCL2 gene and protein expressions, respectively.

Conclusion: These findings revealed that close interaction between MSCs and neuroblastoma cells causes inhibition of the SH-SY5Y cell proliferation and promotes cell senescence via BAX and caspase-3 cascade pathways.

Keywords: Caspase3; Mesenchymal stem cells; Neuroblastoma; Signaling pathways; Telomerase; Telomere.

Figure 1

Expression of adipose tissue derived-MSC cell surface markers by flow cytometry. During this experiment, each antibody was examined separately, and isotope controls served as negative controls; adipose-derived MSCs were positively characterized by CD73 (94%) and CD44 (80%), while CD31 (0.2%) and CD34 (0.04%) were negative. Isotype controls of CD31 and CD34 are depicted as blue diagrams

Figure 2

Co-culturing MSCs and SH-SY5Y cells was analyzed with Annexin-FITC and PI flow cytometric method. The analysis was conducted on two distinct populations of cells marked as: negative cells indicating live cell population, (A) Annexin-V positive cells, showing early apoptosis; (B) PI-positive cells, showing late apoptosis

Figure 3

Proliferation assay of SH-SY5Y cells and SH-SY5Y cells co-cultured with MSCs. Flow cytometry was used to analyze the harvested cells for Ki-67 (A-E) and caspase-3 expression assay (F-J). In this Figure, A and F represent selected cell populations, B and G are isotype controls, C and H are SH-SY5Y cells, finally, D and I show co-cultured SH-SY5Y with MSCs cells. Data is represented as mean ± SD of independent experiments; (*P<0.05, n=3)

Figure 4

aTL measurement of SH-SY5Y cells following co-culture with MSCs. (A) shows the standard curve for calculating genome copies using the 36B4 copy number, and (B) shows the standard curve for calculating the length of telomere sequence per reaction tube (the X-axis represents the number of the cycle and the Y-axis shows the standard’s concentration). (C) For both control and experimental groups, real-time PCR analysis was performed in triplicate to evaluate telomere length. The data were analyzed as kb/reaction and the genome copies/reaction for the telomere and the SCG. Values are mean ± SD from independent experiments (**P<0.01, n=3); (D) Relative telomerase activity measurement of SH-SY5Y cells following co-culture with MSCs. From each group of cells (control and experimental), 1×10⁶ cells were collected per well. In the following step, protein was extracted and PCR-ELISA TRAP assay was carried out in triplicate using the method detailed in the materials and methods section. Values are mean ± SD from independent experiments ;(*P<0.05, n=3)

Figure 5

β-GAL activity assay of SH-SY5Y cells and SH-SY5Y cells co-cultured with MSCs

Figure 6

Effect of MSCs on apoptosis-related gene and protein expression. (A and B) BAX and BCL-2 protein expression (C) BAX/BCL2 ratio, (D) Relative level of mRNA expression of Bax and BCL2. Data are expressed as mean ± SD from three independent experiments ;(*P<0.05, **P<0.01, ***P<0.001, n=3)