The Comparative Experimental Study of Sodium and Magnesium Dichloroacetate Effects on Pediatric PBT24 and SF8628 Cell Glioblastoma Tumors Using a Chicken Embryo Chorioallantoic Membrane Model and on Cells In Vitro

Abstract

In this study, pyruvate dehydrogenase kinase-1 inhibition with dichloroacetate (DCA) was explored as an alternative cancer therapy. The study's aim was to compare the effectiveness of NaDCA and MgDCA on pediatric glioblastoma PBT24 and SF8628 tumors and cells. The treatment effects were evaluated on xenografts growth on a chicken embryo chorioallantoic membrane. The PCNA, EZH2, p53, survivin expression in tumor, and the SLC12A2, SLC12A5, SLC5A8, CDH1, and CDH2 expression in cells were studied. The tumor groups were: control, cells treated with 10 mM and 5 mM of NaDCA, and 5 mM and 2.5 mM of MgDCA. The cells were also treated with 3 mM DCA. Both the 10 mM DCA preparations significantly reduced PBT24 and SF8624 tumor invasion rates, while 5 mM NaDCA reduced it only in the SF8628 tumors. The 5 mM MgDCA inhibited tumor-associated neoangiogenesis in PBT24; both doses of NaDCA inhibited tumor-associated neoangiogenesis in SF8628. The 10 mM DCA inhibited the expression of markers tested in PBT24 and SF8628 tumors, but the 5 mM DCA affect on their expression depended on the cation. The DCA treatment did not affect the SLC12A2, SLC12A5, and SLC5A8 expression in cells but increased CDH1 expression in SF8628. The tumor response to DCA at different doses indicated that a contrast between NaDCA and MgDCA effectiveness reflects the differences in the tested cells' biologies.

Keywords: CAM model; PBT24; SF8628; dichloroacetate; gene expression; pediatric glioblastoma; sex.

Figure 1

Stereomicroscopic and histologic images of PBT24 and SF8628 tumors in vivo on the CAM, the tumor ex ovo, and the H–E stained tumors histologically. EDD9, EDD12, and CAM ex ovo scale bar is 1 mm; H–E preparations’ scale bar is 200 µm.

Figure 2

Fluorescent stereomicroscopic images of PBT24 and SF8628 tumors (fluorescent dextran highlights the tumor and surrounding blood vessels). Scale bar is 1 mm.

Figure 3

The invasion into CAM frequency in the PBT24 and SF8628 tumor groups.

Figure 4

Immunohistochemical PCNA expression of PBT24 and SF8628 in control and DCA-treated tumor groups. (A) Dark brown nuclei indicate PCNA-positive cells (red arrows). The magnification is 40×, scale bar is 20 µm. (B) Percentage of PCNA-positive cells in the tissue of PBT24 and SF8628 tumors.

Figure 5

Immunohistochemical EZH2 expression in control and treated tumor groups. (A) Dark brown nuclei indicate EZH2-positive cells (red arrows). The magnification is 40×, scale bar is 20 µm. (B) The percentage of EZH2-positive cells in PBT24 and SF8628 tumors.

Figure 6

Immunohistochemical expression of p53 in control and DCA-treated tumor groups. (A) Dark brown nuclei indicate p53-positive cells (red arrows). The magnification is 40×, scale bar is 20 µm. (B) The percentage of p53-positive cells in PBT24 and SF8628 tumors.

Figure 7

Immunohistochemical expression of survivin in control and DCA-treated PBT24 and SF8628 tumors. (A) Dark brown nuclei indicate survivin-positive cells (red arrows). The magnification is 40×, scale bar is 20 µm. (B) The percentage of survivin-positive cells in PBT24 and SF8628 tumors.

Figure 8

CDH1 expression in PBT24 and SF8628 control and dichloroacetate-treated cell groups. (A) The graph shows the ΔCT value, it’s mean (long horizontal bar), and SD value (short horizontal bars). (B) Relative CDH1 expression of PBT24 and SF8628 cell groups treated with dichloroacetate preparations. CDH1 expression of the treated group was compared with the corresponding control. The 1.0 line denotes the reference value of gene expression; * p < 0.05.