The Different Temozolomide Effects on Tumorigenesis Mechanisms of Pediatric Glioblastoma PBT24 and SF8628 Cell Tumor in CAM Model and on Cells In Vitro

Abstract

It is necessary to elucidate the individual effects of temozolomide (TMZ) on carcinogenesis and tumor resistance to chemotherapy mechanisms. The study aimed to investigate the TMZ 50 and 100 μM dose effect difference between PBT24 and SF8628 cell line high-grade pediatric glioblastoma (phGBM) xenografts in a chicken chorioallantoic membrane (CAM) model, on PCNA and EZH2 immunohistochemical expression in the tumor and on the expression of NKCC1, KCC2, E- and N-cadherin genes in TMZ-treated and control cell groups in vitro. TMZ at a 100 μg dose reduced the incidence of PBT24 xenograft invasion into the CAM, CAM thickening and the number of blood vessels in the CAM (p < 0.05), but did not affect the SF8628 tumor in the CAM model. The TMZ impact on PBT24 and SF8628 tumor PCNA expression was similarly significantly effective but did not alter EZH2 expression in the studied tumors. The TMZ at 50 μM caused significantly increased RNA expression of the NKCC1 gene in both studied cell types compared with controls (p < 0.05). The expression of the KCC2 gene was increased in PBT24 TMZ-treated cells (p < 0.05), and no TMZ effect was found in SF8628-treated cells. The study supports the suggestion that individual sensitivity to TMZ should be assessed when starting treatment.

Keywords: CAM; EZH2; KCC2; NKCC1; PCNA; pediatric glioblastoma; temozolomide.

Figure 1

Stereomicroscopy of PBT24 and SF8628 tumors in vivo, a chorioallantoic membrane with tumor ex ovo and the histologic images of the study groups. EDD9, EDD12 and CAM ex ovo scale bar—1 mm; hematoxylin and eosin (H–E) stained preparations‘ scale bar—200 µm.

Figure 2

Fluorescent stereomicroscopy assay with fluorescent dextran of PBT24 and SF8628 tumors. Dextran highlighted the tumor and vascular network around it. Scale bar—1 mm.

Figure 3

PBT24 and SF8628 tumor invasion into CAM frequency in control and TMZ-treated groups.

Figure 4

PCNA and EZH2 positively stained tumors of the PBT24 and SF8628 control and 100 µM TMZ-treated study groups. Dark brown nuclei indicate a PCNA-positive (a) and EZH2-positive cell (b). Scale bar—20 µm.

Figure 5

The percentage of PCNA-positive (a) and EZH2-positive (b) cells in PBT24 and SF8628 tumors.

Figure 6

SLC12A2 (a) and SLC12A5 (b) expression in PBT24 and SF8628 control groups and 50 µM TMZ-treated groups. Data are after normalization with the GAPDH gene. Delta threshold cycle (ΔCT) values were used for the graph (the horizontal bars represent the mean; the short horizontal lines show standard deviation (SD) values). SLC12A2 (c) and SLC12A5 (d) relative expression in PBT24 and SF8628 50 µM TMZ-treated groups. The relative gene expression in TMZ-treated groups compared with respective controls. The 1.0 line shows the starting point of gene expression; * p < 0.05.

Figure 7

Comparison of the mean of ΔCT SLC12A5/ΔCT SLC12A2 ratio value with SD among the PBT24 and SF8628 cell study groups.

Figure 8

CDH1 (a) and CDH2 (b) expression in PBT24 and SF8628 control groups and 50 µM TMZ-treated groups. Data are after normalization with GAPDH. Delta threshold cycle (ΔCT) values were used for the graph (the horizontal bars represent the mean; the short horizontal lines show SD values). CDH1 (c) and CDH2 (d) gene expression in PBT24 and SF8628 50 µM TMZ-treated groups. The relative gene expression in TMZ-treated groups as compared with their control groups. The 1.0 line shows the starting point of gene expression.