Abstract
Intensive adjuvant radiotherapy (RT) is a standard treatment for glioblastoma multiforme (GBM) patients; however, its effect on the normal brain tissue remains unclear. Here, we investigated the short-term effects of multiple irradiation on the cellular and extracellular glycosylated components of normal brain tissue and their functional significance. Triple irradiation (7 Gy*3 days) of C57Bl/6 mouse brain inhibited the viability, proliferation and biosynthetic activity of normal glial cells, resulting in a fast brain-zone-dependent deregulation of the expression of proteoglycans (PGs) (decorin, biglycan, versican, brevican and CD44). Complex time-point-specific (24–72 h) changes in decorin and brevican protein and chondroitin sulfate (CS) and heparan sulfate (HS) content suggested deterioration of the PGs glycosylation in irradiated brain tissue, while the transcriptional activity of HS-biosynthetic system remained unchanged. The primary glial cultures and organotypic slices from triple-irradiated brain tissue were more susceptible to GBM U87 cells’ adhesion and proliferation in co-culture systems in vitro and ex vivo. In summary, multiple irradiation affects glycosylated components of normal brain extracellular matrix (ECM) through inhibition of the functional activity of normal glial cells. The changed content and pattern of PGs and GAGs in irradiated brain tissues are accompanied by the increased adhesion and proliferation of GBM cells, suggesting a novel molecular mechanism of negative side-effects of anti-GBM radiotherapy.
Highlights
glioblastoma multiforme (GBM) remains one of the most aggressive and deadly malignant tumors [1], mainly due to its relapse, which develops in 7–9 months after the first surgery [2] and results in disease progression and patient death
The obtained results regarding the negative effects of multiple irradiation on PGs’ expression and GAGs’ composition in normal mouse brain tissue support and extend our previous data on the ability of even a single 7 Gy irradiation to interfere in the transcriptional activity of some PG-coding genes [31]
This dose was chosen from the literature data and validated further in terms of the absence of evident effects on brain tissue morphology in a single-irradiation study
Summary
GBM remains one of the most aggressive and deadly malignant tumors [1], mainly due to its relapse, which develops in 7–9 months after the first surgery [2] and results in disease progression and patient death. The overall transcriptional activity of PG core protein-coding genes in primary glial cells was significantly down-regulated (Figure 6F), mainly due to the decreased expression of predominant brain tissue PGs—syndecan-1 (-7.9-fold), perlecan (-2.8-fold), decorin (-6.5-fold), biglycan (-3.6-fold) and CD44 (-1.4-fold) (Figure 6E). In spite of the decrease in mRNA levels for key brain PGs in irradiated brain tissue (Figure 5E,F), the protein content of decorin and brevican in primary glial cells was increased (+1,5-fold) but the CS content was decreased 1,5-fold, which presupposes a deterioration of the PG core proteins’ glycosylation in brain tissue upon irradiation (Figure 6C,D) In this experiment, two variables (irradiation and presence of U87 GBM cells) developed a complex effect on PG content—decorin expression was increased upon irradiation and unchanged upon the addition of U87 cells, but the combined impact was a significant (5-8-fold) decrease in decorin content. The obtained results demonstrate a significant increase in the ability of U87 GBM cells to adhere and proliferate on the primary glial cultures obtained in vitro from the triple-irradiated mouse brain compared to the control ones. ANOVA and post-hoc Fisher’s LSD test, * p < 0.05, ** p < 0.01
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