Page 167 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

cytotoxic effects of a newly synthesized class of pyrido-dipyrimidine compounds against human cancer cell lines,
aiming to evaluate their therapeutic potential and underlying mechanisms of action.
Material and methods: To evaluate the cytotoxic effects, 25 pyrido-dipyrimidine compounds (C1-C25) were tested on
human cell lines: HeLa (cervical adenocarcinoma), K562 (chronic myeloid leukemia), LS174 (colon adenocarcinoma),
A549 (lung adenocarcinoma), and MRC5 (non-malignant fetal lung fibroblasts). Cells were exposed to graded
concentrations of the compounds and incubated for 24, 48, and 72 hours, followed by assessment of cell survival via
the MTT assay. The most active compounds were selected for further investigation, specifically targeting HeLa and
K562 cell lines. Flow cytometry (propidium iodide staining) and fluorescence microscopy (acridine orange/ethidium
bromide staining) were utilized to analyze cell cycle distribution and morphological changes. Additionally, selective
caspase inhibitors were used to elucidate the apoptotic pathways involved.
Results: Results showed that the compounds exhibited concentration-dependent cytotoxicity. HeLa and K562 cells
demonstrated the highest sensitivity, with C17 and C9 yielding the lowest IC50 values (9.82±1.07 μM and 17.36±1.39
μM, respectively) and displaying high selectivity over MRC5 cells (selectivity coefficients of 12.46 for C17 and 9.20 for
C9). Further analysis revealed that these compounds significantly increased the SubG1 cell cycle phase, suggesting
apoptosis induction. Fluorescence microscopy with AO/EB staining further confirmed characteristic apoptotic
morphological changes. Flow cytometric analysis indicated that apoptosis induced by C17, was inhibited by caspase
inhibitors, implicating caspase-3, -8, and -9 in its apoptotic pathway, while C9 functioned via a caspase-independent
mechanism.
Conclusions: These findings suggest that novel pyrido-dipyrimidine compounds C17 and C9 are potent, selective
cytotoxic agents with distinct apoptotic mechanisms, highlighting their potential as candidates for future cancer
research.

Acknowledgments and funding: This work was supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia Agreements No. 451-03-136/2025-03/200043

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miR-219: A Novel Radiosensitizing Candidate in Glioblastoma Treatment

Maja Petrović1, Simona Lapčević2, Danijela Drakulić2, Tatjana Stanojković1, Danijela Stanisavljević Ninković2

1 Institute for Oncology and Radiology of Serbia, Belgrade, Serbia
2 Institute of Molecular Genetics and Genetic Engineering University of Belgrade, Belgrade, Serbia

Keywords: radiotherapy, GBM, miRNAs, radiosensitization

Background: Glioblastoma multiforme (GBM) is the most prevalent and aggressive form of malignant brain cancer
in adults, with a 5-year overall survival rate of 9.8%. It is characterized by rapid growth and a highly invasive nature.
The standard approach for GBM treatment includes surgical resection followed by radiotherapy combined with
temozolomide therapy. Improving the response of GBM cells to radiotherapy remains one of the major challenges in
clinical oncology, and discovering novel molecules that can enhance radiosensitivity is a promising strategy to overcome
this hurdle. MicroRNAs (miRNAs) regulate key processes in GBM progression and can influence radiosensitivity by
targeting radiation-related pathways, making them promising therapeutic targets. The brain-specific miRNA miR-219 is
already recognized as an important tumor suppressor in GBM. The main aim of this study is to analyze how modulating
miR-219 expression affects the radiosensitivity of GBM cells.
Methods: GBM cell line LN229 was transduced with a miR-219 lentiviral construct for overexpression or with a control
vector. Transduced cells were then seeded into different dishes and, the next day, irradiated with 4 Gy or sham-
irradiated. To analyze the migration potential, a wound scratch assay was performed. The sphere-forming ability was
assessed using a sphere formation assay, including measurements of sphere size and propidium iodide (PI) staining.
Expression of the Vimentin marker, associated with epithelial-to-mesenchymal transition and GBM stem cells, was
analyzed in both 2D and 3D cultures using immunocytochemistry.
Results: LN229 cells overexpressing miR-219 showed a significantly lower migration rate compared to control cells
under both sham-irradiated and irradiated conditions. Spheroids formed by miR-219 overexpressing cells exhibited
a significantly lower growth rate compared to control cells. PI staining confirmed a higher proportion of dead cells in
both sham-irradiated and irradiated conditions, associated with miR-219 overexpression. Also, Vimentin expression
was reduced in cells with miR-219 overexpression in both 2D and 3D models, under sham-irradiated and irradiated

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