Page 160 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

Serbian Association for Cancer Research SDIRSACR

Background: Drug repurposing is a promising strategy in drug development in oncology. A notable example of this
approach is diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) traditionally used for pain relief, which has
shown anticancer potential via COX-dependent and COX-independent mechanisms. In this study, a novel anticancer
compound, a unique carborane derivative of diclofenac with a zwitterionic amidine structure and a nido-carborane
cluster, was developed using fragment-based drug discovery. Designed as an engineered prodrug, it aims to overcome
traditional NSAID limitations and enhance biological effects.
Material and Methods: The anticancer properties of the compound were tested against three cancer cell lines:
murine colon adenocarcinoma (MC38), human colorectal carcinoma (HCT116), and human colorectal adenocarcinoma
(HT29), as well as peritoneal exudate cells (PECs) isolated from healthy mice and on the human fetal lung fibroblast
cell line MRC-5. Furthermore, the effects of the experimental compound were tested on colon cancer mouse-derived
organoids. Cell viability was measured using MTT and CV assays on cell lines and the CellTiter-Glo® assay on organoids.
The mechanism of action of the substance on the MC38 cell culture was evaluated by flow cytometry and fluorescence
microscopy.
Results and Conclusions: The tested compound showed strong cytotoxicity on all cancer lines after 72 h, with IC50
values for PECs and MRC-5 being 2–3 times higher, indicating moderate selectivity towards malignant cells. Treatment
also decreased the viability of organoids. Flow cytometry analysis revealed that the cytotoxicity of the substance is
based on caspase-independent apoptosis preceded by cell division blockage. Fluorescence microscopy after PI staining
confirmed apoptosis, cells with possibly undivided genetic material, and senescence cells. Acridine orange staining
showed increased presence of autophagosomes, but co-treatment with autophagy inhibitors further reduced viability,
suggesting a pro-survival role of autophagy. The production of reactive oxygen and nitrogen species (ROS/RNS) in
the presence of the tested compound was strongly reduced, and it was in concordance with the efficient inhibition
of COX-2. Our findings suggest that carborane-based prodrugs offer a promising route for developing new anticancer
therapies, especially as adjuvant or combination therapies. The presented results open valuable possibilities for further
research.

Acknowledgments and funding: We thank the Ministry of Science, Technological Development, and Innovation of the
Republic of Serbia (No. 451-03-66/2024-03/200007) and the Science Fund of the Republic of Serbia, Prisma Program,
GRANT No. 5456, for financial support. Funding by the Deutsche Forschungsgemeinschaft (DFG) (SA 2902/2-1 (M.B.S.);
PI 304/7-1 (M.L., J.S., and J.P.); HE 1376/54-1 (E.H-H.)) is gratefully acknowledged. Partial support by the DFG (SFB/TRR
205/2 (No. 314061271; J.P.)) is also acknowledged.

P71

Novel aroylacrylic acid phenylamides revealed strong prooxidative activity and consequent cell death
induction in A549 (KRASmut) and Mcf7 (KRASwt) cancer cell lines

Snežana K. Bjelogrlić , Tamara Vujatović-Velimirov , Jelena Grahovac , Maja D. Vitorović-Todorović 2
2
1
1
1Institute for Oncology and Radiology of Serbia, Belgrade, Serbia
2Military Technical Institute, Belgrade, Serbia

Keywords: aroylacrylic acid phenylamides, reactive oxygen species, mitochondria, KRAS

Background: This study was organized to unravel the mechanism responsible for induction of cell death by treatment
with aroylacrylic acid phenylamides. Four novel structural analogues were designed (1a-1d) by means of Michaels’
adduction of seconadary cyclic amines to aroylacrylic acid phenylamides. A549 and Mcf7 cell lines were used as being
KRASmut and KRASwt, respectively. The difference in their KRAS status implies that A549 and Mcf7 cells utilize disparate
metabolic strategies to produce energy, and exhibit different responses to applied treatments.
Material and Methods: Cell death was determined by Annexin V/propidium iodide (PI) staining, mitochondrial
superoxide anions (MtSR) were detected by MitoSOX Red, total reactive oxygen species (ROS) by DCFDA. Morphological
integrity of mitochondria and mitochondrial network was determined by MitoTracker CMX Ros (MTR) staining. Analyses
were performed by flow cytometry and fluorescent microscopy.
Results: Results of the Annexin V/PI staining after 24 h of treatment showed that Mcf7 cells were more sensitive
than A549 cells. Cell death response was reversed by co-incubation with N-acetylcysteine (NAC) in both cell lines
confirming that ROS generation was the mechanism of compounds’ activity. At lower time-frame (6 h), significantly
higher magnitude of cumulative MtSR production was found only in Mcf7 cells treated with 1a, whereas dimmed

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