Page 163 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

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Comparative Transcriptome Analysis of Caspase and Apoptosis Pathway of K562 cells treated with butin,
butein, and sulfuretin from the heartwood of Cotinus coggygria Scop.

Ivana Pašić1, Ahmad Sami2, Miroslav Novaković3, Tatjana Stanojkovic1, Ivana Z. Matić 1*

1Institute for Oncology and Radiology of Serbia, Belgrade, Serbia
2Heidelberg University, Department of Radiation Oncology, Heidelberg, Germany
3University of Belgrade – Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, Belgrade,
Serbia

Keywords: Apoptosis

Background: This research examined the anticancer effects of butin, butein, and sulfuretin, which are derived from
the heartwood of Cotinus coggygria, against K562 cells. In K562, the main treatment approach focuses on targeting
BCR-ABL signaling with TKIs. To explore apoptosis induction as an additional therapeutic approach, the study aimed to
determine whether these natural compounds can enhance or restore apoptotic responses in p53-null leukemia cells.
Material and Methods: The study employed flow cytometry to assess the patterns of apoptosis and caspase activation.
RNA-Seq was used to substantiate the results obtained from the cellular assay.
Results: In the caspase inhibitor assays, each compound exhibited a distinct dependency profile (butin and butein
on caspase-3/-8, and sulfuretin primarily on caspase-3), and the RNA-Seq results provided a matching molecular
“fingerprint” for these differences. Notably, butin and sulfuretin both induced transcriptional upregulation of CASP7
and CASP10, whereas butein did not significantly alter these particular caspases. Conversely, butein and sulfuretin
upregulated CASP9 (mitochondrial pathway). Only sulfuretin led to a significant downregulation of CASP8 transcripts,
consistent with its reduced reliance on the extrinsic initiator caspase. All three treatments induced the upregulation of
pro-apoptotic genes, including BAX, BAK1, and APAF1, as well as FADD, TRAIL, and TNFRSF10B, indicating a pro-apoptotic
transcriptional program. Butein, which influenced the highest number of genes (809 DEGs), triggered both the extrinsic
and intrinsic pathways and exhibited the most extensive enrichment of apoptosis-related genes. Butin predominantly
engaged the extrinsic pathway with a strong executioner caspase response, while showing minimal impact on intrinsic
caspase-9, aligning with its caspase-8 dependence. Sulfuretin, with the fewest DEGs (299), still upregulated CASP9
and CASP7 but uniquely repressed CASP8, implying that it may bypass the extrinsic route. Sulfuretin’s transcriptome
showed strong downregulation of anti-apoptotic factors (e.g., BCL-2 family and IAP genes).
Conclusions: All three investigated compounds, flavanone butin, chalcone butein, and aurone sulfuretin, converge on
the induction of apoptosis, as evidenced by both functional assays and GSEA data.

Acknowledgments and funding: The authors are grateful to the Ministry of Science, Technological Development, and
Innovation of the Republic of Serbia for the financial support (Grant numbers: 451-03-136/2025-03/200043, 451-03-
136/2025-03/200026). The authors would like to thank Tatjana Petrović, and Jasna Popović Basić for their excellent
technical assistance.

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DPP3 knockdown in HeLa cells induces G1 arrest

Marina Oskomić1, Lea Barbarić1, Katja Ester2, Ana Tomašić Paić1, Mihaela Matovina1

1Laboratory for Protein Biochemistry and Molecular Modelling, Division for Organic Chemistry and Biochemistry, Ruđer Bošković
Institute, Zagreb, Croatia
2Laboratory of Experimental Therapy, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia

Keywords: DPP3, CDKN1A, Cell Cycle, Flow Cytometry, HeLa cells, RNA Interference

Background: Dipeptidyl peptidase 3 (DPP3) is a cytosolic zinc-dependent metallopeptidase that contributes to the
intracellular degradation of peptides and the regulation of oxidative stress. DPP3 is overexpressed in a variety of cancers
(endometrial, ovarian, lung, breast, colorectal and esophageal) suggesting its possible role in cancer development.

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