Page 164 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

Serbian Association for Cancer Research SDIRSACR

Because of the interplay between stress signaling and cell cycle checkpoints, the objective of our research was to
investigate DPP3 knockdown effects on cell cycle regulatory pathways in cervical carcinoma cells, so we analysed cell
cycle distribution and the expression of CDKN1A (p21), a cyclin-dependent kinase inhibitor, that is related to cell cycle
arrest and is typically elevated due to cellular stress, after DPP3 knock-down in HeLa cells.
Material and Methods: HeLa cells were subjected to DPP3 depletion by siRNA-mediated knockdown. At 48 hours post-
transfection, cells were harvested for protein isolation and subjected to SDS-PAGE and western blot analysis to assess
knockdown efficiency and p21 protein levels, alongside mRNA quantification by qPCR. For cell cycle analysis, cells were
ethanol fixed, treated with RNase, and stained with propidium iodide. DNA content was analyzed by flow cytometry to
determine cell cycle phase distribution.
Results: DPP3 silencing in HeLa cells resulted in effective knockdown as confirmed by western blot, accompanied
by an increase in CDKN1 (p21) expression at both mRNA and protein levels. Flow cytometry analysis demonstrated
significant G1 phase arrest, indicating an inhibitory effect on cell cycle progression.
Conclusions: Our results indicated that DPP3 knockdown results in G1 phase arrest in HeLa cells by increasing p21
expression, supporting DPP3 as a potential negative regulator of the cell cycle, in relation to a p21-mediated pathways,
and a potential therapeutic target for future cancer therapies.

P76

Sirtuin 3 enhances stress sensitivity and cell cycle disruption in triple-negative breast cancer

Kate Šešelja1, Marija Pinterić1, Iva I. Podgorski1, Marijana Popović Hadžija1, Vedrana Filić1, Ivan Ciganek1, Denis Plese1,
Dora Marčinko1, Tihomir Balog1, Sandra Sobočanec1

1Ruđer Bošković Institute, Zagreb, Croatia

Keywords: Sirtuin 3, MDA-MB-231, breast cancer, DNA damage, cell cycle

Background: Triple-negative breast cancer (TNBC), an aggressive subtype lacking estrogen receptor, progesterone
receptor, and HER2 expression, is associated with high recurrence rates and limited targeted treatment options. Sirtuin
3 (Sirt3) is a mitochondrial NAD⁺-dependent deacetylase that plays a central role in regulating cellular metabolism,
oxidative stress, and mitochondrial function. In breast cancer, Sirt3 has been suggested to have a potential dual
role, acting either as a tumor suppressor or as a promoter of cancer progression. We previously demonstrated
that overexpression of Sirt3 diminishes tumorigenic properties in the TNBC cell line MDA-MB-231, supporting its
potential tumor-suppressive function in this context. In the present study, we further investigated the effects of Sirt3
overexpression in MDA-MB-231 cells under normal and hyperoxic conditions.
Materials and methods: We use MDA-MB-231 breast cancer cells transfected with the FLAG-tagged Sirt3 (MDA-S3)
or empty pcDNA3.1 plasmid (MDA-C). Upon hyperoxic treatment, cells were analyzed for metabolic activity using
MTT assay. Apoptosis and cell cycle distribution are quantified via flow cytometry. Cell morphology and subcellular
localization of proteins of interest, as well as DNA damage were analyzed using confocal microscopy. Western blot was
used to analyze or confirm protein expression.
Results: Our findings reveal that Sirt3 increases metabolic activity in MDA-MB-231 cells while simultaneously promoting
apoptosis and DNA damage. Notably, exposure to hyperoxic conditions further amplified these effects, resulting in
enhanced apoptosis, higher levels of DNA damage, and cell cycle disruption characterized by S-phase arrest. These
results indicate that, despite promoting metabolic activity, Sirt3 sensitizes TNBC cells to hyperoxic stress, ultimately
enhancing cell death.
Conclusions: Taken together, these data expand on previous observations by demonstrating that Sirt3 not only
diminishes tumorigenic potential but also modulates stress susceptibility, genome stability, and cell cycle progression in
TNBC cells. This supports the hypothesis that Sirt3 functions as a tumor suppressor in TNBC and highlights its potential
as a target for therapeutic strategies aimed at increasing vulnerability of cancer cells to metabolic and oxidative stress.

149