Dipeptidyl Peptidase 3 Knockdown in HeLa Cells Induces G0/G1 Cell Cycle Arrest Associated with Upregulation of p21 Protein
Lea Barbarić1, Marina Oskomić1, Anđela Horvat2, Katja Ester2, Nikolina Stojanović3, Ana Tomašić Paić1, Mihaela Matovina1,*
1 Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia
2 Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia
3 Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia
* Corresponding Author: Mihaela Matovina. Email: 
BIOCELL https://doi.org/10.32604/biocell.2026.080282
Received 06 February 2026; Accepted 27 April 2026; Published online 14 May 2026
Abstract
Objectives: Dipeptidyl peptidase 3 (DPP3) is a zinc metallopeptidase involved in peptide turnover and possibly in blood pressure and pain regulation. It also modulates oxidative stress response via the Kelch-like ECH-associated protein 1–nuclear factor erythroid 2-related factor 2 (KEAP1–NRF2) pathway. Although frequently upregulated in cancer, its role in carcinogenesis remains unclear. This study examined the effects of DPP3 knockdown (KD) and overexpression on migration, proliferation, and KEAP1–NRF2 pathway regulation in HeLa cells.
Methods: We assessed the effects of DPP3-KD and overexpression on HeLa cell migration using a wound healing assay, and on NRF2 activity by measuring mRNA levels of NRF2, NQO1, and CDKN1A (p21). Protein expression related to the regulation of the KEAP1–NRF2 pathway and cell proliferation was analyzed by western blot, and cell cycle distribution after DPP3-KD was analyzed by propidium iodide staining and subsequent analysis by flow cytometry.
Results: We found that DPP3-KD in HeLa cells has no significant effect on the regulation of the KEAP1–NRF2 pathway in the basal conditions; however, it decreases cell migration (*
p = 0.0123) and induces G0/G1 arrest (**
p = 0.0014) associated with upregulation of p21 on both mRNA (**
p = 0.0065) and protein levels (**
p = 0.0087). Protein levels of cyclin A and cyclin E1 were decreased (*
p = 0.037 and **
p = 0.0011, respectively) in DPP3-KD, consistent with its effect on cell cycle distribution.
Conclusion: Our results indicate that reduced proliferation of DPP3-KD HeLa cells is associated with upregulation of p21 and downregulation of cyclins A and E1. Investigation of the potential causal relationship between DPP3-KD and p21 upregulation was beyond the scope of this study.
Keywords
Dipeptidyl peptidase 3; cyclin-dependent kinase inhibitor 1; kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 signaling; cell migration; G0/G1 arrest
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