Citation: Zani, A.P.; Zani, C.P.; Din, Z.U.; Rodrigues-Filho, E.; Ueda-Nakamura, T.; Garcia, F.P.; de Oliveira Silva, S.; Nakamura, C.V. Dibenzylideneacetone Induces Apoptosis in Cervical Cancer Cells through Ros-Mediated Mitochondrial Damage. Antioxidants 2023, 12, 317. https://doi.org/10.3390/ antiox12020317 Academic Editors: Vito Pesce and Angela Maria Serena Lezza Received: 14 December 2022 Revised: 16 January 2023 Accepted: 25 January 2023 Published: 30 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Dibenzylideneacetone Induces Apoptosis in Cervical Cancer Cells through Ros-Mediated Mitochondrial Damage Aline Pinto Zani 1 , Caroline Pinto Zani 1 , Zia Ud Din 2 , Edson Rodrigues-Filho 2 ,Tânia Ueda-Nakamura 1 , Francielle Pelegrin Garcia 1 , Sueli de Oliveira Silva 1 and Celso Vataru Nakamura 1, * 1 Laboratory of Technological Innovation in the Development of Pharmaceuticals and Cosmetics, State University of Maringá, Maringá CEP 87020-900, PR, Brazil 2 LaBioMMi, Department of Chemistry, Federal University of São Carlos, CP 676, São Carlos CEP 13565-905, SP, Brazil * Correspondence: cvnakamura@uem.br; Tel.: +55-(044)-3011-5012; Fax: +55-(044)-3011-5046 Abstract: Cervical cancer is a health problem among women worldwide. Considering the limitations of prevention and antineoplastic chemotherapy against cervical cancer, research is needed to discover new, more effective, and safe antitumor agents. In the present study, we investigated the in vitro cytotoxicity of a new synthetic dibenzylideneacetone derived from 1,5-diaryl-3-oxo-1,4-pentadienyl (A3K2A3) against cervical cancer cells immortalized by HPV 16 (SiHa), and 18 (HeLa) by MTT assay. Furthermore, we performed spectrofluorimetry, flow cytometry, and Western blot analyzes to explore the inhibitory mechanism of A3K2A3 in cervical cancer cells. A3K2A3 showed cytotoxic activity against both cell lines. Mitochondrial depolarization and reduction in intracellular ATP levels were observed, which may be dependent on the redox imbalance between increased ROS and reduced levels of the antioxidant defense. In addition, damage to the cell membrane and DNA, and effective blocking of cell division in the G2/M phase were detected, which possibly led to the induction of apoptosis. This result was further confirmed by the upregulation of apoptosis-related proteins Bax, cytochrome C, and caspases 9 and 3. Our results provided the first evidence that A3K2A3 contributes to the suppression of cervical cancer in vitro, showing promise as a possible alternative for the treatment of this cancer. Keywords: dibenzylideneacetone; apoptosis; oxidative stress; HeLa cell; SiHa cell 1. Introduction Cervical cancer is the fourth most common cancer among women worldwide, with an estimated 604,000 new cases and 342,000 deaths in 2020. More than 95% of cervical cancer cases are infection-related with high-risk for human papillomavirus (HPV) [1]. Among the high-risk HPVs, HPV16 and HPV18 are responsible for almost 70% of these cases [2,3]. In the last decade, screening for HPV lesions through HPV and Pap smear tests, and prevention through HPV vaccinations have been recognized as the most effective interventions to control the mortality rate associated with this disease [3,4]. Despite these screening and prevention programs, the incidence of cervical cancer is still one of the leading causes of cancer-related death in women [5]. This scenario may be due to the fact that screening tests are not widely available in many underdeveloped countries, a situation related to poverty and the lack of resources and infrastructure [6]. Further, vaccination is limited to young people to ensure effectiveness [7]. Even with recent advances, the clinical treatments available for cervical cancer, including radiotherapy, chemotherapy, and surgery, are still not wholly adequate due to the frequent occurrence of adverse effects, among other limitations, such as systemic toxicity and chemoresistance [8,9]. The need to identify new, more effective, and safer antitumor agents becomes clearly justifiable. In general, natural products have demonstrated advantages due to their avail- ability and biological potential and, in recent decades, several natural products have been Antioxidants 2023, 12, 317. https://doi.org/10.3390/antiox12020317 https://www.mdpi.com/journal/antioxidants