Combined Hesperidin and Gemcitabine Therapy Modulates Apoptosis and Angiogenesis Pathways in ISHIKAWA Human Endometrial Adenocarcinoma Cells

<i>Background and Objectives:</i> Endometrial adenocarcinoma is among the most prevalent malignancies of the female reproductive system, and therapeutic options remain limited, particularly in advanced stages. In recent years, natural agents, especially flavonoids, have gained considerable interest for their capacity to enhance the effectiveness of chemotherapeutic drugs and modulate tumor-related molecular mechanisms. Hesperidin, a citrus-derived flavonoid, is recognized for its antioxidant and anti-inflammatory effects, while Gemcitabine, a nucleoside analog, is widely used in cancer treatment. Investigating their combined effects on endometrial carcinoma cells could yield novel insights into multimodal therapeutic development. This current study aimed to assess the impact of Hesperidin (Hes) and Gemcitabine (Gem) on ISHIKAWA cells, a human endometrial adenocarcinoma model, with particular attention to pathways associated with hypoxia, angiogenesis, apoptosis, and oxidative stress. <i>Materials and Methods:</i> ISHIKAWA cells were treated with varying concentrations of Hes (50-200 µM) and Gem (10-50 nM), either individually or together, for 24 and 48 h. Cell viability was determined using the MTT assay, while apoptosis was measured by Caspase-3/7 activity and NucBlue nuclear staining. Intracellular reactive oxygen species (ROS) generation was quantified via DCFH-DA fluorescence. Expression levels of HIF-1α, VEGF, Bax, Bcl-2, and Caspase-3 were examined by <i>RT-qPCR</i>. Synergistic interactions were analyzed with the Chou-Talalay combination index. Biological enrichment was further explored using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. <i>Results</i>: Both Hes and Gem significantly decreased ISHIKAWA cell viability in a concentration- and time-dependent manner (<i>p</i> < 0.001). The combined treatment induced stronger apoptotic effects, as reflected by increased Caspase-3/7 activity and nuclear morphological changes. <i>RT-qPCR</i> demonstrated upregulation of Bax and Caspase-3, together with downregulation of Bcl-2, HIF-1α, and VEGF. While Hes reduced intracellular ROS, Gem elevated it; their combination produced a balanced oxidative response. All dose combinations displayed strong synergism (CI < 1). GO and KEGG enrichment confirmed the involvement of apoptosis-, angiogenesis-, and hypoxia-related pathways. <i>Conclusions</i>: Co-treatment with Hes and Gem exhibits synergistic anticancer activity in endometrial cancer cells by promoting apoptosis, suppressing angiogenesis- and hypoxia-related gene expression, and modulating oxidative stress. This combined therapeutic approach highlights its potential as a promising adjuvant option, warranting further evaluation in in vivo and translational studies.