Combined Hesperidin and Doxorubicin Treatment Induces Apoptosis and Modulates Inflammatory Cytokines in HeLa Cervical Cancer Cells

Cervical cancer is a major gynecological malignancy linked to hormonal dysregulation and genetic alterations. Chemotherapy is standard but limited by toxicity and chemoresistance, prompting interest in plant-derived adjuncts. This study examined the anticancer and immunomodulatory effects of Hesperidin (Hes), a citrus flavonoid, with Doxorubicin (DX) in HeLa cervical cancer cells. Cell viability was assessed by MTT assay, apoptotic markers (<i>Bcl-2</i>, <i>Caspase-3</i>) by RT-qPCR, and inflammatory cytokines (<i>IL-1β</i>, <i>IL-6</i>, <i>TNF-α</i>, <i>IFN-γ</i>) by ELISA. Cytokine levels were normalized to 10⁴ viable cells, and mRNA expression of all four cytokines was quantified by RT-qPCR, confirming protein-level changes and showing the strongest <i>IL-6</i> suppression with Hes+DX. Chou-Talalay combination index (CI) analysis demonstrated synergistic interactions (CI < 1.0) between Hes and DX across all tested concentrations, with strong synergism (CI < 0.7) at medium and high doses, particularly at 48 and 72 h. Hes alone showed dose-dependent cytotoxicity, while the combination markedly increased <i>Caspase-3</i>, reduced <i>Bcl-2</i>, and decreased <i>IL-1β</i>, <i>IL-6</i>, and <i>TNF-α</i>, indicating enhanced intrinsic apoptosis and complementary immunomodulation. These results suggest that Hes augments DX's pro-apoptotic and anti-inflammatory effects, potentially allowing lower chemotherapy doses and reduced systemic toxicity in cervical cancer treatment.