Antineoplastic Effects of Ankaferd Hemostat on Hep-2 Laryngeal Cancer Cells: Involvement of Apoptosis and PI3K/Akt/mTOR Signaling

Abstract

To investigate the cellular and molecular effects of Ankaferd Hemostat on human laryngeal squamous carcinoma Hep-2 cells, with particular emphasis on cell viability, apoptosis, oxidative stress, cell cycle progression, migration, invasion, and PI3K/Akt/mTOR signaling. Hep-2 cells were treated with increasing concentrations of Ankaferd Hemostat (50-1600 nL/mL) for 24, 48, and 72 h. Cell viability, clonogenic survival, apoptosis, cell cycle distribution, migration, invasion, nuclear morphology, DNA fragmentation, intracellular reactive oxygen species (ROS) production, and expression of apoptosis- and signaling-related proteins were evaluated using established in vitro assays. Ankaferd Hemostat significantly reduced Hep-2 cell viability and clonogenic survival in a dose- and time-dependent manner. It induced apoptosis, as demonstrated by Annexin V/PI staining, nuclear fragmentation, DNA laddering, and activation of caspase-3 and PARP. Treatment also resulted in G2/M phase cell cycle arrest and increased intracellular reactive oxygen species (ROS) levels. In addition, Ankaferd Hemostat inhibited cell migration and invasion and modulated metastasis-associated proteins by downregulating MMP-2, MMP-9, and VEGF, while upregulating E-cadherin. At the signaling level, Ankaferd Hemostat suppressed the activation of the PI3K/Akt/mTOR pathway. These findings provide preclinical in vitro evidence that Ankaferd Hemostat exerts multifaceted antineoplastic effects on Hep-2 laryngeal cancer cells by modulating apoptosis, oxidative stress, cell cycle progression, migratory and invasive behavior, and intracellular survival signaling. Validation in additional laryngeal cancer models and in vivo systems is required before the translational relevance can be established.