The PI3K Inhibitor HCD Promotes Caspase Activation in Head and Neck Squamous Cells by Upregulating the Extrinsic and Intrinsic Apoptosis Pathways

16-hydroxycleroda-3,13-dien-15,16-olide (HCD), a compound isolated from Polyalthia longifolia var. pendula, has been identified as a PI3K inhibitor. P. longifolia var. pendula is recognized for its diverse pharmacological properties, including antimicrobial, hypotensive, anti-inflammatory, and cytotoxic activities. Considering the pivotal role of PI3K in the regulation of apoptosis in cancer cells, this study aims to investigate the apoptotic effects induced by HCD in head and neck cancer cell models. The study focused on investigating the mechanism through which HCD induces apoptosis in oral squamous cell carcinoma (OSCC) using SCC25 and SCC180 cells. These cells were treated with HCD at IC20, IC50, and IC80 concentrations over periods of 24, 48, and 72 h. Assessments included cell viability, oxidative stress levels using MitoSOX and CellROX, and the analysis of gene and protein expressions related to apoptosis via immunofluorescence staining, flow cytometry, RT-PCR, and western blotting. The results demonstrated that HCD significantly increased the sub-G1 phase cell population, suppressed clonogenic activity, and promoted mitochondrial ROS production, accompanied by a reduction in glutathione levels. HCD also activated cytochrome c (cyto. c) release and Bax expression while downregulating Bcl-2 expression. Notably, in SCC25 cells, HCD induced apoptosis through the TNF-α/TNF-R and FasL/Fas death domains, leading to caspase cascade activation. Moreover, HCD showed dose-dependent growth inhibition of SCC25 cells, both alone and combined with cisplatin, by enhancing caspase-3 expression. This investigation into HCD's molecular mechanisms in OSCC cells offers insight into developing effective anticancer therapies, highlighting its role in apoptosis induction through oxidative stress, mitochondrial pathways, and death receptor signaling.