Repurposing Antimalarials for Oral Cancer: Selective Efficacy of Hydroxychloroquine on Gingival Squamous Cell Carcinoma

Oral cancer, the most common head and neck malignancy, has a high recurrence rate and poor prognosis largely owing to chemotherapy resistance. The adverse effects of conventional therapies have prompted investigations into safer and more effective alternative therapies. Chloroquine (CQ) and hydroxychloroquine (HCQ) have shown potential owing to their roles in autophagy modulation and immune regulation. This study clarifies the selective efficacy of hydroxychloroquine (HCQ) and chloroquine (CQ) in oral squamous cell carcinoma models, emphasizing distinct responses in gingival (Ca9-22) and tongue (SCC-9) carcinoma cells. Non-oncogenic oral epithelial cells (GMSM-K) and oral carcinoma cell lines from the tongue (SCC-9, Cal-27) and gingiva (Ca9-22) were used. Cell viability, cytotoxicity, and colony formation were assessed via MTT, LDH, and crystal violet assays. Flow cytometry was used to measure apoptosis, autophagy, oxidative stress, mitochondrial membrane potential, and DNA damage. The transcriptomic profiles of apoptosis and autophagy-related genes were assessed by qPCR arrays. Bioinformatics analysis allowed estimation of the main gene interaction networks. Pre-screening showed that GMSM-K and Cal-27 cells were non-responsive or exhibited non-specific toxicity at high doses; therefore, subsequent analyses focused on Ca9-22 (GC) and SCC-9 (TC). HCQ significantly reduced viability and colony formation in Ca9-22 cells while moderately affecting SCC-9 cells. Autophagy inhibition was accompanied by compensatory up-regulation of autophagy-related genes, consistent with feedback activation of TFEB and FOXO3a pathways. Gene expression profiling and flow-cytometry analyses revealed cell-type-specific differences in apoptosis, mitochondrial potential, and DNA damage, suggesting HCQ's selective anti-tumor potential in gingival carcinoma. These findings highlight HCQ as a repurposed adjuvant therapy that modulates autophagy and apoptosis to enhance chemosensitivity in oral cancer.