Cisplatin resistance in head and neck squamous cell carcinoma is linked to DNA damage response and cell cycle arrest transcriptomics rather than poor drug uptake

<b>Aim:</b> Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) is thought to involve both reduced drug uptake and altered molecular responses. However, the relative contribution of these mechanisms remains unclear. <b>Methods:</b> Two HNSCC cell lines with differing sensitivity (HNO97 and HNO41) were analyzed using cytotoxicity assays, atomic absorption spectroscopy-based quantification of intracellular cisplatin, caspase 3/7 assays, Western blotting, polymerase chain reaction (PCR)-based transcriptomic analysis of DNA damage response and cell cycle arrest pathways, and RNA-seq data from The Cancer Genome Atlas (TCGA) to characterize the resistance phenotype. <b>Results:</b> HNO97 (IC₅₀ = 440 µM) was 7.6-fold more resistant to cisplatin than HNO41 (IC₅₀ = 57.8 µM; <i>P</i> = 0.0286). After quantifying intracellular uptake (pg Pt/µg protein) and normalizing cytotoxicity to intracellular drug levels, HNO97 (IC₅₀ = 778.9 pg Pt/µg protein) remained 5-fold more resistant than HNO41 (IC₅₀ = 153.5 pg Pt/µg protein), indicating only a partial reduction in resistance (33% decrease, from 7.6-fold to 5-fold; <i>P</i> = 0.0286). At cisplatin concentrations yielding comparable intracellular exposure (HNO97: 440 µM; HNO41: 196 µM; both ≈ 725 pg Pt/µg protein), caspase 3/7 activation and induction of <i>CDKN1A</i>, <i>GADD45A</i>, <i>GADD45G</i>, and <i>PPP1R15A</i> were weaker in HNO97 than in HNO41. Notably, baseline expression of these genes was significantly higher in HNO97. In the TCGA cohort, multivariate analysis showed that high <i>FANCD2</i> expression was associated with unfavorable recurrence-free survival in platinum-treated patients (hazard ratio = 4.0; <i>P</i> = 0.011), but not in those who did not receive platinum chemotherapy. <b>Conclusion:</b> Cisplatin resistance in HNSCC appears to be driven primarily by molecular mechanisms involving DNA damage response and cell cycle arrest pathways, rather than poor drug uptake.