HO-1-u-1

Cetuximab–Toxin Conjugate and NPe6 with Light Enhanced Cytotoxic Effects in Head and Neck Squamous Cell Carcinoma In Vitro

Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and irradiation of a specific wavelength to exert cytotoxic effects. To enhance the antitumor effect against head and neck squamous cell carcinoma (HNSCC), Komatsu et al. developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin (IT) and a PS for PDT and light irradiation.

In this study, they newly investigate the iTAP targeting epidermal growth factor receptor (EGFR) which is widely used as a therapeutic target for HNSCC. First, Cmab and IT-Cmab showed dose-dependent cytotoxicity on SAS cells, which moderately express EGFR, with no significant difference in cytotoxicity between the two (p > 0.05) (Fig. 1a–d). The IC₅₀ of IT-Cmab was about 0.07 nM in SAS (Fig. 1c). Both Cmab and IT-Cmab were ineffective in Sa3, HO-1-u-1, and HSQ-89 cells, likely due to insufficient internalization (Fig. 1a, b, d). Second, to enhance IT-Cmab's endosomal escape, they used NPe6 and its excitation wavelength to generate ROS, disrupting endosomes. The cytotoxicity of NPe6 varied among cell lines and with irradiation. No significant effect was observed in Sa3 (p > 0.05) (Fig. 2a), while significant differences appeared in HO-1-u-1 at 100 µM (p < 0.05) (Fig. 2b), SAS at 0.16 µM (p < 0.05) (Fig. 2c), and HSQ-89 at 20 and 100 µM (p < 0.05) (Fig. 2d). Maximum nonlethal NPe6 concentrations with irradiation were set at 20 µM for Sa3/HO-1-u-1, 0.8 µM for SAS, and 4 µM for HSQ-89 (Fig. 2a–d), which were used in subsequent assays.

Fig. 1. Cytotoxicity of IT-Cmab or Cmab on each HNSCC cell line (Komatsu N, Kosai A, et al., 2024).

Fig. 2. Cytotoxicity of NPe6 and light irradiation on each HNSCC cell line (Komatsu N, Kosai A, et al., 2024).

PTC124 Rescues Nonsense Mutation of Two Tumor Suppressor Genes NOTCH1 and FAT1 to Repress HNSCC Cell Proliferation

Nonsense mutations in tumor suppressor genes like NOTCH1 and FAT1 lead to premature stop codons, resulting in truncated, non-functional proteins. These mutations have a significant presence in head and neck squamous cell carcinoma (HNSCC). PTC124 (Ataluren), a drug known to promote readthrough of such mutations, has potential in restoring the expression of these tumor suppressors. Wu's team explored the ability of PTC124 to rescue nonsense mutations in NOTCH1 and FAT1, thereby inhibiting HNSCC cell proliferation.

To test if PTC124 affects HES and YAP transactivation, they cloned firefly luciferase reporters with 2X NOTCH1/HES (CGTGGGAA) and 2X YAP1/TEAD (ACATTCCA) consensus sequences. Since PTC124 can stabilize luciferase, they washed cells three times before adding the lysis buffer to prevent interference. PTC124 activated the 2X NOTCH1/HES reporter in DOK cells (Fig. 3A) and repressed the 2X YAP1/TEAD reporter in HO-1-u-1 cells (Fig. 3B). PTC124's effect on growth inhibition in DOK cells was counteracted by the NOTCH inhibitor FLI-06 (Fig. 4A), while it reversed TT-10-driven growth in HO-1-u-1 cells (Fig. 4B). Therefore, PTC124 was able to rescue nonsense mutation of NOTCH1 and FAT1 to repress HNSCC cell proliferation.

Fig. 3. PTC124 can moderate HES and TEAD transactivation function (Wu M-H, Lu R-Y, et al., 2022).

Fig. 4. PTC124 can control HNSCC cell growth in DOK and HO-1-u-1 cells (Wu M-H, Lu R-Y, et al., 2022).