Dual anticancer activity of Aspergillus nidulans pigment and Ionizing γ-Radiation on human larynx carcinoma cell line

**Background:** Laryngeal cancer accounts for approximately 211,000 new cases and 126,000 deaths annually. Radiotherapy is a mainstay treatment, but radiosensitizers can enhance tumor cell killing while sparing normal tissues. Fungal pigments, particularly carotenoids, have antioxidant and anticancer properties. This study investigates whether the pigment from Aspergillus nidulans can act as a radiosensitizer against human larynx carcinoma (HEp-2) cells when combined with γ-radiation.

**Methods:** Aspergillus nidulans was cultured on potato dextrose agar (PDA) broth at pH 7.0 and 28±1°C for 15 days. Pigment was extracted from mycelial mats using 90% ethanol and characterized by UV-visible spectrophotometry (peaks at 450–550 nm) and GC-MS, which identified rhodopin (a carotenol) among other compounds. HEp-2 cells were treated with: (1) γ-radiation alone at 5, 10, or 15 Gy; (2) pigment alone at concentrations from 15.63 to 500 µg/mL; or (3) pigment plus radiation at the same doses. Cytotoxicity was measured by MTT assay after 24 h. Normal Vero cells were similarly tested with pigment alone or pigment plus 10 Gy. Apoptosis was quantified by flow cytometry using Annexin V-FITC/PI staining. Gene expression of P53, Caspase 3, and Bcl-2 was measured by RT-qPCR using the ΔΔCt method with GAPDH as housekeeping gene. Statistical analysis used one-way ANOVA with Tukey–Kramer post-hoc test (significance at p<0.05).

**Key Results:** The pigment alone had an IC50 of 208±0.46 µg/mL against HEp-2 cells. When combined with 10 Gy γ-radiation, the IC50 decreased to 115±0.76 µg/mL. No significant antitumor activity was observed with pigment plus 5 Gy or 15 Gy, nor with radiation alone at any dose (IC50 >500 µg/mL for all). On normal Vero cells, the pigment alone had an IC50 of 223±0.53 µg/mL, and pigment plus 10 Gy had an IC50 of 215±0.32 µg/mL, indicating lower toxicity than on cancer cells. Flow cytometry showed that pigment alone (208 µg/mL) induced 50%±1.0 total apoptosis, while pigment (115 µg/mL) plus 10 Gy induced 47.41%±1.7 total apoptosis, both significantly higher than untreated cells (p<0.05). Gene expression analysis revealed that pigment alone upregulated P53 (2.3-fold) and Caspase 3 (1.84-fold) and downregulated Bcl-2 (0.63-fold) relative to untreated cells. The combination of pigment (115 µg/mL) plus 10 Gy produced even greater changes: P53 2.53-fold, Caspase 3 2.0-fold, and Bcl-2 0.61-fold (all p≤0.05 vs. control).

**Clinical Implications:** This study demonstrates that the natural carotenoid pigment from Aspergillus nidulans, rhodopin, has intrinsic anticancer activity against laryngeal carcinoma cells and can be enhanced by combining with 10 Gy γ-radiation. The combination achieves a lower IC50 (115 vs. 208 µg/mL) while maintaining low cytotoxicity to normal Vero cells. The mechanism involves induction of apoptosis via upregulation of P53 and Caspase 3 and downregulation of Bcl-2. These findings suggest that A. nidulans pigment could serve as a radiosensitizer in radiotherapy for laryngeal cancer, potentially allowing lower radiation doses with improved tumor cell killing and reduced side effects. However, further in vivo studies and clinical trials are needed to confirm safety and efficacy.