Gingerol acts as a potent radiosensitizer in head and neck squamous cell carcinoma

Abstract

Treatment options for advanced head and neck squamous cell carcinoma (HNSCC) are limited and often cause severe toxicity and debilitating long-term impacts. Developing effective and safer treatments is warranted. Several plant extracts have shown their effectiveness, but a comprehensive comparison between plant extracts in HNSCC has not been reported. Our aim was to investigate the effect of different plant extracts on the proliferation and viability of HNSCC cell lines. In addition, we investigated the efficacy of combining cytotoxic plant extracts with radiation. Since RT is a cornerstone in the treatment and management of HNSCC, it is desirable to enhance its efficacy through combination with cytotoxic agents that have minimal side effects. HNSCC cell lines were treated with various plant extracts at different concentrations. MTT assays were performed to identify the most potent anti-tumor plant extract. Colony-formation assays were performed to determine the radiosensitization effect. To investigate the effect on migration, transwell migration assays were performed. Annexin V staining was performed to analyze cell apoptosis. 6-gingerol resulted in the most significant dose-dependent inhibition in all cell lines compared to other plant extracts. Colony-formation assays showed a significant radiosensitizing effect when 6-gingerol was combined with radiation. In addition, the combination of 6-gingerol with radiation resulted in a significant decrease in HNSCC cell migration. Mechanistically, Annexin V staining showed that the combination of 6-gingerol and RT induces a synergistic apoptotic effect in MOC1, MOC2 and SCC9 cells compared to RT alone. In conclusion, 6-gingerol enhances the effect of radiation in HNSCC cell lines and could be a suitable candidate for combination therapy in HNSCC.

Keywords: 6-gingerol; Apoptosis; Head and neck squamous cell carcinoma; Radiation.

Fig. 1

6-Gingerol has antiproliferative effects in HNSCC cells. Effect of plant extracts on cancer cell proliferation. MOC1, MOC2, Cal27 and SCC9 cells were treated with 0, 30 or 300 uM for 72 h. One-way ANOVA followed by Tukey’s multiple comparisons test was performed to assess significance among the groups. Only statistically different findings are shown by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001)

Fig. 2

6-gingerol reduces HNSCC cell migration. A Schematic illustration of experimental setup. B MOC1, MOC2, Cal27 and SCC9 cells were treated with DMSO or 100 uM 6-gingerol for 48 h. Two-tailed t-test was performed to assess significance. **** < 0.0001

Fig. 3

Clonogenic assay for radiosensitivity. Clonogenic survival of HNSCC (MOC1, MOC2, Cal27, SCC9) cell lines after exposure to DMSO and 6-gingerol. TOP: Data shows the mean survival fraction of 3–4 independent experiments. Error bars represent standard deviation. Connecting lines show the linear-quadratic fit of the data points. Two-tailed t-test was performed to assess significance at each data point between the groups. BOTTOM: Table summarizing the survival fractions at 2 Gy, 4 Gy and 6 Gy for RT + DMSO and RT + Gingerol. % Enhancement represents the % decrease in SF in the RT + Gingerol relative to RT + DMSO

Fig. 4

Effect of 6-gingerol and RT on apoptosis in HNSCC cells. Cells were seeded in 6-well plates and treated with DMSO, 6-gingerol (100uM), radiation (4 Gy) alone or pretreated with 6-gingerol for 4 h before being exposed to radiation. Cells were stained with Annexin V/PI and detected by flow cytometry 24 h post-irradiation: A MOC1, B MOC2, C Cal27, D SCC9. E–F Analysis of Caspase-3 protein expression by Western blot. Representative images are shown above quantification graphs. Two-way ANOVA was used to assess significance. (* p < 0.05; ** p < 0.01)