New Propargyloxy Derivatives of Galangin, Kaempferol and Fisetin-Synthesis, Spectroscopic Analysis and In Vitro Anticancer Activity on Head and Neck Cancer Cells

Abstract

Head and neck cancer (HNC) therapy is limited; therefore, new solutions are increasingly being sought among flavonoids, which exhibit numerous biological properties, including potential anticancer activity. However, because they are mostly insoluble in water, are unstable and have low bioavailability, they are subjected to chemical modification to obtain new derivatives with better properties. This study aimed to synthesize and analyze new propargyloxy derivatives of galangin, kaempferol and fisetin, and to evaluate their anticancer activity against selected HNC cell lines. The obtained derivatives were assessed by spectroscopic analysis; next, their anticancer activity was evaluated using a flow cytometer and real-time cell analysis. The results showed that only the fisetin derivative was suitable for further analysis, due to the lack of crystal formation of the compound. The fisetin derivative statistically significantly increases the number of cells in the G2/M phase (p < 0.05) and increases cyclin B1 levels. A statistically significant increase in the number of apoptotic cells after being exposed to the tested compound was also observed (p < 0.05). The data indicate that the obtained fisetin derivative exhibits anticancer activity by affecting the cell cycle and increasing apoptosis in selected HNC lines, which suggests its potential use as a new medicinal agent in the future.

Keywords: anticancer; apoptosis; cyclin; flavonol; flow cytometry; head and neck cancer.

Figure 1

The synthesis route of propargyloxy derivatives of flavone.

Figure 2

Cytotoxic and antiproliferative effect of fisetin derivative on Detroit 562, A-253, UCSF-OT-1109, FaDu and Cal-27 cell lines. The calculated IC₅₀ values of propargyloxy derivatives (fisetin, galangin and kaempferol) for 24 and 48 h of cell incubation are presented in Table. Compounds with SI index ≥ 3 are marked in green (A). RTCA measurement of cell index values during cancer cells incubation with fisetin derivative (B).

Figure 3

Cell cycle analysis via flow cytometry: Analysis of DNA content in cells treated with fisetin derivative for 24 h was compared with control cells. The analysis showed that the fisetin derivative induces cell cycle arrest in the G0/G1 phase in cells of the A-253, Cal-27 and UCSF-OT-1109 lines. The right side of the figure shows a bar graph depicting quantitative values of flow cytometry data. The data shown are representative of a single experiment. The graphs show the mean ± SD from three independent experiments. Statistical analysis was performed using Student’s t-test.

Figure 4

Fisetin derivative enhances cyclin B1 protein expression in cells of A-253, Cal-27 and UCSF-OT-1109 lines. Data shown are representative of a single experiment.

Figure 5

Representative scoring plots of PI (y-axis) against annexin V (x-axis). Representative scoring plots of PI (y-axis) against annexin V (x-axis). Variable effects of fisetin derivative on apoptosis induction are noted. Lower left quadrant (FITC−/PI−) = viable cells, lower right quadrant (FITC+/PI−) = early apoptotic cells, upper right quadrant (FITC+/PI+) = late apoptotic cells and upper left quadrant (FITC−/PI+) = necrotic cells.

Figure 6

Apoptosis assay using flow cytometry after staining with annexin V-FITC/propidium iodide (PI). Percentage of annexin (−) and annexin (+) cells. Data are presented as the means ± SD of triplicate experiments. Statistical analysis was performed using Student’s t-test.