Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells

Abstract

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

Keywords: anticancer activity; apoptosis; cartilage; peptide; skate (Raja porosa).

Figure 1

Proliferation inhibition of HeLa cell lines treated by FIMGPY for 24 h. All data are presented as the mean ± standard deviation (SD) of three experiments. ^a–f Values with same letters indicate no significant difference for each group of samples at the same concentration (p > 0.05).

Figure 2

Morphological observation with AO/EB staining at 400× actual magnification. HeLa cells were treated with FIMGPY at (A-1) 0, (A-2) 3, (A-3) 5, and (A-4) 7 mg/mL for 24 h. (A-1) Cell indicated by the arrow indicates viable cell; (A-2 and A-3) Cells indicated by the arrow indicates early apoptotic cells; (A-4) Cell indicated by the arrow indicates late apoptotic cell. Each experiment was performed in triplicate and generated similar morphological features.

Figure 3

Flow cytometry analysis of HeLa cells by double-labeling with Annexin-V and PI. Quadrants: lower left-live, cells; upper left, necrotic cells; lower right, early apoptotic cells; upper right, late apoptotic cells. The percentages of early apoptotic cells were (A-1) 4.54% in the blank control cells; (A-2) 8.64% in the 3-mg/mL FIMGPY-treated cells; (A-3) 11.72% in the 5-mg/mL FIMGPY-treated cells; and (A-4) 19.25% in the 7-mg/mL FIMGPY-treated cells. All data are presented as the mean ± standard deviation (SD) of three experiments.

Figure 3

Flow cytometry analysis of HeLa cells by double-labeling with Annexin-V and PI. Quadrants: lower left-live, cells; upper left, necrotic cells; lower right, early apoptotic cells; upper right, late apoptotic cells. The percentages of early apoptotic cells were (A-1) 4.54% in the blank control cells; (A-2) 8.64% in the 3-mg/mL FIMGPY-treated cells; (A-3) 11.72% in the 5-mg/mL FIMGPY-treated cells; and (A-4) 19.25% in the 7-mg/mL FIMGPY-treated cells. All data are presented as the mean ± standard deviation (SD) of three experiments.

Figure 4

Expression of the apoptosis-associated proteins Bax and Bcl-2 in HeLa cells treated with FIMGPY for 24 h. (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns for Bax and Bcl-2 and (B) the Bax/Bcl-2 ratio. * p < 0.05 and ** p< 0.01 vs. control.

Figure 5

Caspase-3 expression in HeLa cells treated with FIMGPY for 24 h. (A) SDS-PAGE pattern of caspase-3 and (B) the relative intensity of caspase-3. * p < 0.05 and ** p < 0.01 vs. control.

Figure 6

DNA fragmentation assay of HeLa cells treated with different concentrations of FIMGPY for 24 h. (A) Blank control; (B) 3 mg/mL; (C) 5 mg/mL; (D) 7 mg/mL, and the MV 2000 marker.