Cell cycle arrest and mechanism of apoptosis induction in H400 oral cancer cells in response to Damnacanthal and Nordamnacanthal isolated from Morinda citrifolia

Abstract

Oral cancer is the eleventh most prevalent cancer worldwide. The most prevalent oral cancer is oral squamous cell carcinoma (OSCC). Damnacanthal (DAM) and nordamnacanthal (NDAM), the anthraquinone compounds, are isolated from the root of Morinda citrifolia L. (Noni), which has been used for the treatment of several chronic diseases including cancer. The objectives of this study were to evaluate the cytotoxicity, cell death mode, cell cycle, and the molecular mechanism of apoptosis induced by DAM and NDAM on OSCC. The cytotoxic effects of these compounds against OSCC cell lines were determined by MTT assay. The cell death mode was analysed by DNA laddering and FITC-annexin V/PI flow cytometric assays. In addition, the mechanism of apoptosis induced by DAM and NDAM was detected using mitochondrial membrane potential, Cytochrome c, and caspases assays. Finally, the effect of DAM and NDAM on cell cycle phase distribution of OSCC cells was detected by flow cytometry. In the present study, DAM and NDAM showed cytotoxicity towards OSCC cell lines and the maximum growth inhibition for both compounds was observed in H400 cells with IC50 value of 1.9 and 6.8 μg/ml, respectively, after 72 h treatment. The results also demonstrated the inhibition of H400 OSCC cells proliferation, internucleosomal cleavage of DNA, activation of intrinsic apoptosis pathway, and cell cycle arrest caused by DAM and NDAM. Therefore, these findings suggest that DAM and NDAM can be potentially used as antitumor agents for oral cancer therapy.

Keywords: Apoptosis; Cytochrome c; Damnacanthal and nordamnacanthal; Human oral squamous cell carcinoma (OSCC); Mitochondrial membrane potential.

Fig. 1

The molecular structure of Damnacanthal (a) and Nordamnacanthal (b)

Fig. 2

Apoptotic DNA fragmentation in H400 cells treated with IC₅₀ and IC₇₅ concentrations of DAM and NDAM for 24, 48 and 72 h. Images are representative of two independent experiments. M 1 kb DNA marker, C control cells, Lane 1 Treatment with IC₅₀ concentration for 24 h, Lane 2 Treatment with IC₇₅ concentration for 24 h, Lane 3 Treatment with IC₅₀ concentration for 48 h, Lane 4 Treatment with IC₇₅ concentration for 48 h, Lane 5 Treatment with IC₅₀ concentration for 72 h, Lane 6 Treatment with IC₇₅ concentration for 72 h

Fig. 3

Flow cytometric analysis of apoptosis in H400 cells treated with IC₅₀ concentration of DAM and NDAM for 12, 24 and 48 h using FITC-annexin V/PI double staining. Early and late apoptosis were examined on fluorescence 2 (FL2 for propidium iodide) versus fluorescence 1 (FL1 for Annexin) plots. Images are representative of three independent experiments. R3 damaged/dead cells, R4 late apoptotic/secondary necrotic cells, R5 viable cells and R6 early apoptotic cells. Statistically significant differences between control and treated cells were set at **P < 0.01

Fig. 3

Flow cytometric analysis of apoptosis in H400 cells treated with IC₅₀ concentration of DAM and NDAM for 12, 24 and 48 h using FITC-annexin V/PI double staining. Early and late apoptosis were examined on fluorescence 2 (FL2 for propidium iodide) versus fluorescence 1 (FL1 for Annexin) plots. Images are representative of three independent experiments. R3 damaged/dead cells, R4 late apoptotic/secondary necrotic cells, R5 viable cells and R6 early apoptotic cells. Statistically significant differences between control and treated cells were set at **P < 0.01

Fig. 4

Caspase-3/7, 8 and 9 activities in H400 cells treated with IC₂₅, IC₅₀ and IC₇₅ concentrations of DAM and NDAM for 24 h. a Treatment with IC₂₅ concentration; b treatment with IC₅₀ concentration; c treatment with IC₇₅ concentration. Data are presented as mean ± SEM from three individual experiments (n = 3). Statistically significant differences between control and treated cells were set at *P < 0.05, **P < 0.01. RLU Relative luminescence units

Fig. 5

Mitochondrial membrane potential disruption in H400 cells treated with IC₅₀ concentration of DAM and NDAM for 24 h. Data are presented as mean ± SEM from three individual experiments (n = 3). Statistically significant differences between control and treated cells were set at *P < 0.05, **P < 0.01

Fig. 6

a Standard curve associated with Cytochrome c concentration in the samples. Natural Cytochrome c from cell lysate was serially diluted (0–5 ng/mL) in Standard Diluent Buffer. The optical density (OD) of each dilution was plotted against the Cytochrome c standard curve. Parallelism of Cytochrome c demonstrated by the figure, showed that the standard precisely reflected Cytochrome c content in the samples. Linear regression analysis provided a correlation coefficient of 0.995. b Quantitative detection of Cytochrome c in cell lysates of H400 OSCC cells treated with IC₅₀ concentration of DAM and NDAM for 24 h. Cytochrome c levels were examined in duplicate using ELISA kit. Data are shown as mean ± SEM of two independent experiments. Significant difference from the control was at *P < 0.05, **P < 0.01

Fig. 7

Flow cytometric analysis of cell cycle in H400 cells treated with IC₅₀ concentration of DAM and NDAM for 24 and 48 h. Images are representative of three independent experiments. The x and y axes represent DNA content and cell number respectively

Fig. 8

The effect of DAM and NDAM on cell cycle phase distribution of H400 cells after 24 and 48 h treatment. The values are expressed as mean ± SEM from three independent experiments. Significant difference from the control was at *P < 0.05, **P < 0.01