In Vitro Antiproliferative Effect of Arthrocnemum indicum Extracts on Caco-2 Cancer Cells through Cell Cycle Control and Related Phenol LC-TOF-MS Identification

Abstract

This study aimed to determinate phenolic contents and antioxidant activities of the halophyte Arthrocnemum indicum shoot extracts. Moreover, the anticancer effect of this plant on human colon cancer cells and the likely underlying mechanisms were also investigated, and the major phenols were identified by LC-ESI-TOF-MS. Results showed that shoot extracts had an antiproliferative effect of about 55% as compared to the control and were characterised by substantial total polyphenol content (19 mg GAE/g DW) and high antioxidant activity (IC50 = 40 μ g/mL for DPPH test). DAPI staining revealed that these extracts decrease DNA synthesis and reduce the proliferation of Caco-2 cells which were stopped at the G2/M phase. The changes in the cell-cycle-associated proteins (cyclin B1, p38, Erk1/2, Chk1, and Chk2) correlate with the changes in cell cycle distribution. Eight phenolic compounds were also identified. In conclusion, A. indicum showed interesting antioxidant capacities associated with a significant antiproliferative effect explained by a cell cycle blocking at the G2/M phase. Taken together, these data suggest that A. indicum could be a promising candidate species as a source of anticancer molecules.

Figure 1

(a) Measurements of cell proliferation using MTT assay in human carcinoma Caco-2 cell line treated with A. indicum shoot extracts. Cells at 2 × 10⁴ cells/mL were left untreated or were treated with 0.01, 0.1, 1, 10, 50, or 100 μg/mL of A. indicum for 72 h. Values represent the results of three independent experiments. (b) DAPI staining of Caco-2 cells treated with A. indicum. Cells at 2 × 10⁴ cells/mL were incubated with 100 μg/mL of A. indicum for 72 h. Control cells were incubated with 0.4% DMSO. After 72 h, the nuclear morphologies of cells were examined using a fluorescent DNA-binding agent, DAPI. The DNA was analyzed using fluorescence microscopy. The arrow indicates mitotic cells with chromatin distribution. Results shown (a) and (b) are typical of 3 independent experiments.

Figure 2

(a) Western analysis of the cell cycle regulatory proteins. Cells at 2 × 10⁴ cells/mL were treated with 100 μg/mL of A. indicum for 72 h. After protein extraction, the same blot was incubated with the appropriate antibodies. The analyzed MAP kinases were cyclin B1, Erk1/2, pErk1/2, p38, pp38, Chk1, pChk1, Chk2, and pChk2. Results shown are typical of 3 independent experiments. (b) Relative intensities of detected bands of cyclin B1, Erk1/2, pErk1/2, p38, pp38, Chk1, pChk1, Chk2, and pChk2.

Figure 3

Total ions chromatogram (TIC) of A. indicum methanolic extract obtained by LC-ESI-TOF-MS. Peaks designation: (1) gallic acid, (2) 3-hydroxy-4′-methoxyflavone, (3) cyanidin, (4) chrysoeriol, (5) quercetin, (6) catechol, syringic acid, and luteolin.

Figure 4

Extract ions chromatogram/mass spectra of each protonated molecule (positive mode): gallic acid ((A)/(A′)), 3-hydroxy-4′-methoxyflavone ((B)/(B′)), cyanidin ((C)/(C′)), chrysoeriol ((D)/(D′)), quercetin ((E)/(E′)), and catechol, syringic acid, and luteolin ((F)/(F′)).