The Effects of 5,6,7,8,3′,4′-Hexamethoxyflavone on Apoptosis of Cultured Human Choriocarcinoma Trophoblast Cells

5,6,7,8,3,4'-Hexamethoxyflavone, also called nobiletin (NOB), widely found in the citrus peel, is one of the main byproducts in citrus processing. NOB is considered safe, but its safety for women during pregnancy is unknown. Therefore, the effect of NOB on apoptosis in human choriocarcinoma trophoblast cells (BeWo cells) was evaluated. Cells were divided into four groups and cultured with different concentrations of NOB (0, 10, 33, and 100 μM) for 12, 24, 36, and 48 h respectively. Cell viability was detected by CCK-8 assay, cell morphology was detected by a Cell Imaging Multi-Mode Reader, and cell cycle and apoptosis were detected by flow cytometry. Cleaved PARP level, the expressions of B cell lymphoma 2 (BCL2) family proteins, and p53 pathway proteins were detected by Western blot. The results showed that after 48 h of cell culture, the cell viability was decreased significantly, but apoptosis was significantly increased. Compared to the cells without NOB treatment, the cells treated with NOB at 10 or 33 μΜ showed no significant differences in the number of suspended cells or late apoptosis rate, except the increase of cell viability. Treatment of NOB at the concentration of 100 μM improved cell viability, attenuated apoptosis, decreased suspended cells, and did not alter the G1 phase arrest, compared with the non-NOB-treated group after 48 h of culturing. The 100 μΜ NOB treatment increased the levels of BCL2 and BCLX_L, and decreased p53 accumulation in BeWo cells at 48 h, but had no effect on the expression of BAX, BAK, BAD, p21, and G1 phase arrest. These findings provide evidence that NOB (10, 33, and 100 μΜ) was safe for BeWo cells. NOB at the concentration of 100 μΜ could attenuate apoptosis in BeWo cells, which might be helpful to prevent pregnancy-related diseases caused by apoptosis.

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