Key words licorice acid human breast cancer cells (MCF-7 apoptosis of intracellular Ca2 +
Licorice acid (Glycyrrhizin, GL), is one of the most important active ingredients in licorice studies [1,2] that glycyrrhizin on human cancer cell proliferation significantly inhibited and induced apoptosis. This study explores the relationship between apoptosis and intracellular Ca2 + concentration changes licorice acid-induced human breast cancer cells (MCF 7).
1 Materials and methods
1.1 cell line MCF 7 introduced by the United States ; licorice acid monoamine (99.7% purity) the Zhejiang potenline Institute of pharmaceutical companies.
1.2 inhibition of cell proliferation was measured by MTT determination.
1.3 Determination of apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP end labeling and Annexin V flow cytometry and single cell gel electrophoresis (comet assay) assay apoptotic cells. 7.5mmol / L glycyrrhizic acid-treated cells 24h, Fura 2 fluorescence load method  Determination of [Ca2 +] i changes.
1.4 statistical methods t test, the semi-proliferation inhibitory concentration (IC50) is calculated using the weighted linear regression method.
2.1 glycyrrhizin on MCF 7 cell proliferation inhibition 5mmol / L glycyrrhizic acid concentration from cell proliferation significant difference (P <0.01), and the degree of inhibition in a dose-dependent manner, IC50 to 15.84 mmol / L 5mmol / L, 7.5mmol / L and 10 mmol / L glycyrrhizic acid proliferation inhibition rates were 22.5%, 32.9% and 40.2% (P <0.01), these concentrations were selected as apoptosis test drug concentration, as shown in Figure 1. 0.05，*P<0.05，**P<0.01″>Table 1 glycyrrhizic acid induced apoptosis of MCF 7 compared with control group: △ P> 0.05, * P <0.05, ** P <0.01
2.2 licorice acid-induced apoptosis of MCF 7 MCF 7 cells 24 h after the detection of apoptosis results are shown in Table 1, we can see 7.5mmol / L and 10 mmol / L glycyrrhizic acid glycyrrhizic acid concentration selected processing apoptosis was significantly increased (P <0.01 and P <0.05), and a dose-dependent manner. In the same concentration effect, the result of three methods to detect little difference.
2.3 licorice acid-induced MCF 7 cell apoptosis [Ca2 +] i of change 7.5mmol / L glycyrrhizic acid treatment of MCF 7 cells after 24h, measured glycyrrhizic acid treated [Ca2 +] i (58.59 ± 1.03) nmol / L the control group [Ca2 +] i as (129.17 ± 11.45) nmol / L, licorice acid treated in [Ca2 +] i was significantly lower than the control group (P <0.05).
The role of of 2.4 intracellular Ca2 + chelator repressor glycyrrhizic acid induced MCF 7 cell apoptosis 100μmol / L of intracellular Ca2 + chelator BAPTA AM and 7.5mmol / L of glycyrrhizic acid combination processing MCF 7 cells 24 h after the single cell gel The electrophoresis assay apoptosis results in Table 2, the apoptosis rate of the combination treatment group was significantly lower than the simple 7.5mmol / L glycyrrhizic acid treatment group (P <0.05). 0.05，*P<0.05″>Table 2 intracellular Ca2 + chelator role of repressor induced MCF 7 apoptosis compared with control group: △ P> 0.05, * P <0.05
The results of this study show that glycyrrhizic acid inhibition of human breast cancer MCF 7 cell proliferation. Cancer cell proliferation inhibition by a variety of mechanisms leading to cancer cell apoptosis which is the focus of attention in recent years. In this study, using the alkaline single-cell gel electrophoresis assay apoptosis rate detection reflect the characteristics of early apoptotic cell membrane valgus phosphatidylserine Annexin V flow cytometry method, as well as with high-sensitivity detection of late The apoptotic characteristics that apparent consistency of the results of DNA fragmentation of apoptotic cells by TUNEL method. The results showed that the apoptosis rate measured by the three methods under the same the glycyrrhizic acid concentrations role basically the same, which may reflect the rate of glycyrrhizic acid induces apoptosis cells DNA breaks soon. In addition, glycyrrhizic acid induced MCF 7 cell apoptosis apoptosis rate, cell proliferation inhibition rate only slightly lower than the corresponding effect of licorice acid concentration, visible the glycyrrhizic acid induced apoptosis plays an important role in inhibition of MCF 7 cell proliferation in . Currently, the majority of the literature [3,4] reported intracellular Ca2 + levels is closely related with the induction of apoptosis, Kluck et al  and Bansal  with EGTA and other Ca2 + complexing agent deal with NS 1 mouse myeloma cells and CEM C7 human T lymphocytes, accompanied by apoptosis [Ca2 +] i decline occurred, they think [Ca2 +] i decreased inhibition of DNA and protein synthesis, the impact of many of Ca2 +-dependent protein activity, cell activity and gene expression . The results of this study show the the the glycyrrhizic acid induced human breast cancer MCF-7 cell apoptosis group Ca2 + levels were significantly lower than the control group, this result Kluck et al  and Bansal et al  reported consistent, we speculated that glycyrrhizic acid induced breast cancer MCF 7 apoptosis may be the decreased intracellular Ca2 + levels. 18β glycyrrhetinic acid but weakened by intracellular Ca2 + chelator BAPTA AM complexation cell intracellular free Ca2 + induced apoptosis of MCF 7, suggesting that glycyrrhizic acid-induced MCF 7 apoptosis is not wholly dependent on the intracellular Ca2 + levels down, may also be associated with other signal transduction pathways induce apoptosis in cancer cells, specific licorice acid induced tumor cell apoptosis mechanism remains to be further explored.