Anti-proliferative effects of ellagic acid depend on the status of estrogen receptor in human breast cancer cell lines

Abstract

Pomegranate (Punica granatum) has anti-inflammatory effects, anti-oxidant effects, anti-angiogenic potential and anti-proliferative effects. Epidemiologically, some reports showed that the extract of pomegranate has chemopreventive and anticancer effect for breast cancer. Pomegranate contains numerous phytochmicals like ellagic acid, quercetin, kaempferol and luteolin glycosides. In these components, ellagic acid has been demonstrated that it can inhibit tumor growth through apoptosis. However, the mechanism of ellagic acid elicits is poorly understood. Although most of the reports about ellagic acid showed that the anti-proliferative effect of it is appeared through the apoptotic pathway, it was also reported that ellagic acid acts via the estrogen receptor. Our object in this study was to evaluate the biological activity of ellagic acid by comparing the proliferative effect of ellagic acid according to the estrogen receptor negative(ER-) and estrogen receptor positive (ER+) human breast cancer cells, by examining its estrogenic effect in ER(+) human breast cancer cells. As cell models, the ER(+) cell line MCF-7 and the ER(-) cell line MDA-MB231 were used. The ellagic acid was tested in each time and concentration. Cell cycle was analyzed by flow cytometry. Expressions of bcl-xL, cytochrome c and caspase 3 were evaluated to investigate the mitochondrial apoptotic pathway. To show effects of ellagic acid to IAP family, expressions of XIAP and survivin were investigated. Expressions of bcl-xL , cytochrome c, and survivin were evaluated by western blotting. Expressions of caspase 3 and XIAP were examined by semiquantitative RT-PCR. In MCF-7 cells, anti-estrogenic effect was evaluated using MTT assay and western blotting of pS2 and c-fos. The ellagic acid in ER(-) MDA-MB-213 cells showed significant anti-proliferative effects with dose dependent pattern. The anti-proliferative effects in ER (+) MCF-7 cells were observed in only high concentration. Anti-proliferation effect was observed in ER-negative cells after treatment with dose-time dependent pattern. However, there was no time dependent result showed anti-proliferative effect in MCF-7 cells. The effects of ellagic acid on cell cycle in MDA-MB-231 and MCF-7 cells were determined busing flow cytometry. The results indicated that ellagic acid has no effect on cell cycle in both breast cancer cells. In MDA-MB-231, expression of bcl-xL was decreased with the decreasing of concentration of ellagic acid. And the expression of caspase 3 showed no change. Ellagic acid decreased the expression of XIAP and survivin. In MCF-7, expressions of bcl-xL and cytochrome c showed no change after treatment of ellagic acid even in high dose. However, expressions of XIAP and survivin were decreased and expression of caspase 3 has tendency to increase. Anti-estrogenic activity was experienced. Ellagic acid prevented the estradiol-induced cell proliferation. It also competed with ICI 182,780 for binding to ER which acts as estrogen antagonist. Ellagic acid can inhibit cancer cell growth which was induced by E2, and that effect looks like to be mediated by ER. We have specifically studied the expression of c-fos and pS2 expression in MCF-7 cells that treated with ellagic acid. pS2 and c-fos were examined for sensitivity to ligand structure in MCF-7 cells. The result indicated that ellagic acid increased c-fos and pS2 and the stimulation by ellagic acid is more powerful than that by E2. In summary, ellagic acid has anti-proliferative effect in ER(-) MDA-MB-231 cells. This effect of ellagic acid is through the decrease of expression of bcl-xL, XIAP and survivin in MDA-MB-231 cells. However, the expression of bcl-xL showed no change in MCF-7 cells. It should be the different point of response to ellagic acid between MDA-MB-231 and MCF-7 cells. Our results indicated that ellagic acid can directly suppress ER-dependent pathway. It inhibits the cell proliferation which was induced by E2 and stimulate the synthesis of c-fos and pS2 protein through ER. Ellagic acid has a potency as a chemotherapeutic agent in ER(-) breast cancer and a selective estrogen receptor modulator in ER(+) breast cancer.;배경 및 목적: 석류는 항염증작용, 항산화작용, 혈관신생의 억제 및 종양 세포에서 세포 분열을 억제하는 효능이 있다. 역학적으로 석류의 섭취는 유방암의 발생 억제와 연관이 있는 것으로 알려져 있다. 이러한 효능을 일으키는 석류의 가장 중요한 성분은 ellagic acid이다. 하지만, 어떤 과정을 통하여 이러한 작용이 일어나는지에 대하여는 잘 알려진 바가 없다. 대부분의 연구는 ellagic acid가 세포자연사를 일으켜 항암작용을 한다고 보고하고 있으나, 최근의 몇몇 연구는 ellagic acid가 여성호르몬 수용체와 결합하여 이러한 작용을 일으킨다고 주장하였다. 본 연구의 목적은 유방암 세포주에서 여성호르몬 수용체 유무에 따른 ellagic acid의 생화학적 작용을 연구하여 보며, 여성호르몬 수용체 양성의 유방암 세포주에서 ellagic acid가 항 여성호르몬 작용을 일으키는가에 대하여 조사하여보고자 하였다. 방법: 여성호르몬 수용체 양성의 세포주로서 MCF-7 세포주를, 여성호르몬 수용체 음성의 세포주로서 MDA-MB-231 세포주를 선택하였다. Ellagic acid의 작용은 농도별, 시간별로 실험하였다. 세포 주기는 flow cytometry를 이용하여 확인하였으며, bcl-xL, cytochrome c, caspase 3, XIAP 및 survivin 의 발현을 조사하여보았다. MCF-7 세포주에서의 ellagic acid의 항 여성호르몬 작용을 연구하기 위하여 ellagic acid와 17β-estradiol(E2), ICI 182,780의 영향을 MTT-assay로 확인하였다. 또한 pS2와 c-fos 단백질의 발현도 같이 분석하였다. 결과: MDA-MB-213 세포주에서 ellagic acid는 통계학적으로 유의하게 농도와 시간의 증가에 따라 세포의 증식을 억제하였다. 그러나 MCF-7 세포주에서는 이러한 세포 증식 억제의 효과가 단지 고농도에서만 나타났으며, 시간에 따른 차이도 나타나지 않았다. 세포주기는 양 세포군 모두에서 ellagic acid에 의한 변화가 나타나지 않았다. MDA-MB-231 세포주에서는 bcl-xL의 발현이 ellagic acid의 농도가 증가함에 따라 감소하였지만, MCF-7 세포주는 ellagic acid의 농도가 증가하여도 bcl-xL의 발현에 차이가 없었다. 양 세포군 모두에서 XIAP와 survivin이 감소하는 경향을 보였지만, 감소의 폭은 MDA-MB-231에서 훨씬 크게 나타났다. MCF-7 세포주에서 ellagic acid는 E2에 의해 유발되는 세포 증식을 억제하였으며, 여성호르몬 수용체 억제제인 ICI 182,780과 상호 경쟁적으로 작용하였다. ellagic acid에 의한 pS2와 c-fos의 발현은 E2에 의한 발현보다도 오히려 더 강하게 나타났다. 결론 : ellagic acid는 MDA-MB-231 세포주에서 항증식효과를 나타내었으며, 이러한 효과는 ellagic aicd에 의한 bcl-xL, XIAP, survivin의 발현 억제와 연관이 있을 것으로 생각된다. MCF-7 세포주에서는 항증식 효과가 거의 나타나지 않았으며, 이는 bcl-xL의 발현이 ellagic acid에 영향을 받지 않은 것과 연관이 있을 것으로 생각된다. MCF-7 세포주에서 ellagic acid는 여성호르몬에 직접적으로 작용하여 항여성호르몬 작용을 일으킨다. Ellagic acid는 여성호르몬 수용체 음성 유방암에서는 항암효과를 지닌 약물로서, 여성호르몬 수용체 양성 유방암에서는 선택적 여성호르몬 수용체 조정자(selective estrogen receptor modulators)로서 사용되어질 가능성이 있는 물질로 사료된다.