Inhibitory Effect Of (3R, 6R)-Bassiatin (1) On Cell Proliferation Of Human Breast Cancer MCF-7 Cells And Its Mechanisms

Clinical and experimental data have demonstrated that estrogen plays an important role in the growth of ERαpositive breast cancer and cultured breast cancer cells, especially in MCF-7 cells. The proliferative effects of estrogen are mediated through estrogen receptors, especially ERα. When E2 binds to estrogen receptor ERα, both genomic and nongenomic estrogen-responsive pathways could be activated. This is followed by the activation of several signaling pathways and up-regulation of some genes related to cell proliferation such as CyclinD1, PS2, and PR. Increased expression of these genes enhances mammary epithelial cell proliferation and tumorigenesis.Anti-hormone therapy has become an effective strategy in the treatment of breast cancers. Antiestrogen tamoxifen inhibits E2-stimulated cell proliferation and has become the most frequently prescribed antiestrogen agent in women with estrogen receptor-positive breast cancer. Tamoxifen is thought to act primarily as a competitive inhibitor of estrogen binding to estrogen receptor (ER), and block the upstream cascades. However, some ERαpositive breast cancers fail to respond or eventually develop resistance to tamoxifen, leading to disease progression. Thus, identification of novel antiestrogen agents may provide alternative options for breast cancer patients.(3R,6R)-Bassiatin (1) was isolated from the endogenous fungus Fusarium oxysporum J8-1-2. In the present study, (3R,6R)-bassiatin was found to exert antiestrogen activities by inhibiting E2-stimulated cell proliferation, inducing cell apoptosis, and down-regulating ERαand estrogen-responsive gene (cyclinD1 and PR) expression in ERα-positive breast cancer cells. Our observations strongly suggest that (3R, 6R) -bassiatin may be a novel antiestrogen agent. Considering that ERαplayed a crucial role in the proliferative effect of estrogen, we observed the effect of (3R, 6R)-bassiatin treatment on ERαexpression. It was found that ERαexpression was down-regulated effeciently by (3R,6R) -bassiatin at both mRNA and protein levels in the presence of E2, and that the expression of ERαwas also reduced in the absence of E2, but the effect was weak as compared to that in the presence of E2. The results showed that (3R,6R)-bassiatin could down-regulate ERαexpression, thus suppressing the estrogen-responsive signaling.CyclinD1 plays a prominent role in driving cell cycle progression compared with other members of the CyclinD family. It was found in this study that (3R,6R)-bassiatin induced MCF-7 cell cycle arrest in G0/G1 phase in the presence of E2 in a time- dependent manner. However, the anti-proliferative effect of (3R,6R)-bassiatin was minimal in the absence of E2. We also found that (3R,6R)-bassiatin decreased E2-stimulated cyclinD1 mRNA and protein expression in the presence of E2, while it had a minimal effect in the absence of E2.Another typical estrogen-responsive gene, PR (progesterone receptor, an indicator of a functional ER pathway) showed the same phenomenon when it was treated with (3R,6R)-bassiatin: the increased mRNA level stimulated by E2 could be reduced by co-treatment of (3R,6R)-bassiatin, suggesting that the genomic pathway of estrogen activated by E2 may be blocked by co-treatment with (3R,6R)-bassiatin. Another form of CyclinD1 regulation is phosphorylation-dependent degradation through the ubiquitin-dependent pathway. This process requires CyclinD1 phosphorylation at Thr286. We found that (3R,6R)-bassiatin could stimulate phosphorylation of CyclinD1 at Thr286 and decrease the CyclinD1 protein. This (3R,6R)-bassiatin-induced phosphorylation was strengthened in the presence of E2. These results suggest that (3R,6R)-bassiatin may play a role in the degradation of CyclinD1.In addition, (3R,6R)-bassiatin could induce MCF-7 cell apoptosis, activate ERK-MAPK and suppress GSK3βpathway whether in the presence of 17β-estradiol or not. But whether the apoptosis of MCF-7 cell was caused by ERK-MAPK pathway and why the activity of GSK3βwas suppressed by (3R,6R)- bassiatin need more research.Collectively, our results suggest that (3R,6R)-bassiatin exerted antiestrogen effects by down-regulating the expression of ERαat the mRNA and protein levels, reducing the increased mRNA expression of estrogen-responsive genes (PR and CyclinD1) and inhibiting E2-stimulated cell proliferation. In addition, (3R,6R)- bassiatin could also up-regulate the phosphorylation of CyclinD1 at Thr286, leading to CyclinD1 degradation.The result of molecular docking also showed that the compound (3R,6R)-bassiatin could compete with 17β-estradiol for the crucial His524 of the active sites so as to form a pair of hydrogen bonds.Whether (3R,6R)-bassiatin could be used as a potential drug target for antiestrogen therapy in ERα-positive breast cancer needs more experimental clinical research.