Figure 4.

Purified glyceollin I preferentially inhibits LTED cells and induces apoptosis. (A) The Eleanor RNA cloud was inhibited with glyceollin I purified from activated soybeans, equivalently to a known inhibitor, resveratrol¹⁰. LTED cells were treated as indicated and subjected to RNA FISH to visualize Eleanor foci (green). The nucleus was counterstained with DAPI (blue). Scale bar, 10 µm. (B) Transcription of ESR1 mRNA was inhibited with glyceollin I. Quantitative RT-PCR was performed to measure relative ESR1 mRNA levels in LTED cells treated as indicated. Values were normalized against GAPDH mRNA, and values for cells treated with DMSO (control) were set to 1. The bars represent the means ± S.D. n >3, *p < 0.05; **p < 0.01. (C) LTED cell proliferation was inhibited by glyceollin I. LTED cells were treated as indicated, and cell viabilities were measured. The values represent the means ± S.D. n >3, *p < 0.05. (D) LTED cell growth was inhibited by glyceollin I in a dose-dependent manner. The inhibition by glyceollin I was more effective than that by resveratrol. The value of DMSO treatment (control) was set to 100. The values represent the means ± S.D. n = 3. (E) Glyceollin I preferentially inhibited growth of LTED cells. IMR-90 (normal human fibroblast derived from fetal lung), MCF7 and LTED cells were treated as indicated, and cell viabilities were measured with a colorimetric assay at Ab 490 nm (Kit-8). *p < 0.05; **p < 0.01. (F) Apoptosis was induced in LTED cells by glyceollin I. Cells were treated with indicated compounds for 24 h, stained with Annexin V. FACS analysis showed that glyceollin I and resveratrol induced apoptosis, as estradiol. The values represent the means ± S.D. n = 3, *p < 0.05; **p < 0.01, ***p < 0.001. Corresponding FACS data are shown in Supplementary Fig. S4.