t47d kbluc reporter cells  (ATCC)

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy

doi: 10.1016/j.jsbmb.2019.105415

Figure Lengend Snippet: Biologic activity of selected SERD candidates. A) Downregulation of ER protein. ER-positive MCF-7 cells were treated in phenol-red free RPMI 1640 without FBS and containing vehicle control (CON) or 100 nM concentrations of either fulvestrant (FX) or antiestrogens 105, 109, 121, 140, 151, 160 or JD128 in vitro. After 4 hours, cells were harvested and processed for PAGE and Western immunoblots using ERα antibody (1D5, Thermofisher Scientific). RPL13A was used as a loading control. B) Specific [3H]estradiol-17β (E2) binding and competition for binding by antiestrogen JD128 or fulvestrant (FX) at 10 nM was assessed in human MCF-7 breast cancer cells using methods as described before [36, 41]. C) Response of the ERE-luciferase T47D reporter construct to estrogen antagonists fulvestrant (10 nM) or JD128 (10 nM) in combination with 2nM 17β-estradiol as compared to treatment with 17β-estradiol alone (E2; 2 nM). Cells were dosed with either E2 alone or with SERDs combined with E2 in phenol red-free medium with 0.1% dextran-coated charcoal-treated FBS in luminometer plates. Data are presented as relative light units (RLU) relative to that of E2 alone in three replicate assays (4 wells per replicate) + SEM. Treatment with E2 alone induced a 12-fold induction of ER-dependent luciferase activity quantified as RLU relative to vehicle control-treated samples.

Article Snippet: A stable ER-positive T47D ERE luciferase reporter cell line, in which the ERE and the reporter luciferase gene are consistently expressed in the cell line were used in this study (Signosis).

Techniques: Activity Assay, Control, In Vitro, Western Blot, Binding Assay, Luciferase, Construct

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy

doi: 10.1016/j.jsbmb.2019.105415

Figure Lengend Snippet: Steroid-like SERD 128 inhibits estrogen-induced BC cell proliferation in vitro and in vivo. A) ER-positive MCF-7, T47D and ZR75 cells were grown in phenol red-free media with 1% DCC-FBS for 48 hr., then treated 48 hr. with 2 nM estradiol-17β alone (control) or in combination with 10 nM doses of JD128. Note that MCF-7 cell populations included cells with no HER2-overexpression (MCF-7/PAR), cells with HER2-overexpression (MCF-7/HER2) and MCF-7 cells with tamoxifen resistance (MCF-7/TMR). Cell proliferation is shown as % of that in estradiol-treated controls (n=3 experiments). Inhibition of cell proliferation in MCF-7/PAR, MCF-7/HER2, MCF-7/TMR, T47D and ZR75 cells averaged 98%, 85%, 94%, 97% and 98% as compared to estradiol-treated controls. JD128 significantly blocked proliferation in all BC cell models in vitro (P<0.001). Of note, E2 alone stimulated cell proliferation several-fold in each cell line as compared to cells treated only with vehicle (not shown). B) JD128 inhibits growth of human breast tumor xenografts in vivo. MCF-7 human breast cancer cells were subcutaneously inoculated in nude mice previously primed with estradiol pellets. When animals developed tumors of comparable size they were randomized to treatment with vehicle control (control) or JD128 at 15 and 75 mg/kg once a day by oral gavage for 28 days. Tumors were measured every 3 days, and tumor volume was calculated as V= (l ×w × w)/2). Results are expressed as mean ± SEM. *** P < 0.001 as compared to control group.

Article Snippet: A stable ER-positive T47D ERE luciferase reporter cell line, in which the ERE and the reporter luciferase gene are consistently expressed in the cell line were used in this study (Signosis).

Techniques: In Vitro, In Vivo, Control, Over Expression, Inhibition