The Bik BH3-only protein is induced in estrogen-starved and antiestrogen-exposed breast cancer cells and provokes apoptosis

Abstract

Evidence has been accumulating that some estrogen-dependent human breast cancers require estrogen for not only proliferation but also survival. To obtain insights into the molecular mechanisms of apoptosis of breast cancer cells subjected to estrogen starvation or exposed to antiestrogens, we characterized changes in the gene expression profile of MCF-7/BUS human breast cancer cells and revealed a strong induction of Bik, a member of the BH3-only proapoptotic proteins. The Bik mRNA transcript and protein were strongly induced by estrogen starvation or exposure to fulvestrant, a pure antiestrogen that competes with the natural estrogens for binding to the estrogen receptors. This Bik induction preceded apoptotic cell death, which was blocked by zVAD-fmk, a pancaspase inhibitor. Amounts of the Bcl-2-related proteins, such as Bcl-2, Bcl-XL, or Bax, showed only marginal changes in the presence or absence of estrogens or antiestrogens. Suppression of Bik expression by using the small interfering RNA effectively blocked the fulvestrant-induced breast cancer cell apoptosis. These results indicate that Bik is induced in MCF-7/BUS cells in the absence of estrogen signaling and plays a critical role in the antiestrogen-provoked breast cancer cell apoptosis.

Fig. 1.

Estrogen depletion provokes MCF-7/BUS cell apoptosis. (A and C) Estrogen starvation induces apoptosis. Cells were cultured in the presence of indicated concentrations of E2 for 72 h with or without 50 μM zVAD-fmk. The appearance of the culture was photographed (A), and numbers of apoptotic cells (round-shaped and detached form the dish) and live cells were counted (C Left; mean ± SEM, n = 3). (B and C) Fulvestrant-induced apoptosis. Cells were cultured in the presence or absence of 100 nM fulvestrant for 24–72hin the medium containing ≈60 pM serum-derived E2. The culture was photographed (B), and numbers of apoptotic cells and live cells were counted (C Right; mean ± SEM, n = 3).

Fig. 2.

Effects of E2 and antiestrogen on expression of the mRNA transcripts for Bcl-2 family proteins in breast cancer cells. (A and B) DNA microarray data. (C) RT-PCR. MCF-7/BUS cells were cultured in the presence of indicated concentrations of E2 for 48 h (A and C) or in the absence of E2 for indicated periods of time (B and C), and the relative amounts of the mRNA transcripts were determined by the Affymetrix DNA microarray. Each datum point represents mean ± SEM (n = 5).

Fig. 3.

Effects of E2 and antiestrogen on expression of the Bcl-2 family proteins in breast cancer cells. (A–E) Western blotting. Actin or GAPDH was visualized on the same blot as control. (A) Suppression of Bik expression by E2. MCF-7/BUS cells were cultured in the presence of indicated concentrations of E2 for 48 h. (B) Induction of Bik by estrogen starvation. MCF-7/BUS cells were subjected to estrogen starvation for up to 2 days in the presence or absence of 50 μM zVAD-fmk. (C) Induction of Bik by fulvestrant. MCF-7/BUS cells were cultured in the presence of indicated concentrations of fulvestrant for 24–72 h. The medium contained ≈60 pM serum-derived E2. (D) Effects of E2 on expression of other Bcl-2 family proteins. MCF-7/BUS cells were cultured in the presence (+) or absence (–) of 30 pM E2 for 48 h. (E) Diminished induction of Bik in E8CASS, an estrogen-independent MCF-7 derivative. Cells were cultured in the presence or absence of E2 for 48 h. (F) Expression of the Bik mRNA transcripts (RT-PCR) or protein (Western blotting) in human breast cancer cells. Cells were cultured for 48 h in the medium containing ≈60 pM serum-derived E2 with or without 100 nM fulvestrant.

Fig. 4.

Bik is necessary for the fulvestrant-induced MCF-7/BUS cell apoptosis. (A) Suppression of Bik expression by siRNA. Cells were transiently transfected with Bik-specific (BIK) or sequence-scrambled control (CON) siRNA and then cultured in the presence or absence of 100 nM fulvestrant for 48 h, followed by evaluation of expression of Bik and actin (as control) by Western blotting. (B) Suppression of fulvestrant-induced cell death by Bik-specific siRNA. Cells were transiently transfected with Bik-specific (BIK) or sequence-scrambled control (CON) siRNA and then cultured in the presence or absence of 100 nM fulvestrant for 72 h. The phase-contrast images of the cultures show apoptotic cells that are detached from the dish and round-shaped. (C) Suppression of fulvestrant-induced PARP cleavage by Bik-specific siRNA. Cells were transiently transfected with Bik-specific (BIK) or sequence-scrambled control (CON) siRNA and then cultured in the presence or absence of 100 nM fulvestrant for 72 h. The full-length (116 kDa) and apoptosis-signature fragment (85 kDa) of PARP were detected by Western blotting.