Plumbagin sensitizes breast cancer cells to tamoxifen-induced cell death through GRP78 inhibition and Bik upregulation

Abstract

The glucose regulated protein 78 (GRP78) is a major chaperone of the endoplasmic reticulum, and a prosurvival component of the unfolded protein response. GRP78 is upregulated in many types of cancers, including breast cancer. Research has suggested that GRP78 overexpression confers chemoresistance to anti-estrogen agents through a mechanism involving the inhibition of a pro-apoptotic BH3-only protein, Bik. In the present research the role of plumbagin, a naturally occurring naphthoquinone, in GRP78-associated cell death inhibition was examined. The results demonstrated that plumbagin inhibits GRP78 activity and GRP78 inhibition contributes to plumbagin-mediated cell death induction. Furthermore, Bik upregulation was associated with plumbagin-induced cell death and an increase in plumbagin-mediated Bik induction was observed upon GRP78 downregulation. Plumbagin sensitized estrogen-positive breast cancer cells to tamoxifen and the association of GRP78 inhibition and Bik upregulation in plumbagin-mediated cell sensitization was shown. Collectively, the results of this research suggest that plumbagin inhibits the antiapoptotic activity of GRP78 leading to Bik upregulation and apoptosis induction, which contributes to the sensitization of breast cancer cells to tamoxifen.

Figure 1. Cytotoxic and apoptosis-inducing activity of plumbagin towards ER-positive breast cancer cells.

(A) Cytotoxic activity of plumbagin. MCF-7 and T47D cells were treated with plumbagin (0–5 μM) for 24 h with plumbagin and cell survival was assessed with the MTT assay. (B) Apoptotic changes in plasma membrane induced by plumbagin. Cells were treated with plumbagin (0–5 μM) for 24 h, stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Values represent mean ± SE of three independent experiments. p < 0.05 (*) indicates differences between control and plumbagin-treated cells. (C) Effects of plumbagin on the expression levels of Bik, Bax, Bak and Bcl-2. Cells were treated with plumbagin (0–2.5 μM) for 24 h and protein levels were determined with Western blot analysis.

Figure 2. The role of GRP78 in plumbagin-mediated apoptosis induction in estrogen-positive breast cancer cells.

(A) Effects of plumbagin on the expression levels of GRP78. MCF-7 and T47D cells were treated with plumbagin (0–2.5 μM) for 24 h and protein levels were determined with Western blot analysis. (B) Silencing of GRP78 in MCF-7 and T47D cells. Cells were transiently transfected with GRP78 siRNA and after 24 h GRP78 levels were determined with Western blot analysis. (C) Overexpression of GRP78 was performed by transiently transfecting cells with a GRP78 CRISPR/dCas9 activation plasmid. 24 h after transfection, GRP78 levels were determined with Western blot analysis. (D) Influence of GRP78 silencing and (E) GRP78 upregulation on the induction of apoptosis by plumbagin. 24 h-post transfection cells were treated with plumbagin (0.5 and 1 μM) for 24 h after which cells were stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Values represent mean ± SE of three independent experiments. p < 0.05 (*) indicates differences between control and GRP78-downregulated and upregulated cells treated with plumbagin.

Figure 3. Plumbagin sensitizes breast cancer cells to tamoxifen.

(A) Influence of plumbagin on tamoxifen-induced apoptosis. Cells were incubated with a combination of plumbagin (0.5 or 1 μM) and tamoxifen (1 and 5 μM) for 24 h after which cells were stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Values represent mean ± SE of three independent experiments. p < 0.05 (*) indicates differences between single agent-treated and combination-treated cells. (B) Synergistic effect of plumbagin and tamoxifen on estrogen-positive breast cancer cell viability. MCF-7 and T47D cells were exposed to combinations of plumbagin and tamoxifen. Cell viability was assessed 24 h after exposure with the MTT assay.

Figure 4. The involvement of GRP78 and Bik in the induction of apoptosis by combination treatment of breast cancer cells with plumbagin and tamoxifen.

MCF-7 and T47D cells were transiently transfected with (A) GRP78 siRNA or (B) Bik siRNA. 24 h-post transfection cells were treated for an additional 24 h with plumbagin and tamoxifen. Cells were stained with Annexin V-PE/7-AAD and analyzed by flow cytometry. Values represent mean ± SE of three independent experiments. p < 0.05 (*) indicates differences between combination-treated control and GRP78 or Bik-downregulated cells. (C) Influence of combination treatment with plumbagin and tamoxifen on expression levels of GRP78 and Bik. Cells were treated for 24 h with plumbagin (0.5 μM) and tamoxifen (1 μM) and GRP78 or Bik levels were determined with Western blot analysis. (D) Influence of GRP78 silencing on plumbagin-mediated Bik induction. 24 h-post transfection cells were treated with plumbagin (0.5 μM) for 24 h and the expression levels of Bik were determined with Western blot analysis.