Suppression of the death gene BIK is a critical factor for resistance to tamoxifen in MCF-7 breast cancer cells

Abstract

Apoptosis is controlled by the BCL-2 family of proteins, which can be divided into three different subclasses based on the conservation of BCL-2 homology domains. BIK is a founding member of the BH3-only pro-apoptotic protein family. BIK is predominantly localized in the endoplasmic reticulum (ER) and induces apoptosis through the mitochondrial pathway by mobilizing calcium from the ER to the mitochondria. In this study, we determined that suppression of the death gene Bik promotes resistance to tamoxifen (TAM) in MCF-7 breast cancer cells. We utilized small interfering (siRNA) to specifically knockdown BIK in MCF-7 cells and studied their response to tamoxifen. The levels of cell apoptosis, the potential mitochondrial membrane (∆Ψ(m)), and the activation of total caspases were analyzed. Western blot analysis was used to determine the expression of some BCL-2 family proteins. Flow cytometry studies revealed an increase in apoptosis level in MCF-7 cells and a 2-fold increase in relative BIK messenger RNA (mRNA) expression at a concentration of 6.0 μM of TAM. BIK silencing, with a specific RNAi, blocked TAM-induced apoptosis in 45 ± 6.78% of cells. Moreover, it decreased mitochondrial membrane potential (Ψm) and total caspase activity, and exhibited low expression of pro-apoptotic proteins BAX, BAK, PUMA and a high expression of BCl-2 and MCL-1. The above suggests resistance to TAM, regulating the intrinsic pathway and indicate that BIK comprises an important factor in the process of apoptosis, which may exert an influence the ER pathway, which regulates mitochondrial integrity. Collectively, our results show that BIK is a central component of the programmed cell death of TAM-induced MCF-7 breast cancer cells. The silencing of BIK gene will be useful for future studies to establish the mechanisms of regulation of resistance to TAM.

Figure 1.

The messenger RNA (mRNA) and protein levels of Bik were increased in MCF-7 cells treated with tamoxifen (TAM). (A) MCF-7 cells were treated with TAM by determining Ec₅₀. (B) MCF-7 cells were treated with TAM for 24 h and subjected to RT-PCR analyses. (C) Cell lysates were prepared and subjected to western blot analyses for Bik and β-actin as a control; one representative of at least three independent experiments with similar results is shown. Each value represents the mean + standard error of the mean (SE) (bars) of three independent experiments. ^*p<0.05, according to the Student’s t-test.

Figure 2.

Transfection and silencing of Bik in the human breast cancer line MCF-7. (A and B) Cells were transiently transfected with Bik-specific (BIK) or sequence-scrambled control and them cultured in the presence or absence of 6.0 μM of tamoxifen (TAM) for 24 h, followed by evaluation of expression of Bik and β-actin (as control) by real-time RT-PCR and western blot analysis. The groups were analyzed by one-way ANOVA with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated. Representative figures of three independent experiments carried out in triplicate are shown.

Figure 3.

Suppression of Bik expression does not alter the mitochondrial membrane potential. (A) MCF-7 cells [non-treated, scrambled, siRNAiBIK, siRNAiBIK/tamoxifen (TAM), and TAM] were stained with JC-1 for loss of ΔΨ_m using flow cytometry. JC-1 fluorescence is seen in both the FL-2 (red) and FL-1 channels (green). In FL-1 channels are cells with low levels of ΔΨ_m, while Fl-2 channel indicates cells with stable levels of ΔΨ_m. (B) Representative graphic of fluorescence intensities. One representative of at least three independent experiments with similar results is shown. The groups were analyzed by one-way analysis of variance (ANOVA) with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated. Representative figures of three independent experiments carried out in triplicate are shown.

Figure 4.

Silencing of Bik does not promote the release of cytochrome in response to tamoxifen. (A) Cell lysates were prepared and subjected to western blot analyses for cytochrome c (Cyt C) and β-actin as control. (B) Densitometry analysis of the expression levels of Cyt C. One representative of at least three independent experiments with similar results is shown. The groups were analyzed by one-way analysis of variance (ANOVA) with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated. Representative figures of three independent experiments carried out in triplicate are shown.

Figure 4.

Silencing of Bik does not promote the release of cytochrome in response to tamoxifen. (A) Cell lysates were prepared and subjected to western blot analyses for cytochrome c (Cyt C) and β-actin as control. (B) Densitometry analysis of the expression levels of Cyt C. One representative of at least three independent experiments with similar results is shown. The groups were analyzed by one-way analysis of variance (ANOVA) with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated. Representative figures of three independent experiments carried out in triplicate are shown.

Figure 5.

Suppression of the Bik gene promotes inhibition of caspase activity. (A) MCF-7 [non-treated, scrambled, siRNAiBIK, siRNAiBIK/tamoxifen (TAM), and TAM] were then labeled with the FAM-VAD-FMK probe and PI for determination of caspase activation. The samples were analyzed on a flow cytometer with 488 nm excitation using 530 nm band pass and 670 nm long pass emission filters. (B) Representative graphic of fluorescence intensities. Results are representative of three independent experiments. The groups were analyzed by one-way ANOVA with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated. Representative figures of three independent experiments carried out in triplicate are shown.

Figure 6.

Suppression of the Bik gene promotes resistance in the process of apoptosis. (A) For corroborate apoptosis MCF-7 cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry. (B) Representative graphic of fluorescence intensities for apoptosis. Results are representative of three independent experiments. The groups were analyzed by one-way ANOVA with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated. Representative figures of three independent experiments carried out in triplicate are shown.

Figure 7.

Anti-apoptotic proteins are highly expressed in MCF cells transfected with BIK and treated with TAM. (A and B) MCF-7 cells were incubated for 48 h, cell lysates were prepared from the cells, and subjected to western blot analyses for Bcl-2, and Mcl-1. One representative of at least three independent experiments with similar results is shown. (C and D) Band intensity in Bcl-2, and Mcl-1 was quantified by densitometric analysis. The groups were analyzed by one-way analysis of variance (ANOVA) with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated. Representative figures of three independent experiments carried out in triplicate are shown.

Figure 8.

Suppression of BIK regulated negatively pro-apoptotic proteins in MCF-7 cells treated with TAM. (A) MCF-7 cells were incubated for 48 h, cell lysates were prepared from the cells, and subjected to western blot analyses for BAX, BAK and PUMA. One representative of at least three independent experiments with similar results is shown. (B, C and D) Band intensity in BAX, BAK and PUMA was quantified by densitometric analysis. The groups were analyzed by one-way analysis of variance (ANOVA) with the Tukey’s test. ^*p<0.05; ^**p<0.01; ^***p<0.001 vs. non-treated; n.s., not significant. Representative figures of three independent experiments carried out in triplicate are shown.