Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR

<div><p>Although drug resistance is often observed in metastatic recurrence of breast cancer, little is known about the intrinsic drug resistance in such metastases. In the present study, we found, for the first time, that MDA-MB-231BR, a brain metastatic variant of a human breast cancer cell line, was refractory to treatment with 5-fluorouracil (5-FU) even without chronic drug exposure, compared to its parent cell line, MDA-MB-231, and a bone metastatic variant, MDA-MB-231SCP2. Both the mRNA and protein levels of COX-2 and BCL2A1 in MDA-MB-231BR were significantly higher than those in MDA-MB-231 or MDA-MB-231SCP2. Neither the COX-2 inhibitor celecoxib nor the NF-κB inhibitor BAY11-7082 could sensitize MDA-MB-231BR to 5-FU, indicating that COX-2 plays little, if any, role in the resistance of MDA-MB-231BR to 5-FU. Although BCL2-family inhibitor ABT-263 failed to sensitize MDA-MB-231BR to 5-FU at a dose at which ABT-263 is considered to bind to BCL2, BCL2-xL, and BCL2-w, but not to BCL2A1, ABT-263 did sensitize MDA-MB-231BR to 5-FU to a level comparable to that in MDA-MB-231 at a dose of 5 μM, at which ABT-263 may disrupt intracellular BCL2A1 protein interactions. More importantly, <i>BCL2A1</i> siRNA sensitized MDA-MB-231BR to 5-FU, whereas the overexpression of <i>BCL2A1</i> conferred 5-FU-resistance on MDA-MB-231. These results indicate that BCL2A1 is a key contributor to the intrinsic 5-FU-resistance in MDA-MB-231BR. It is interesting to note that the drug sensitivity of MDA-MB-231BR was distinct from that of MDA-MB-231SCP2 even though they have the same origin (MDA-MB-231). Further investigations pertinent to the present findings may provide valuable insight into the breast cancer brain metastasis.</p></div

Effect of celecoxib on the cytotoxicity of 5-FU against MDA-MB-231BR.

<p>Cytotoxicity of 5-FU to MDA-MB-231BR in the presence of 50 μM celecoxib. Blue broken lines parallel to x-axis represent the cell viability of MDA-MB-231 (Parent) treated with 30 μM 5-FU. Data represent the mean with SEM of four independent samples.</p

Effect of ABT-263 on the cytotoxicity of 5-FU against MDA-MB-231BR.

<p>Cytotoxicity of 5-FU to MDA-MB-231BR in the presence of 0.3 or 5 μM ABT-263. Blue broken lines parallel to x-axis represent the cell viability of MDA-MB-231 (Parent) treated with 30 μM 5-FU. Data represent the mean with SEM of four independent samples (*<i>P</i><0.05, BR treated with 30 μM 5-FU <i>vs</i>. BR treated with 30 μM 5-FU and 5 μM ABT-263).</p

Morphological differences among MDA-MB-231 cell lines, and their cytotoxic response to 5-FU.

<p>(A-C) Photomicrographs of (A) MDA-MB-231, (B) MDA-MB-231SCP2, and (C) MDA-MB-231BR. Scale bar, 100 μm. Scale bars in the insets indicate 50 μm. (D) Cytotoxicity of 5-FU toward MDA-MB-231 (Parent), MDA-MB-231SCP2 (SCP2), and MDA-MB-231BR (BR). The inset shows the IC₅₀ of 5-FU against each cell line. Data represent the mean with SEM of at least six independent samples (***<i>p</i><0.001 <i>vs</i>. Parent and SCP2).</p

Protein expression of BCL2A1 in various human cell lines.

<p>Protein expression of BCL2A1 in various human cell lines.</p

Effect of <i>BCL2A1</i> overexpression on 5-FU-resistance in MDA-MB-231 and T-47D.

<p>BCL2A1 protein expression of (A) MDA-MB-231 and (C) T-47D after transfection with a pCMV-Myc-DDK Entry vector (Vector) or a pCMV-Myc-DDK <i>BCL2A1</i> vector (<i>BCL2A1</i>). Cytotoxicity of 5-FU toward (B) MDA-MB-231 transfected with a <i>BCL2A1</i> vector and (D) T-47D transfected with a <i>BCL2A1</i> vector. The insets show the IC₅₀ of 5-FU against cell lines transfected with vectors. Data represent the mean with SEM of at least three independent samples (***<i>p</i><0.001 <i>vs</i>. MDA-MB-231 transfected with a control vector; *<i>p</i><0.05 <i>vs</i>. T-47D transfected with a control vector).</p

Gene expressions and breast cancer stemness in MDA-MB-231 and its metastatic variants.

<p>(A) Gene expression levels in MDA-MB-231 (Parent), MDA-MB-231SCP2 (SCP2), and MDA-MB-231BR (BR). Candidate genes for drug resistance were screened by RNA sequencing. The intensity of gene expression in the heat map is shown relative to that in the Parent. (B-D) Flow cytometry analysis of MDA-MB-231 and its metastatic variants. (B) MDA-MB-231. (C) MDA-MB-231SCP2. (D) MDA-MB-231BR. Both scatter plots and histograms demonstrate that the CD44⁺/CD24^- populations of the two metastatic variants were not different from those of their parent cell line.</p

Effect of BAY11-7082 on MDA-MB-231BR as an NF-κB inhibitor.

<p>(A) Cytotoxicity of 5-FU to MDA-MB-231BR in the presence of 1 μM BAY11-7082. Blue broken lines parallel to x-axis represent the cell viability of MDA-MB-231 (Parent) treated with 30 μM 5-FU. Data represent the mean with SEM of four independent samples. (B) Changes in protein expressions of COX-2 and BCL2A1 in MDA-MB-231BR in response to 1 μM BAY11-7082.</p

Elevated gene and protein expressions of COX-2 and BCL2A1 in MDA-MB-231BR.

<p>(A) Gene expression levels in MDA-MB-231 (Parent), MDA-MB-231SCP2 (SCP2), and MDA-MB-231BR (BR). Candidate genes for drug resistance were screened by RNA sequencing. The intensity of gene expression in the heat map is shown relative to that in the Parent. (B) COX-2 and BCL2A1 mRNA and protein expression levels in Parent, SCP2, and BR. mRNA expression levels in the SCP2 and BR are shown relative to those in the Parent. Data represent the mean with SEM of at least three independent samples (***<i>P</i><0.001 <i>vs</i>. Parent and SCP2) for mRNA expressions.</p

Additional file 1 of Peripheral-central network analysis of cancer cachexia status accompanied by the polarization of hypothalamic microglia with low expression of inhibitory immune checkpoint receptors

Supplementary Material 1