The role of miRNAs 34a, 146a, 320a and 542 in the synergistic anticancer effects of methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC) with doxorubicin in breast cancer cells

Abstract

Combination Index (CI) analysis suggested that MBIC and doxorubicin synergistically inhibited up to 97% of cell proliferation in ER⁺/PR⁺MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. Moreover, treatment of the breast cancer cells with the combined drugs resulted in lower IC₅₀ values in contrast to the individual drug treatment. Small noncoding microRNAs (miRNA) may function as non-mutational gene regulators at post-transcriptional level of protein synthesis. In the present study, the effect of the combined treatment of MBIC and doxorubicin on the expression level of several miRNAs including miR-34a, miR-146a, miR-320a and miR-542 were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. These miRNAs have the potential to alter the protein level of survivin, the anti-apoptotic protein and reduce the metastatic activity in human breast cancer cell lines by interfering with the nuclear accumulation of NF-κB. Our results demonstrated the several fold changes in expression of miRNAs, which is drug and cell line dependent. This finding demonstrated a functional synergistic network between miR-34a, miR-320a and miR-542 that are negatively involved in post-transcriptional regulation of survivin in MCF-7 cells. While in MDA-MB-231 cells, changes in expression level of miR-146a was correlated with inhibition of the nuclear translocation of NF-κB. The overall result suggested that alteration in protein level and location of survivin and NF-κB by miR-34a, miR-320a, miR-146a and miR-542, remarkably influenced the synergistic enhancement of combined MBIC and doxorubicin in treatment of aggressive and less aggressive human breast cancer cell lines.

Keywords: Breast cancer; NF-κB; Survivin; Synergism; microRNA.

Figure 1. The role of miRNAs (miRs) miR-34a and miR-146a in synergistic effect of MBIC with doxorubicin in MCF-7 cells.

Total RNA was extracted and reverse transcribed with their specific designed primers. The level of miRs were normalized to a small RNA (U6) and compared with this endogenous control (set as one-fold). Individual miRNA profiling was done using qRT-PCR analysis. The expression level of miR-34a (A & B) and miR-146a (C & D) in MCF-7 cells following 24 h treatment with MBIC, doxorubicin (A & C) and combination (B & D) of these two drugs are shown. The bars show the fold change of each treated group compared with untreated group (Cont.) as a horizontal dashed line. * p < 0.05. ** p < 0.01, *** p < 0.001, **** p < 0.0001 and “ns” indicates not significant compared with untreated control (Cont.). Results are mean + standard deviation of three independent experiments.

Figure 2. The role of miRNAs (miRs) miR-320a and miR-542 in synergistic effect of MBIC with doxorubicin in MCF-7 cells.

Total RNA was extracted and reverse transcribed with their specific designed primers. The level of miRs were normalized to a small RNA (U6) and compared with this endogenous control (set as one-fold). Individual miRNA profiling was done using qRT-PCR analysis. The expression level of miR-320a (A & B) and miR-542a (C & D) in MCF-7 cells following 24 h treatment with MBIC, doxorubicin (A & C) and combination (B & D) of these two drugs are shown. The bars show the fold change of each treated group compared with untreated group (Cont.) as a horizontal dashed line. * p < 0.05. ** p < 0.01, **** p < 0.0001 and “ns” indicates not significant compared with untreated control (Cont.). Results are mean + standard deviation of three independent experiments.

Figure 3. The role of miRNAs (miRs) miR-34a and miR-146a in synergistic effect of MBIC with doxorubicin in MDA-MB-231 cells.

Total RNA was extracted and reverse transcribed with their specific designed primers. The level of miRs were normalized to a small RNA (U6) and compared with this endogenous control (set as one-fold). Individual miRNA profiling was done using qRT-PCR analysis. The expression level of miR-34a (A & B) and miR-146a (C & D) in MDA-MB-231 cells following 24 h treatment with MBIC, doxorubicin (A & C) and combination (B & D) of these two drugs are shown. The bars show the fold change of each treated group compared with untreated group (Cont.) as a horizontal dashed line. * p < 0.05. ** p < 0.01, *** p < 0.001, **** p < 0.0001 and “ns” indicates not significant compared with untreated control (Cont.). Results are mean + standard deviation of three independent experiments.

Figure 4. The role of miRNAs (miRs) miR-320a and miR-542 in synergistic effect of MBIC with doxorubicin in MDA-MB-231 cells.

Total RNA was extracted and reverse transcribed with their specific designed primers. The level of miRs were normalized to a small RNA (U6) and compared with this endogenous control (set as one-fold). Individual miRNA profiling was done using qRT-PCR analysis. The expression level of miR-320a (A & B) and miR-542a (C & D) in MDA-MB-231 cells following 24 h treatment with MBIC, doxorubicin (A & C) and combination (B & D) of these two drugs are shown. The bars show the fold change of each treated group compared with untreated group (Cont.) as a horizontal dashed line. * p < 0.05. ** p < 0.01, *** p < 0.001, **** p < 0.0001 and “ns” indicates not significant compared with untreated control (Cont.). Results are mean + standard deviation of three independent experiments.

Figure 5. Illustration of elevated or reduced expression level of miRNAs.

Four miRNAs including miR-34a, miR-146a, miR-320a and miR-542 in presence or absence of MBIC and doxorubicin in monotherapy or in combination. X shape represents no change in expression level. Up-arrows and down-arrows represent elevation and reduction of expression level respectively.

Figure 6. Protein level of survivin following treatment with MBIC and doxorubicin, individually or in combination in MCF-7 and MDA-MB-231 human breast cancer cell lines.

(A & B) Representative images from three independent experiments showing western blot analysis to assess the differences in the effect of MBIC, doxorubicin and their combination on the protein level of survivin. MCF-7 (A) and MDA-MB-231 (B) cell lines were treated with MBIC and doxorubicin at 2 × IC₅₀ concentration including 1.5 µM of MBIC against MCF-7 cells; 40 µM of MBIC against MDA-MB-231 cells; 11 µM of doxorubicin against MCF-7 cells, and 20 µM of doxorubicin against MDA-MB-231 cells. Selected concentrations in combination therapy were twice the sufficient concentrations of both drugs to cause 50% of cell death. These concentrations were selected to be compared with the expression level of miRNAs at 2 × IC₅₀. (C & D) The relative intensity of survivin was normalized with β-actin as internal standard. **** p < 0.0001 and “ns” indicate not significant versus untreated control (Cont.).

Figure 7. NF-κB activation and its correlation with expression level of miR-146a in MCF-7 cell line.

(A & B) Representative images from three independent experiments showing western blot analysis to assess the differences in the effect of MBIC, doxorubicin and their combination on the cytoplasmic (A) and nuclear (B) accumulation of NF-κB. MCF-7 cells were treated with MBIC and doxorubicin at 2 × IC₅₀ concentration. 1.5 µM of MBIC and 11 µM of doxorubicin. Selected concentrations in combination therapy were twice the sufficient concentrations of both drugs to cause 50% of cell death. These concentrations were selected to be compared with the expression level of miRNAs at 2 × IC₅₀. GAPDH and Lamin B1 proteins were selected as endogenous normalizer reference proteins. These two endogenous normalizers were also probed as negative controls for the opposing fractions (GAPDH for the nuclear and Lamin B1 for the cytosolic fractions). (C & D) The relative intensity of NF-κB was normalized with GAPDH (in cytoplasmic extraction) and Lamin B1 (in nuclear extraction). **** p < 0.0001 versus untreated control (Cont.).

Figure 8. NF-κB activation and its correlation with expression level of miR-146a in MDA-MB-231 cell-line.

(A & B) Representative images from three independent experiments showing western blot analysis to assess the differences in the effect of MBIC, doxorubicin and their combination on the cytoplasmic (A) and nuclear (B) accumulation of NF-κB. MDA-MB-231 cells were treated with MBIC and doxorubicin at 2 × IC₅₀ concentration. 40 µM of MBIC and 20 µM of doxorubicin. Selected concentrations in combination therapy were twice the sufficient concentrations of both drugs to cause 50% of cell death. These concentrations were selected to be compared with the expression level of miRNAs at 2 × IC₅₀. GAPDH and Lamin B1 proteins were selected as endogenous normalizer reference proteins. These two endogenous normalizers were also probed as negative controls for the opposing fractions (GAPDH for the nuclear and Lamin B1 for the cytosolic fractions). (C & D) The relative intensity of NF-κB was normalized with GAPDH (in cytoplasmic extraction) and Lamin B1 (in nuclear extraction). *** p < 0.001 and **** p < 0.0001 versus untreated control (Cont.).