Empagliflozin and Doxorubicin Synergistically Inhibit the Survival of Triple-Negative Breast Cancer Cells via Interfering with the mTOR Pathway and Inhibition of Calmodulin: In Vitro and Molecular Docking Studies

Abstract

Triple-negative breast cancers (TNBCs) comprise 10-15% of all breast cancers but with more resistance affinity against chemotherapeutics. Although doxorubicin (DOX) is the recommended first choice, it has observed cardiotoxicity together with apparent drug resistance. The anti-hyperglycemic drug, empagliflozin (EMP), was recently indicated to have in vitro anticancer potential together with its previously reported cardioprotective properties related to calmodulin inhibition. In this study, we carried out molecular docking studies which revealed the potential blocking of the calmodulin receptor by EMP through its binding with similar crucial amino acids compared to its cocrystallized inhibitor (AAA) as a proposed mechanism of action. Moreover, combination of DOX with EMP showed a slightly lower cytotoxic activity against the MDA-MB-231 cell line (IC₅₀ = 1.700 ± 0.121) compared to DOX alone (IC₅₀ = 1.230 ± 0.131), but it achieved a more characteristic arrest in the growth of cells by 4.67-fold more than DOX alone (with only 3.27-fold) in comparison to the control as determined by cell cycle analysis, and at the same time reached an increase in the total apoptosis percentage from 27.05- to 29.22-fold, compared to DOX alone as indicated by Annexin V-FITC apoptosis assay. Briefly, the aforementioned in vitro studies in addition to PCR of pro- and antiapoptotic genes (mTOR, p21, JNK, Bcl2, and MDR1) suggest the chemosensitization effect of EMP combination with DOX which can reduce the required therapeutic dose of DOX in TNBC and eventually will decrease its toxic side effects (especially cardiotoxicity), along with decreasing the chemoresistance of TNBC cells to DOX treatment.

Figure 1

Graphical representation showing that adding EMP enhances the anticancer effects of DOX at a reduced concentration, suggesting that EMP could minimize adverse effects of DOX while preserving its therapeutic value.

Figure 2

2D and 3D representations for the redocked cocrystallized inhibitor (AAA) inside the calmodulin receptor pocket. The cocrystallized inhibitor is represented in green and the redocked one is represented in red.

Figure 3

3D representations and positionings for the docked cocrystallized calmodulin antagonist (AAA) inside A and B subunits of the calmodulin receptor.

Figure 4

3D representations and positionings for empagliflozin inside A and B subunits of the calmodulin receptor.

Figure 5

Graphical representations showing (A) the cytotoxic effects of DOX, EMP, and DOX/EMP combination, (B) IC₅₀ of each treatment, (C) combination index (CI) of EMP and DOX, and (D) dose-reduction index (DRI) of DOX as analyzed with CompuSyn software. Data are average of three independent experiments ± SD.

Figure 6

Impact of conjugates DOX, EMP, and DOX/EMP combination on the cell cycle phases of MDA-MB-231 cells. Data are average of three independent experiments ± SD. *, §, and # represent significance (p < 0.5) compared with control, EMP, or DOX alone, respectively.

Figure 7

Combined effect of DOX and EMP on the apoptosis of MDA-MB-231 cells. Representative dot plot presenting the flow cytometry analysis of cells stained with Annexin V and PI following treatment with vehicle alone (A), 1.3 μM DOX alone (B), 50 μM EMP alone (C), or a combination of 1.3 μM DOX +50 μM EMP (D) for 24 h. Data are representative of three individual experiments.

Figure 8

Distribution of apoptotic cells in the AnnexinV-FITC/PI apoptosis assay in MDA-MB-231 cells after treatment with DOX, EMP, and DOX/EMP combination. Data are average of three independent experiments ± SD. *, §, and # represent significance (p < 0.5) compared with control, EMP, or DOX alone, respectively.

Figure 9

Analysis of gene expression profiles of mTOR (A), Bcl2 (B), JNK (C), and p21 (D), and of MDA-MB-231 cells treated with DOX, EMP, and DOX/EMP combination compared to blank by using real-time PCR. Data are average of three independent experiments ± SD. *, §, and # represent significance (p < 0.5) compared with control, EMP, or DOX alone, respectively.

Figure 10

Analysis of gene expression profiles of the multidrug resistance gene MDR1 in MDA-MB-231 after treatment with DOX, EMP, and DOX/EMP combination compared to blank by using real-time PCR. Data are average of three independent experiments ± SD. * and § represent significance (p < 0.5) compared with control, EMP, or DOX alone, respectively.