Induction of cell apoptosis by biliverdin reductase inhibitor in MCF-7 and MDA-MB-468 breast cancer cell lines: Experimental and in silico studies

Abstract

Biliverdin reductase, biliverdin and bilirubin are known as important components of cellular signaling pathways that play major roles in cell proliferation and apoptosis, although their physiological relevance is still under evaluation. This study was designed to investigate the expression and activity of BVR-A and its apoptotic effect in the breast cancer cell lines, MCF-7 and MDA-MB-468. The expression of BVR-A was examined by real-time PCR and western blot analysis. Bilirubin concentration was measured by HPLC and molecular docking was performed to identify an appropriate inhibitor for BVR-A. To detect cell apoptosis, annexin V-PI staining, caspase-3, -8, and -9 activities were evaluated. Cell viability was reduced by biliverdin, in a dose-dependent manner, and an intrinsic apoptotic response occurred which was evidenced by caspase-3 and -9 activities. The intra- and extracellular concentrations of bilirubin were higher in MCF-7 cells than those of MDA-MB-468 cells. The expression of BVR-A, at mRNA and protein levels, in MCF-7 was also higher than that of MDA-MB-468 cells. Treatment of both cell lines with biliverdin plus DTNB, a BVR-A inhibitor, increased the cell death significantly when compared with biliverdin alone. Using annexin V-PI staining and assessment of caspase-3 activity, it was confirmed that biliverdin together with DTNB increases apoptosis in breast cancer cells. In conclusion, biliverdin has an important role in cell apoptosis and inhibition of biliverdin reductase increases the apoptotic effect of biliverdin.

Keywords: biliverdin reductase-A; breast cancer cell lines; caspase activity; high-performance liquid chromatography; molecular docking.

Table 1. Primer sequences for qRT-PCR analysis

Table 2. Grid box coordinates for BVR-A

Table 3. Compounds were used as ligands in the pharmacophore model.

Table 4. The binding energy of selected ligands against the BVR-A

Figure 1. Figure 1: Three-dimensional conformation and close-up view of the BVR-A binding site with biliverdin (A), and DTNB (B).

Figure 2. Two-dimensional scheme of interactions of BVR-A with the docked molecule: A) Disulfiram, B) Fluphenazine dihydrochloride, C) DTNB, and D) Montelukast, obtained by Ligplot+ software. Receptor residues involved in hydrophobic interactions are represented by black semicircular arcs, and hydrogen bonding shows in green dotted lines. Carbon, oxygen, nitrogen, and fluorine atoms are displayed in filled black, red, blue, and green circles, respectively.

Figure 3. Figure 3: Induction of cell death by exogenous biliverdin in breast cancer cell lines. MCF-7 (A) and MDA-MB-468 (B) cells were treated with different concentrations of biliverdin (from 25 to 250 µM) for 24 and 48 hrs. The cell viability was evaluated by MTT assay. The data are presented as mean ± SD (three separate experiments). **P<0.01, ***P<0.001, and ****P<0.0001, denote means significantly different from control cells.

Figure 4. Induction of different modes of cell death by biliverdin, biliverdin plus DTNB, and DTNB in MCF-7 and MDA-MB-468 cell lines. (A) MCF-7 and MDA-MB-468 cell line treated with biliverdin for 24 hrs. DTNB was pretreated for 12hrs prior to biliverdin treatment. After 24 hrs of incubation with biliverdin, the percentage of cell death (early and late apoptosis) was significantly increased in both cell lines. Necrosis was also increased in these cell lines, although not significantly in MDA-MB-468 cells. Pretreatment with DTNB has significantly decreased cell death after biliverdin treatment. (B) Quantification of Figure A. Data is represented as mean ± SD (three separate experiments). *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 denotes a mean significantly different from control cells, ns not significantly different from control cells (BV= biliverdin)

Figure 5. Specific activities of caspases in the cell lysate of breast cancer cell lines, MCF-7 and MDA-MB-468 cells, treated with biliverdin for 24 hrs. DTNB was pretreated for 12 hrs prior to biliverdin treatment after incubation of MCF-7 (A) and MDA-MB-468 (B) cells with Biliverdin, the activity of caspase-3, -9, and -8 were increased significantly, using an enzymatic assay. In contrast, biliverdin did not affect the activation of caspase‐8. Pretreatment with DTNB significantly increased the activity of caspase-3 after biliverdin treatment. The results were presented as the mean values ± SD. **P < 0.01, ***P < 0.001 denotes a mean significantly different from control cells.

Figure 6. mRNA level of BVR-A and AhR (A) and protein level of BVR-A (B) in the biliverdin-treated breast cancer cell lines. β-actin was used for normalization in real-time-PCR analysis and western blot. *P<0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 denotes a mean significantly different from control cells.

Figure 7. HPLC chromatograms of bilirubin in intra- and extracellular of in control cells (A) and treated cells (B). Detection wavelength of 453 nm. (C) Quantification of intra- and extracellular levels of bilirubin. The data are expressed as the mean ± SD, *P<0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 denotes a mean significantly different from control cells.

Figure 8. Inhibition of BVR-A by DTNB in breast cancer cell lines. MCF-7 and MDA-MB-468 cells were treated with different concentrations of DTNB (from 125 to 1000 µM) for 12 hrs. The data are presented as mean ± SD (three separate experiments). **P<0.01, ***P<0.001 and ****P<0.0001, denote means significantly different from control cells.