Acridine-Based Chalcone 1C and ABC Transporters

Abstract

Chalcones, potential anticancer agents, have shown promise in the suppression of multidrug resistance due to the inhibition of drug efflux driven by certain adenosine triphosphate (ATP)-binding cassette (ABC) transporters. The gene and protein expression of chosen ABC transporters (multidrug resistance protein 1, ABCB1; multidrug resistance-associated protein 1, ABCC1; and breast cancer resistance protein, ABCG2) in human colorectal cancer cells (COLO 205 and COLO 320, which overexpress active ABCB1) was mainly studied in this work under the influence of a novel synthetic acridine-based chalcone, 1C. While gene expression dropped just at 24 h, compound 1C selectively suppressed colorectal cancer cell growth and greatly lowered ABCB1 protein levels in COLO 320 cells at 24, 48, and 72 h. It also reduced ABCC1 protein levels after 48 h. Molecular docking and ATPase tests show that 1C probably acts as an allosteric modulator of ABCB1. It also lowered galectin-1 (GAL1) expression in COLO 205 cells at 24 h. Functional tests on COLO cells revealed ABCB1 and ABCC1/2 to be major contributors to multidrug resistance in both. Overall, 1C transiently lowered GAL1 in COLO 205 while affecting important functional ABC transporters, mostly ABCB1 and to a lesser extent ABCC1 in COLO 320 cells. COLO 320's absence of GAL1 expression points to a possible yet unknown interaction between GAL1 and ABCB1.

Keywords: ABCB1; ABCC1; ABCG2; chalcone; colorectal carcinoma; drug efflux; expression; galectin-1; multidrug resistance; transporter.

Figure 1

(A) Effect of 1C on the viability of COLO 205, COLO 320, and BJ-5ta, as determined by the MTT assay. (B) Effect of 1C on the viability of HT-29 and FHC cells, as determined by the SRB assay. Results are presented as the mean ± standard deviation from three independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 for treated vs. the control.

Figure 2

Western blot analysis of 1C effects on the protein expression of ABC transporters ((A) ABCB1, (C) ABCC1, and (B) ABCG2) and (D) galectin-1 (GAL1) after 12, 24, and 48 h of incubation. Graphs depicting a change in the expression of proteins in cells exposed to the 1C chalcone derivative. Significance levels are indicated as follows: * p < 0.05 and ** p < 0.01 in comparison to the DMSO-treated control group. β-actin was used as a loading control. ABCB1 was not detected in the COLO 205 cell line, and no GAL1 signals were observed in COLO 320 cells.

Figure 3

Immunofluorescence analysis of the presence of ABCB1 in COLO 205 and COLO 320 cells. Green indicates the presence of the transporter; blue indicates the nuclei. Representative images. Magnification 10×. Scale bar = 200 μm.

Figure 4

(A) Result of the ATPase assay displaying the dose–response curve of the 1C chalcone derivative on ABCB1-linked ATPase. Activity of ATPase is measured through the concentration of released inorganic phosphate (Pi). (B) The half-maximal effective concentration (EC₅₀) for the 1C derivative was determined to be 5.53 ± 0.97 µmol/L.

Figure 5

Docking results of compound 1C and the human ABCB1 protein in different visualizations: (A) transmembrane region of ABCB1 and (B) M binding site residues interacting with 1C. The protein is represented by gray cartoons or sticks; the compound is represented by colored sticks.

Figure 6

Multidrug resistance activity factors (MAFs) of ABC transporters. The red line represents the threshold, below which the transporter is considered non-functional. Results were obtained from at least three independent experiments. The graph was constructed using the geometric means of MAF. Significance level ** p < 0.01.

Figure 7

Histograms from the EFLUXX-ID^® Green multidrug resistance assay for ABC transporter activity. The histograms illustrate changes in the retention of the fluorescent substrate (Efflux-ID Green dye). The non-tinted histograms represent the fluorescence of untreated COLO 320 and COLO 205 cells, while the tinted histograms correspond to cells treated with the specific ABC transporter inhibitors: verapamil (for ABCB1), MK-571 (for ABCC1/2), and novobiocin (for ABCG2). Representative histograms of 3 experiments per group.