Unveiling the Intricacies of Monoamine Oxidase-A (MAO-A) Inhibition in Colorectal Cancer: Computational Systems Biology, Expression Patterns, and the Anticancer Therapeutic Potential

Abstract

Colorectal cancer (CRC) remains a significant health burden globally, necessitating a deeper understanding of its molecular intricacies for effective therapeutic interventions. Elevated monoamine oxidase-A (MAO-A) expression has been consistently observed in CRC tissues, correlating with advanced disease stages and a poorer prognosis. This research explores the systems biology effects of MAO-A inhibition with small molecule inhibitor clorgyline regarding CRC. The applied systems biology approach starts with a chemocentric informatics approach to derive high-confidence hypotheses regarding the antiproliferative effects of MAO-A inhibitors and ends with experimental validation. Our computational results emphasized the anticancer effects of MAO-A inhibition and the chemogenomics similarities between clorgyline and structurally diverse groups of apoptosis inducers in addition to highlighting apoptotic, DNA-damage, and microRNAs in cancer pathways. Experimental validation results revealed that MAO inhibition results in antiproliferative antimigratory activities in addition to synergistic effects with doxorubicin. Moreover, the results demonstrated a putative role of MAO-A inhibition in commencing CRC cellular death by potentially mediating the induction of apoptosis.

Figure 1

Workflow for studying the systems biology effects of MAO-A inhibitor clorgyline. GS is the gene signature; CMap is the connectivity map.

Figure 2

Pathway enrichment and genomic alterations for MAO-A and five colon cancer cell lines. (A) Pathways analysis for the MAO-A protein–protein interaction network. (B) MAO-A genomic alterations in cancer were determined using cBioPortal. (C) Affected cancer pathways were across five different colon cancer cell lines. The affected cancer pathways are shown on the x-axis. The number of affected genes in each pathway is shown on the y-axis. The NRF2 pathway is not shown because it was not perturbed in any of the analyzed colon cancer cell lines.

Figure 3

(A) Time-dependent inhibition of HCT116, Caco2, SW480, SW620, and HT-29 colorectal cancer cells at 24, 48, and 72 h treatment duration with clorgyline. The data shown represent the GI₅₀ ± SD. GI₅₀ was calculated by using Prism software. Each experiment was performed in triplicate and repeated at least twice independently. P-value < 0.05 indicates statistical significance in comparison to GI₅₀ at 24 h treatment, while asterisk: ns (not significant) P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (according to GraphPad prism 9). μM: micromolar. (B) Effects of doxorubicin alone and in combination with MAO-A inhibitor clorgyline treatment in HCT116, Caco2, SW620, and HT-29 colorectal cancer cells. The data shown represent mean percentages of cell viability ± SD. Each experiment was performed in duplicate in three independent trials (n = 6). P-value < 0.05 expresses significantly different from respective doxorubicin treatment; asterisk: ns (not significant) P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (according to GraphPad prism 9). μM: micromolar.

Figure 4

Effects of MAO-A inhibitor clorgyline on cellular migration of HCT116 and HT29. (A) Wound pictures at 0 time and 24 h post-treatment captured at 10× magnification using EVOS XL Core imaging system. NA: not applicable; h: hour; μM: micromolar. (B) Quantitative comparison of HCT116 and HT29 cells. Each experiment was performed in duplicate in three independent trials (n = 6). P-value < 0.05 express significantly different from respective untreated condition; while asterisk: ns (not significant) P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (according to GraphPad prism 9). μM: micromolar.

Figure 5

Apoptotic and necrotic effects of clorgyline on HCT116 and Caco2. (A) Dot plot for annexin V-FITC/PI staining expressing the effect of MAO-A inhibitor, clorgyline 400 μM for 24 h (double GI₅₀) treatment on apoptosis of colorectal cancer cell lines, HCT116 and Caco2. Q3 shows healthy viable cells, Q1 necrotic cells, Q2 late apoptotic, and Q4 early apoptotic cells. The experiment was performed in duplicate and repeated in two independent trials (n = 4). h: hour; μM: micromolar. (B) Percentages of healthy, apoptotic, and necrotic cells expressed as mean ± SD expressing the effect of MAO-A inhibitor, clorgyline 400 μM for 24 h (double GI₅₀) treatment on apoptosis of colorectal cancer cell lines, HCT116 and Caco2. The experiment was performed in duplicate and repeated in two independent trials (n = 4). P-value < 0.05 express significantly different from respective untreated cells’ status; while asterisk: ns (not-significant) P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (according to GraphPad prism 9).

Figure 6

(A) Baseline gene expression variation between HCT116 colorectal cancer cell line and normal human dermal fibroblast cell line. MAOA: gene for monoamine oxidase-A enzyme; Bcl-2: gene for Bcl-2; VEGF: gene for Vascular endothelial growth factor. Fold difference was expressed as mean ± SD and was measured using ΔΔ the Ct method explained in eq 3. All experiments were run in duplicates and with two independent experiments. P-value <0.05 express significantly different from respective normal cells’ status; while asterisk: ns (not-significant) P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (according to GraphPad prism 9). (B) Effect of MAO-A inhibitor, Clorgyline, on multiple RNA expression of specific genes in the HCT116 colorectal cancer cell line. MAOA: the gene for monoamine oxidase-A enzyme; Bcl-2: the gene for Bcl-2; VEGF: the gene for Vascular endothelial growth factor; Fold difference expressed as mean ± SD and was measured using ΔΔCt method explained in eq 3. All experiments were run in duplicates and with two independent experiments. P-value <0.05 express significantly different from respective untreated cells’ status; while asterisk: ns (not-significant) P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (according to GraphPad prism 9). h: hour; μM: micromolar.