The Proapoptotic Effect of MB-653 Is Associated with the Modulation of Metastasis and Invasiveness-Related Signalling Pathways in Human Colorectal Cancer Cells

Abstract

Colorectal cancer is one of the most common cancers worldwide and has a high mortality rate. In this study, we investigated the cytotoxic, proapoptotic, and anti-invasive effects of the synthetic indole phytoalexin MB-653. The antiproliferative effect was determined using an MTT assay, showing IC₅₀ values of 5.8 ± 0.3 μmol/L for HCT116 cells and 6.1 ± 2.1 μmol/L for Caco2 cells. Flow cytometry and Western blot analysis were employed to investigate the molecular mechanisms underlying cytotoxicity, proapoptotic action, and anti-invasion effects. The proapoptotic activity was evidenced by the activation of caspases 3 and 7, mitochondrial dysfunction, and an increased number of apoptotic cells, confirmed by annexin V/PI and AO/PI staining. Additionally, MB-653 induces dose-dependent G2/M phase cell cycle arrest, the cause of which could be cyclin B1/CDC2 complex dysfunction and/or a decrease in α-tubulin protein expression. Another important observation was that MB-653 modulated several signalling pathways associated with various cellular activities, including survival, proliferation, tumour invasiveness, metastasis, and epithelial-mesenchymal transition (EMT). We further demonstrated its safety for topical and parenteral application. To sum up, our results indicate the real potential of MB-653 in treating colorectal cancer.

Keywords: EMT; apoptosis; colorectal cancer; indole phytoalexins; metastasis.

Figure 1

MB-653(trans-(±)-N,N’-bis [1-(tert-butoxycarbonyl)-2-methoxy-spiro{indoline-3,5′-[4′,5′]dihydrotriazol-2′-yl}]benzene-1,4-diamine).

Figure 2

Western blot analysis of cell cycle-associated proteins affected by MB-653 in HCT116 and Caco2 cells after 24, 48, and 72 h incubation. Representative figure.

Figure 3

Densitometric analysis of phospho-Rb (A), phospho-cdc2 (B), Cyclin D1 (C), Cyclin B1 (D), phospho-Cyclin B1 (E) and α-Tubulin (F). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure 4

Effect of MB-653 on the loss of mitochondrial membrane potential in HCT116 (A) and Caco 2 (B) cells after 24, 48, and 72 h exposure. Data were obtained from 3 independent acquisitions and are presented as the mean ± standard deviation. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure 5

Flow cytometric analysis of activated caspase-3/7 after 24, 48, and 72 h of MB-653 treatment in HCT116 (A) and Caco2 (B) cells. Data were obtained from 3 independent acquisitions and are presented as the mean ± standard deviation. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure 6

Fluorescence microscopic analysis of MB-653 induced apoptosis in HCT116 (A) and Caco2 (B) cell lines after 24, 48, and 72 h of treatment (10 µmol/L for HCT116 and Caco2) using AO/PI staining. Green shows live cells, yellow shows cells in the initial phase of apoptosis, orange shows cells in the advanced stage of apoptosis, and red indicates dead or necrotic cells. This image is a representative example from three separate experiments. Magnification is set at 100×.

Figure 7

Western blot analysis of the effect on proliferation-associated proteins affected by MB-653 in HCT116 and Caco2 cells after 24, 48, and 72 h incubation. Representative figure. Data were obtained from 3 independent acquisitions.

Figure 8

Densitometric analysis of NF-κ1 B p50 (A), NF-κ1 B p105 (B), Non-phospho (Active) β-Catenin (C), ß-Catenin (D), phospho-mTOR (E) and mTOR (F). Data were obtained from 3 independent acquisitions and are presented as the mean ± standard deviation. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure 9

Western blot analysis of proteins responsible for invasiveness and metastasis affected by MB-653 in HCT116 and Caco2 cells after 24, 48, and 72 h incubation. Representative figure. Data were obtained from 3 independent acquisitions.

Figure 10

Densitometric analysis of Snail—1,2,3 (A), MMP-9 (B), N-Cadherin (C) and E-Cadherin (D). Data were obtained from 3 independent acquisitions and are presented as the mean ± standard deviation. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure 11

Substance MB-653 in concentration 50 µmol/L has no irritant activity at 30 s, 120 s, and 300 s after application. Data were obtained from 3 independent acquisitions.

Figure 12

Measured angiogenesis parameters—vascular zone (A), number of branching points (B), total length (C), average thickness of vessels (D) after application of 10, 25, and 50 μM MB-653 according to software IKOSA. Px—pixels. Data were obtained from 3 independent acquisitions.