Assessment of an Anticancer Effect of the Simultaneous Administration of MM-129 and Indoximod in the Colorectal Cancer Model

Abstract

(1) Background: The purpose of the given study was to examine the antitumor activity of the simultaneous administration of MM-129, a 1,2,4-triazine derivative, and indoximod (IND), the kynurenine pathway inhibitor, toward colon cancer. (2) Methods: The efficiency of the co-administration of the studied compounds was assessed in xenografted zebrafish embryos. Then, the effects of the combined administration of compounds on cellular processes such as cell viability, apoptosis, and intracellular signaling pathways were evaluated. In vitro studies were performed using two colorectal cancer cell lines, namely, DLD-1 and HT-29. (3) Results: The results indicated that the simultaneous application of MM-129 and indoximod induced a stronger inhibition of tumor growth in zebrafish xenografts. The combination of these compounds intensified the process of apoptosis by lowering the mitochondrial potential, enhancing the externalization of phosphatidylserine (PS) and activation of caspases. Additionally, the expression of protein kinase B (AKT) and indoleamine 2,3-dioxygenase-(1IDO1) was disrupted under the applied compound combination. (4) Conclusions: Simultaneous targeting of ongoing cell signaling that promotes tumor progression, along with inhibition of the kynurenine pathway enzyme IDO1, results in the enhancement of the antitumor effect of the tested compounds against the colon cancer cells.

Keywords: MM-129; colon cancer; immunooncology; indoximod; kynurenine pathway; zebrafish.

Figure 1

Impact of MM-129 (10 µM), IND (200 µM) or a combination of these agents (MM-129 + IND) on tumor development in DLD-1 (a), and HT-29 (b) zebrafish xenografts, and the expression of human GAPDH mRNA was determined at 120 hpf. Data were presented as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA). n = 20 each group; ** p < 0.01, *** p < 0.001 vs. CON; ^ p < 0.05, ^^^ p < 0.001 vs. MM-129; # p < 0.05 vs. IND.

Figure 2

Effect of MM-129 (10 µM), IND (200 µM), and compound combination (MM-129 + IND) on cell division in the zebrafish embryo. Zebrafish eggs after 0, 1, 1.25, and 2 h of exposure to tested compounds; n = 20, hpf: hours post-fertilization.

Figure 3

Viability of DLD-1 (a) and HT-29 (b) colon cancer cells treated for 24 h with MM-129 (10 µM), IND (200 µM), and compound combination (MM-129 + IND). Results are presented as mean values ± standard deviation (SD) from three independent experiments performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON; ### p < 0.001 vs. IND.

Figure 4

Representative dot-plots illustrating the loss of mitochondrial membrane potential, ΔΨm. (a,b) Flow cytometry dot-plots for the annexin V-FITC-propidium iodide assay (c,d) and quantitative chart illustrating the distribution of live, early, and late apoptotic and necrotic cells (e,f) of DLD-1 (a,c,e) and HT-29 (b,d,f) after 24 h exposition to MM-129 (10 µM), IND (200 µM), and MM-129 + IND. Values were obtained from three independent experiments.

Figure 5

Flow cytometric analysis of caspase-3/7, caspase-10, and caspase-8 activation in DLD-1 (a–c) and HT-29 (d–f) colon cancer cells exposed to MM-129 (10 µM), IND (200 µM), and MM-129 + IND for 24 h. Values from three independent experiments performed in duplicate were presented as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA). ** p < 0.01, *** p < 0.001 vs. CON; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001vs. MM-129; ### p < 0.001 vs. IND; && p < 0.01, &&& p < 0.001 IND vs. MM-129.

Figure 6

The downregulation of protein kinase B (AKT) (a,b) and indoleamine 2,3-dioxygenase-1 (IDO1) (c,d) exerted by MM-129 (10 µM), IND (200 µM), or MM-129 + IND toward DLD-1 and HT-29 colorectal cancer cells after 24 h exposition. The values were obtained from three independent experiments performed in duplicate. * p < 0.05, *** p < 0.001 vs. CON; ^^^ p < 0.01 vs. MM-129; ## p < 0.01, ### p < 0.001 vs. IND. Images and quantification were obtained by capillary protein separation and immunodetection. The uncropped blots are shown in the supplemental materials.