Unveiling the antimetastatic activity of monoterpene indole alkaloids targeting MMP9 in cancer cells, with a focus on pharmacokinetic and cellular insights

Abstract

Distant metastasis, together with acquired resistance, limits the therapeutic impact of chemotherapy and molecularly targeted therapies. The properties of the tumor microenvironment determine how sensitive or resistant various cancers are to specific pharmacological treatments. Matrix metalloproteinase 9 (MMP9) is widely known for its ability to break down the extracellular matrix and it also modulates the motility of cancer cells. Here, our goal was to identify compounds that target MMP9 and evaluate their capacity to inhibit the motility of cancer cells. The antimetastatic effect of monoterpene indole alkaloids (MIAs) on cell viability and motility was evaluated by methyl thiazolyl tetrazolium assay, migration assay, invasion assay, quantitative real-time polymerase chain reaction, pathway-focused expression analysis, Western blotting, reporter assay, molecular docking simulation, and target prediction. MIA compounds target MMP9. MIAs inhibited the expression of phospho-epidermal growth factor receptor, phospho-Akt, phospho-JNK, and cyclin D1. Additionally, MIAs had predicted favorable pharmacokinetic profile and drug-like properties. Furthermore, among the MIA compounds, lyaloside and 5(S)-5-carbomethoxystrictosidine had low cytotoxicity and regulated cancer-related signaling, including cell migration, cell invasion, epithelial-mesenchymal transition, and immune evasion. Our findings demonstrated that the MIAs used in this study have potential antimetastasis properties that occur via MMP9-mediated regulation of cancer signaling and have the potential to be used therapeutically at safe doses.

Keywords: 5(S)-5-carbomethoxystrictosidine; Anticancer; Lyaloside; Matrix metalloproteinase 9; Monoterpene indole alkaloids.

Fig. 1

Target identification of MIAs. Human target prediction using SwissTargetPrediction of (A) lyaloside (1), (B) 5(S)-5-carbomethoxystrictosidine (2), (C) strictosamide (3), and (D) mitraphylline (4) and molecular docking of (E) lyaloside (1), (F) 5(S)-5-carbomethoxystrictosidine (2), (G) strictosamide (3), and (H) mitraphylline (4) to MMP9 (PDB 1GKC). The predicted binding poses of MMP9 are given. The MMP9 protein showed significant interactions (eg, van der Waals interactions, hydrophobic interactions, and hydrogen bonding) with the compounds using the BIOVIA Discovery Studio visualizer. Docking scores were calculated by CB dock, and the docking scores are given in the figure. (I) HEK293T cells were cotransfected with the pRL-TK (Renilla) plasmid and the MMP9 reporter plasmid (pGL4-MMP9). After 24 hours, transfected cells were treated with 1 or 2.5 µM lyaloside (1), 5(S)-5-carbomethoxystrictosidine (2), strictosamide (3), mitraphylline (4), or DMSO and incubated for 24 hours. Data are represented as the mean ± SD. *P < .05; **P < .01; ***P < .001. (J) AGS, (K) CaCo2, and (L) H1975 cells were treated with the indicated compound for 48 hours. Protein levels of MMP9, p-EGFR, p-Akt, p-JNK, and cyclin D1 were analyzed by Western blotting.

Fig. 2

Physicochemical and pharmacokinetic properties of the monoterpene indole alkaloids. The bioavailability of (A) lyaloside (1), (B) 5(S)-5-carbomethoxystrictosidine (2), (C) strictosamide (3), and (D) mitraphylline (4) was determined using the SwissADME predictor. The ideal range for each property (lipophilicity, size, polarity, solubility, saturation, and flexibility) is shown by the pink area. (E) The ADME parameters. MW, molecular weight; TPSA, topological polar surface area; GI, gastrointestinal absorption; P-gp, P-glycoprotein substrate.

Fig. 3

The effects of lyaloside (1) and 5(S)-5-carbomethoxystrictosidine (2) on the cell viability and motility of AGS, Caco2, and H1975 cells. (A-C) The cell viability of AGS, CaCo2, and H1975 cells treated with lyaloside and 5(S)-5-carbomethoxystrictosidine for 48 hours. (D and E) Representative images of each wound area and quantitative data of the relative wound density in the scratch wound migration assay using the IncuCyte system. (F and G) Representative images of each insert and the relative number of invaded cells in the Transwell invasion assay. Cells were treated with 1 and 2.5 μM lyaloside and 5(S)-5-carbomethoxystrictosidine at concentrations for 24 hours. Data are represented as the mean ± SD. *P < .05; **P < .01; ***P < .001.

Fig. 4

Lyaloside and 5(S)-5-carbomethoxystrictosidine modulated the expression of EMT effectors and transcription factors. (A-C) The mRNA expression levels of EMT effectors (CDH1, CDH2, VIM) and EMT transcription factors (TWIST, SNAIL, SLUG, ZEB1, and ZEB2) were analyzed by qRT-PCR. The mRNA levels were normalized against β-actin. Data are represented as the mean ± SD. *P < .05; **P < 0.01; ***P < .001.

Fig. 5

Lyaloside and 5(S)-5-carbomethoxystrictosidine suppress the expression of cell motility–related genes. We used pathway-focused gene expression profiling in AGS cells to investigate the signaling pathways through which the compounds affect cell motility using a human cell motility RT² Profiler PCR array. Cells were treated with 2.5 µM lyaloside and 5(S)-5-carbomethoxystrictosidine for 48 hours. Cells were then harvested, and the total RNA was extracted and converted to cDNA. (A) The hierarchical clustering of gene signatures was determined using the results from the RT² Profiler PCR array. The treatment groups were DMSO, lyaloside (1), and 5(S)-5-carbomethoxystrictosidine (2). (B-C) A scatter-plot comparison of genes from the PCR array between the DMSO-lyaloside (1)- and 5(S)-5-carbomethoxystrictosidine (2)-treated groups. (D-F) Quantitative analysis of mRNA expression levels of CAPN1 and MMP9 in AGS, CaCo2, and H1975 cells. (G-I) Relative activities of promoters related to AP-1, STAT, and NF-kB signaling pathways. HEK293T cells were cotransfected with the pRL-TK (Renilla) plasmid and the AP-1-luc (g), STAT-luc (h), and NF-kβ-luc (i) reporter plasmids (firefly). After 24 hours, transfected cells were treated with 1 or 2.5 µM lyaloside (1) and 5(S)-5-carbomethoxystrictosidine (2) or DMSO and incubated for 24 hours. The relative firefly luciferase activity of treated vs control groups is shown. Data are represented as the mean ± SD. *P < .05; **P < .01; ***P < .001. (J) AGS cells were treated with EGF (40 ng/mL) or TGF-β (10 ng/mL) together with lyaloside and 5(S)-5-carbomethoxystrictosidine for 48 hours, then cells were harvested and lyzed. Western blot analysis was conducted for the indicated marker. β-actin served as a loading control. The relative protein levels were given under the bands. (K) A schematic representation of the proposed mechanism of action of lyaloside and 5(S)-5-carbomethoxystrictosidine. Both compounds inhibit multiple signaling pathways to inhibit CAPN1 and MMP9 expression and suppress cancer cell motility. AP-1, activator protein-1; NF-kB, nuclear factor-κB; STAT, signal transducer and activator of transcription.