Investigation of Novel Small Molecular TRPM4 Inhibitors in Colorectal Cancer Cells

Abstract

(1) Background: Transient receptor potential melastatin (TRPM4) ion channel aberrant expression or malfunction contributes to different types of cancer, including colorectal cancer (CRC). However, TRPM4 still needs to be validated as a potential target in anti-cancer therapy. Currently, the lack of potent and selective TRPM4 inhibitors limits further studies on TRPM4 in cancer disease models. In this study, we validated novel TRPM4 inhibitors, CBA, NBA, and LBA, in CRC cells. (2) Methods: The potency to inhibit TRPM4 conductivity in CRC cells was assessed with the whole-cell patch clamp technique. Furthermore, the impact of TRPM4 inhibitors on cellular functions, such as viability, proliferation, and cell cycle, were assessed in cellular assays. (3) Results: We show that in CRC cells, novel TRPM4 inhibitors irreversibly block TRPM4 currents in a low micromolar range. NBA decreases proliferation and alters the cell cycle in HCT116 cells. Furthermore, NBA reduces the viability of the Colo205 cell line, which highly expresses TRPM4. (4) Conclusions: NBA is a promising new TRPM4 inhibitor candidate, which could be used to study the role of TRPM4 in cancer disease models and other diseases.

Keywords: TRPM4; ion channels; patch clamp; small molecule inhibitors.

Figure 1

Novel TRPM4 inhibitors block TRPM4 currents in HCT116 cells. (A) Chemical structure of CBA. (B) Chemical structure of LBA. (C) Chemical structure of NBA. (D) Whole-cell patch clamp data from HCT116. Currents were evoked with 10 µM Ca2⁺ in the patch pipette and normalized to cell size. Current density (CD), displayed as mean +/− SEM, is plotted versus time and 10 µM CBA was applied from 280–400 s. Inset: IV curves at t = 276 s and t = 396 s (n = 8). (E) Same as (D) for LBA (n = 5). (F) Same as (D) for NBA (n = 6). (G) Average TRPM4 CD +/− SEM was plotted against CBA concentrations (n = 9 for 0.01 µM, n = 7 for 0.1 µM, n = 9 for 1 µM, n = 10 for 3 µM, and n = 8 for 10 µM). Dose–response curve was fitted with a Hill equation and IC₅₀ value was 1.84 µM. (H) Average TRPM4 CD +/− SEM was plotted against LBA concentrations (n = 5 for 0.1 µM, n = 9 for 0.3 µM, n = 13 for 1 µM, n = 8 for 3 µM, and n = 5 for 10 µM). Dose–response curve was fitted with a Hill equation and IC₅₀ value was 1.84 µM. (I) Average TRPM4 CD +/− SEM was plotted against NBA concentrations (n = 6 for 0.001 µM, n = 9 0.01 µM, n = 9 for 0.03 µM, n = 6 for 0.1 µM, n = 13 for 0.3 µM, n = 5 for 1 µM, and n = 6 for 10 µM). Dose–response curve was fitted with a Hill equation and IC₅₀ value was 1.84 µM.

Figure 2

Viability of HCT116 and T4KO cell lines after treatment with NBA. Cell viability in HCT116, T4KO 1, and T4KO 2 cells was evaluated using a RealTime-Glo MT assay. Cells were treated with 0.1 µM, 1 µM, 10 µM, 50 µM NBA, or DMSO control. Four independent experiments were performed. (A) Mean of relative luminescence was plotted versus time for HCT116 cells. (B) Same for T4KO 1 cells. (C) Same for T4KO 2 cells. (D) Scatter plot and bar diagram of data (mean + SD) at 24 h from four independent experiments in (A–C). (E) Scatter plot and bar diagram of slope steepness between 5 and 10 h (mean + SD) from data in (A–C). One-way ANOVA was used to determine statistical significance in (D,E).

Figure 3

Effect of CBA on HCT116 cells’ proliferation. Cell proliferation was determined with an xCELLigence^® system. Cells were treated with 0.1 µM, 1 µM, 10 µM, 50 µM CBA, or DMSO control. A total of 3–4 independent experiments were performed. (A) Mean of cell index was plotted versus time for HCT116 cells. (B) Same as (A) for T4KO 1 cells. (C) Same as (A) for T4KO 2 cells. (D) Scatter plot and bar diagram of data (mean + SD) at 24 h from the experiment in (A–C). (E) Scatter plot and bar diagram of slope steepness between 5 and 15 h (mean + SD) from data in (A–C). One-way ANOVA was used to determine statistical significance (* p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001) in (D,E).

Figure 4

Effect of NBA on HCT116 cells’ proliferation. Cell proliferation was determined with an xCELLigence^® system. Cells were treated with 0.1 µM, 1 µM, 10 µM, 50 µM NBA, or DMSO control. A total of 3–4 independent experiments were performed. (A) Mean of cell index was plotted versus time for HCT116 cells. (B) Same as (A) for T4KO 1 cells. (C) Same as (A) for T4KO 2 cells. (D) Scatter plot and bar diagram of data (mean + SD) at 24 h from the experiment in (A–C). (E) Scatter plot and bar diagram of slope steepness between 5 and 15 h (mean + SD) from data in (A–C). One-way ANOVA was used to determine statistical significance (* p < 0.05) in (D,E).

Figure 5

Effect of NBA on cell cycle in HCT116 and T4KO cells. FACS-based cell cycle analysis. The experiment was repeated four times with from one to two replicates in each experiment. (A) Representative histograms of PI staining in HCT116 without inhibitor treatment, treated with DMSO and treated with 10 µM and 50 µM NBA, with cell distribution in % for one specific measurement. (B) Scatter plot and bar diagram (mean + SD) for cell cycle distribution of HCT116 without inhibitor treatment, treated with DMSO and treated with 10 µM and 50 µM NBA. (C) Scatter plot and bar diagram (mean + SD) for cell cycle distribution of T4KO 1 without inhibitor treatment, treated with DMSO and treated with 10 µM and 50 µM NBA. (D) Scatter plot and bar diagram (mean + SD) for cell cycle distribution of T4KO 2 without inhibitor treatment, treated with DMSO and treated with 10 µM and 50 µM NBA. One-way ANOVA was used to determine statistical significance (** p < 0.005, ns—non significant) in (B–D).

Figure 6

Effect of NBA on CRC cells’ viability. Cell viability in LS180, HCT15, and Colo205 cells was evaluated using a RealTime-Glo MT assay. Cells were treated with 0.1 µM, 1 µM, 10 µM, 50 µM NBA, or DMSO control. Three independent experiments were performed. (A) Mean of relative luminescence was plotted versus time for LS180 cells. (B) Same for HCT15 cells. (C) Same for Colo205 cells. (D) Scatter plot and bar diagram of data (mean + SD) at 24 h from three independent experiments in (A–C). (E) Scatter plot and bar diagram of slope steepness between 5 and 10 h (mean + SD) from data in (A–C). One-way ANOVA was used to determine statistical significance (* p < 0.05, ** p < 0.005) in (D,E).