Role of the transient receptor potential melastatin 4 in inhibition effect of arsenic trioxide on the tumor biological features of colorectal cancer cell

Abstract

Background: To investigate the effects of arsenic trioxide (ATO) on human colorectal cancer cells (HCT116) growth and the role of transient receptor potential melastatin 4 (TRPM4) channel in this process.

Methods: The viability of HCT116 cells was assessed using the CCK-8 assay. Western blot analysis was employed to examine the protein expression of TRPM4. The apoptosis of HCT116 cells was determined using TUNEL and Flow cytometry. Cell migration was assessed through the cell scratch recovery assay and Transwell cell migration assay. Additionally, Transwell cell invasion assay was performed to determine the invasion ability of HCT116 cells.

Results: ATO suppressed the viability of HCT116 cells in a dose-dependent manner, accompanied by a decline in cell migration and invasion, and an increase in apoptosis. 9-phenanthroline (9-Ph), a specific inhibitor of TRPM4, abrogated the ATO-induced upregulation of TRPM4 expression. Additionally, blocking TRPM4 reversed the effects of ATO on HCT116 cells proliferation, including restoration of cell viability, migration and invasion, as well as the inhibition of apoptosis.

Conclusion: ATO inhibits CRC cell growth by inducing TRPM4 expression, our findings indicate that ATO is a promising therapeutic strategy and TRPM4 may be a novel target for the treatment of CRC.

Keywords: Arsenic trioxide; Colorectal cancer; HCT116 cells; TRPM4 channel.

Figure 1. ATO inhibited the viability of HCT116 cells.

Quantitative analysis of cell viability after treatment with ATO at different concentrations. ^∗∗∗P < 0.001 vs Ctl group; n = 3. Data are represented as the mean ± SD. One-way ANOVA followed by Tukey’s test for post-hoc comparisons was used. n, number of independent cells.

Figure 2. ATO increased the expression of TRPM4 protein in HCT116 cells.

(A-B) Representative images of western blot and quantification of TRPM4 in ATO treated HCT116 cells after 9-Ph stimulation. (C) Quantitative analysis of cell viability in ATO treated HCT116 cells after 9-Ph (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 μM) stimulation. (D) Quantitative analysis of cell viability after treatment with 9-Ph at different concentrations. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs Ctl group, ^#P < 0.05, ^### P < 0.001 vs ATO group; n = 3. Data are represented as the mean ± SD. One-way ANOVA followed by Tukey’s test for post-hoc comparisons was used. n, number of independent cells.

Figure 3. TRPM4 inhibition abolished the ATO-induced decrease in migration and invasion of HCT116 cells.

(A) Representative images of wound healing assay and quantification of migration in ATO treated HCT116 cells after 9-Ph stimulation. scale bar = 50 µm. (B–C) Representative images of Transwell assay and quantification of migration and invasion in ATO treated HCT116 cells after 9-Ph stimulation. scale bar = 50 µm. ^∗∗ P < 0.01, ^∗∗∗ P < 0.001 vs. Ctl group, ^##P < 0.01, ^###P < 0.001 vs. ATO group; n = 5. Data are represented as the mean ± SD. One-way ANOVA followed by Tukey’s test for post-hoc comparisons was used. n, number of independent cells.

Figure 4. ATO promotes apoptosis of HCT116 cells.

(A) Representative images of TUNEL staining assay and quantification in ATO treated HCT116 cells after 9-Ph stimulation. Scale bar = 50 µm . (B) Representative images of flow cytometry and quantification of positive cell in ATO treated HCT116 cells after 9-Ph stimulation. ^∗P < 0.05, ^∗∗∗ P < 0.001 vs. Ctl group, ^# P < 0.05, ^###P < 0.001 vs ATO group; n = 3. Data are represented as the mean ± SD. One-way ANOVA followed by Tukey’s test for post-hoc comparisons was used. n, number of independent cells.