Activation of TRPM7 by naltriben enhances migration and invasion of glioblastoma cells

Abstract

Glioblastoma (GBM), the most common and aggressive brain tumor in the central nervous system, remains a lethal diagnosis with a median survival of < 15 months. Aberrant expression of the TRPM7 channel has been linked to GBM functions. In this study, using the human GBM cell line U87, we evaluated the TRPM7 activator naltriben on GBM viability, migration, and invasiveness. First, using the whole-cell patch-clamp technique, we showed that naltriben enhanced the endogenous TRPM7-like current in U87 cells. In addition, with Fura-2 Ca2+ imaging, we observed robust Ca2+ influx following naltriben application. Naltriben significantly enhanced U87 cell migration and invasion (assessed with scratch wound assays, Matrigel invasion experiments, and MMP-2 protein expression), but not viability and proliferation (evaluated with MTT assays). Using Western immunoblots, we also detected the protein levels of p-Akt/t-Akt, and p-ERK1|2/t-ERK1|2. We found that naltriben enhanced the MAPK/ERK signaling pathway, but not the PI3k/Akt pathway. Therefore, potentiated TRPM7 activity contributes to the devastating migratory and invasive characteristics of GBM.

Keywords: TRPM7; U87; glioblastoma; migration; naltriben.

Figure 1. Naltriben activated TRPM7-like currents in U87 cells

(A) Representative current density-voltage trace (1 is bath solution; 2 is perfusion of 50 μM naltriben, note that maximal activation was observed ~1 min after naltriben application; 3 is wash). (B) Representative time course of the outward current of TRPM7 at +80 mV. (C) Summary chart comparing the outward current at +80 mV between control versus activation of TRPM7 with naltriben. ***represents p < 0.001 (Student's t-test, n = 6/group). (D) Assessing the pharmacological inhibition of naltriben-potentiated TRPM7-like currents using carvacrol (300 μM). Representative current density-voltage trace (1 is bath solution; 2 is perfusion of 50 μM naltriben; 3 is simultaneous perfusion of both naltriben and 300 μM carvacrol). (E) Representative time course of the outward current of TRPM7 at +80 mV. (F) Summary chart comparing the naltriben-potentiated outward current at +80 mV before and after application of carvacrol. **represents p < 0.01 (Student's t-test, n = 3/group).

Figure 2. Naltriben induces Ca²⁺ influx, which might account for reduction in U87 cell viability

(A) Summary chart of MTT assays, which were used to evaluate U87 cell viability. Cells were treated with vehicle (0.1% DMSO; control; n = 24) or with naltriben (25–100 μM; n = 18) for 24 h. **** represents p < 0.0001 (1-way ANOVA; compared with control). (B) Fura-2 ratiometric Ca²⁺ imaging experiments. U87 cells were pre-loaded with Fura-2 AM (2 μM) in the dark for 30 min at room temperature. Fura-2 Ca²⁺ signal was acquired at alternate excitation wavelengths of 340 and 380 nm. [Top] Representative raw images of U87 cells before (resting basal), during (start naltriben application; continue naltriben application), and after (naltriben peak) application of 50 μM naltriben. White scale bars represent 25 μm. Signal intensity color bar represents the 340/380 ratio. [Bottom Left] Representative 340/380 trace of Fura-2 ratiometric Ca2+ imaging experiments (1 is first application of 50 μM naltriben, 2 is second application of naltriben after wash). [Bottom Right] Summary chart of Fura-2 Ca²⁺ imaging experiments showing no significant difference between the Ca²⁺ responses of the initial exposure to naltriben (n = 24) and the subsequent perfusion following washout (n = 21).

Figure 3. U87 cell migration and invasion were enhanced by naltriben

(A) Representative images of wound healing following induction of scratch. Cells were treated with vehicle (0.1% DMSO; control; n = 6) or with naltriben (50 μM; n = 10), then images were captured at 0, 4, 8, and 12 hours, and gap closure was analyzed. (B) The wound closure of naltriben-treated cells at 4, 8, and 12 hours were significantly higher compared to the control group at the corresponding time points. ** represents p < 0.01 (Student's t-test). (C) Representative images from Corning Biocoat Matrigel invasion assays to detect cell invasion in vitro. Cells were treated with vehicle (0.1% DMSO; control; n = 6) or with naltriben (50 μM; n = 6) for 12 hours. (D) Invasion of naltriben-treated cells at 12 hours was significantly higher compared to the control group. ** represents p < 0.01 (Student's t-test).

Figure 4. Naltriben upregulated the expression of MMP-2, and increased the phosphorylation of ERK1/2 in U87 cells

(A) Representative images of Western immunoblotting. Cells were treated with naltriben (50 μM) for 24 h. Protein expression was then determined by Western immunoblot. Comparison between naltriben-treated cells and the control group was summarized in: (B) ratio of MMP-2/β-actin; (C) ratio of p-ERK1|2/β-actin; (D) ratio of t-ERK1|2/β-actin; (E) ratio of p-ERK1|2/t-ERK1|2; (F) ratio of p-Akt/β-actin; (G) ratio of t-Akt/β-actin; and (H) ratio of p-Akt/t-Akt. * represents p < 0.05 (Student's t-test, n = 3/group).

Figure 5. Schematic model illustrating the cellular signaling involved in the effects of TRPM7 potentiation on U87 migration and invasion

The TRPM7 channel can respond to extracellular or cytosolic stimuli through regulation of Ca²⁺ and Mg²⁺ influx. In addition, TRPM7 contains a self-phosphorylating α-type Ser/Thr protein kinase domain that also modulates the phosphorylation of other cytosolic substrates such as PLC, which in turn, can regulate MAPK/ERK signaling. Consequently, functional gene transcription and translation are affected. Naltriben potentiates TRPM7, which upregulates MAPK/ERK signaling, and ultimately enhances U87 migration and invasion.