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. 2015 Jun 30;6(18):16321-40.
doi: 10.18632/oncotarget.3872.

Inhibition of TRPM7 by carvacrol suppresses glioblastoma cell proliferation, migration and invasion

Wen-Liang Chen  1   2 , Andrew Barszczyk  2 , Ekaterina Turlova  1   2 , Marielle Deurloo  2 , Baosong Liu  1   2 , Burton B Yang  3 , James T Rutka  1 , Zhong-Ping Feng  2 , Hong-Shuo Sun  1   2   4   5
Affiliations

Inhibition of TRPM7 by carvacrol suppresses glioblastoma cell proliferation, migration and invasion

Wen-Liang Chen et al. Oncotarget. .

Abstract

Glioblastomas are progressive brain tumors with devastating proliferative and invasive characteristics. Ion channels are the second largest target class for drug development. In this study, we investigated the effects of the TRPM7 inhibitor carvacrol on the viability, resistance to apoptosis, migration, and invasiveness of the human U87 glioblastoma cell line.The expression levels of TRPM7 mRNA and protein in U87 cells were detected by RT-PCR, western blotting and immunofluorescence. TRPM7 currents were recorded using whole-cell patch-clamp techniques. An MTT assay was used to assess cell viability and proliferation. Wound healing and transwell experiments were used to evaluate cell migration and invasion. Protein levels of p-Akt/t-Akt, p-ERK1/2/t-ERK1/2, cleaved caspase-3, MMP-2 and phosphorylated cofilin were also detected.TRPM7 mRNA and protein expression in U87 cells is higher than in normal human astrocytes. Whole-cell patch-clamp recording showed that carvacrol blocks recombinant TRPM7 current in HEK293 cells and endogenous TRPM7-like current in U87 cells. Carvacrol treatment reduced the viability, migration and invasion of U87 cells. Carvacrol also decreased MMP-2 protein expression and promoted the phosphorylation of cofilin. Furthermore, carvacrol inhibited the Ras/MEK/MAPK and PI3K/Akt signaling pathways.Therefore, carvacrol may have therapeutic potential for the treatment of glioblastomas through its inhibition of TRPM7 channels.

Keywords: TRPM7; carvacrol; cell viability; glioblastoma; invasion; migration.

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Conflict of interest statement

COMPETING INTERESTS

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Increased expression of TRPM7 mRNA and protein in U87 cells compared to NHA cells
A, TRPM7 mRNA in NHA and U87 cells was detected by RT-PCR. TRPM7 mRNA expression was normalized to GAPDH. The results showed that TRPM7 mRNA level in U87 cells increased compared to NHA cells (*, p<0.05, Student's t-test, n=6). B, TRPM7 protein expression in NHA and U87 cells was measured by western blotting from three different passages. After normalized to β-actin, the results showed that TRPM7 protein expression in U87 cells was higher than in NHA cells (*, p<0.05, Student's t-test, n=3). C, TRPM7 protein in situ expression in NHA and U87 cell was detected by immunofluorescence. Images were captured by a laser scanning confocal microscope and representative images are shown. The fluorescence intensity of TRPM7 staining was normalized to β-tubulin. 150 cells were chosen randomly from each experiment for analysis. Analysis showed that TRPM7 protein expression in U87 cells was more abundant than in NHA cells (white scale bar = 20 nm, *, p<0.05, Student's t-test, n=3).
Figure 2
Figure 2. Carvacrol (CAR) blocked TRPM7 currents in HEK293 cells overexpressing TRPM7 and TRPM7-like currents in U87 cells
All currents were recorded as described in the methods section. A, Carvacrol blocked TRPM7 currents in tetracycline (Tet)-induced TRPM7-overexpressing HEK293 cells. Left panel: representative I-V trace (1 is bath solution, 2 is 300μM carvacrol perfusion). Middle panel: representative time course of the outward current of TRPM7 at +80 mV. Right panel: analysis of outward current at +80 mV comparing perfusion with bath solution to carvacrol perfusion. TRPM7 currents were blocked by carvacrol and restored after washout of carvacrol (*, p<0.05, Student's t-test, n=8 cells). B, Carvacrol did not significantly affect background TRPM7 currents in HEK293 cells without tetracycline treatment. Left: representative current–voltage (I-V) trace of TRPM7 current (1 is bath solution, 2 is 300 μM carvacrol perfusion). Middle panel: typical time course of outward currents of TRPM7 at +80 mV. Right panel: analysis of TRPM7 outward current in bath solution and CAR perfusion. These data indicate that carvacrol perfusion does not have significant effects on background TRPM7 currents in HEK293 cells without tetracycline treatment (p>0.05, Student's t-test, n=6 cells). C, carvacrol blocks TRPM7-like currents in U87 cells. Left panel is the representative I-V trace (1 is trace of bath solution, 2 is trace of perfusion with 500 μΜ carvacrol, 3 is trace of washout of carvacrol). Middle and right panels: outward and inward currents of TRPM7 at +100mV and −100 mV. Carvacrol (500 μM) significantly blocked TRPM7-like currents in U87 cells (*, p<0.05, Student's t-test, n=3 cells).
Figure 3
Figure 3. Carvacrol reduced U87 cell viability and proliferation, and induced apoptosis
A, Carvacrol dose-dependently reduced the viability of U87 cells. U87 cells were treated with carvacrol from 125 to 1000 μM for 24 hours. An MTT assay was used to evaluate the cell viability and IC50 was calculated (n=8). B, carvacrol inhibited cell proliferation of U87 cells. U87 cells were treated with carvacrol (125-1000 μM) for 24, 48 and 72 hours and then an MTT assay was used to measure the proliferation. Carvacrol (250μM) significantly inhibited U87 cell proliferation at 48 and 72 hours (“a” indicates p<0.05 versus vehicle group, one-way ANOVA with subsequent Newman-Keuls test, n=8). Carvacrol (500-1000μM) significantly inhibited U87 cell proliferation at 24, 48 and 72 hours (“b,c,d” indicate p<0.05 versus vehicle group, one-way ANOVA with subsequent Newman-Keuls test, n=8). C, carvacrol induced U87 cell nuclear condensation. DAPI staining was used to observe nuclear condensation (arrows) as amorphological alteration indicating apoptosis. Representative images are from three independent experiments. D, carvacrol increased cleaved caspase-3 protein level. Western blotting was used to detect cleaved caspase-3 levels in U87 cells. Carvacrol treatment for 24 hours dose-dependently and significantly increased cleaved caspase-3 protein level in U87 cells (*, versus control, p<0.05, one-way ANOVA with subsequent Newman-Keuls test, n=6).
Figure 4
Figure 4. Carvacrol inhibited U87 cell migration, invasion and MMP-2 protein expression
A, carvacrol inhibited U87 migration. The representative images of wound healing were displayed. After being scratched with a 200μL pipette tip, U87 cells were treated with CAR (500 μM) or vehicle (0.1% DMSO), then images were captured at 0, 6, 12, and 24 hours, and gap closure was analyzed B. The wound closure of carvacrol treatment groups at 6, 12 and 24 hours was significantly different compared to the control group at the corresponding time-point (*, p<0.05, Student's t-test, n=4). C, carvacrol inhibited U87 cell invasion. Representative images are from transwell experiments to detect cell invasion in vitro. D, analysis of transwell experiments (* versus control, p<0.05, Student's t-test, n=3). E, carvacrol dose-dependently reduced MMP-2 protein expression in U87 cells. U87 cells were treated with carvacrol (500 and 750 μM) for 24 hours. Western blotting was carried out to detect MMP-2 protein expression and β-actin was used as a loading control (* versus control, p<0.05, one-way ANOVA with subsequent Newman-Keuls test, n=6).
Figure 5
Figure 5. Carvacrol increased p-cofilin level and reduced F-actin polymerization in U87 cells
A-D, Western blotting results of p-cofilin and t-cofilin protein expression in U87 cells. It showed that carvacrol (500μM) treatment for 24 hours increased p-cofilin level but not t-cofilin expression in U87 cells (*, versus control, p<0.05, one-way ANOVA with subsequent Newman-Keuls test, n=6). E and F, representative images from rhodamine phalloidin staining. U87 cells without carvacrol treatment displayed abundant bright actin clusters as indicated by arrows. G, analysis of the average number of bright actin clusters. The number of F-actin-rich bright actin clusters in U87 cells were significantly reduced by carvacrol (500 μM) treatment for 24 hours (* versus control, p<0.05, Student's t-test, n=3).
Figure 6
Figure 6. Carvacrol reduced p-Akt and p-ERK1/2 protein levels in U87 cells
U87 cells were treated with carvacrol (250 and 500 μM) for 24 hours, and then protein expression was detected by western blotting. A, Representative images of western blotting results. B, Carvacrol (250 and 500 μM) significantly reduced p-Akt protein level in a dose-dependent manner. * p<0.05, one-way ANOVA with subsequent Newman-Keuls test, n=6. C, Carvacrol did not significantly affect t-Akt protein expression (p>0.05, n=6). D, Ratio of p-Akt/t-Akt decreased in the carvacrol (250 and 500 μM) group in a dose-dependent manner. *, p<0.05, one-way ANOVA with subsequent Newman-Keuls test, n=6. E, Carvacrol (500μM) significantly decreased p-ERK1/2 protein level. * p<0.05, one-way ANOVA with subsequent Newman-Keuls test, n=6. F, Carvacrol did not significantly affect t-ERK1/2 protein expression (p>0.05, n=6). G, Ratio of p-ERK1/2/t-ERK1/2 decreased in the carvacrol (500 μM) group. *, p<0.05, one-way ANOVA with subsequent Newman-Keuls test, n=6.
Figure 7
Figure 7. Silencing TRPM7 reduced cell viability, migration and invasion
A, U87 cells were transfected with siRNA for 72 hours, then western blotting was carried out. TRPM7 siRNA significantly decreased TRPM7 protein expression. *, p<0.05, Student's t-test, n=4. B, Whole-cell patch-clamp experiments were performed after siRNA transfection for 72 hours. It showed the representative current–voltage (I-V) traces of TRPM7-like current in U87 cells with NC and siRNA transfection. C, TRPM7-like currents in U87 cells were significantly inhibited by TRPM7 siRNA. * versus NC, p<0.05, Student's t-test, from 5 cells in NC group and 9 cells in siRNA group. D, An MTT assay shows that TRPM7 silencing significantly reduced U87 cell viability. *, p<0.05, Student's t-test, n=5. E and F, Wound healing experiments show that TRPM7 silencing significantly inhibited U87 cell migration. *, p<0.05, Student's t-test, n=3. G, Transwell experiments show that TRPM7 silencing significantly reduced cell invasion. *, p<0.05, Student's t-test, n=3.
Figure 8
Figure 8. TRPM7 silencing reduced p-Akt, p-ERK1/2 and MMP-2 protein levels in U87 cells
U87 cells were transfected with TRPM7 siRNA for 72 hours, and then protein expression was detected by western blotting. A, Representative images of western blots (n=4). B, TRPM7 silencing significantly decreased p-Akt protein level. *, p<0.05, Student's t-test, n=4. C, TRPM7 silencing did not significantly affect t-Akt protein expression (p>0.05, n=4). D, The ratio of p-Akt/t-Akt is reduced in the TRPM7-silenced group. *, p<0.05, Student's t-test, n=4. E, TRPM7 silencing significantly decreases p-ERK1/2 protein level. *, p<0.05, Student's t-test, n=4. F, TRPM7 silencing did not significantly regulate t-ERK1/2 protein expression (p>0.05, n=4). G, Ratio of p-ERK1/2/t-ERK1/2 is reduced in the TRPM7 silencing group. *, p<0.05, Student's t-test, n=4. H, TRPM7 silencing significantly decreased MMP-2 protein expression. *, p<0.05, Student's t-test, n=4.
Figure 9
Figure 9. Schematic diagram of signaling mechanisms involved in the effects of carvacrol on proliferation, migration and invasion of U87 cells
TRPM7 is constitutively active in resting cells. When exposed to various extracellular or cytosolic stress or stimuli, TRPM7 regulates entry of Mg2+ and Ca2+. Moreover, TRPM7 has an α-type serine/threonine protein kinase domain that can phosphorylate itself, as well as cytosolic substrates such as modulating phosphorylation of PLC and subsequently regulating PI3K/Akt and MEK/MAPK signaling pathways, leading to functional gene transcription and translation. As a consequence, the TRPM7 channel modulates cellular proliferation, migration and invasion. It indicates that carvacrol exerts its anti-glioblastoma effects by inhibiting TRPM7 and thus PI3K/Akt and MEK/MAPK signaling pathways.
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