Carvacrol Alleviates Prostate Cancer Cell Proliferation, Migration, and Invasion through Regulation of PI3K/Akt and MAPK Signaling Pathways

TRPM7 is a potential therapeutic target for treatment of prostate cancer. In this study, we investigated the effects of nonselective TRPM7 inhibitor carvacrol on cell proliferation, migration, and invasion of prostate cancer PC-3 and DU145 cells. Our results showed that carvacrol blocked TRPM7-like currents in PC-3 and DU145 cells and reduced their proliferation, migration, and invasion. Moreover, carvacrol treatment significantly decreased MMP-2, p-Akt, and p-ERK1/2 protein expression and inhibited F-actin reorganization. Furthermore, consistently, TRPM7 knockdown reduced prostate cancer cell proliferation, migration, and invasion as well. Our study suggests that carvacrol may have therapeutic potential for the treatment of prostate cancer through its inhibition of TRPM7 channels and suppression of PI3K/Akt and MAPK signaling pathways.


Introduction
Prostate cancer (PCa) is the second leading cause of cancerrelated death in men [1][2][3]. Although multiple treatment options are available, it is currently lack of effective therapies for the treatment of androgen-independent prostate cancer which often arises after hormonal deprivation or ablation therapy [4].
Transient receptor potential melastatin-like 7 channel (TRPM7) is a member of melastatin-like transient receptor potential (TRPM) subfamilies, widely expressed in mammalian cells [5]. It is permeable to Ca 2+ and Mg 2+ and other divalent cations and has an alpha-kinase domain [6]. It is found that TRPM7 is highly expressed in a number of human cancer tissues and cell lines to regulate cell proliferation, migration, and invasion, such as glioblastoma [7], ovarian cancer [8], and breast cancer [9]. Increasing Ca 2+ and Mg 2+ influx promotes the proliferation of prostate cancer cells through activating TRPM7 [10]. Moreover, cholesterol activates TRPM7 and thus increases Ca 2+ entry, regulating proliferation, migration, and viability of human prostate cells [11]. Inhibition of TRPM7 enhances TNF-related apoptosis inducing-ligand-(TRAIL-) induced apoptosis in PC-3 cells [12], indicating that TRPM7 contributes to the pathogenesis of prostate cancer and serves as a potential therapeutic target for prostate cancer [13]. So far, several signaling pathways were reported to be regulated by TRPM7, including signal Transducer and Activator of Transcription 3 (STAT3), Notch, PI3K/Akt, and MAPK signaling pathways [14,15]. In prostate cancer cells, knockdown TRPM7 by shRNA inhibited cholesterol-induced Akt or ERK phosphorylation [11]. Hence, it suggests that both PI3K/Akt and MAPK signaling pathways are the downstream mechanisms of TRPM7 functions in prostate cancer.
Carvacrol (CAR) is a natural-bioactive monoterpenoid phenol with multiple uses. It is used as flavor agent in cosmetic and food products and the most active constituent of thyme EOs extracted from many plants, including fruits, vegetables, spices, and herbs. Carvacrol also exhibits antifungal, antiviral, antitumor, and anti-inflammatory activities [16]. Carvacrol was first reported by Parnas et al. as a nonselective TRPM7 inhibitor [17]. The inhibitory effects of carvacrol on TRPM7 and TRPM7-like currents in HEK293 cells and glioblastoma cell line were further confirmed [7]. However, the pharmacological effects of carvacrol on the proliferation, migration, and invasion of prostate cancer cells have not yet been investigated.
In this study, we compared the TRPM7 protein expression between control prostate cells and PCa cells. We further evaluated the effects of carvacrol on TRPM7-like currents, proliferation, migration, and invasion in PC-3 and DU145 cells and investigated the potential underlying mechanisms involved in these effects.

Cell Culture and Reagents.
Nonneoplastic human prostatic epithelial cells (RWPE-1) using as control prostate cell line as well as prostate cancer cell lines DU145 (HTB-81) and PC-3 (CRL1435) were obtained from the American Type Culture Collection (Manassas, VA). PWPE-1 cells were maintained in defined keratinocyte serum-free medium (K-SFM) containing 50 g/mL bovine pituitary extract and 5 ng/mL EGF (Invitrogen, USA). DU145 and PC-3 cells were cultured in DMEM with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 ng/mL) and maintained at 37 ∘ C with 95% humidified air and 5% CO 2 and passaged as needed. Culture medium was changed twice weekly. Cell culture related materials were purchased from Gibco Life Technologies Corporation (USA). All other reagents used were purchased from Sigma-Aldrich (USA) unless mentioned otherwise.

RNAi Assay.
Lentivirus plasmids were obtained from Addgene (Cambridge, MA) in pLKO.1 cloning vector and contained either nonspecific control shRNA (sh-Control) or shRNA specific for human TRPM7 (GeneBank: AY032950). According to the other study, the sequences for TRPM7 and control shRNA were as follows: GTCTTGCCATGAAAT-ACTC and TGTGCTCCGAACGTGTAGT [18]. ShRNA viruses were packaged and produced following the protocol provided online by Addgene Company (http://www.addgene .org/tools/protocols/plko/). PC-3 cells were infected with either lentiviral-sh-Control or lentiviral-sh-TRPM7 (MOI = 40). Culture medium was changed to the fresh medium 24 h after infection. TRPM7, p-Akt, and p-ERK1/2 protein expression were determined at 72 h after infection, and TRPM7like current was determined as well. In the meantime, cells with infection for 72 h were digested and seeded into the corresponding culture plate to carry out CCK-8 assay, wound healing, and Transwell assay. Briefly, cells were seeded on 96-well plates at a density of 0.5 × 10 5 cells/well and grown for additional 24 h prior to the experiment. Cells were treated as indicated concentration of carvacrol and corresponding vehicle for 24, 48, and 72 h. Then CCK-8 solution (10 L) was added to each well and incubated for additional 1 h. Absorbance at 450 nm was measured using a microplate reader (Syngery H1, Biotek, USA). Cell viability was expressed as a percentage of the vehicle control.

Colony Formation.
Colony formation experiments were carried out according to our previous study [19,20]. PC-3 and DU145 cells (300 cells/well) were seeded in 6-well plates overnight and subsequently treated with carvacrol (500 M) for 24 h, and then it was replaced with fresh culture medium without carvacrol. After that, culture medium was changed every 5 days. After 10 days of culture, cells were fixed with 100% ice-cold Methanol for 10 minutes and stained with 0.5% crystal violet solution for 10 min, then washed with water, and air-dried. Cell colonies images were captured using a digital camera connected to a phase-contrast Olympus microscope (×10 objectives). Colony numbers (containing >50 cells) were determined using Image-Pro Plus software. Data were presented as a percentage of vehicle control.

Wound Healing.
Wound healing experiments were carried out according to our previous study [21,22]. Briefly, cells were seeded in 6-well plates (5 × 10 5 /well) and grown to about 80% confluence, and then the monolayer of cells was scratched with a 200 L pipette tip to create a wound gap and treated with either carvacrol (500 M) or corresponding vehicle control for 24 and 48 h. Cells were allowed to migrate in serum-free medium as indicated time point. Cell images and the scratches were photographed using a phasecontrast Olympus microscope (10x objective). Throughout experiments, the same visual field was used. The gap lengths were measured by Image-Pro Plus software.

Transwell Assay.
Invasion experiments were carried out according to our previous study [14,23]. BioCoat Matrigel invasion chambers (8 m polycarbonate Nucleopore filters, Cat. 354480) were used. Briefly, PC-3/DU145 cells were treated with carvacrol (500 M) or equivalent vehicle for 24 h. Then 100 L of cells (2.5 × 10 4 cells/mL) in FBS-free DMEM was plated in the upper chamber and the lower chamber contained medium with 10% FBS/DEEM. After incubating for 24 h at 37 ∘ C in 5% CO 2 , nonmigrated cells in upper chamber were scraped from the upper surface of the membrane using cotton swab. Migrated cells remaining on the bottom surface were fixed with 75% ethanol and stained with crystal violet (0.1%). Finally, images of the invaded cells were photographed and invading cells were counted using Image-Pro Plus software.

Immunofluorescent
Staining. Immunofluorescent staining experiments were carried out according to our previous study [24,25]. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature (RT) and then permeabilized for 30 min with 0.1% Triton X-100 in PBS. Rhodamine phalloidin staining was performed following the manufacturer's instructions. Cells were incubated with rhodamine phalloidin (1 : 50; Molecular Probes, USA) to label F-actin and with DAPI (1 g/mL, Sigma-Aldrich, USA) to label nucleic acid, for 20 min at RT. Immunofluorescent images were captured from at least 6 randomly chosen areas using Zeiss confocal microscope.

Patch Clamp Recording.
Patch clamp experiments were carried out according to Sun et al. 's report [10]. Whole cell currents were recorded using an Axopatch 200B (Axon Instruments, Inc.), with holding potential of 0 mV, 100 ms voltage ramps ranging from −100 to +100 mV, and 2-s intervals at 2 kHz. pClamp 9.2 software was used for data acquisition and analysis. The bath solution contained 145 mm NaCl, 5 mm CsCl, 1 mm MgCl 2 , 1 mm CaCl 2 , 10 mm Hepes, 10 mm glucose, and pH 7.4 (NaOH). Patch pipette resistance was between 3-5 megaohms after filling with pipette solution containing 150 mm cesium methane sulfonate, 8 mm NaCl, 10 mm Hepes, 10 mm EGTA, and pH 7.2 (CsOH). All recordings were carried out at RT.

Statistical
Analysis. Data are presented as means ± SEM. Two-way unpaired Student's t-test was used to compare the statistical significance between two groups, and ANOVA with subsequent Newman-Keuls test was used for multiple comparisons. < 0.05 was considered statistically significant for all tests.

Carvacrol Inhibits PC-3 and DU145 Cell Proliferation.
Then, we evaluated the effects of carvacrol on the proliferation of PCa cells. As shown in Figure 2

Carvacrol Reduces PCa Cell Migration.
Wound healing assay was carried out to detect cell migration. As shown in Figures 3(a) and 3(b), after treatment of 24 h, wound closures of PC-3 and DU145 in control group were 56.4±8.5 and 38.7± 5.9, respectively. And after treatment of 48 h, wound closure of vehicle control in PC-3 and DU145 cells increased to 83.9 ± 4.2% and 92.5 ± 7.1%, respectively. Carvacrol significantly inhibited cell wound healing of PC-3 and DU145 cells ( < 0.05, = 6), as the wound closure of PC-3 and DU145 cells in carvacrol treatment group was 31.8 ± 9.2 and 21.6 ± 4.1 at 24 h and 42.4 ± 8.6% and 35.6 ± 7.9% at 48 h, respectively. Thus, compared with vehicle control, carvacrol (500 M) significantly reduced PC-3 and DU145 cell migration.
As cell migration is related to reorganization of the actin cytoskeleton, we also measured the cytoskeletal actin organization by staining F-actin with phalloidin in PC-3 cells and DU145 cells. As shown in Figures 3(a ) and 3(b ), F-actin was condensed at the leading edge within structures resembling fans or protrusions in vehicle group. After treatment with carvacrol, less F-actin was condensed in dot-like structures at the margins of the cells, compared to vehicle control cells. The data suggest that inhibition of cell migration by carvacrol might be related to its prevention of F-actin reorganization.

Carvacrol Suppresses PI3K/Akt and MAPK Signaling
Pathways. Next, we studied the underlying signaling pathway involved in the anti-PCa effects of carvacrol. As shown in Figure 5, in carvacrol treatment group, phosphorylation of p-Akt and p-ERK in PC-3 cells was significantly reduced to 21.2 ± 4.5% and 36.4 ± 7.9% of vehicle control ( < 0.06, = 6). In the meantime, similar results were observed in DU145 cells. The data suggest that PI3K/Akt and MAPK signaling pathways are involved in anti-PCa effects of carvacrol.
The protein levels of p-Akt and p-ERK1/2 were reduced by TRPM7 knockdown as well (Figure 6(h)).

Discussion
In the present study, we demonstrated that carvacrol inhibited TRPM7-like currents in PCa cells and reduced cell proliferation, migration, and invasion of PCa cell. Furthermore, we found that carvacrol treatment decreased MMP-2, p-Akt, and p-ERK protein expression and blocked F-actin reorganization in PCa cells. Consistently, TRPM7 knockdown inhibited PC-3 cell proliferation, migration, and invasion. It also suppressed p-Akt and p-ERK protein expression in PC-3 cells as well. TRPM7 channels are widely expressed in a variety of cells including prostate tissues [28]. Activation of TRPM7 promotes prostate cancer cell proliferation, migration, and viability [10,11], while inhibition of TRPM7 by Gd 3+ or 2-aminoethoxy diphenylborate (2-APB) enhances TRAILinduced PC-3 cell apoptosis [12]. Carvacrol, an approved food  flavor additive by the United States Food and Drug Administration (FDA), with oral LD 50 is 810 mg/kg in rats [29] which was reported as a nonspecific TRPM7 inhibitor with IC 50 of 306 ± 65 M [17]. Our data showed the inhibitory effects of carvacrol on TRPM7-like currents in PC-3 and DU145 cells, which was consistent with another researcher's study [7]. Furthermore, our results showed that both carvacrol treatment and TRPM7 knockdown significantly suppressed cell proliferation, migration, and invasion of PCa cells. These results suggest that blocking TRPM7 by carvacrol plays a key role in PCa growth and metastasis.
Cell adhesion and spreading properties directly regulate the cellular motility and invasiveness. F-actin dynamics is essential for alteration of cytoskeleton during cell migration and invasion [30]. We found that carvacrol treatment inhibited F-actin condensing at the leading edge of PCa cells, indicating that carvacrol reduced PCa cell motility through blocking F-actin-mediated cytoskeleton alteration. Matrix metalloproteinase-2 (MMP-2) is essential for focal extracellular matrix (ECM) degradation and invasion of the surrounding tissue. MMP-2 expression decreased in glioblastoma cells by treatment with carvacrol [7]. Consistently, we also found that carvacrol reduced MMP-2 protein expression in both PC-3 and DU145 cells. Hence, we could speculate that suppression of PCa functions by carvacrol might be closely related to regulation of F-actin and MMP-2 expression.
PI3K/Akt and MAPK signaling pathways are important in the PCa growth and metastasis [31,32]. Phosphorylation of Akt and ERK is the key proteins regulating both signaling pathways, respectively. TRPM7 downexpression decreases the phosphorylation of p-Akt in ovarian cancer cells and lung fibroblasts and also decreases the phosphorylation of p-ERK1/2 in breast cancer cells [8,33,34]. In this study, our results showed that carvacrol reduced levels of p-Akt and p-ERK in both PC-3 and DU145 cells, which is consistent with another study [7].
Taken together, carvacrol treatment represses cell proliferation, migration, and invasion in both PC-3 and DU145 PCa cell lines, likely by blocking TRPM7-like current and reducing MMP-2 protein expression and F-actin dynamics. Moreover, both the PI3K/Akt and MEK/MAPK signaling pathways are involved in these antiprostate cancer effects. Our findings indicate that carvacrol has antiprostate cancer effects in vitro.