This article is mentioned in:

Introduction

Angiogenesis is a biological process defined as the formation of new blood vessels from pre-existing ones (1). Angiogenesis is involved in various physiological processes, including tumor metastasis, tissue repair, wound healing and embryonic development (2,3). Endothelial cells constitute the inner wall of blood vessels (4) Upon pro-angiogenic stimulation, endothelial cells are activated and secrete proteolytic enzymes to degrade the vascular basement membrane (5). Subsequently, the endothelial cells proliferate and migrate, thus contributing to the formation and growth of blood vessels (6,7). Increasing evidence has demonstrated that angiogenesis is associated with various diseases including cancer, inflammatory disease, cardiovascular disease and diabetes (8,9). Previous studies have demonstrated that tumor angiogenesis is regulated by multiple cytokines, including prostaglandin E2, transforming growth factor-β (TGF-β), fibroblast growth factor and vascular endothelial growth factor (10,11).

Magnesium, one of the most important minerals, is essential for physiological processes and cellular metabolism (12). Magnesium ions stabilize the structures of cell membranes, nucleic acids and proteins, and enhance the activities of ribozymes and enzymes (13). Zinc is an essential trace element for humans, as it is a constituent of various enzymes, regulating their catalyzing activity (14). Zinc ions are easily absorbed and do not harm vital organs (15). Zinc is also a cofactor for many transcription factors, proteins and enzymes that are involved in DNA repair, cell apoptosis, cell cycle regulation and oxidative stress (16). It has been reported that calcium and magnesium ions serve vital roles in the growth, mineralization and angiogenesis of bone tissues (17).

It has been demonstrated that cell migration and cytoskeletal reorganization in endothelial cells is closely associated with angiogenesis (18). The epithelial-mesenchymal transition (EMT) is a process during which cells lose their epithelial features and acquire mesenchymal properties, including reduced adhesive and enhanced invasion abilities (19,20). During EMT, E-cadherin expression is downregulated, while expression of N-cadherin, vimentin, fibronectin and transcription factors are upregulated (21,22). EMT is associated with multiple biological processes, including organ fibrosis, tissue regeneration, wound repair and tumor progression (23,24). Angiogenesis is a multi-step process that includes proliferation, migration and tube formation of endothelial cells (25). HUVECs have been widely used to investigate the effect of drugs on angiogenesis (26). However, the effects of MgCl2 and ZnCl2 on the biological characteristics of human umbilical vein endothelial cells (HUVECs) are not fully understood. In the present study, human HUVECs were cultured in vitro and the possible roles and molecular mechanisms of MgCl2 and ZnCl2 in the metastasis and EMT of HUVECs were investigated.

Materials and methods

Cell culture

HUVECs were obtained from the China Center for Type Culture Collection (Wuhan, China) and cultured in Dulbecco's modified Eagle medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in an incubator (HF-90; Shanghai Lishen Scientific Equipment Co., Ltd., Shanghai, China) containing 5% CO2. The culture medium was supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA).

MTT assay

HUVECs were plated at a density of 5×103 cells/well onto 96-well plates and cultured for 24 h at 37°C. Following this, cells were incubated at 37°C with various doses of MgCl2 (1.25, 2.5, 5, 6.25, 12.5, 25, 50 and 100 mM) or ZnCl2 (1, 25, 50, 100, 300 and 500 µM; both Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Untreated HUVECs served as a control. Following 24 h treatment, MTT solution (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to the cells and they were incubated at 37°C for 4 h. Subsequently, the supernatant was discarded and 200 µl dimethylsulfoxide (Sigma-Aldrich; Merck KGaA) was added to dissolve the formazan crystals. The absorbance was recorded at 490 nm using an ELX-800 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Migration and invasion assays

The upper chamber of a Transwell chamber (Corning Life Sciences, Tewksbury, MA, USA) was coated with 45 µl diluted Matrigel (BD Biosciences, San Jose, CA, USA) and placed in a 24-well plate for the invasion assay. HUVECs were cultured in DMEM supplemented with 10% FBS. At 90% confluence, the medium was replaced with serum-free DMEM and cells were incubated with 10 µg/ml mitomycin C (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C. Following this, cells were digested with 0.25% trypsin and resuspended in serum-free DMEM to prepare a single cell suspension. Cells were seeded into the upper chamber uncoated (8,000 cells in 200 µl cell suspension for migration assay) or coated with Matrigel (2×104 cells in 200 µl cell suspension for invasion assay) and DMEM supplemented with 20% FBS was added into the lower chamber. Subsequently, the cells were incubated with 12.5 mM MgCl2 or 100 µM ZnCl2. After 24 h cell culture at 37°C, the cells on the upper side of the filters were wiped with cotton swabs. The migrated or invaded cells were fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 20 min at room temperature and stained with crystal violet (Amresco, LLC, Cleveland, OH, USA) for 5 min. The migrated or invaded cell number was counted in five random fields under an AE31 inverted microscope (Motic, Xiamen, China) at magnification, ×200, and the average was calculated.

Western blotting analysis

HUVECs were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1% phenylmethanesulfonyl fluoride protease inhibitor (ST506; Beyotime Institute of Biotechnology) on ice for 5 min and centrifuged at 10,005 × g for 10 min at 4°C. The supernatant was collected and protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (40 µg/lane) were separated by 8 or 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1 h. Subsequently, membranes were incubated with antibodies against matrix metalloproteinase (MMP)-2 (1:400; BA0569; Wuhan Boster Biological Technology, Ltd., Wuhan, China), MMP-9 (1:400; BA0573; Wuhan Boster Biological Technology, Ltd.), vimentin (1:500; bs-8533R; BIOSS, Beijing, China), Snail (1:500; bs-1371R; BIOSS), N-cadherin (1:400; BA0673; Wuhan Boster Biological Technology, Ltd.), Wnt1 (1:200; sc-5630; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-catenin (1:400; BA0426; Wuhan Boster Biological Technology, Ltd.) overnight at 4°C. Subsequent to washing with Tris-buffered saline-Tween-20, membranes were incubated with goat anti-rabbit horseradish peroxidase-labeled immunoglobulin G (1:5,000; A0208; Beyotime Institute of Biotechnology) for 45 min at 37°C and visualized using a high sensitivity enhanced chemiluminescence reagent kit (WLA003; Wanleibio Co., Ltd., Shenyang, China). The experiment was repeated three times and results were quantified using Gel-Pro Analyzer v.4 (Media Cybernetics, Inc., Rockville, MD, USA).

ELISA

The supernatant of the cell culture was harvested by centrifugation (1,000 × g for 10 min) following 24 h treatment with MgCl2 or ZnCl2 at 37°C. The expression levels of MMP-2 and MMP-9 in the supernatant were measured using ELISA kits (DRE11368 and DRE10154; Shanghai WHB Biotech Co., Ltd., Shanghai, China), according to the manufacturer's instructions. The plates were read on a microplate reader (BioTek Instruments, Inc.) at 450 nm.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from HUVECs using RL lysis buffer containing DNase, according to the manufacturer's protocol (BioTeke Corp., Beijing, China). RNA was then reverse transcribed into cDNA using M-MLV reverse transcriptase (BioTeke Corp.). Template RNA (1 µg) was mixed with 1 µl Oligo (dT)15, 1 µl random primer, and 2 µl dNTP and ddH2O (final volume of 14.5 µl), incubated at 70°C for 5 min and then put on ice for 2 min. The mixture was subsequently incubated with 0.5 µl RNasin, 4 µl reaction buffer and 1 µl (200 U) M-MLV reverse transcriptase (final volume of 20 µl) at 25°C for 10 min, followed by 42°C for 50 min and 95°C for 5 min. The primer sequences for qPCR were as follows: E-cadherin, forward 5′-AGAACGCATTGCCACATACA-3′ and reverse 5′-TAAGCGATGGCGGCATTGTA-3′; N-cadherin, forward 5′-CAACACACTCGCAGACGCTCA-3′ and reverse 5′-AAGACGGCTCCAGGCAGTTT-3′; and β-actin, forward 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′ and reverse 5′-CTGTCACCTTCACCGTTCCAGTTT-3′. qPCR was performed using an Exicycler™ 96 Real-Time Quantitative PCR system (Bioneer Corporation, Daejeon, Korea). The cycling conditions were as follows: 10 min at 95°C; followed by 40 cycles of 10 sec at 95°C, 20 sec at 60°C and 30 sec at 72°C (20 µl reaction volume comprising 1 µl cDNA template, 0.5 µl forward primer, 0.5 µl reverse primer, 10 µl SYBR Green PCR Mastermix and 8 µl ddH2O). The experiment was repeated three times and β-actin was used as an internal control. The relative mRNA expression levels were normalized to β-actin, following the 2−ΔΔCq method (27).

Gelatin zymography

HUVECs were treated with MgCl2 or ZnCl2 for 24 h and subsequently lysed by repeated freezing and thawing. Following centrifugation of the homogenate at 10,005 × g for 10 min at 4°C, the supernatant was harvested and the protein concentration was determined by the BCA method (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg/lane) were separated by 10% SDS-PAGE containing 1 ml gelatin (10 mg/ml) (Sigma-Aldrich; Merck KGaA). Following this, the gels were washed twice with elution buffer (40 min; 2.5% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl2 and 1 µM ZnCl2; pH 7.6) at room temperature and then twice with washing buffer (20 min; 50 mM Tris-HCl, 5 mM CaCl2 and 1 µM ZnCl2; pH 7.6). Subsequently, the gels were stained with Coomassie Brilliant Blue R-250 (Amresco, LLC) for 3 h. Gels were then destained with methanol and acetic acid, and imaged using a gel documentation system (WD-9413B; Beijing Liuyi Instrument Factory, Beijing, China).

Cytoskeletal staining

HUVECs were seeded in coverslips and incubated with 12.5 mM MgCl2 or 100 µM ZnCl2 for 24 h at 37°C. Following this, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 15 min at room temperature and washed three times with PBS. Subsequently, coverslips were incubated with Acti-stain™ 488 Fluorescent Phalloidin (F-actin staining; Cytoskeleton, Inc., Denver, CO, USA) for 1 h. Then, 4′,6-diamidino-2-phenylindole (DAPI; Biosharp, Hefei, China) was added to counterstain the nuclei of the cells. Images were captured under a fluorescent microscope (magnification, ×400; BX53; Olympus Corp., Tokyo, Japan) and analyzed using Image Pro Plus software v.6 (Media Cybernetics, Inc.).

Immunofluorescence staining

HUVECs were plated on slides, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. Subsequent to blocking with goat serum (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) for 15 min at room temperature, the slides were incubated with antibody against E-cadherin (1:200; BA0474; Wuhan Boster Biological Technology, Ltd.) at 4°C overnight and then with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature (1:200; A0562; Beyotime Institute of Biotechnology). Following this, the slides were stained with DAPI and imaged under a fluorescent microscope (magnification, ×400; BX53; Olympus Corp.).

Statistical analysis

Results were presented as the mean ± standard deviation. The differences were analyzed by one-way analysis of variance followed by Bonferroni post hoc tests using GraphPad Prism v.5 software (GraphPad Software, Inc., La Jolla, CA, USA) or Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

MgCl2 and ZnCl2 do not significantly influence cell viability

HUVECs were incubated with various doses of MgCl2 (0, 1.25, 2.5, 5, 6.25, 12.5, 25, 50 and 100 mM) and ZnCl2 (0, 1, 25, 50, 100, 300 and 500 µM) for 24 h. The cytotoxicity of MgCl2 or ZnCl2 was measured by MTT assay. The results demonstrated that the cell viability of HUVECs was not significantly affected following exposure to all concentrations of MgCl2 and ZnCl2 (Fig. 1). Therefore, 12.5 mM MgCl2 and 100 µM ZnCl2 were selected for further experiments.

Figure 1. Cytotoxic effect of MgCl2 and ZnCl2 on HUVECs. HUVECs were exposed to various concentrations of MgCl2 (0, 1.25, 2.5, 5, 6.25, 12.5, 25, 50 and 100 mM) and ZnCl2 (0, 1, 25, 50, 100, 300 and 500 µM) for 24 h. Cells were then subjected to MTT assay and cell viability was determined. Data are presented as mean ± standard deviation. HUVECs, human umbilical vein endothelial cells.

MgCl2 and ZnCl2 promote the migration and invasion of HUVECs in vitro

Cell migration and invasion assays using Transwell chambers were performed to investigate the effect of MgCl2 and ZnCl2 on the migration and invasion capabilities of HUVECs. The results demonstrated that both MgCl2 and ZnCl2 significantly enhanced the migration and invasion abilities of HUVECs compared with the control group (Fig. 2A). The extracellular matrix and basement membrane provide the major physical barriers to cell invasion. MMPs are important proteolytic enzymes able to degrade the extracellular matrix and basement membrane and serve critical roles in the invasion process (28). MgCl2 and ZnCl2 treatment significantly increased the expression of MMP-2 and MMP-9 proteins, as determined by western blotting (Fig. 2B) and ELISA (Fig. 2C). RT-qPCR determined that the expression of MMP-2 and MMP-9 mRNA was significantly increased (Fig. 2D). The gelatin zymography assay demonstrated that MMP-2 and MMP-9 activity was significantly enhanced by MgCl2 and ZnCl2 (Fig. 2E).

Figure 2. Effects of MgCl2 and ZnCl2 on the migration and invasion of HUVECs. (A) The migration and invasion capabilities of HUVECs following MgCl2 and ZnCl2 stimulation were examined by Transwell assays. The numbers of migrated and invaded cells were counted in five random fields and the average was calculated. The migrated and invaded cells were stained with crystal violet. Scale bar, 100 µm (magnification, ×200). (B) Total proteins were extracted from the MgCl2- and ZnCl2-treated HUVECs. Levels of MMP-2 and MMP-9 proteins were measured by western blotting. β-actin was used as an internal control. (C) MMP-2 and MMP-9 levels in the supernatant of cell cultures were also determined by ELISA. (D) Reverse transcription-quantitative polymerase chain reaction analysis of MMP-2 and MMP-9 mRNA levels. (E) MMP-2 and MMP-9 activities were measured using gelatin zymography. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. the control. HUVECs, human umbilical vein endothelial cells; MMP, matrix metalloproteinase.

MgCl2 and ZnCl2 induce cytoskeletal reorganization in HUVECs

Subsequently, the effects of MgCl2 and ZnCl2 on cytoskeletal reorganization in HUVECs were investigated. The results demonstrated that MgCl2 and ZnCl2 treatment promoted cytoskeletal reorganization, with an increased long axis/short axis dimension ratio compared with the control (Fig. 3).

Figure 3. Effects of MgCl2 and ZnCl2 on cytoskeletal reorganization in HUVECs. The effect of MgCl2 and ZnCl2 on cytoskeletal dynamics was analyzed using F-actin staining. The nuclei were then counterstained with DAPI. The long axis dimension and the short axis dimension were measured. Scale bar, 50 µm (magnification, ×400). Data are presented as the mean ± standard deviation. *P<0.05 vs. the control. HUVECs, human umbilical vein endothelial cells; DAPI, 4′,6-diamidino-2-phenylindole.

MgCl2 and ZnCl2 promote EMT

To further investigate whether MgCl2 and ZnCl2 were able to modulate EMT, the expression of several EMT-related genes were measured using RT-qPCR, western blotting and immunofluorescence staining assays. The results demonstrated that the expression of E-cadherin was downregulated following MgCl2 and ZnCl2 stimulation; however, this difference was only significant following ZnCl2 stimulation (Fig. 4A). N-cadherin mRNA and protein expression was significantly upregulated following MgCl2 and ZnCl2 stimulation compared with the control (Fig. 4A and B). Furthermore, the results demonstrated that MgCl2 and ZnCl2 treatment significantly increased the expression of vimentin and Snail at both mRNA and protein levels (Fig. 4A and B). The immunofluorescence staining assay further demonstrated that E-cadherin was downregulated following stimulation with MgCl2 and ZnCl2 (Fig. 4C).

Figure 4. Effects of MgCl2 and ZnCl2 on EMT. (A) Levels of E-cadherin, N-cadherin, vimentin and Snail mRNA were measured using reverse transcription-quantitative polymerase chain reaction and normalized to β-actin expression. (B) Levels of N-cadherin, vimentin and Snail protein were quantified by western blotting. (C) E-cadherin expression was analyzed by immunofluorescence staining. The nuclei were stained with DAPI. Scale bars, 50 µm (magnification, ×400). Data are presented as the mean ± standard deviation. **P<0.01 vs. the control. EMT, epithelial-mesenchymal transition; DAPI, 4′,6-diamidino-2-phenylindole.

MgCl2 and ZnCl2 activate the Wnt/β-catenin signaling pathway

HUVECs were incubated with MgCl2 and ZnCl2 for 24 h and the expression of Wnt1 and β-catenin were measured by western blotting analysis. The expression of Wnt1 and β-catenin protein were significantly increased in MgCl2- and ZnCl2-treated HUVECs compared with the control (Fig. 5).

Figure 5. Effects of MgCl2 and ZnCl2 on the Wnt/β-catenin pathway. Levels of Wnt1 and β-catenin protein were determined by western blotting and β-actin was used as an internal control. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. the control.

Discussion

In the present study, HUVECs were treated with various concentrations of MgCl2 and ZnCl2. Cell viability was measured by MTT assay and the optimum concentrations of MgCl2 and ZnCl2 were determined. The effect of MgCl2 and ZnCl2 on the migration, invasion, cytoskeletal dynamics and EMT of HUVECs was then investigated in vitro and the mechanisms involved were also studied. To the best of our knowledge, the present study was the first to demonstrate that MgCl2 and ZnCl2 enhanced the migration and invasion abilities of HUVECs, stimulated cytoskeletal reorganization and induced EMT via the Wnt/β-catenin signaling pathway.

The impact of MgCl2 and ZnCl2 on the motility of HUVECs was examined. It was determined that MgCl2 and ZnCl2 promoted the migration and invasion of HUVECs. MMPs are key modulators of various biological processes, including the EMT process, cancer, angiogenesis, skeletal formation, inflammation and cell migration (29). Notably, both MMP-2 and MMP-9 are crucial gelatinases that regulate angiogenesis in endothelial cells (30). It has been observed that ZnCl2 reverses the inhibitory effects of ellagic acid on MMP-2 expression, MMP-2 activity and the migration of HUVECs (31). Mg2+ is the most abundant divalent cation in cells in the human body and has been demonstrated to be associated with various cell functions (32). It has been determined that high Mg2+ levels enhance microvascular endothelial cell (1G11 cell) migration and induce angiogenesis (33). A study by Takatani-Nakase et al (34) demonstrated that Zn2+ contributes to the promotion of cell migration in breast cancer cells following exposure to high glucose. However, the effects of MgCl2 and ZnCl2 treatment on HUVEC motility, MMP-2 and MMP-9 levels and activities are not fully understood. The present study indicated that MgCl2 and ZnCl2 promoted HUVEC migration and invasion, as determined by Transwell assays. Additionally, MgCl2 and ZnCl2 treatment increased MMP-2 and MMP-9 expression at the mRNA and protein levels. Meanwhile, MMP-2 and MMP-9 activities were markedly enhanced. The results suggest that MgCl2 and ZnCl2 may promote HUVEC migration and invasion by regulating the expression and activities of MMPs.

EMT is characterized by a loss of cell-cell adhesion and increase in cell motility (35,36). Cytoskeletal reorganization is involved in the process of EMT and is a crucial hallmark of EMT (37,38). In the present study, it was determined that MgCl2 and ZnCl2 induced reorganization of the actin cytoskeleton in HUVECs. Various genes are associated with the EMT process, including epithelial markers, mesenchymal markers and transcription factors (39,40). During EMT, E-cadherin and cytokeratin (epithelial markers) are downregulated, whereas fibronectin, N-cadherin and vimentin (mesenchymal markers) are upregulated (41). E-cadherin is a transmembrane glycoprotein that regulates cell-cell adhesion (42). It is a major epithelial marker and its expression is reduced in cells that have undergone EMT (39). N-cadherin is a calcium-dependent adhesion molecule and in the presence of Ca2+, N-cadherin resists hydrolysis by protease and promotes tumor cell metastasis (43). Snail, a zinc finger transcription factor, promotes EMT of microvascular endothelial cells (43). Vimentin is a type III intermediate filament protein with a molecular weight of 57 kDa and is expressed in non-epithelial cells, particularly in mesenchymal cells (44,45). A study by Xiao et al (46) indicated that Zn2+ induces EMT in human gastric adenocarcinoma cells via the gastrin gene. The present study demonstrated that MgCl2 and ZnCl2 incubation led to a significant decrease in E-cadherin and increases in N-cadherin, vimentin and Snail expression. These results suggest that MgCl2 and ZnCl2 may promote the EMT of HUVECs by regulating the expression of EMT markers.

It has been demonstrated that various signaling pathways participate in the EMT process, including the Wnt signaling pathway (47). A study by Scheel et al (48) determined that the Wnt signaling pathway and TGF-β induces EMT in mammary epithelial cells. Furthermore, it has been determined that Wnt signaling is the earliest event in the EMT process and cell invasion (49). Thus, the present experiments also investigated on the activation of the Wnt/β-catenin signaling pathway. The results demonstrated that the expression of Wnt1 and β-catenin protein were significantly increased following MgCl2 and ZnCl2 treatment, indicating that MgCl2 and ZnCl2 may promote the migration and invasion of HUVECs via the Wnt/β-catenin pathway. These results provide a potential therapeutic strategy for the inhibition of HUVEC migration, invasion, EMT and angiogenesis.

In conclusion, the results of the present study suggest that MgCl2 and ZnCl2 may promote cell migration and invasion and stimulate cytoskeletal reorganization and EMT by activating the Wnt/β-catenin pathway. Further studies are required to verify these observations.

References