Research ArticleExperimental Studies

Phenethyl Isothiocyanate (PEITC) and Benzyl Isothiocyanate (BITC) Inhibit Human Melanoma A375.S2 Cell Migration and Invasion by Affecting MAPK Signaling Pathway In Vitro

YI-SHIH MA, YUNG-TING HSIAO, JEN-JYH LIN, CHING-LUNG LIAO, CHIN-CHUNG LIN and JING-GUNG CHUNG
Anticancer Research November 2017, 37 (11) 6223-6234;

Abstract

Background/Aim: Numerous evidence has shown that PEITC and BITC inhibit cancer cell migration and invasion. In this study, we investigated the anti-metastatic mechanisms of PEITC and BITC in human melanoma cancer A375.S2 cells in vitro. Materials and Methods: We used a cell viability assay, an in-vitro scratch wound healing assay, a transwell assay for cell migration and invasion, a gelatin zymography assay, western blotting and EMSA to examine the anti-metastatic mechanisms of PEITC and BITC in A375.S2 cells. Results: Sublethal concentrations of PEITC (0, 1, 2 and 2.5 μM) and BITC (0, 0.5, 1 and 2 μM) inhibited mobility, migration and invasion of A375.S2 cells that were assayed by wound healing and Transwell filter. PEITC and BITC inhibited MMP-2 activity in A375.S2 cells, as assessed by gelatin zymography assay. Results from western blotting indicated that PEITC (2.5 μM) and BITC (2 μM) decreased the levels of p-p38 following 24 and 48 h treatment. PEITC (1-2.5 μM) reduced the levels of p-JNK1/2 proteins following 48-h treatment but BITC increased p-JNK1/2 levels following 24-h treatment. PEITC (2.5 μM) reduced the levels of p-ERK1/2 proteins following 48-h treatment but BITC (0.5-2 μM) increased p-ERK1/2 levels following 24- and 48-h treatment. PEITC and BITC affect cell migration and invasion of A375.S2 cells via MAPK pathway. PEITC and BITC inhibited MMP-2 activity. PEITC increased NF-κB expression but BITC decreased NF-κB expression in the nucleus. Furthermore, NF-κB p65 binding to DNA was decreased following 2.5 μM PEITC treatment, but increased following treatment with 1-2 μM. However, 0.5-2 μM BITC treatment decreased the binding of NF-κB to DNA in A375.S2 cells, as assessed by electrophoretic mobility shift (EMSA) assay. Conclusion: Based on these observations, we suggest that PEITC and BITC can be used as anti-metastastic agents of human melanoma cells in the future.

Skin cancer is one of the most common malignant neoplasms in the white population. Melanoma is much more common in whites than in other ethnic groups (1). In USA, the incidence of melanoma is the fifth and seventh most common cancer among men and women, respectively (2). Worldwide, the incidence rate of melanoma skin cancer is increasing (3). At ages over the age of 75 years old, the incidence rate is almost three times higher in males than that in females (4, 5). In the past years, metastatic melanomas have been increased and led to a high rate of death for patients with melanoma (6). The treatment of melanoma includes surgical resection, chemotherapy in non-resectable cases, and immunotherapy; however, the cure rate is still unsatisfying. Therefore, there is urgent need to find and identify new chemicals from natural products for melanoma treatment.

PEITC and BITC both are members of isothiocyanates (ITCs), and have been reported to induce cell death through the induction of cell apoptosis in many human cancer cell lines such as cervical cancer cells (7), non-small cell lung cancer cells (8), prostate cancer cells (9), bladder carcinoma cells (10), breast cancer cells (11), pancreatic carcinoma cells (12) melanoma A375.S2 cells (13).

In human prostate cancer cells, PEITC suppressed the nuclear factor-κB (NF-κB)-regulated gene expression (14) and activated the Atg5-mediated autophagy (15). Recently, we found that PEITC alters gene expression and cell cycle associated protein expression in human glioblastoma GBM 8401 cells (16). We also found that BITC affected cell cycle associated gene expression and apoptosis in human glioblastoma GBM 8401 cells (17). PEITC has been clinically assessed against human lung cancer (18). BITC-induced cell death, involves the induction of ROS production, autophagy and apoptosis in human prostate cancer cells (19). Our previous studies also showed that PEITC and BITC inhibited migration and invasion of human colon cancer HT29 cells through NF-κB and inhibition of MMP-2, -7 and -9 (20, 21). They also have been found to inhibit migration and invasion of human brain glioblastoma GBM 8401 through the inhibition of uPA, Rho A, and Ras as well as inhibition of MMP-2, -7 and -9 gene expression (22). BITC inhibited the gene expression of MMP-2, -7 and -9, FAK, ROCK1 and Rho A in AGS cells (23). BITC inhibited hepatocellular carcinoma cell migration and invasion via decreasing MMP-2 and -9, survivin and CXCR4 expression (24). Although numerous studies have shown that PEITC and BITC inhibit cancer cell migration and invasion, there is no study on the effect of PEITC and BITC on migration and invasion of human melanoma A375.S2 cells. Herein, we focused on the effects of PEITC and BITC on A375.S2 cell metastasis in vitro and we found that PEITC and BITC suppressed the migration and invasion of A375.S2 cells.

Materials and Methods

Test chemicals, reagents and culture medium. Phenethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), Tris-HCl, Trypsin, Trypan blue, dimethyl sulfoxide (DMSO) and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). Minimum essential medium (MEM) medium, fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). PEITC and BITC were dissolved in DMSO.

Cell line and culture. Human melanoma A375.S2 cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan) and were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) and 2 mM L-glutamine at 37°C in a 5% CO2 humidified atmosphere (25, 26).

Cell viability assay. A375.S2 cells were plated at a density of 1×105 cells/well in 12 well plate, with MEM overnight and were treated with PEITC or BITC in triplicate with the final concentrations (0, 1, 1.5, 2, 2.5 and 5 μM) and (0, 0.5, 1, 2, 4 and 8 μM) for 48 h, respectively. At the end of incubation, cells were harvested, washed with PBS and were stained with PI (5 μg/ml). The total percentage of cell viability were quantitated by using flow cytometry (Becton-Dickinson, San Jose, CA, USA) as described previously (22).

In vitro scratch wound healing assay. A375.S2 cells (2×105 cells/well) were seeded into 6 well plate for 24 h and grown until reaching a confluent monolayer, after that media was replaced with serum-free MEM medium. Plates were extensively washed with PBS and then were scratched using a sterile 200 μl pipette tip and washed with PBS to remove cell debris. Cells in each well were incubated with PEITC (0, 1, 2 and 2.5 μM) and BITC (0, 0.5, 1 and 2 μM) at 37°C in a 5% CO2 humidified atmosphere for 0 or 24 h and photographed under phase contrast microscopy. Scratch experiments were repeated thrice and representative pictures are presented in this study (27).

Transwell assay for cell migration and invasion examinations. The examination of cell migration and invasion in vitro were performed by using Cell Migration and Invasion System as described previously (28, 29). To assay cell migration, A375.S2 cells (5×104 cells/well) were cultured in serum-free MEM and placed in the upper chamber (8 mm pore size; Millipore, Temecula, CA, USA) coated with collagen. MEM with 10% FBS was placed in the lower chamber. The cells were then incubated with PEITC (0, 1, 2 and 2.5 μM) and BITC (0, 0.5, 1 and 2 μM). Cells penetrated the filter to the lower surface (invasive cells) were fixed with 4% formaldehyde in PBS followed by 2% crystal violet for staining and all samples were examined and photographed under light microscopy at x200. We counted total cells and measured percentage of inhibition based on cell numbers in each picture. To assay for cell invasion, applied the same protocol with the exception of the use of matrigel instead of collagen on the filter membrane as described previously (25, 30).

Gelatin zymography assay. A375.S2 cells (5×105 cells/well) were seeded into 6-well plate for 24 h at 80% confluency and serum-free MEM medium containing PEITC or BITC was individually added to each plate for 24 or 48 h. At the end of incubation, the conditioned medium was harvested and sample underwent electrophoresis on 8% SDS-PAGE gel containing 0.2% gelatin, and then soaked the gel in 2.5% Triton X-100 in dH2O for 30 min two times at room temp. Gels were soaked in substrate buffer (50 mM Tris HCl, 5 mM CaCl2, 0.02% NaN3 and 1% triton X-100, pH 8.0) at 37°C for 24 h by shaking, stained with 0.2% Coomassie blue (Bio-Rad, Hercules, CA, USA) in 10% acetic acid and 50% methanol and then photographed as described previously (31).

Western blotting analysis. A375.S2 cells were maintained in 10 cm dish at the density of 1.5×106 cells and were incubated with PEITC (0, 1, 2 and 2.5 μM) or BITC (0, 0.5, 1 and 2 μM) for 24 or 48 h. At the end of treatment, cells were collected and the whole-cell lysates were subjected to Western blot analysis using anti-p-ERK1/2, p-p38, p-JNK1/2, PKC, PI3K, p-AKT(Thr308), p-AKT(Ser473), PCNA, p65, TIMP-1, MMP-2, MMP-9, 14-3-3-σ, GRB2, Ras, SOS-1, N-cadherin, E-cadherin, p-FAK, Lamin A/C and β-actin. Following washing the membrane was incubated with diluted secondary antibodies (diluted 1: 5,000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After the final washing, signals were revealed by enhanced chemiluminescence using the ECL detection system (Amersham ECL™; GE Healthcare, Chicago, IL, USA) and recorded on film. The relative photographic density was quantitated as described previously (32).

Figure 1.
Figure 1.

PEITC and BITC affect the cell viability of A375.S2 cells. Cells (1×105 cells/well) were incubated with various concentrations of PEITC (0, 1, 1.5, 2, 2.5 and 5 μM) and BITC (0, 0.5, 1, 2, 4 and 8 μM) for 48 h. Cells were collected for total percentage of total viable cells, as described in Materials and Methods. *p<0.05, **p<0.01 and ***p<0.001, significant difference between PEITC- or BITC-treated groups and the control as analyzed by the Student's t-test.

Figure 2.
Figure 2.

PEITC and BITC affect in vitro wound closure of A375.S2 cells. Cells (2×105 cells/well) were maintained in 6-well plates for 24 h and were wounded with a scratch and incubated with PEITC (0, 1, 2 and 2.5 μM) or BITC (0, 0.5, 1 and 2 μM) for 24 h. The relative wound closures were photographed using phase contrast microscopy, as described in Materials and Methods.

Figure 3.
Figure 3.

PEITC and BITC suppressed cell migration and invasion of A375.S2 cells in vitro. Cells (5×104 cells/well) were placed on filter (coated with collagen for migration or matrigel for invasion) and were treated with PEITC (0, 1, 2 and 2. 5 μM) or BITC (0, 0.5, 1 and 2 μM) for 48 h. A375.S2 cells penetrated through to the lower surface of the filter and were stained with crystal violet and photographed under a light microscope at ×200 (A and B) and cells were counted, as described in Materials and Method. Results were obtained from three independent experiments. *p<0.05 and ***p<0.001, significant difference between PEITC- or BITC-treated groups and the control, as analyzed by the Student's t-test.

Figure 4.
Figure 4.

PEITC and BITC affect the activities of MMP-2 in A375.S2 cells. Cells (5×105 cells/well) were incubated with PEITC (0, 1, 2 and 2.5 μM) or BITC (0, 0.5, 1 and 2 μM) for 24 and 48 h and then conditioned media were harvested for gelatin zymography assay, as described in Materials and Methods.

Electrophoretic mobility shift assay (EMSA). After treatment, cells were collected and nuclear extracts were prepared by the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, Illinois, USA). 5 μg nuclear extract proteins were used to perform EMSA with a LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, Illinois, USA) according to the protocol of the manufacturer as described previously (33).

Statistical analysis. Results are presented as mean±SD. A significant difference between the PEITC and BITC-treated and control groups were compared by Student's t-test. A p<0.05 was considered as an indication of statistical significance.

Results

PEITC and BITC decrease viability of A375.S2 cells. As indicated in Figure 1, PEITC (1-5 μM) and BITC (0.5-8 μM) treatment for 48 h exhibited a strong cytotoxic activity in A375.S2 cells in vitro. Therefore, we selected 1, 2.5 and 5 μM of PEITC and 0.5, 1 and 2 μM of BITC concentrations for scratch wound healing assay, cell migration and invasion, western blotting and EMSA experiments.

PEITC and BITC inhibited cell mobility of A375.S2 cells. As indicated in Figure 2 which was obtained from wound healing assay, 24 h treatment of A375.S2 cells with PEITC and BITC inhibited the closure rate of the scratch, compared to the control group. The effect of PEITC was dose dependent but that of BITC was not (Figure 2).

PEITC and BITC inhibited cell migration and invasion of A375.S2 cells. To study migration and invasion, transwell chambers coated with collagen or matrigel were used, respectively, and the results are shown in Figure 3. Figure 3A indicates that PEITC (2 and 2.5 μM) and BITC (1 and 2 μM) significantly inhibited the migration of A375.S2 cells. PEITC inhibited cell invasion of A375.S2 cells in dose-dependent manner but BITC at 1 and 2 μM significantly inhibited the invasion of A375.S2 cells (Figure 3B).

PEITC and BITC inhibited MMP-2 activities in A375.S2 cells. After A375.S2 cells were treated with various concentrations of PEITC and BITC for 24 or 48 h, culture medium were harvested to assay MMP-2 activities by using gelatin zymography and the results are shown in Figure 4. Results indicate that following 24 h treatment, only 2.5 μM of PEITC inhibited MMP-2 activity. However, following 48 h treatment, 1, 2 and 2.5 μM of PEITC inhibited MMP-2 activity. 1 and 2 μM of BITC treatment for 24 h inhibited MMP-2 activity. 0.5-2 μM treatment for 48 h with BITC inhibited MMP-2 activity.

PEITC and BITC affect key metastasis-related proteins in A375.S2 cells. In order to understand whether PEITC and BITC inhibited A375.S2 cell migration and invasion through inhibition of metastasis associated protein expression, cells were treated with PEITC (0, 1, 2 and 2.5 μM) and BITC (0, 0.5, 1 and 2 μM) for 24 or 48 h and cell extracts were harvested for Western blotting. The results revealed that MAPK associated signaling proteins such as p-p38, p-JNK1/2 and p-ERK1/2 are involved. The levels of p-p38 were decreased at PEITC (2.5 μM) and BITC (2 μM) at both time treatments (Figure 5A). PEITC (1-2.5 μM) and BITC (0.5-2 μM) decreased the levels of p-JNK1/2 proteins at 48 h treatment (Figure 5A). PEITC (2.5 μM) reduced p-ERK1/2 proteins at 48 h treatment (Figure 5A). However, BITC (0.5-2 μM) treatment at 24 and 48 h increased p-ERK1/2 levels (Figure 5A).

PEITC (1-2.5 μM) inhibited PKC and PI3K proteins at 24 and 48 h treatment (Figure 5B), however, BITC (2 μM) treatment decreased PKC and BITC (0.2-5 μM) treatment increased PI3K levels at 24 and 48 h (Figure 5B). PEITC (1-2.5 μM) increased p-AKT (Ser473) levels at 24 h treatment but decreased them at 48 h (Figure 5B). BITC (2 μM) treatment at both time periods increased p-AKT (Ser473) levels (Figure 5B). PEITC and BITC treatment at both time periods decreased p-AKT (Thr308) (Figure 5B). PEITC (1-2.5 μM) increased PCNA and p65 at 24 and 48 h treatment. BITC only at 24 h treatment increased PCNA, but decreased PCNA levels at 48 h treatment (Figure 5B) and decreased p65 at 2 μM at both time periods (Figure 5B).

Figure 5.
Figure 5.
Figure 5.

PEITC and BITC affect the levels of associated proteins in migration and invasion of A375.S2 cells. Cells (1.5×106 cells/dish) were treated with PEITC (0, 1, 2 and 2.5 μM) and BITC (0, 0.5, 1 and 2 μM) for 24 and 48 h. Cells were collected and total protein was determined for SDS-PAGE gel electrophoresis, as described in Materials and Methods. The levels of p-p38, p-ERK1/2 and p-JNK1/2 (A); PKC, PI3K, p-AKT(Ser473), p-AKT(Thr308), PCNA and p65 (B); TIMP1, MMP-2 and MMP-9 (C); 14-3-3σ, GRB2, Ras and SOS-1 (D); N-cadherin, E-cadherin and p-FAK (E) were estimated by western blotting.

PEITC (1-2.5 μM) increased TIMP-1 at 24 and 48 h treatment, but 2 μM BITC treatment at 24 and 48 h decreased TIMP-1 (Figure 5C). PEITC (1-2.5 μM) decreased MMP-2 at 24 and 48 h treatment but BITC (0.5-2 μM) treatment at 24 and 48 h increased MMP-2 (Figure 5C). PEITC (2.5 μM) decreased MMP-9 at 24 and 48 h treatment but BITC (0.5-2 μM) treatment at 24 increased MMP-9 and 48 h treatment decreased MMP-9 (Figure 5C).

PEITC (1-2.5 μM) decreased 14-3-3σ only at 48 h treatment but BITC (0.5-2 μM) decreased 14-3-3σ at both 24 and 48 h (Figure 5D). PEITC (1-2.5 μM) decreased GRB2 at 24 and 48 h treatment but BITC (2 μM) only at 48 h decreased GRB2 (Figure 5D). PEITC (2-2.5 μM) increased Ras levels at 24 h and decreased them at 48 h, but BITC (0.5-2 μM) increased Ras at both time periods (Figure 5D). PEITC (1-2.5 μM) increased SOS-1 for both time periods, but BITC only at 0.5 and 1 μM increased SOS-1 at 24 h and decreased of all doses at 48 h (Figure 5D).

PEITC (1-2.5 μM) decreased levels of N-cadherin at 24 h but increased them at 48 h (Figure 5E). BITC (0.1-2 μM) increased N-cadherin at both time periods (Figure 5E). PEITC (1-2.5 μM) increased E-cadherin at 24 and 48 h, however, BITC (0.5-2 μM) at 24 h decreased and at 48 h increased levels of E-cadherin (Figure 5E). PEITC (1-2.5 μM) decreased p-FAK at 24 h but at 48 h treatment only at 2.5 μM decrease p-FAK levels (Figure 5E). 0.5 μM of BITC at 24 h and 0.5-2 μM of BITC at 48 h increased p-FAK, respectively (Figure 5E).

PEITC and BITC affected the binding of NF-κB p65 to DNA in A375.S2 cells. For further investigating whether or not PEITC and BITC affect NF-κB expression and the binding of NF-κB p65 to DNA in A375.S2 cells, cells were treated with PEITC (0, 1, 2.5 and 5 μM) and BITC (0, 0.5, 1 and 2.5 μM) for 48 h and were collected for Western blotting and EMSA assay. The results are shown in Figure 6. The levels of nuclear NF-κB protein were increased at (1-2.5 μM) treatment with PEITC but BITC treatment at all doses decreased NF-κB expression (Figure 6). Furthermore, NF-κB p65 bind to DNA was decreased following PEITC treatment at 2.5 μM but 1-2 μM treatment increased binding; however, BITC treatment at 0.5-2 μM decreased binding of NF-κB to DNA in A375.S2 cells (Figure 6).

Discussion

Although there reports indicating that about 90% of cancer deaths are via cancer cell metastasis, so far, there is no complete understanding regarding the exact pathogenetic mechanisms involved in metastasis (34). Despite the fact that numerous studies have already demonstrated that PEITC and BITC induce apoptotic cell death in many cancer cell lines, the molecular mechanisms by which PEITC and BITC affect cell migration and invasion in human melanoma A375.S2 cells are unclear. Herein, we investigated the effect of PEITC and BITC on migration and invasion in A375.S2 cells. Results indicated that PEITC and BITC display cytotoxic effects on A375.S2 cells in a dose depedent manner (Figure 1) (1, 2.5 and 5 μM of PEITC and 0.5, 1 and 2 μM of BITC). PEITC displayed a dose-dependent inhibition of mobility of A375.S2 cells (Figure 2), however, BITC did not (Figure 2). PEITC and BITC have significantly suppressed the migration of A375.S2 cells (Figure 3A) and inhibited cell invasion of A375.S2 cells in a dose-dependent manner (Figure 3B). PEITC and BITC at examined doses significantly inhibited MMP-2 activity in A375.S2 cells (Figure 4). PEITC and BITC have affected MAPK signaling associated proteins such as p-ERK1/2, p-p38 and p-JNK1/2 (Figure 5A). The PEITC (1-2.5 μM) decreased PKC and PI3K (Figure 5B) but BITC decreased PKC only at 2 μM and both time treatments led to increased PI3K (Figure 5B) in A375.S2 cells. PEITC increased PCNA at 24 and 48 h treatment but BITC only increased at 24 h treatment and decreased at 48 h (Figure 5B). PEITC reduced MMP-2 protein levels but BITC increased them. Thus, our findings indicate that PEITC and BITC have anti-metastatic activity in melanoma.

We used wound healing assay and transwell chamber coated with collagen or matrigel to assay for cell migration and invasion. Results indicate that PEITC and BITC suppressed cell mobility, migration and invasion of A375.S2 cells at non-cytotoxic concentrations (Figures 2 and 3). Both assays are well known for measuring cell migration and invasion in vitro (20, 22, 23). We also used gelatin zymography assay for measuring MMP-2 activity in A375.S2 cells after exposure to PEITC and BITC. Numerous studies have used gelatin zymography assay for measuring MMP-2 activity in cancer cells in vitro (20, 22, 23). It is well-documented that MMPs play an important role in degrading the extracellular matrix (ECM) which could be a mechanical barrier to cell movement. Furthermore, the proteolytic activities of MMPs have been shown to be involved in the process of metastasis including cell adhesion, migration and invasion (35-38). Therefore, the inhibition of MMPs has been recognized to lead in suppression of cancer cell metastasis (28, 29) and MMPs have been shown to be present in melanoma (39) and many cancer cells express high levels of MMPs that facilitate cancer invasion and metastasis (40). We evaluated MMP-2 and MMP-9 activities and the levels of both proteins which are associated with reduced survival in human malignancies (41, 42) and with metastatic cancer and tumor-induced angiogenesis (43).

We found that PEITC inhibited MMP-2 activities (Figure 4) and secretion (Figure 5C) as assayed by gelatin zymography and western blotting, respectively. However, BITC inhibited MMP-2 activity but increased MMP-2 protein levels at all examined doses following 24 or 48 h treatment. It has been reported that MMP-2 is associated with tumor invasion and angiogenesis and that inhibited MMP-2 could suppress tumor metastasis (44, 45).

It is well-documented that mitogen-activated protein kinase (MAPK) (p38, ERK1/2 and JNK1/2) pathway is involved cell death and survival (46, 47) and is also involved in the regulation of the expression of MMP-2 and MMP-9 (48). Herein, we found that PEITC (2.5 μM) and BITC (2 μM) decreased the levels of p-p38 at both time treatments. PEITC (1-2.5 μM) significantly inhibited p-JNK1/2 proteins at 48 h treatment but BITC treatment increased p-JNK1/2 levels at 24 h treatment (Figure 5A). PEITC (2.5 μM) inhibited p-ERK1/2 proteins at 48 h treatment (Figure 5A) but BITC (0.5-2 μM) treatment at 24 and 48 h increased p-ERK1/2 levels (Figure 5A). Thus, PEITC and BITC may affect cell migration and invasion of A375.S2 cells via MAPK pathway. MAPK pathway regulated cellular invasion and metastasis (49) and suppression of MAPK pathway could prevent cancer's invasion and metastasis including in melanoma (50, 51).

Figure 6.
Figure 6.

PEITC and BITC affect the binding of NF-κB p65 on DNA in A375.S2 cells. Cells were treated with PEITC (0, 1, 2 and 2.5 μM) and BITC (0, 0.5, 1 and 2 μM) for 48 h and then were assayed using western blotting and EMSA, as described in Materials and Methods.

We found that PEITC inhibited PKC and PI3K proteins (Figure 5B) and BITC decreased PKC only at 2 μM and increased PI3K levels at 24 and 48 h (Figure 5B). PEITC (1-2.5 μM) increased p-AKT (Ser473) at 24 h treatment but decreased them at 48 h (Figure 5B). BITC (2 μM) increased p-AKT (Ser473) levels (Figure 5B). PEITC and BITC treatment at both time periods led to decreased p-AKT (Thr308) (Figure 5B). It has been reported that AKT activation could induce cancer invasion and metastasis by stimulating secretion of MMPs (52, 53). Furthermore, activated PI3K/AKT signaling is involved in tumor cell invasion and oncogenesis (54, 55) and it is also involved melanoma cells invasion (56), however, the inhibition of the PI3K/AKT pathway led to decreased invasion of melanoma cells (52, 53). PEITC (1-2.5 μM) increased E-cadherin at 24 and 48 h treatment, but BITC (0.5-2 μM) treatment decreased E-cadherin at 24 h but increased at 48 h (Figure 5E). AKT and PI3K have been reported to suppress transcription of the E-cadherin gene (57).

NF-κB has been shown to be linked to tumor cell proliferation, survival, invasion, and metastasis (58) and chemo- and radio-resistance (59, 60). Herein, we found that PEITC increased the levels of nuclear NF-κB protein but BITC decreased NF-κB expression (Figure 6). We also used EMSA assay to show that PEITC (1-2 μM) promoted DNA bindings but BITC (0.5-2 μM) inhibited DNA binding of NF-κB in A375.S2 cells (Figure 6). BITC reduction of binding of NF-κB was accompanied by inhibition of the nuclear protein expression of this factor in A375.S2 cells. From the cytoplasm NF-κB translocates to the nucleus for binding on the promoter region of MMP-9 gene to elevate gene expression (58, 61).

Taken together, our results indicate that PEITC and BITC significantly inhibit cell mobility, migration and invasion of A375.S2 cells in vitro. Western blotting assays also indicated that PEITC and BITC inhibited expression of metastasis associated protein such as MAPKs, MMP-2, MMP-9, E-cadherin and NF-κB in A375.S2 cells. NF-κB translocates to nuclei for binding to promoter of target gens. These findings suggest that both PEITC and BITC can be considered to be co-chemotherapeutic treatments for melanoma cells in the future.

Acknowledgements

This work was supported by the China Medical University, Taichung, Taiwan [grant numbers CMU106-ASIA-01]. This work was supported by the National Science Council, Taipei, Taiwan [grant numbers NSC103-2320-B-039-037-].

Footnotes

  • ↵* These Authors contributed equally to this work.

  • This article is freely accessible online.

  • Conflicts of Interest

    The Authors do not have any conflicts of interest to disclose.

  • Received August 23, 2017.
  • Revision received September 15, 2017.
  • Accepted September 18, 2017.

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Phenethyl Isothiocyanate (PEITC) and Benzyl Isothiocyanate (BITC) Inhibit Human Melanoma A375.S2 Cell Migration and Invasion by Affecting MAPK Signaling Pathway In Vitro
YI-SHIH MA, YUNG-TING HSIAO, JEN-JYH LIN, CHING-LUNG LIAO, CHIN-CHUNG LIN, JING-GUNG CHUNG
Anticancer Research Nov 2017, 37 (11) 6223-6234;
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