Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro

  • Authors:
    • Kuang-Chi Lai
    • Yung-Ting Hsiao
    • Jiun-Long Yang
    • Yi-Shih Ma
    • Yi-Ping Huang
    • Tai-An Chiang
    • Jing-Gung Chung
  • View Affiliations

  • Published online on: July 27, 2017     https://doi.org/10.3892/ijo.2017.4084
  • Pages: 832-840
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC) have been demonstrated to induce anticancer function in many human cancer cells and also inhibit cancer cell migration and invasion. However, there are no studies that show BITC and PEITC to inhibit cell migration and invasion in mouse melanoma B16F10 cells. In this study, we investigated anti-metastasis effects of BITC and PEITC in melanoma cancer cells in vitro. Under sub-lethal concentrations (from 1, 2.5 up to 5 µM), BITC and PEITC significantly inhibited cell mobility, migration and invasion nature of B16F10 cells. Gelatin zymography assay also showed that BITC and PEITC inhibited matrix metalloproteinase-2 (MMP-2) activity in B16F10 cells. PEITC reduced MAPK signaling associated proteins such as p-ERK1/2, p-p38 and p-JNK1/2 but BITC increased those MAPK signaling associated proteins. BITC and PEITC both suppressed the expression of RhoA, Ras, and SOS-1, however, PEITC increased FAK and GRB2 but BITC increased FAK at 48 h. Furthermore, PEITC decreased the expression of MMP-2 and tissue inhibitors of matrix metalloproteinases (TIMP) but BITC increased them. PEITC inhibited NF-κB protein levels and DNA binding which was confirmed by electrophoretic mobility shift (EMSA) assay. Based on these observations, we suggest that BITC and PEITC can be used in anti-metastasis of melanoma cells in the future.

Introduction

Melanoma has been recognized to be one of the most malignant tumors with the most aggressive and treatment-resistant form of human skin cancer. Currently its incidence is still increasing worldwide. The treatment for this disease after it spread beyond the primary site is difficult (1). Melanoma has rapid proliferation rate (2), however, the exact mechanisms of the rapid proliferation of melanoma cells was unclear (3). Melanoma cell metastasis is also a cause for difficulty in curing this disease. Although melanoma treatment has shown some breakthroughs in targeted and immunotherapy (4), it is still urgently needed to identify new targets for melanoma treatment.

PEITC, a member of isothiocyanates (ITCs), have been shown to induce cell cycle arrest PC-3 human prostate cancer cells (5), oral cancer cells (6), gastric cancer cells (7) and gastric cancer cells (8). Furthermore, several studies have shown that PEITC induced human cancer cell apoptosis (911) and it also inhibited nuclear factor-κB (NF-κB)-regulated gene expression (12) and activation of Atg5-mediated autophagy (13) in human prostate cancer cells. PEITC is also used in clinical trials for lung cancer (14). It was reported that PEITC inhibits the invasion of EGF-stimulated SAS oral cancer cells via targeting EGFR and also to induce its downstream signaling molecules for reducing the expression and enzymatic activities of both matrix metalloproteinase-2 (MMP-2) and MMP-9 (15).

BITC, also one of ITCs, has been shown to induce cell apoptosis in many human cancer cell lines such as bladder cancer cells (16), breast cancer cells (17), ovary cancer cells (18), prostate cancer cells (19) and melanoma A375.S2 cells (20). Furthermore, BITC induced cell cycle arrest and apoptosis in human leukemia cells through the downregulation of myeloid cell leukemia-1 (Mcl-1) (21) and alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells (22).

Metastasis, a multistep process, which is often resistant to conventional therapies such as chemotherapy and radiation therapy, is involved in cell motility, cellular adhesion and invasiveness, entry to blood circulation, and stays in other tissues for new colonization of a distant site (23). One of the major steps for cancer cell metastasis is the breakdown of connective tissue barriers which is involved with proteolytic enzymes such as MMPs to mediate ECM breakdown and facilitate invasion (24). ITCs inhibit the invasion and migration via blocking FAK/JNK-mediated MMP-9 expression in mouse C6 glioma cells (25). In lung cancer cells, BITC and PEITC inhibit cell metastasis potential via the modulation of metastasis-related gene expression and the inhibition of Akt/NF-κB pathway (26). However, there is no available report to show BITC and PEITC suppressing the migration and invasion of mouse melanoma cells. Thus, we investigated the effects of BITC and PEITC on the B16F10 cell metastasis and we found that BITC and PEITC suppressed the migration and invasion of B16F10 cells in vitro.

Materials and methods

Test compound, reagents and culture medium

Benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), dimethyl sulfoxide (DMSO), propidium iodide (PI), Tris-HCl, Trypsin and Trypan blue were purchased from Sigma Chemical Co. (St. Louis, MO, USA). BITC and PEITC were dissolved in DMSO as a carrier solvent and control cultures 0.5% DMSO. DMEM medium, fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA).

Cell culture

Murine melanoma B16F10 cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in 75 cm2 flask with DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in 5% CO2 humidified incubators at 37°C.

Cell viability assay

B16F10 cells (1×105 cells/well) were maintained in 12-well plates with DMEM for 48 h and then PEITC were added to cells at final concentrations (0, 1, 2.5, 5, 10 and 15 µM) and BITC were added to cells at final concentrations (0, 1, 2.5, 5 and 10 µM) for 48 h. After incubation, cells were collected from each treatment, washed with PBS and were stained with PI (5 µg/ml). All samples were analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA) for percentage of viable cells as previously described (27).

Scratch wound healing assay

To investigate the wound healing effect of PEITC and BITC on murine melanoma cells, B16F10 cells (2×105 cells/well) were placed in 6-well plate for 24 h and after the cells formed a confluent monolayer, they were scratched using a sterile pipette tip to create a wound at confluence and washed in PBS to remove cell debris. Cells in each well were incubated with PEITC and BITC at the final concentrations (0, 1, 2.5 and 5 µM) at 37°C with 5% CO2 at time = 0 and 24 h and were photographed by phase contrast microscopy. The relative wound size at each time point of treatment was quantified by ImageJ software. Cell migration inhibition rate (%) = new scratch width/original scratch width × 100% as previously described (28,29).

Cell migration and invasion assay

Matrigel Cell Migration Assay and Invasion System were used for measuring cell migration and invasion in vitro as previously described (30). Cell migration was performed with Transwell cell culture chambers (8-mm pore size; Millipore, Temecula, CA, USA). B16F10 cells (5×104 cells/well) were added in serum-free DMEM and were placed in the upper chamber which was coated with collagen of the Transwell insert and incubated with PEITC and BITC (0, 1, 2.5 and 5 µM). DMEM with 10% FBS was placed in the lower chamber and were incubated for 48 h. The invasive cells (penetrated the filter in the lower surface) were fixed with 4% formaldehyde in PBS and stained with 2% crystal violet and were examined and photographed under light microscopy at ×200 followed by counting for the percentage of inhibition (30). Cell invasion experiment was performed similarly to cell migration assay, using matrigel collagen to replace collagen on the filter membrane (30).

Gelatin zymography assay

B16F10 cells (5×105 cells/well) were maintained in 6-well culture plates for approximately 80% confluency. The serum-free medium with PEITC or BITC was added to each dish for 24 h culture. After incubation, the conditioned medium was collected from each treatment for measuring the total proteins; a 50 µg of protein from each treatment was electrophoresis on 10% SDS-PAGE containing 0.2% gelatin. Gel was washed and soaked in 2.5% Triton X-100 in dH2O twice at 25°C for 30 min twice. Gels were soaked in substrate buffer (50 mM Tris HCl, 5 mM CaCl2, 0.02% NaN3 and 1% Triton X-100, pH 8.0) while shaking for 18 h at 37°C. Gels were stained with 0.2% Coomassie blue (Bio-Rad, Hercules, CA, USA) in 10% acetic acid and 50% methanol (30,31) and were photographed on a light box. Proteolysis was detected as a white zone (MMP-2 gelatinolytic activities) in a dark blue field.

Protein extraction and western blot analysis

B16F10 cells (1×106 cells) in 10-cm dish were incubated with PEITC or BITC (0, 1, 2.5 and 5 µM) for 24 and 48 h. After incubation, cells were collected and lysed in ice-cold potassium phosphate buffer (pH 7.4) containing 2 mM EDTA and 0.1% Triton X-100 for sonication and centrifuged and total protein was measured by Bio-Rad protein assay kit (Bio-Rad) with bovine serum albumin (BSA) as the standard as previously described (30). The protein from each treatment of total cells was separated by 12% SDS-polyacrylamide gel electrophoresis, and transferred onto PVDF nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat milk in TBS-T buffer (10 mmol/l Tris-HCl, 150 mmol/l NaCl, and 0.05% Tween-20, pH 7.8) at room temperature fort 1 h followed by washing in TBS-T buffer. The membrane was incubated with monoclonal antibodies such as anti-p-ERK1/2, anti-p-p38, anti-p-JNK1/2, anti-PKC, anti-phosphatidylinositol 3 kinase (PI3K), anti-p-AKT (Thr308), anti-p-AKT(Ser473), anti-PCAN anti-p-FAK, anti-RhoA, anti-Ras, anti-GRB2, anti-SOS-1, anti-MMP-2, anti-MMP-9, anti-uPA, anti-TIMP-1, anti-NF-κBp65, anti-NF-κBp50 and anti-E-cadherin. Membranes were washed and incubated with the diluted secondary antibodies (goat anti-mouse immunoglobulin G (IgG), diluted 1:5000, Santa Cruz Biotechnology Inc., Dallas, TX, USA; goat anti-rabbit IgG, diluted, 1:5,000, Santa Cruz Biotechnology Inc.).

Investigated proteins on the membrane were visualized using the enhanced chemiluminescence detection system (ECL®, Millipore, Temecula, CA, USA) (30,32).

Electrophoretic mobility shift assay (EMSA)

A 5×105 cells/well of B16F10 cells were placed in a 12-well and were treated with 0, 1, 2.5 and 5 µM of PEITC and BITC for 48 h. Cells were collected for nuclear extracts by the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA). Nuclear extract protein (5 µg) was performed for EMSA with a LightShift Chemiluminescent EMSA kit (Pierce) according to the manufacturer's protocol. 5′-Biotin-GATCCAGGGGACTTTCCCTAGC-3′ (biotin end-labeled oligonucleotide sequences) corresponding to the consensus of NF-κB was developed as previously described (33). Both biotin end-labeled duplex DNA were then incubated with a nuclear extract for further electrophoresis in 6% polyacrylamide native gel and then a 100-fold excess of unlabeled double stranded oligonucleotide was added to the reaction for competition. Both samples (DNA) were transferred to a positive nylon membrane. They were UV cross-linked and probed with biotin-HRP conjugate for incubating with the substrate of ECL kit (Millipore) as previously described (33).

Statistical analysis

All data represent at least 3 independent experiments and are expressed as mean ± SD. A significant difference between the PEITC and BITC-treated and control groups were compared by Student's t-test. *P<0.05 and ***P<0.001 were considered as an indication of statistical significance.

Results

PEITC and BITC decrease the viability of B16F10 cells

In order to understand the possible concentrations for inhibiting cell migration and invasion of B16F10 cells, cells were incubated with PEITC (0–15 µM) and BITC (0–10 µM) for 48 h. After incubation, cells from each treatment were collected for measuring cell viability by flow cytometric assay and results are shown in Fig. 1. Results indicated that PEITC and BITC significantly reduced total cell viability from 5 to 15 µM and 1 to 10 µM, respectively in B16F10 cells. Therefore, we selected 1, 2.5, and 5 µM for scratch wound healing assay, cell migration and invasion experiments.

PEITC and BITC inhibit cell mobility in B16F10 cells

Scratch wound healing assay was used to investigate the inhibition of PEITC and BITC on cell mobility of B16F10 cells in vitro and results are shown in Fig. 2. One of the representative figures was present and wound healing images (cell mobile capabilities) were undertaken at the same magnification and time (0 and 24 h) after PEITC and BITC treatments in B16F10 cells. PEITC and BITC decreased the closure rate of the scratch, dose-dependently, when compared to the control group at 24 h treatment. Based on the results, it indicated that BITC at 1–2.5 µM has higher inhibition of cell mobility than that of PEITC (Fig. 2).

PEITC and BITC suppress migration and invasion of B16F10 cells

In order to further investigate PEITC and BITC suppressed cell migration and invasion in B16F10 cells, the Transwell chamber coated with collagen for cell migration and coated with matrigel for cell invasion were performed and the results are shown in Fig. 3. Fig. 3A indicated that PEITC (1–5 µM) significantly suppressed the migration of B16F10 cells dose-dependently; however, BITC only at 1 µM induced the inhibition of cell migration. PEITC suppressed cell migration was greater than that of BITC. Fig. 3B indicated that PEITC (1–5 µM) significantly suppressed the invasion of B16F10 cells dose-dependently; however, BITC only at 5 µM induced the inhibition of cell invasion. PEITC suppressed cell invasion was greater than that of BITC.

PEITC and BITC inhibit MMP-2 activities in B16F10 cells

B16F10 cells were incubated with various concentrations of PEITC and BITC for 24 and 48 h and were collected for MMP-2 activities by using the gelatin zymography assay and the results are shown in Fig. 4. Results indicated that PEITC and BITC suppressed the activities of MMP-2 in B16F10 cells. The inhibition rate between PEITC and BITC are not significantly different; however both compounds are significantly different when compared to control groups.

PEITC and BITC affect key metastasis-related proteins in B16F10 cells

For further investigating PEITC and BITC suppressed cell invasion and migration involved in the inhibition of metastasis-associated protein expression in B16F10 cells, cells after incubation with PEITC and BITC (0, 1, 2.5 and 5 µM) for 24 and 48 h were harvested for western blotting and the results are shown in Fig. 5. The results revealed several depressed key metastasis-related proteins, such as p-ERK1/2, P-p38 and p-JNK1/2 underwent significant reduction at 24 and 48 h treatment by PEITC (Fig. 5A). However, BITC treatment occurred at both time periods and only p-ERK1/2 was significantly reduced (Fig. 5A). At PEITC and BITC treatment at 24 and 48 h, the p-AKT (Thr308), p-AKT (Ser473) and PCNA were significantly reduced when compared to control, however, PKC and PI3K were significantly increased when compared to control in PEITC treatment, and PKC and PI3K were reduced at 24 h treatment of BITC but it increased the expression of PKC and PI3K at 48 h treatment of BITC (Fig. 5B). The RhoA, Ras and SOS-1 were reduced at 24 and 48 h treatment of PEITC, p-FAK was reduced at 24 h treatment of PEITC, GRB2 was increased at 48 h treatment but p-FAK was increased at 48 h treatment of PEITC (Fig. 5C). However, for BITC treatment only GRB2 at 24 h treatment was increased and p-FAK, RhoA, Ras and SOS-1 were decreased at 24 and 48 h treatment including GRB2 at 48 h treatment of BITC (Fig. 5C). The MMP-2, MMP-9, uPA and TIMP were reduced at 24 and 48 h treatment of PEITC (Fig. 5D), however, at BITC treatment for 24 and 48 h, MMP-2 and MMP-9 were reduced, the TIMP-1 was reduced at 24 h treatment of BITC, uPA was increased at both time periods of treatment and TIMP-1 was increased at 48 h treatment of BITC (Fig. 5D). The NF-κBp65 and NF-κBp50 were reduced at 24 and 48 h treatment of PEITC, however, BITC at both treatment periods, NF-κBp65 were increased but NF-κBp50 was reduced at 48 h treatment but 24 h treatment was increased (Fig. 5E). The E-cadherin was increased at 24 and 48 h treatment of PEITC, however BITC treatment at both time periods were increased (Fig. 5F).

PEITC and BITC decreased the binding of NF-κBp65 on DNA in B16F10 cells

In order to understand the effects of PEITC and BITC on the binding of NF-κBp65 on DNA in B16F10 cells, cells were treated with 0, 1, 2.5 and 5 µM of PEITC and BITC for 48 h and were assayed by using EMSA. The results are shown in Fig. 6. The results indicated that NF-κBp65 bind on DNA was decreased in PEITC treatment, however, BITC treatment was increased.

Discussion

Numerous studies have shown that phenethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) significantly induced cytotoxic effects on many human cancer cell lines. The cytotoxic effects include decreased percentage of viable cell number through cell cycle arrest and apoptosis. However, currently how PEITC and BITC affect mouse melanoma B16F10 cell migration and invasion are unclear. Thus, in the present studies, we investigated PEITC and BITC effect on cell migration and invasion in B16F10 cells in vitro and we found that 1) PEITC has lower cytotoxic effects than that of BITC (Fig. 1), thus, we selected 1, 2.5 and 5 µM for further experiments; 2) wound healing assay showed that PEITC have lower inhibition of wound healing (mobility) than that of BITC except at high dose (5 µM) PEITC inhibited cell mobility more than that of BITC (Fig. 2); 3) results obtained from Transwell chamber coated with collagen or matrigel for cell migration and invasion, respectively, PEITC inhibited cell migration more than that of BITC (Fig. 3A) and PEITC inhibited cell invasion more than that of BITC (Fig. 3B); 4) the inhibition of MMP-2 activity was not significantly different between PEITC and BITC (Fig. 4) western blot examination demonstrated that PEITC has reduced MAPK signaling-associated proteins such as p-ERK1/2, p-p38 and p-JNK1/2 (Fig. 5A), but BITC treatment increased all those MAPK signaling associated proteins (Fig. 5A).

The PEITC increased PI3K but BITC decreased PI3K at both time periods of treatment (Fig. 5B). PEITC and BITC both suppressed the expression of RhoA, Ras, and SOS-1, however, PEITC increased FAK and GRB2, but BITC increased FAK at 48 h (Fig. 5C). PEITC decreased the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) but BITC increased them (Fig. 5D). At 48 h treatment, PEITC decreased NF-κBp65 and NF-κBp50 but BITC increased both (Fig. 5E) and both increased E-cadherin (Fig. 5F); 5) EMSA also confirmed that PEITC inhibited NF-κB binding DNA but BITC increased NF-κB binding to DNA in B16F10 cells (Fig. 6). All these observations are compatible with PEITC and BITC have anti-metastasis capabilities. Thus, our findings may prove that PEITC and BITC have potential as anti-metastatic in melanoma.

It is well known that approximately 90% of cancer deaths are caused by metastasis but the exact pathogenesis and mechanisms involved are not completely clear (34). Herein, we used wound healing and Transwell filter to show B16F10 cell suppression of the cell migration and invasion (Figs. 2 and 3). It is well documented that matrix metalloproteinases (MMPs) are involved in cell metastasis (24) and inhibition of MMPs can lead to suppression of cancer cell metastasis (35). Thus, one of the strategies for inhibiting cancer metastasis is to suppress the regulation of MMP proteins. Increased MMP-2 and MMP-9 activities and expression levels are correlated with reduced survival and poor prognosis in human malignancies (36,37) and in many pathological processes including metastatic cancer and tumor-induced angiogenesis (38). In the present study, we used gelatin zymography assay which demonstrated that PEITC and BITC inhibited activities of MMP-2 in B16F10 cells (Fig. 4). PEITC and BITC inhibited MMP-2 production (Fig. 5D) and activity (Fig. 4) is also evident from the inhibition of collagen matrix invasion in B16F10 cells in vitro. MMP-2 (gelatinases) involved in tumor invasion and angiogenesis and chemical suppression of MMP-2 may lead to the inhibition of tumor metastasis (39,40). Results from western blotting indicated that PEITC and BITC at 24 and 48 h treatment significantly reduced the protein levels of MMP-2 (Fig. 5D) and PEITC inhibited TIMP-1 at 24 and 48 h treatment but BITC at 48 h treatment led to increased TIMP-1 (Fig. 5D). PEITC suppressed urokinase-type plasminogen activator (uPA) at 24 and 48 h treatment but BITC increased it at both treatment times (Fig. 5D). The uPA protein expression levels have also been considered as promising targets of anticancer drugs (41) because it is involved in cell invasion and metastasis. It was reported that uPA gene transcription is involved in motifs that upstream sequences which correspond to NF-κB, AP-1, and PEA3-binding sites (42,43).

In the current study, the effects of PEITC and BITC on NF-κB transcription activity (DNA binding) were examined by using EMSA assay and results (Fig. 6) indicated that PEIT reduce the binding of NF-κB to DNA in DNA-binding domains but BITC elevated the binding of NF-κB to DNA in DNA-binding domains (Fig. 6). Based on the results from western blotting indicated that PEITC suppressed the expression of MMP-2 and TIMP-1 but BITC increased them (Fig. 5D) and TIMPs act as natural inhibitors of MMPs by tightly binding the MMP in a 1:1 stoichiometric ratio (44). At 48 h treatment of PEITC, it decreased NF-κBp65 and NF-κBp50 but BITC increased both (Fig. 5E) and both increased E-cadherin (Fig. 5F). This reduced binding activity was accompanied by inhibition of the nuclear protein expression of this factor in B16F10 cells that was confirmed by western blotting. These results indicated that NF-κB binding activity suppression was also possibly implicated in the inhibition of MMP or uPA synthesis. It is well known that cancer cells can express high levels of MMPs, cathepsins and uPA, which degrade tissue extracellular matrix (ECM) and facilitate cancer invasion and metastasis (45). NF-κB is a complex family, thus, further investigations are needed in the future. These MMPs have been shown to be present in different types of cancer cells, including melanoma, lung and breast (46).

Numerous studies have shown that in many physiological and pathological settings, the mitogen-activated protein kinase (MAPK) pathway involved in regulating cell death and survival (47,48) plays a central role in regulating the expression of MMP-2 and MMP-9 (49). MMPs are partly mediated by the MAPK pathway (50,51). MAPK includes ERK1/2, c-Jun NH2-terminal kinase, and p38 and other factors. The MAPK pathway regulated various cellular activities such as proliferation, invasion, metastasis, and death (52). It was suggested that agents to inhibit the MAPK pathway might lead to prevent cancer angiogenesis, proliferation, invasion, and metastasis including melanoma (49,53). Results from western blotting indicated that PEITC and BITC treatment at 24 and 48 h significantly reduced the expression of p-ERK1/2, p-p38 and p-JNK1/2 when compared to control (Fig. 5A) which means PEITC and BITC suppressed MAPK signal pathway in B16F10 cells. Fig. 5B indicated that p-AKT(Thr308), p-AKT(Ser473) and PCAN were reduced in PEITC and BITC treatment, however, both agents increased AKT expression in B16F10 cells. Activated PI3K and its downstream target Akt have been recognized to be involved with tumor cell invasion, and oncogenesis (54,55). The downregulation of the PI3k/Akt pathway have been reported to decrease the invasion of melanoma cells (53,56). In melanoma cells, the activation of the PI3K-Akt signaling pathway promoting cell invasion has been shown (57). AKT, downstream of PI3K has been reported to be suppressed by the transcription of the E-cadherin gene (58). Ras regulating RhoA has been recognized to affect tumor cells transmigration through mesothelial monolayer (59). Results (Fig. 5C) indicated that PEITC and BITC significantly reduced the expression of RhoA and Ras in B16F10 cells at both treatment times. Thus, PEITC and BITC inhibited B16F10 cell migration and invasion may also be via the inhibition of RhoA and Ras.

In conclusion, our results indicated that PEITC and BITC reduced the viable cell number of B16F10 cells. We selected the low concentrations (1, 2.5 and 5 µM) of PEITC and BITC bufalin for examining the effects of cancer cell metastasis and we found that PEITC and BITC significantly inhibited cell mobility, migration and invasion of B16F10 cells in vitro. We also used western blot assay and found that PEITC and BITC inhibited many metastasis-associated protein molecules including MMP-2, MAPKs, E-cadherin, Ras, RhoA and NF-κB. NF-κB also was confirmed by confocal laser microscopy examination. This finding suggests that PEITC and BITC are potential candidates for the development of chemotherapeutic treatments for melanoma cells in the future.

Acknowledgments

The present study was supported by grants NSC103-2320-B-039-037 from the National Science Council, Taipei, Taiwan.

References

1 

Soengas MS and Lowe SW: Apoptosis and melanoma chemo-resistance. Oncogene. 22:3138–3151. 2003. View Article : Google Scholar : PubMed/NCBI

2 

Hersey P, Zhuang L and Zhang XD: Current strategies in overcoming resistance of cancer cells to apoptosis melanoma as a model. Int Rev Cytol. 251:131–158. 2006. View Article : Google Scholar : PubMed/NCBI

3 

Sykes EK, Mactier S and Christopherson RI: Melanoma and the Unfolded Protein Response. Cancers (Basel). 8:82016. View Article : Google Scholar

4 

Sharma A, Shah SR, Illum H and Dowell J: Vemurafenib: Targeted inhibition of mutated BRAF for treatment of advanced melanoma and its potential in other malignancies. Drugs. 72:2207–2222. 2012. View Article : Google Scholar : PubMed/NCBI

5 

Xiao D, Johnson CS, Trump DL and Singh SV: Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells. Mol Cancer Ther. 3:567–575. 2004.PubMed/NCBI

6 

Yeh YT, Yeh H, Su SH, Lin JS, Lee KJ, Shyu HW, Chen ZF, Huang SY and Su SJ: Phenethyl isothiocyanate induces DNA damage-associated G2/M arrest and subsequent apoptosis in oral cancer cells with varying p53 mutations. Free Radic Biol Med. 74:1–13. 2014. View Article : Google Scholar : PubMed/NCBI

7 

Øverby A, Zhao CM, Bones AM and Chen D: Naturally occurring phenethyl isothiocyanate-induced inhibition of gastric cancer cell growth by disruption of microtubules. J Gastroenterol Hepatol. 29(Suppl 4): 99–106. 2014. View Article : Google Scholar : PubMed/NCBI

8 

Chou YC, Chang MY, Wang MJ, Liu HC, Chang SJ, Harnod T, Hung CH, Lee HT, Shen CC and Chung JG: Phenethyl isothiocyanate alters the gene expression and the levels of protein associated with cell cycle regulation in human glioblastoma GBM 8401 cells. Environ Toxicol. 32:176–187. 2017. View Article : Google Scholar

9 

Huong D, Shim JH, Choi KH, Shin JA, Choi ES, Kim HS, Lee SJ, Kim SJ, Cho NP and Cho SD: Effect of β-phenylethyl isothiocyanate from cruciferous vegetables on growth inhibition and apoptosis of cervical cancer cells through the induction of death receptors 4 and 5. J Agric Food Chem. 59:8124–8131. 2011. View Article : Google Scholar

10 

Hwang ES and Lee HJ: Effects of phenylethyl isothiocyanate and its metabolite on cell-cycle arrest and apoptosis in LNCaP human prostate cancer cells. Int J Food Sci Nutr. 61:324–336. 2010. View Article : Google Scholar : PubMed/NCBI

11 

Yan H, Zhu Y, Liu B, Wu H, Li Y, Wu X, Zhou Q and Xu K: Mitogen-activated protein kinase mediates the apoptosis of highly metastatic human non-small cell lung cancer cells induced by isothiocyanates. Br J Nutr. 106:1779–1791. 2011. View Article : Google Scholar : PubMed/NCBI

12 

Xu C, Shen G, Chen C, Gélinas C and Kong AN: Suppression of NF-kappaB and NF-kappaB-regulated gene expression by sulforaphane and PEITC through IkappaBalpha, IKK pathway in human prostate cancer PC-3 cells. Oncogene. 24:4486–4495. 2005. View Article : Google Scholar : PubMed/NCBI

13 

Bommareddy A, Hahm ER, Xiao D, Powolny AA, Fisher AL, Jiang Y and Singh SV: Atg5 regulates phenethyl isothiocyanate-induced autophagic and apoptotic cell death in human prostate cancer cells. Cancer Res. 69:3704–3712. 2009. View Article : Google Scholar : PubMed/NCBI

14 

Kelloff GJ, Crowell JA, Hawk ET, Steele VE, Lubet RA, Boone CW, Covey JM, Doody LA, Omenn GS, Greenwald P, et al: Strategy and planning for chemopreventive drug development: Clinical development plans II. J Cell Biochem. (Suppl 26): 54–71. 1996. View Article : Google Scholar

15 

Chen HJ, Lin CM, Lee CY, Shih NC, Amagaya S, Lin YC and Yang JS: Phenethyl isothiocyanate suppresses EGF-stimulated SAS human oral squamous carcinoma cell invasion by targeting EGF receptor signaling. Int J Oncol. 43:629–637. 2013.PubMed/NCBI

16 

Tang L and Zhang Y: Dietary isothiocyanates inhibit the growth of human bladder carcinoma cells. J Nutr. 134:2004–2010. 2004.PubMed/NCBI

17 

Xiao D, Powolny AA and Singh SV: Benzyl isothiocyanate targets mitochondrial respiratory chain to trigger reactive oxygen species-dependent apoptosis in human breast cancer cells. J Biol Chem. 283:30151–30163. 2008. View Article : Google Scholar : PubMed/NCBI

18 

Batra S, Sahu RP, Kandala PK and Srivastava SK: Benzyl isothiocyanate-mediated inhibition of histone deacetylase leads to NF-kappaB turnoff in human pancreatic carcinoma cells. Mol Cancer Ther. 9:1596–1608. 2010. View Article : Google Scholar : PubMed/NCBI

19 

Liu KC, Huang YT, Wu PP, Ji BC, Yang JS, Yang JL, Chiu TH, Chueh FS and Chung JG: The roles of AIF and Endo G in the apoptotic effects of benzyl isothiocyanate on DU 145 human prostate cancer cells via the mitochondrial signaling pathway. Int J Oncol. 38:787–796. 2011.PubMed/NCBI

20 

Huang SH, Wu LW, Huang AC, Yu CC, Lien JC, Huang YP, Yang JS, Yang JH, Hsiao YP, Wood WG, et al: Benzyl isothiocyanate (BITC) induces G2/M phase arrest and apoptosis in human melanoma A375.S2 cells through reactive oxygen species (ROS) and both mitochondria-dependent and death receptor-mediated multiple signaling pathways. J Agric Food Chem. 60:665–675. 2012. View Article : Google Scholar

21 

Zhou T, Li G, Cao B, Liu L, Cheng Q, Kong H, Shan C, Huang X, Chen J and Gao N: Downregulation of Mcl-1 through inhibition of translation contributes to benzyl isothiocyanate-induced cell cycle arrest and apoptosis in human leukemia cells. Cell Death Dis. 4:e5152013. View Article : Google Scholar : PubMed/NCBI

22 

Tang NY, Chueh FS, Yu CC, Liao CL, Lin JJ, Hsia TC, Wu KC, Liu HC, Lu KW and Chung JG: Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells. Oncol Rep. 35:2089–2096. 2016.PubMed/NCBI

23 

Chambers AF, Groom AC and MacDonald IC: Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2:563–572. 2002. View Article : Google Scholar : PubMed/NCBI

24 

Cruz-Munoz W and Khokha R: The role of tissue inhibitors of metalloproteinases in tumorigenesis and metastasis. Crit Rev Clin Lab Sci. 45:291–338. 2008. View Article : Google Scholar : PubMed/NCBI

25 

Lee CS, Cho HJ, Jeong YJ, Shin JM, Park KK, Park YY, Bae YS, Chung IK, Kim M, Kim CH, et al: Isothiocyanates inhibit the invasion and migration of C6 glioma cells by blocking FAK/JNK-mediated MMP-9 expression. Oncol Rep. 34:2901–2908. 2015.PubMed/NCBI

26 

Wu X, Zhu Y, Yan H, Liu B, Li Y, Zhou Q and Xu K: Isothiocyanates induce oxidative stress and suppress the metastasis potential of human non-small cell lung cancer cells. BMC Cancer. 10:2692010. View Article : Google Scholar : PubMed/NCBI

27 

Shih YL, Chou J, Yeh MY, Chou HM, Chou HC, Lu HF, Shang HS, Chueh FS, Chu YL, Hsueh SC, et al: Casticin induces DNA damage and inhibits DNA repair-associated protein expression in B16F10 mouse melanoma cancer cells. Oncol Rep. 36:2094–2100. 2016.PubMed/NCBI

28 

Chen YY, Lu HF, Hsu SC, Kuo CL, Chang SJ, Lin JJ, Wu PP, Liu JY, Lee CH, Chung JG, et al: Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK-Hep1 cells through the inhibitions of NF-κB and matrix metalloproteinase-2/-9-signaling pathways. Environ Toxicol. 30:74–2. 2015. View Article : Google Scholar

29 

Huang YP and Chang NW: PPARα modulates gene expression profiles of mitochondrial energy metabolism in oral tumorigenesis. Biomedicine (Taipei). 6:32016. View Article : Google Scholar

30 

Huang AC, Yang MD, Hsiao YT, Lin TS, Ma YS, Peng SF, Hsia TC, Cheng YD, Kuo CL and Chung JG: Bufalin inhibits gefitinib resistant NCI-H460 human lung cancer cell migration and invasion in vitro. J Ethnopharmacol. 194:1043–1050. 2016. View Article : Google Scholar : PubMed/NCBI

31 

Chan CY, Lien CH, Lee MF and Huang CY: Quercetin suppresses cellular migration and invasion in human head and neck squamous cell carcinoma (HNSCC). Biomedicine (Taipei). 6:152016. View Article : Google Scholar

32 

Lin M-C, Tsai S-Y, Wang F-Y, Liu F-H, Syu J-N and Tang F-Y: Leptin induces cell invasion and the upregulation of matrilysin in human colon cancer cells. BioMedicine. 3:pp. 174–180. 2013, http://biomedicine.cmu.edu.tw/doc/9-5.pdfurisimplehttp://biomedicine.cmu.edu.tw/doc/9-5.pdf. View Article : Google Scholar

33 

Kuo TC, Yang JS, Lin MW, Hsu SC, Lin JJ, Lin HJ, Hsia TC, Liao CL, Yang MD, Fan MJ, et al: Emodin has cytotoxic and protective effects in rat C6 glioma cells: Roles of Mdr1a and nuclear factor kappaB in cell survival. J Pharmacol Exp Ther. 330:736–744. 2009. View Article : Google Scholar : PubMed/NCBI

34 

Wu Y and Zhou BP: New insights of epithelial-mesenchymal transition in cancer metastasis. Acta Biochim Biophys Sin (Shanghai). 40:643–650. 2008. View Article : Google Scholar

35 

Jia W, Gao XJ, Zhang ZD, Yang ZX and Zhang G: S100A4 silencing suppresses proliferation, angiogenesis and invasion of thyroid cancer cells through downregulation of MMP-9 and VEGF. Eur Rev Med Pharmacol Sci. 17:1495–1508. 2013.PubMed/NCBI

36 

Kallakury BV, Karikehalli S, Haholu A, Sheehan CE, Azumi N and Ross JS: Increased expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinases 1 and 2 correlate with poor prognostic variables in renal cell carcinoma. Clin Cancer Res. 7:3113–3119. 2001.PubMed/NCBI

37 

Yoshizaki T, Maruyama Y, Sato H and Furukawa M: Expression of tissue inhibitor of matrix metalloproteinase-2 correlates with activation of matrix metalloproteinase-2 and predicts poor prognosis in tongue squamous cell carcinoma. Int J Cancer. 95:44–50. 2001. View Article : Google Scholar : PubMed/NCBI

38 

Hsieh YS, Chu SC, Hsu LS, Chen KS, Lai MT, Yeh CH and Chen PN: Rubus idaeus L. reverses epithelial-to-mesenchymal transition and suppresses cell invasion and protease activities by targeting ERK1/2 and FAK pathways in human lung cancer cells. Food Chem Toxicol. 62:908–918. 2013. View Article : Google Scholar : PubMed/NCBI

39 

Li D, Qu X, Hou K, Zhang Y, Dong Q, Teng Y, Zhang J and Liu Y: PI3K/Akt is involved in bufalin-induced apoptosis in gastric cancer cells. Anticancer Drugs. 20:59–64. 2009. View Article : Google Scholar : PubMed/NCBI

40 

Rajoria S, Suriano R, George A, Shanmugam A, Schantz SP, Geliebter J and Tiwari RK: Estrogen induced metastatic modulators MMP-2 and MMP-9 are targets of 3,3′-diindolylmethane in thyroid cancer. PLoS One. 6:e158792011. View Article : Google Scholar

41 

Chu SC, Yu CC, Hsu LS, Chen KS, Su MY and Chen PN: Berberine reverses epithelial-to-mesenchymal transition and inhibits metastasis and tumor-induced angiogenesis in human cervical cancer cells. Mol Pharmacol. 86:609–623. 2014. View Article : Google Scholar : PubMed/NCBI

42 

D'Orazio D, Besser D, Marksitzer R, Kunz C, Hume DA, Kiefer B and Nagamine Y: Cooperation of two PEA3/AP1 sites in uPA gene induction by TPA and FGF-2. Gene. 201:179–187. 1997. View Article : Google Scholar : PubMed/NCBI

43 

Mori Y, Akita K, Tanida S, Ishida A, Toda M, Inoue M, Yashiro M, Sawada T, Hirakawa K and Nakada H: MUC1 protein induces urokinase-type plasminogen activator (uPA) by forming a complex with NF-κB p65 transcription factor and binding to the uPA promoter, leading to enhanced invasiveness of cancer cells. J Biol Chem. 289:35193–35204. 2014. View Article : Google Scholar : PubMed/NCBI

44 

Offenberg H, Brünner N, Mansilla F, Orntoft Torben F and Birkenkamp-Demtroder K: TIMP-1 expression in human colorectal cancer is associated with TGF-B1, LOXL2, INHBA1, TNF-AIP6 and TIMP-2 transcript profiles. Mol Oncol. 2:233–240. 2008. View Article : Google Scholar

45 

Pulyaeva H, Bueno J, Polette M, Birembaut P, Sato H, Seiki M and Thompson EW: MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells. Clin Exp Metastasis. 15:111–120. 1997. View Article : Google Scholar : PubMed/NCBI

46 

Heppner KJ, Matrisian LM, Jensen RA and Rodgers WH: Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response. Am J Pathol. 149:273–282. 1996.PubMed/NCBI

47 

Eisenmann KM, VanBrocklin MW, Staffend NA, Kitchen SM and Koo HM: Mitogen-activated protein kinase pathway-dependent tumor-specific survival signaling in melanoma cells through inactivation of the proapoptotic protein bad. Cancer Res. 63:8330–8337. 2003.PubMed/NCBI

48 

Junttila MR, Li SP and Westermarck J: Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival. FASEB J. 22:954–965. 2008. View Article : Google Scholar

49 

Lee KR, Lee JS, Kim YR, Song IG and Hong EK: Polysaccharide from Inonotus obliquus inhibits migration and invasion in B16-F10 cells by suppressing MMP-2 and MMP-9 via down-regulation of NF-κB signaling pathway. Oncol Rep. 31:2447–2453. 2014.PubMed/NCBI

50 

Mori T, Wang X, Aoki T and Lo EH: Downregulation of matrix metalloproteinase-9 and attenuation of edema via inhibition of ERK mitogen activated protein kinase in traumatic brain injury. J Neurotrauma. 19:1411–1419. 2002. View Article : Google Scholar : PubMed/NCBI

51 

Shin DY, Lu JN, Kim GY, Jung JM, Kang HS, Lee WS and Choi YH: Anti-invasive activities of anthocyanins through modulation of tight junctions and suppression of matrix metalloproteinase activities in HCT-116 human colon carcinoma cells. Oncol Rep. 25:567–572. 2011.

52 

Sun Y, Liu WZ, Liu T, Feng X, Yang N and Zhou HF: Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis. J Recept Signal Transduct Res. 35:600–604. 2015. View Article : Google Scholar : PubMed/NCBI

53 

Lin BW, Jiao ZL, Fan JF, Peng L, Li L, Zhao ZG, Ding XY and Li HJ: Inhibitory effect of melanoma differentiation associated gene-7/interleukin-24 on invasion in vitro of human melanoma cancer cells. J Korean Med Sci. 28:833–839. 2013. View Article : Google Scholar : PubMed/NCBI

54 

Kim D, Kim S, Koh H, Yoon SO, Chung AS, Cho KS and Chung J: Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J. 15:1953–1962. 2001. View Article : Google Scholar : PubMed/NCBI

55 

Shukla S, Maclennan GT, Hartman DJ, Fu P, Resnick MI and Gupta S: Activation of PI3K-Akt signaling pathway promotes prostate cancer cell invasion. Int J Cancer. 121:1424–1432. 2007. View Article : Google Scholar : PubMed/NCBI

56 

Shi H, Wu Y, Wang Y, Zhou M, Yan S, Chen Z, Gu D and Cai Y: Liquiritigenin potentiates the inhibitory effects of cisplatin on invasion and metastasis via downregulation MMP-2/9 and PI3K/AKT signaling pathway in B16F10 melanoma cells and mice model. Nutr Cancer. 67:761–770. 2015. View Article : Google Scholar

57 

Thang ND, Yajima I, Kumasaka MY, Iida M, Suzuki T and Kato M: Deltex-3-like (DTX3L) stimulates metastasis of melanoma through FAK/PI3K/AKT but not MEK/ERK pathway. Oncotarget. 6:14290–14299. 2015. View Article : Google Scholar : PubMed/NCBI

58 

Qiao M, Sheng S and Pardee AB: Metastasis and AKT activation. Cell Cycle. 7:2991–2996. 2008. View Article : Google Scholar : PubMed/NCBI

59 

Narumiya S, Tanji M and Ishizaki T: Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion. Cancer Metastasis Rev. 28:65–76. 2009. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

September-2017
Volume 51 Issue 3

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Lai K, Hsiao Y, Yang J, Ma Y, Huang Y, Chiang T and Chung J: Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro. Int J Oncol 51: 832-840, 2017
APA
Lai, K., Hsiao, Y., Yang, J., Ma, Y., Huang, Y., Chiang, T., & Chung, J. (2017). Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro. International Journal of Oncology, 51, 832-840. https://doi.org/10.3892/ijo.2017.4084
MLA
Lai, K., Hsiao, Y., Yang, J., Ma, Y., Huang, Y., Chiang, T., Chung, J."Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro". International Journal of Oncology 51.3 (2017): 832-840.
Chicago
Lai, K., Hsiao, Y., Yang, J., Ma, Y., Huang, Y., Chiang, T., Chung, J."Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro". International Journal of Oncology 51, no. 3 (2017): 832-840. https://doi.org/10.3892/ijo.2017.4084