Cancer is the second-most common fatal disease after cardiovascular disease [1
], and several studies have shown that some dietary components exert cancer-preventing effects [3
]. Gastric cancer is the fifth most common cancer worldwide in both men and women [5
]. The most common form of gastric cancer is gastric adenocarcinoma, which has a risk of relapse and metastasis even after surgery, which is the only curative treatment for gastric cancer and is supplemented with adjuvant chemotherapy and/or chemoradiation [6
]. Currently, targeted molecular therapy for gastric cancers is emphasized to establish a standard chemotherapy regimen with decreased resistance and lower non-selective toxicity [7
Benzyl isothiocyanate, BITC, is one of the isothiocyanates, which are breakdown products of glucosinolates and are found in various edible plants belonging to the Brassicaceae family, such as broccoli, cabbage, and water cress [8
]. Clinical evidence from China has demonstrated that isothiocyanates can protect against gastric cancer [9
]. BITC has been studied in various cancers, such as oral cancer [10
], pancreatic cancer [11
], brain cancer [12
], melanoma [13
], and gastric cancer [15
], to evaluate its potential for cytotoxic effects on cancer cells. Additionally, BITC has been shown to have anthelmintic [16
], anti-inflammatory [17
], and anti-adipogenic effects [18
]. Due to the large number of therapeutic advantages that have been found in various studies, BITC has gained interest as a novel therapeutic candidate for gastric cancer, with potential chemopreventive effects.
Deregulation of the apoptotic machinery is considered to be a hallmark of cancer and fixing the deregulation can contribute to cancer cell death/removal [19
]. Apoptosis can be characterized by morphological features, such as cell shrinkage, nuclear chromatin condensation, and cell detachment and budding, and by biochemical hallmarks like internucleosomal DNA fragmentation and cysteine-aspartic proteases (caspases) activation [20
]. It has been well documented that apoptosis is mediated by the extrinsic death receptor-regulated apoptotic pathway and the intrinsic mitochondria-involved apoptotic pathway [19
In apoptosis modulation, Caspase-8 and Caspase-9 are the two main potential initiator caspases that regulate the activation of Caspase-3, which, in turn leads to the disruption of several cellular processes [22
]. It has also been shown that Caspase-8 is activated by the interaction between the death effector domains of proactive Caspase-8 (Pro-Cas-8) and an adaptor protein, Fas-associated protein with death domain, which is recruited by the clustering of TNF-related apoptosis-inducing ligand (TRAIL) receptors, death receptor DR4/TRAIL-R1 and DR5/TRAIL-R2 [23
]. On the contrary, proactive Caspase-9 (Pro-Cas-9) has been reported to be activated by the formation of a complex between cytochrome c (Cyt c) and apoptotic protease activating factor-1 (Apaf-1), which is initiated by the B-cell lymphoma 2 (Bcl-2)-regulated mitochondria-mediated apoptotic pathway [24
It has been proposed that the Bcl-2-regulated apoptotic pathway can be promoted by reactive oxygen species (ROS), which are known to interrupt mitochondrial membrane integrity and cause oxidative injury [25
]. As a result of mitochondrial depolarization, the levels of the anti-apoptotic protein Bcl-2, which is localized to the outer mitochondrial membrane, are dampened [13
]. On the contrary, the proapoptotic effector protein Bcl-2-associated X protein (Bax) translocates from the cytosol to the mitochondria, and releases Cyt c from the mitochondrial intermembrane space into the cytosol [26
The aim of the present study is to explore the underlying molecular mechanism of BITC-induced cell death in the human gastric adenocarcinoma cell line, AGS cells (KCLB 21739). We hypothesized that BITC is a potential candidate for the treatment of gastric cancer as it triggers apoptosis in AGS cells. We tried to find whether BITC induces AGS cell death by eliciting the ROS-initiated mitochondria-mediated pathway and the death receptor-mediated signaling pathway.
2. Materials and Methods
2.1. Materials and Reagents
BITC was purchased from Selleckchem (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO) to create a 10 mM stock solution. A glutathione (GSH) (G4705, Sigma-Aldrich Inc., Darmstadt, Germany) stock solution was prepared by dissolving GSH in HPLC-grade water (Fisher Scientific Korea Ltd., Gangnam-gu, Seoul, Korea), and this stock solution was diluted in the culture medium before treatment of cells to obtain the final working solution. RPMI-1640 medium was purchased from Welgene Inc. (Daegu, Korea), and fetal bovine serum (FBS) was purchased from Corning Inc. (Corning, NY, USA). Antibodies against cleaved Caspase-3 (c-Cas-3) (9664), Caspase-8 (9746), Caspase-9 (9508), poly (ADP-ribose) polymerase (PARP) (9532), X-linked inhibitor of apoptosis protein (XIAP) (14334), DR4 (42533) and DR5 (8074) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP antibodies were bought from Enzo Life Sciences (Farmingdale, NY, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-32233), Bcl-2 (sc-492), Bax (sc-493) and Cyt c (sc-13156) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.2. Cell Culture and Morphological Observation
The human gastric cancer cell line AGS (KCLB 21739) was obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI-1640 medium containing 10% FBS and 5% antibiotics, which contained 1% penicillin-streptomycin and 0.1% amphotericin B, at 37 °C in a humidified atmosphere with 5% CO2 and 95% air. Cells were seeded in a petri dish as a monolayer at a 1:10 ratio (cell:total media) and were passaged every time 90% confluence was reached.
To observe the changes in cell morphology, the cells were seeded in culture dishes. When 70% confluence was achieved, the cells were treated with different concentrations of BITC (i.e., 1, 5, or 10 μM) for 48 h. Then, changes in cell morphology were observed using a fluorescence microscope (Leica, Wetzlar, Germany).
2.3. Cell Viability Measurement (MTT Assay)
To determine cell viability regarding the apoptosis induced by BITC, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed. AGS cells were seeded in 24-well plates and maintained in RPMI-1640 media supplemented with 10% FBS and 5% antibiotics. When the cells reached 70% confluence, the medium was removed by careful aspiration, and fresh culture medium containing different concentrations of BITC (i.e., 1, 5, or 10 μM) was added to evaluate dose-dependent responses and culture medium containing 5 μM BITC was added to evaluate time-dependent responses (24–72 h). After the AGS cells were treated with specific BITC doses for the desired time periods, the cells were washed twice with 1× phosphate buffered saline (1× PBS), and MTT solution (0.5 mg/mL in 1× PBS) was added to each well. After incubation for 4 h with the MTT reagent at 37 °C in the dark, the supernatant was slowly removed, and 250 μL DMSO was added. Formazan crystals were dissolved in DMSO for 30 min in the incubator, and the solution was transferred to a 96-well plate. Absorbance was measured at 570 nm [27
] using a Synergy H1 Hybrid Multi-Mode microplate reader (BioTek, Winooski, VT, USA).
2.4. Measurement of Intracellular ROS
Intracellular ROS generation was measured through 2′,7′-dichlorofluorescin diacetate (DCFDA; Sigma-Aldrich Inc., Darmstadt, Germany) staining [28
]. Briefly, the cells were seeded in a 6-well plate. After reaching 80% confluence, the cells were washed with 1× PBS and incubated with 15 μM DCFDA for 30 min to determine time-dependent ROS analysis, and 15 μM DCFDA with or without 10 µg/mL 4′,6′-diamidino-2-phenylindole (DAPI; Roche Diagnostics, Indianapolis, IN, USA) for 30 min at 37 °C in the dark to determine BITC dose-dependent ROS analysis, which was followed by another wash with 1× PBS. The cells were, then, treated with 5 μM BITC at different time points (i.e., 2.5, 4.5, and 6 h) for time-dependent ROS production analysis and cells were treated with 100 μM hydrogen peroxide (H2
) and 1, 5, or 10 μM BITC for BITC concentration-dependent ROS analysis. Fluorescent dichlorofluorescin (DCF) was examined with a fluorescence microscope (JULITM
Smart fluorescent cell analyzer, NanoEnTek Inc., Seoul, Korea) or (Leica, Wetzlar, Germany). Next, the AGS cells were collected by trypsinization and resuspended in 500 μL of 1× PBS. The collected cells were transferred to a 96-well plate, and DCF fluorescence was measured using a fluorescence microplate reader (Synergy H1 Hybrid Multi-Mode Reader; BioTek, Winooski, VT) at excitation and emission wavelengths of 485 and 535 nm, respectively, and a fix gain-100 at each time point.
2.5. Cell Viability Inhibition Assay
Changes in cell viability in the presence or absence of GSH were observed by measuring dye absorbance at 570 nm, as previously described. Briefly, AGS cells were incubated in a 24-well plate. After achieving 60% confluence, cells were preincubated with 1 mM GSH for 1 h. Cells were then either treated with 5 or 10 μM BITC for 48 h and control cells were incubated with DMSO and water for the same period. Cells were washed with 1× PBS and MTT reagent was, then, added for 4 h. The resulting yellow formazan crystals were dissolved in DMSO for 30 min in an incubator and the absorbance was, then, measured with a microplate reader (Synergy H1 Hybrid Multi-Mode Reader; BioTek, Winooski, VT, USA).
2.6. Preparation of Cellular Extracts and Western Blot Analysis
Human gastric adenocarcinoma cells were grown until 80% confluence was achieved and, then, treated with the desired concentrations of 5 or 10 μM BITC in RPMI-1640 medium containing 3% FBS for the 24 h BITC treatment; cells were treated with 1, 5, or 10 μM BITC for the 48 h treatment. After treatment, the cells were washed twice with ice-cold 1× PBS after the floating cells were collected following centrifugation at 1000 rpm for 5 min. Then, the cells were lysed using NP-40 lysis buffer [29
] and harvested by scraping on ice. The lysates were incubated on ice for 30 min and vortexed four times for 10 s. Afterwards, the lysates were centrifuged at 14,000 rpm at 4 °C for 30 min. Protein quantification was carried out with a Pierce BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The lysates (30–50 μg) were subjected to 10–15% sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, IRL) using a Power Pac power supply (Bio-Rad, Melville, NY, USA). After blocking with 5% skim milk or 5% bovine serum albumin for 1 h, the membranes were incubated with the designated primary antibodies overnight at 4 °C at a 1:1000 dilution, with an exception of the primary antibody against Bcl-2, for which a 1:700 dilution was used. The membrane was, then, washed three times with tris-buffered saline containing 0.1% Tween 20 and incubated with secondary HRP-labeled antibodies at a 1:5000 dilution to detect the expression of the proteins of interest. Protein bands were examined using an enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and exposed to X-ray photographic films in a dark room. Image J was used to analyze the data.
2.7. Statistical Analysis
All data were expressed as the mean ± the standard error of the mean (SEM) of at least three independent experiments. Statistical differences among the groups were analyzed by Student’s t-test. A p-value of 0.05 or less was considered to be statistically significant.
Apoptosis, or programmed cell death, is critical for the success of chemotherapies against various cancers and cancer cell resistance [32
]. Failure in gastric cancer therapy may be the result of defective apoptosis process accompanied with the underlying mechanisms of Bcl-2 protein overexpression, hypoxia, and tumor microenvironment remodeling [33
]. Nowadays, gastric cancer cells are acquiring resistance to TRAIL and various chemotherapy approaches have been investigated to increase TRAIL sensitivity in gastric cancer cells [35
]. Thus, TRAIL, which can alter the tumor microenvironment resulting in cancer cell death [37
], can be a target to reduce the chemotherapeutic resistance in gastric cancer. BITC has been reported to exert chemopreventive effects in several cancer cells by inducing anti-angiogenesis, apoptosis, cell-cycle arrest, anti-metastasis, and anti-tumorigenesis effects, and has been described to have synergistic effects with TRAIL, cisplatin, sulforaphane, phenethyl isothiocyanate, and radiation treatments [38
]. BITC has been shown to inhibit AGS cell proliferation [15
], but information relating to its chemotherapeutic potential in gastric adenocarcinoma has been too inconclusive to progress to its clinical use. This study was designed to explore the molecular mechanisms underlying BITC-induced AGS cell cytotoxicity.
We investigated BITC-induced ROS generation in AGS cells and its role in BITC-triggered AGS cell death. ROS generation in BITC-induced AGS cell death was examined, given that ROS production had been found to be triggered by BITC in prostate cancer cells and rat liver epithelial cells [25
]. To the best of our knowledge, this is the first study to report that BITC-treated AGS cells produce intracellular ROS, as demonstrated by the increased number of DCF-positive cells observed upon time-dependent BITC treatment (Figure 2
A,B) and dose-dependent BITC treatment (Figure 2
D–F). To evaluate the impact of ROS on BITC-triggered AGS cell death, we utilized GSH to suppress BITC-induced ROS accumulation. According to our MTT assay results, AGS cell viability was restored in the presence of GSH, indicating that BITC-induced AGS cell death is truly correlated with intracellular ROS generation. Recent studies have also provided information to support the hypothesis that cytotoxic cell death is associated with intracellular ROS accumulation [40
]. For example, apoptotic cell death has been shown to be promoted by ROS but inhibited by the antioxidant GSH [42
]. Other studies that have used different cell lines have found that cell cytotoxicity ceases after treatment with GSH similarly to our data regarding the inhibition of BITC-triggered AGS cell apoptosis [11
]. Based on these data, it is reasonable to conclude that BITC-triggered AGS cell apoptosis is promoted by ROS.
We also found that BITC modulated the expression of mitochondrial apoptosis-related proteins in AGS cells via western blot analysis. Bcl-2, a mitochondrial membrane stabilizing protein, has been shown to inhibit apoptosis by binding to pro-death proteins, such as Bax, Bcl-2-associated agonist of cell death, and Bcl-2 homologous antagonist/killer [24
]. When we evaluated the levels of Bcl-2 expression, we found that Bcl-2 protein levels decreased significantly after treatment with BITC, although Bax was not affected. Concomitantly, the release of Cyt c was observed with the dose-dependent increase in protein expression compared to that in the control group. According to recent studies, Bcl-2 was found to be downregulated while Bax was upregulated in the presence of an apoptotic signal, and Cyt c was observed to be released as a result of altered mitochondrial membrane permeability [13
]. Bcl-2 was shown to phosphorylate without the expression alteration of Bax levels in human bladder cancer cells, which is similar to our study that Bax level was not changed by BITC [45
]. It has further been suggested that this discrepancy in the modulation of the Bcl-2 associated proteins by isothiocyanates may be correlated with the concentrations of the treatments. However, an accepted explanation for the dissociation of Bcl-2 family proteins in BITC-triggered apoptosis remains unknown. The results of the previous study [45
] may explain why we found that the Bax expression level did not change upon exposure to BITC in AGS cells, while a downregulation of the Bcl-2 expression level was observed. Our data demonstrate that BITC favors a dose-dependent decrease in the Bcl-2: Bax ratio and the release of Cyt c in AGS cells. Therefore, we hypothesize that BITC triggers AGS cell death by initiating the Bcl-2-regulated apoptotic signaling pathway.
The regulation of the expression of the proactive form of the caspase initiator of the intrinsic apoptotic pathway, Pro-Cas-9, may be one of the important checkpoints of mitochondrial dysfunction in BITC-triggered apoptosis. When we evaluated Pro-Cas-9 expression after BITC treatment, we found that it was downregulated. On the contrary, the expression of XIAP, which may inhibit the cleavage of the Caspase-9 initiator and the Caspase-3 executor [46
], was reduced in a dose-dependent manner with the reduction in the levels of Pro-Cas-9 expression and elevation in the levels of c-Cas-3 expression. This hyperactivation of the caspases has been found to contribute to membrane blebbing in apoptotic cells [23
]. Similar results can be found in a previous study [45
] that supports the premise that BITC-induced mitochondrial damage is involved in the apoptotic machinery. The data produced in this study clearly suggest that mitochondrial dysfunction plays a role in BITC-triggered AGS cell apoptosis.
Caspases activation may be mediated by an another pathway, the death-receptor-mediated apoptotic pathway [48
], and the elevated expressions of DR4 and DR5 have been found to be correlated with the cellular sensitivity to TRAIL-mediated apoptosis [49
]. In the current study, we showed that BITC initiates caspases activation via the extrinsic apoptotic pathway by regulating DR4, DR5, and Caspase-8. When we observed membrane-bound TRAIL receptors activation during BITC-induced apoptosis, we found that BITC upregulated both DR4 and DR5 expressions. This hyperactivation of the death receptors initiated the caspases cascade. PARP, a downstream caspase substrate, can be cleaved by c-Cas-3, which results in the disruption of several cellular processes and leads to apoptosis [50
]. As such, BITC-induced AGS cell death was triggered by the DR4- and DR5-modulated apoptotic pathway. This result is supported by previous studies that poncirin and tangeretin also induced apoptosis through the death receptor-mediated apoptotic pathway [51
]. Other studies have reported similar results that show that the molecular mechanisms of apoptosis in gastric cancer cells are related to DR4 [53
] and DR5 [54
]. These data support the hypothesis that the regulation of DR4 and DR5 is a key molecular mechanism underlying apoptosis in AGS cells.
Interestingly, a study on glioma cells found that the apoptosis signaling cascade was initiated by the specific death receptor DR5, but not DR4 [55
]. However, in human cancer stem-like cells, upregulation of both DR4 and DR5 was found to contribute to apoptosis [56
]. Therefore, death receptor activation that is initiated by a cell death stimulus appears to be specific for particular cell types [57
]. On the contrary, DR4 is considered to play a pivotal role in apoptosis and its dysfunction can promote tumor cell metastasis [58
]. Hence, the upregulation of both DR4 and DR5 in BITC-induced AGS cell death may be advantageous for enhancing the responsiveness of AGS cells to TRAIL ligands.
Overall, our findings highlight the importance of the apoptotic machinery in BITC-triggered AGS cell death. Moreover, our results provide evidence that BITC provokes DR4/DR5-dependent and mitochondria-mediated apoptosis, which is accompanied by the intracellular generation of ROS. These conclusions are supported by a recent study that used different cell lines to determine the involvement of extrinsic and intrinsic apoptotic pathways in BITC-induced cell death [13
]. These new findings greatly improve our understanding of the molecular mechanisms of BITC-induced AGS cell death and pave the way for designing novel chemotherapeutic strategies for gastric cancer. However, the link between the different signaling pathways that promote BITC-triggered AGS cell death requires further investigation. Previous studies showing that chemotherapeutic efficacy can be obtained with BITC doses below the genotoxic levels, both in vitro and in vivo [59
], and negligible or absence of BITC toxicity in normal PBMCs [60
], normal breast MCF-10A cells [61
], and normal HPDE-6 cells [63
] indicate that BITC could be a novel chemotherapeutic agent with low toxicity in normal cells. According to our data, BITC could be helpful to gastric cancer patients because it induces apoptosis in gastric cancer cells. As BITC is a dietary phytochemical, it can be easily supplemented as a diet or pill. BITC may enhance the effects of chemotherapies in gastric cancer patients when it is given in combination with chemotherapies or it may be helpful for gastric cancer prevention when it is ingested daily as a food supplement.