Gastric adenocarcinoma is a cancer originating from glandular epithelium of the gastric mucosa and accounts for approximately 90-95% of total gastric cancer incidence (Smyth
Recent studies showed that targeting autophagy is a promising approach for chemotherapy (Dalby
Studies have reported that autophagy is involved in the regulatory process of cancer cells (Feng
Benzyl isothiocyanate (BITC) is an isothiocyanate compound found in plants of the mustard family, including mustard, radish, broccoli, cabbage, water cress, etc. (Moy
As in other cancers, BITC also has a chemotherapeutic effect on gastric cancer. Clinical evidence from China has demonstrated that isothiocyanates can protect against gastric cancer (Moy
All of the basic cell culture reagents were purchased from Invitrogen (Grand Island, NY, USA), Corning Inc. (NY, USA), and Welgene Inc (Daegu, Korea). To prepare the cellular extracts, RIPA lysis and extraction buffer were purchased from Thermofisher Scientific (Waltham, MA, USA), and protease and phosphatase inhibitors were purchased from Sigma Chemical Co (St. Louis, MO, USA). For cell viability assay, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were also purchased from Sigma Chemical Co. Antibodies against LC3B, Atg5, BECLIN1, p62/SQSTM1, PI3K class III, mTOR, p-mTOR, cathepsin B, and cathepsin D antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cathepsin L, anti-LAMP2, anti-GAPDH, and anti-actin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology. Lipofactamine RNAiMAX reagent and Opti-MEM reduced serum medium were purchased from Thermofisher Scientific. All western blotting reagents were purchased from ELPIS-BIOTECH (Daejeon, Korea) and Bio-Rad (Hercules, CA, USA). PVDF membranes were purchased from Koma Biotech (Seoul, Korea) and NC membranes were purchased from Bio-Rad. X-ray films were purchased from Thermofisher Scientific. PCR primers for hLC3B, hSQSM1 and β-actin were purchased from BIONEER (Daejeon, Korea). Nucleozol reagent was purchased from MACHEREY-NAGEL (Düren, North Rhine-Westphalia, Germany). The ReverTra Ace qPCR RT Master Mix kit was purchased from TOYOBO (Osaka, Japan), and the iQTM SYBR® Green Supermix was obtained from Bio-Rad. Benzyl isothiocyanate (BITC), a pan-caspase inhibitor, Z-VAD-FMK, and the PI3K inhibitor, wortmannin were purchased from Selleckchem (Houston, TX, USA). Bafilomycin A1 was purchased from TOCRIS Bioscience (Bristol, UK). Omnicathepsin fluorogenic substrate (Z-FR-AMC) was purchased from ENZO Life Sciences (Farmingdale, NY, USA). 4’-6-Diamidine-2’-phenylindole dihydrochloride (DAPI) was purchased from Roche (Mannheim, Baden-Württemberg, Germany) and acridine orange (AO) was purchased from Sigma Chemical Co.
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 (Welgene Inc.) containing 10% FBS and 5% antibiotics, which consisted of 1% penicillin-streptomycin and 0.1% amphotericin B. The culture was kept at 37°C in a humidified atmosphere with 5% CO2 and 95% air. Cells were seeded as a monolayer in a petri dish in a 1:10 ratio of cells to total media. They were passaged every time 90% confluence was reached.
For treatment, the cells were seeded to 70-80% confluency and incubated with different concentrations of BITC (5, 10, or 15 μM) for a specified duration. The changes in cellular morphology were observed using a fluorescence microscope (Leica, Wetzlar, Germany).
To determine the cell viability, an MTT assay was performed. AGS cells were plated at a density of 1×105 cells/well in 24-well plates overnight in RPMI-1640 medium supplemented with 10% FBS and 5% antibiotics. The next day, the cells were treated with different concentrations of treatment agents. When desired treatment duration (24, 48 or 72 h) was reached, the treatment medium was removed. Then, the cells were washed twice with PBS, and MTT solution (final concentration, 0.5 mg/mL) was added into each well. After incubation for 4 h at 37°C, the medium was slowly removed, and 300 μL DMSO was added to dissolve the formazan crystals. The percent cell viability was calculated based on the absorbance measured at 570 nm by using a microplate reader (Molecular Devices, Sunnyvale, CA, USA), and normalized to control.
To detect the changes in nuclear morphology after BITC treatment, the AGS cells were seeded on coverslips in a 24-well plate. After treatment, they were fixed with 4% paraformaldehyde for 30 min. Next, they were washed with PBS twice and stained with 300 nM DAPI solution for 5 min at room temperature. The stain solution was then removed, and the cells were washed with PBS twice. The nuclear morphological changes were observed and imaged with a fluorescent microscope (Leica).
After treatment, the AGS cells were collected by trypsinization and lysed with cold RIPA lysis buffer. Protein quantification was conducted with a Pierce BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The lysates (20-40 μg) were subjected to 7.5-15% sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and transferred to PVDF or NC membranes using a Power Pac power supply (Bio-Rad). After blocking with 5% skim milk or 5% BSA for 1 h, the membranes were incubated with the designated primary antibodies overnight at 4°C in a 1:1000 dilution. Next, they were washed three times with TBST (1% Tween 20) and incubated with secondary HRP-labeled antibodies in a 1:5000 dilution to detect the expression of the proteins of interest. Protein bands were examined using an ECL reagent (Santa Cruz Biotechnology) by the Bio-Rad Universal Hood II Gel doc system or X-ray film method. Image J (NIH Image, MD, USA) was used to analyze the data.
The total RNA of the cells was extracted using Nucleozol reagent (MACHEREY-NAGEL). For first-strand cDNA synthesis, 1 µg RNA was reverse transcribed with ReverTra Ace qPCR RT Master Mix (TOYOBO) according to the manufacturer’s instructions. Next, the synthesized cDNAs were subjected to quantitative real-time PCR analysis. The sequences of the primers used in the current study were the following: hLC3B, 5’-GTG TCC GTT CAC CAA CAG GAA G-3’ (reverse), and 5’-GAG AAG CAG CTT CCT GTT CTG G-3’ (forward); hSQSTM1, 5’-AGT GTC CGT GTT TCA CCT TCC G -3’ (reverse) and 5’-TGT GTA GCG TCT GCG AGG GAA A-3’ (forward); β-actin, 5’-TAC AGG CCG GGG AGC ATC GT-3’ (reverse) and 5’-CGC CAC CAG TTC GCC ATG GA-3’ (forward). Real-time PCR was performed using a qPCR instrument (CFX Connect Optics Module, Bio-Rad Laboratories, Inc, CA, USA) using iQTM SYBR® Green Supermix (Bio-Rad) according to the manufacturer’s instructions. All of the samples were analyzed in duplicate. GAPDH was used as an internal control. Gene expression was calculated using the comparative threshold cycle (2–∆∆CT) method.
Immunocytochemistry for LC3B and LAMP2 was performed after the AGS cells were fixed with cold 100% methanol. After blocking with normal goat serum, the cells were incubated with the primary antibody overnight at 4°C followed by incubation with Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature. Then, the nuclei were stained with 300 nM of DAPI for 5 min at room temperature. The cells were observed and imaged using a fluorescent microscope (Leica) or a confocal microscope (Zeiss, Oberkochen, Germany).
AGS cells were seeded with a complete medium at a density of approximately 3×105 cells in each confocal dish overnight. The next day, the cells were treated with different concentrations of BITC for 24 h and washed with PBS (10%FBS) once. After 20 min staining with 1 µg/mL acridine orange dye at room temperature, the fluorescence pattern was observed within 1 h under a confocal microscope (Zeiss).
The supernatant from AGS cell lysates was incubated with omnicathepsin fluorogenic substrate (Z-FR-AMC, 100 µM) for 30 min at 37°C. The fluorescence emitted by released AMC was quantified in a plate reader (exc: 380 nm; em: 440 nm) and normalized by total protein content.
AGS cells were transfected with either 50 nM siRNA against LC3B or control siRNA using lipofectamine RNAiMAX reagent according to the manufacturer’s instructions. The cells were then incubated with 10 μM BITC, and further analysis was conducted.
Data were expressed as the mean ± SEM of at least three independent experiments. Statistical differences between the groups were calculated using the Student’s
The cytotoxic effect of BITC in AGS cells was initially confirmed by an MTT assay. AGS cells were treated with 2.5, 5, 10, 20, or 40 µM BITC for 24 or 48 h. Then, the MTT assay was conducted to quantify the reduction in AGS cell viability. As shown in Fig. 1A, BITC reduced the AGS cell viability in time and concentration-dependent manners. The IC50 measurements for 24 h and 48 h were approximately 10 µM and 5 μM, respectively. Next, the cellular morphology was observed under a fluorescent microscope, and apoptotic features such as cell shrinkage, cell rounding, and apoptotic bodies were detected after treating the cells with BITC for 6 h (Fig. 1B). The nuclear morphological changes were also observed with a nuclear dye: DAPI under a fluorescent microscope (Fig. 1C). As a result, the number of the cells with condensed nuclei was increased significantly in a concentration-dependent manner.
To explore the effect of BITC on autophagy, the levels of the commonly-applied autophagy marker proteins LC3B and p62/SQSTM1 were detected by western blot and qPCR. Since autophagy is a dynamic process, the levels of both proteins were examined not only in concentration-based conditions but also in time-based conditions. As shown in Fig. 2A and 2B, BITC significantly increased the expression levels of both LC3B II and p62 in time and concentration-based conditions. BITC treatment also increased their mRNA levels (Fig. 2C). Treatment with 10 µM BITC increased the LC3B mRNA level by approximately three-fold, but this did not occur with 5 and 15 µM BITC. Treatment with 5 and 10 µM BITC increased the p62 mRNA level up to approximately three-fold, but this did not occur with 15 µM BITC.
Since autophagy is as a multi-step mechanism, the other autophagy-related proteins involved in different steps of autophagy, such as Beclin1, Atg5-Atg12 complex, and mTOR phosphorylation were analyzed by western blotting. As shown in Fig. 3, BITC decreased the expression levels of Beclin1 and Atg5-Atg12. There was a concentration-dependent decrease in Beclin1 expression. Approximately 50% and 90% of Beclin1 expression were depleted in 10 μM and 15 μM BITC-treated groups, respectively, compared to the control. For Atg5-Atg12 complex, only a high concentration 15 μM BITC significantly decreased its expression to half, compared to control group. On the other hand, statistically significant data was not obtained for mTOR phosphorylation detection.
The expression level of class III PI3K was measured after treating the cells with BITC for 24 h, and a potent PI3K inhibitor called wortmannin was utilized to observe the changes in LC3B conjugation level. The results showed that 15 μM BITC significantly reduced the class III PI3K protein level by approximately 50% compared to the control group (Fig. 4A). In the presence of wortmannin, the class III PI3K protein expression was significantly decreased by approximately 80%, compared to BITC monotherapy (Fig. 4A). An MTT assay was also performed to examine the cytotoxicity of wortmannin, and 24-h treatment of the typical working concentrations of wortmannin (0.2 to 1 µM) showed a 20% maximum cell death in AGS cells (Fig. 4B). Next, the LC3B conjugation (LC3B II level) was detected in the BITC-treated cells with or without wortmannin. As shown in Fig. 4C, wortmannin could not block the BITC-induced LC3B conjugation, and in fact, it unexpectedly enhanced the expression of LC3B II.
The degradation step of autophagy was investigated in AGS cells under BITC treatment. The activity of the cathepsin proteins was examined in BITC-treated AGS cells to evaluate the function of lysosomes. The cathepsin activity was quantified with an omnicathepsin assay in concentration-based or time-based conditions. As shown in Fig. 5A and 5B, BITC decreased the cathepsin activity of AGS cells in a concentration dependent manner, but not in a time-dependent manner. Bafilomycin A1 was used as a positive control to inhibit lysosomal degradation. AGS cells were treated with BITC in the presence or absence of bafilomycin A1, and the cathepsin activity was compared. As shown in Fig. 5C, bafilomycin A1 alone could reduce the basal cathepsin activity of AGS cells by approximately 60%. BITC treatment alone reduced the cathepsin activity by approximately 40% compared to the control group. In the combined treatment groups, the cathepsin activity significantly declined by approximately 80% or 90% compared to the control.
Next, lysosomal stability was detected via AO staining. It was predicted that BITC would decrease lysosomal stability. However, BITC did not reduce the red fluorescence or increase the green fluorescence of AO. In addition, the sizes of the red dots, which represent the acidic vesicles, were noticeably larger (Fig. 5D).
To confirm the lysosomal swelling, the expression level of lysosomal membrane protein LAMP2 was analyzed by western blotting and immunocytochemistry staining. Under the same conditions as the previously described experiments, AGS cells were exposed to 5, 10, or 15 μM BITC for 24 h, and the expression level of LAMP2 was measured by western blotting. For immunostaining, the cells were treated with BITC for 7 h. DAPI was used as a counterstain to stain the nuclei. The western blot analysis showed that 24-h BITC treatment significantly increased the expression level of LAMP2 in a concentration-dependent manner (Fig. 6A), whereas no difference was visualized through immunostaining in the 7-h BITC-treated cells (Fig. 6B).
To further confirm the lysosomal dysfunction induced by BITC, the expression levels of cathepsin proteins D, L, and B were measured by western blotting. As shown in Fig. 7A, after 24-h BITC treatment, pro-cathepsin D expression dramatically increased up to 4-fold. Prepro-cathepsin D expression seemed to increase slightly, but no statistically significant data was obtained. Mature-cathepsin D level was decreased by approximately 2%, 24%, and 28% after BITC treatments of 5, 10 and 15 μM, respectively, compared to the control. Similarly, 5 μM BITC increased the expression of pro-cathepsin L to almost twice that of the control group. On the other hand, 10 and 15 μM BITC treatments reduced mature-cathepsin L expression by approximately 40% compared to the control group (Fig. 7B). As shown in Fig. 7C, BITC decreased the expression level of mature-cathepsin B by approximately 10%, 20%, and 70% in 5, 10, and 15 μM BITC-treated groups, respectively, compared to the control group.
Next, the lysosomal degradation inhibitor bafilomycin A1 was utilized, and the LC3B II accumulation pattern was assessed by western blotting and immunocytochemistry staining. As shown in Fig. 8A and 8B, the combined treatment of BITC and bafilomycin A1 showed a remarkable additive effect on LC3B II accumulation. Compared to 10 μM BITC treatment alone, LC3B II level increased by approximately 1.5 times in the presence of bafilomycin A1 in western blotting. The cytotoxicity of bafilomycin A1 was evaluated by an MTT assay, and it was confirmed that the applied concentration and duration of bafilomycin A1 treatment (100 nM, 24 h) did not induce cytotoxicity to AGS cells (Fig. 8C).
After confirming the BITC-induced autophagy inhibition through lysosomal dysfunction, the role of basal autophagy in AGS cells was determined using an MTT assay. The difference in cell viability level was detected by further inhibiting autophagy with different autophagic stage inhibitors: wortmannin to inhibit autophagy initiation, and Bafilomycin A1 to block the autophagy degradation. As shown in Fig. 9A and 9B, wortmannin significantly increased BITC-induced cell death, but bafilomycin A1 did not.
Next, the probable relationship between BITC-induced LC3B accumulation and cell death was investigated. Specific siRNA was used to knock down the LC3B gene. The cells were transfected with the LC3B siRNA for 12 h, followed by an 8-h BITC treatment. Transfection efficacy was confirmed by the almost depleted LC3B protein expression observed via western blotting. A control siRNA was used as a negative control (Fig. 10A). An MTT assay was continuously conducted, and the changes in cell viability in the 24-h BITC-treated cells with and without LC3B siRNA were determined. As shown in Fig. 10B, the knockdown of LC3B did not influence the cell death induced by BITC.
BITC was known to have anticancer effect, and our previous study demonstrated that the mechanism of anticancer effect results from inducing apoptosis (Han
Autophagy has a dual role in gastric cancer as either a tumor suppressor or tumor promoter, depending on the stress conditions (Cao
In our previous study, we found that BITC induced both intrinsic and extrinsic apoptosis in gastric cancer cells. In addition to apoptosis, the present study found that BITC increased the protein expression levels of both LC3B II and p62 in time and concentration-dependent manners. LC3 and p62 are well known and commonly applied autophagy markers (Mizushima
Meanwhile, BITC did not alter mTOR phosphorylation, but it significantly decreased the levels of both Beclin1 and Atg5-Atg12 proteins in AGS cells. mTOR is an upstream negative regulator of autophagy, and the inhibition of mTOR phosphorylation enhances autophagy (Dossou and Basu, 2019; Wang and Zhang, 2019). On the other hand, Beclin1 is involved in the initiation step of autophagy and is essential for autophagy (Liang
Furthermore, the results showed that 15 μM BITC decreased the class III PI3K protein expression in AGS cells. Notably, the LC3B II accumulation induced by BITC was enhanced in the presence of wortmannin, a potent PI3K inhibitor. Evidence has shown that class III PI3K is essential for LC3 conjugation (conversion of LC3 I to LC3 II) to initiate the autophagy process (Jaber and Zong, 2013; Ge
Lysosomes play a key role in autophagic degradation (Yim and Mizushima, 2020). In this study, BITC reduced the cathepsin activity of AGS cells in concentration and time-dependent manners. Moreover, a synergistic decrease in cathepsin activity was found in the combined treatment of BITC and bafilomycin A1, a specific inhibitor of V-ATPase that strongly inhibits the acidification of lysosomes and blocks lysosomal degradation (Yoshimori
In addition, lysosomal dysfunction is one of the reasons for the LC3 II accumulation (Rossi
Furthermore, BITC induced synergistic cell death with the PI3K inhibitor wortmannin, but not with bafilomycin A1, a lysosomal degradation inhibitor. This suggests that wortmannin and BITC may complement each other’s function to inhibit autophagy by inhibiting the autophagy initiation step and degradation step, respectively. Therefore, as autophagy inhibition increases, there would be a greater quantity of dead cells if the basal autophagy in AGS cells has a pro-survival role. For bafilomycin A1, it is possible that its mechanism overlaps with BITC, and competition may have occurred. Nevertheless, the increased cell death with wortmannin indicated the tumor-promoting role of basal autophagy in AGS cells. This suggests that the BITC induced autophagy inhibition may be beneficial for gastric cancer treatment.
In this study, the relative toxicity of BITC in normal cells was not examined due to limited time and resources. However, toxicity testing of BITC in normal cells has already been conducted in several studies. In one example, BITC inhibited cell growth, promoted G2/M phase arrest, and triggered apoptosis of oral cancer OC2 cells, with a minimal toxicity to normal PBMCs (Yeh
A previous study reported on the novel role of LC3-induced non-autophagic cell death in 15d-PGJ2 treated cancer cells (Kar
In summary, the BITC-induced AGS cell death mechanism involves autophagy inhibition in addition to its apoptotic effect. BITC could inhibit the cytoprotective autophagy of AGS cells by decreasing both the autophagy initiator proteins and the lysosomal degradation. The results of this study provide a basic for BITC as a novel anti-cancer agent and support autophagy as a promising target for gastric cancer treatment (Fig. 11).
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology [Grant NRF-2019R1F1A1062070], the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.NRF-2021R1F1A1050004), and the Chung-Ang University Young Scientist Scholarship (CAYSS) program in 2017.
There is no conflict of interest to declare.