Alisma canaliculatum Extract Affects AGS Gastric Cancer Cells by Inducing Apoptosis

The anti-cancer effects of Alisma canaliculatum extracts (ACE) were identified in AGS gastric cancer cells. Our results showed that ACE inhibited the growth of AGS cells, increased the proportion of sub-G1 phase cells, and depolarized the membrane potential of mitochondria. ACE-induced gastric cancer cell death was associated with Bcl-2, survivin and Bax level changes, and it activated caspase-3 and -9. In addition, it was involved in the activation of MAPKs and increased the reactive oxygen species (ROS). These results suggest that ACE induces apoptosis in AGS gastric cancer cells, and therefore, ACE may have the potential to treat gastric cancer.


Introduction
Gastric cancer places a heavy burden on people worldwide and is becoming the most common cancer globally [1,2]. Cancer is almost impossible to cure if not detected early, and as the disease progresses, the pain and economic losses of patients and their families become enormous, resulting in immense social distress [3]. Therefore, the importance of cancer prevention and its early treatment has increased recently, since the quality of life, which involves living a pleasant and healthy life, has gained significance over merely extending the life span of patients [4].
Alisma canaliculatum is a commonly used traditional medicine in China, Japan, and Korea [5]. It exhibits various pharmacological properties, such as anti-bacterial and anti-cancer effects, and protects the liver cells [6][7][8]. It is also effective in treating postmenopausal osteoporosis [9,10] and suppressing breast cancer metastasis [11].
Apoptosis plays a vital role in maintaining the balance between cell death and cell division. Apoptosis induced abnormalities can result in uncontrolled cell division, leading to diseases such as cancer [12]. Thus, apoptosis has become a key tool in cancer treatment, and the mechanism underlying cancer cell growth has become an important target for cancer cell therapy [13]. However, the anti-cancer roles of A. canaliculatum on gastric cancer cells have not yet been studied. Thus, we investigated the mechanisms underlying the anti-cancer effects of A. canaliculatum extract (ACE) on AGS gastric cancer cells.

HPLC analysis
A stock solution of ACE and alisol B monoacetate was prepared by separately dissolving each of them in methanol. Standard calibration solutions were prepared, ranging from 6.25 to 100 μg/mL. The HPLC analysis of the extract was performed using a JASCO HPLC system equipped with a binary pump, an auto-sampler, a column oven, and a UV detector. An aliquot containing 20 μL of standard or ACE sample solutions was directly injected onto a Symmetry300 TM C18 column (4.6 × 250 mm × 5 μm). The mobile phase consisted of a mixture of (A) 0.1% phosphoric acid in distilled water and (B) ACN. The gradient running condition was as follows: 40-40% A for 0-10 min, 40-30% A for 10-20 min, 30-20% A for 20-30 min, 20-40% A for 30-35 min, with an assay determination run time of 35 min.

Measurement of cell cycle
AGS cells were treated with ethyl alcohol and vortexed. Samples were centrifuged for 5 min and the supernatant was discarded. Cell pellets were resuspended in propidium iodide (PI) staining solution containing RNase and incubated for 40 min in the dark at room temperature. Samples were analyzed using a fluorescence-activated cell sorter (FACScan; Becton-Dickinson, Mountain View, CA, USA).

Measurement of mitochondrial depolarization assay
AGS cells were treated with 50 nM tetramethylrhodamine methyl ester (TMRM; Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Fluorescence intensities were measured using a BD FACSCANTO II (BD Biosciences, Sunnyvale, CA, USA) at the excitation and emission wavelengths of 510 and 580 nm, respectively.

Caspase assay
Caspase-3 and -9 assay kits (Cellular Activity Assay Kit Plus; BioMol, Plymouth, PA, USA) were used. After resuspending the cells in ice-cold cell lysis buffer, the supernatant was removed. Supernatant samples were incubated with caspase substrate (400-lM Ac-DEVD-pNA; 50 μl) at 37°C and then, samples were read at 405 nm.

Statistics
One-way ANOVA with Tukey's post hoc comparison was used for multiple comparisons. The analysis was performed using Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA) and Origin 8.0 (OriginLab Corporation, Northampton, MA, USA) software. Data were expressed as the mean ± standard error of the mean (SEM), and P values < 0.05 were considered statistically significant.

Quantification of alisol B monoacetate in ACE by HPLC-UV
A previous study reported that alisol B monoacetate is a major component in ACE [14,15]. To quantify alisol B monoacetate in ACE, HPLC-UV was performed ( Fig. 1A and 1B). The results showed that alisol B monoacetate was present at a concentration of 8.68 ± 0.1 mg/g in ACE (Fig. 1C).

Relationship of ACE-induced apoptosis with mitochondrial pathway activation in AGS gastric cancer cells
We also analyzed whether the anti-apoptotic protein, Bcl-2, and pro-apoptotic protein, Bax, were involved in apoptosis following ACE treatment. Western blot analysis revealed that the levels of Bcl-2 decreased after treatment with ACE, whereas those of Bax increased (Fig. 4A-4C). In addition, the expression of survivin, an inhibitor of apoptosis, was decreased after ACE treatment (Fig. 4D and 4E). These results indicate that ACE-induced apoptosis was related to mitochondrial pathway activation in AGS cells.

Relationship of ACE-induced apoptosis with caspase activation in AGS gastric cancer cells
Caspases are important mediators of apoptosis. Moreover, the cleavage product of poly (ADP-ribose) polymerase (PARP) functions as a sign of apoptosis [16]. ACE increased the caspase-3 and -9 activation dose-dependently, and zVAD-fmk inhibited this activation (Fig. 5A). In addition, western blot analysis suggested that ACE downregulated the expression levels of pro-caspase-3 and -9 and upregulated the levels of not only their active forms but also PARP cleavage (Fig. 5B). These results indicate that ACE-induced apoptosis was related to caspase activation in AGS cells.

Regulation of the mitogen-activated protein kinase (MAPK) signaling pathway by ACE in AGS gastric cancer cells
To investigate the effects of ACE on the MAPK signaling pathway, we investigated the phosphorylation of MAPK proteins (extracellular signal regulated kinase (ERK), JNK, and p38) with western blotting. The phosphorylation of p38 increased with ACE treatment (200 μg/ml) (Fig. 6). These results indicate that ACE induced the apoptosis of AGS cells by modulating the MAPK signaling pathway.

Regulation of intracellular ROS generation by ACE in AGS gastric cancer cells
Since ROS also play a key role in apoptosis, we investigated whether ACE increased ROS levels in AGS cells. Flow cytometry results showed that ACE increased ROS levels (Fig. 7). These results indicate that ACE induced AGS cell apoptosis by ROS generation.

Discussion
Traditional medicine is an ancient form of therapy that treats not only an illness or ailment but also the entire body holistically [17]. A. canaliculatum is a plant used as a traditional medicine in Asia [5] and exerts many positive effects [6][7][8][9][10]. It is also effective against cancer cells and can inhibit the metastasis of breast cancer [11]. In addition, the present study shows that ACE induces apoptosis in AGS gastric cancer cells.
Apoptosis is mediated in eukaryotic cells through extrinsic and intrinsic pathways, and caspase cascade plays a key role in this process. The extrinsic pathway is initiated by the activation of caspase-8, which is triggered by the combination of death ligand to the death receptor present in the cell membrane, followed by the activation of caspase-3 and -7 [18,19]. In contrast, the intrinsic pathway, which begins around the mitochondria, forms an apoptosome and then promotes the activity of caspase-9 and caspase-3/-7 to cause apoptosis [20,21]. The inhibition of apoptosis is induced by terminating the activity of caspases, which is elicited by proteins belonging to the inhibitor of apoptosis protein family [22,23]. In the present study, ACE suppressed the proliferation of AGS cells (Fig. 2), induced the increase proportion of the sub-G1 phase, and depolarized the membrane potential of mitochondria (Fig. 3). ACE-induced cell death was associated with Bax increase and Bcl-2 decrease levels (Fig. 4); it also activated caspase-3 and -9 (Fig. 5). In addition, ACE reduced the expression levels of survivin (Fig. 4). Moreover, ACE-induced apoptosis was related to the MAPK signaling pathway (Fig. 6). ACE also increased the production of ROS (Fig. 7). These observations show that ACE induced apoptosis is dependent on caspase and mitochondrial pathways in AGS cells.
Ion channels play an important role in the formation and death of cancer. They are related with cancer cell growth, migration, and metastasis. Ion channels are also known to be critical components in apoptosis [24,25]. Among the various ion channels, TRPM7, TRPM2, and TRPC6, are known to be involved in AGS cell apoptosis [26][27][28][29]. Thus, these ion channels may be targeted for gastric cancer treatment. TRPM7 is essential for the survival of AGS cells [26,27], and TRPM2 has the ability to control the invasion of gastric cancer cells [28]. In addition, the suppression of TRPC6 is known to inhibit gastric cancer formation [29]. However, only a few studies have investigated the relevance of ion channels in the control mechanism of traditional medicine in gastric cancer apoptosis. Therefore, in the future, we should study the role of ion channel in gastric cancer apoptosis by ACE.
MAPKs are related with different cellular mechanisms including apoptosis [30]. Therefore, they are known to be involved in cancer cell therapy mechanisms [31]. In the present study, we found that ACE activated the p38 pathway, and it suggests that the modulation of MAPK cascades induces the apoptosis of AGS cells.
In conclusion, ACE inhibits the growth of AGS cells, induces the increase proportion of sub-G1 phase, and depolarizes the membrane potential of mitochondria. We found ACE-induced apoptosis to be associated with BCl-2 and survivin decreases and Bax increase levels; it also activated caspase-3 and -9 and MAPKs. Moreover, ACE increased the production of ROS. Thus, the present study on ACE may markedly contribute to the development of gastric cancer treatments.