MATERIALS AND METHODS

Water extraction of DS: The DS is composed of 9 species of herbal plants, each of which were purchased from Kwangmyungdang Natural Pharmaceutical Co., Ulsan, Korea. The formula of DS is described in Table 1 and 60 g of DS were boiled in 1 L of distilled water in an Herb Extractor (Dae-Woong Co, Korea) for 2 h, yielding final 200 mL of DS extract. The supernatant was harvested in sterile condition by centrifugation and lyophilized through evaporation at -80°C, yielding final 4.6 g.

Table 1:	Composition of dagkwisoo-san

The lyophilized DS extract was dissolved in an appropriate volume of sterile PBS prior to administrating to cells. The water extract of DS (Voucher No. MH2014-0001) has been deposited at the Division of Longevity and Biofunctional Medicine, School of Korean Medicine, Pusan National University (Gao et al., 2013).

Cell: The AGS lines were used. The AGS cell lines were established at the Cancer Research Center, College of Medicine, Seoul National University, Korea. The cell lines were propagated in RPMI-1640 medium (Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum and 20 μg mL^-1 penicillin and streptomycin in an atmosphere of 5% CO₂ at 37°C.

Flow cytometric analysis: In order to investigate whether the cell cycle of AGS cells was redistributed, flow cytometric analysis was used with Propidium Iodine (PI) stain (Hellein et al., 2012; Nicoletti et al., 1991). The 1x10⁶ cells were placed in an e-tube. About 700 μL of a ice-cold fixation buffer (ethyl alcohol) was slowly added with vortexing. Tubes were sealed with parafilm and incubated at 4°C overnight. Samples were spun for 3 min at 106xg at 4°C and the supernatant was aspirated and discarded. The cell pellet was resuspended by 200 μL of PI staining solution (PI [5 mg mL^-1] 2 μL and RNase 2 μL in PBS 196 μL) at 20817 g for 5 sec. After 30 min in the dark at room temperature, samples were analyzed in a fluorescence activated cell sorter (FACScan; Becton-Dickinson, Moutain View, CA, USA) at λ = 488 nm using Cell-Quest software (Becton-Dickinson). The DNA content distribution of normal growing cells is characterized by two peaks, the G1/G0 and G2/M phases. The G1/G0 phase comprises the normal functioning and resting state of the cell cycle with the most diploid DNA content, while the DNA content in the G2/M phase is more than diploid. Cells in the sub-G1 phase have the least DNA content in cell cycle distribution; this is termed hypodiploid. The hypoploid DNA contents represent the DNA fragmentation (Wang et al., 2005).

MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay: Cell viability was assessed by using a MTT assay. The AGS cells were seeded into each well of 12-well culture plates and then cultured in Roswell Park Memorial Institute medium (RPMI)-1640 supplemented with other reagents for 72 h. After incubation, 100 μL of MTT solution (5 mg mL^-1 in Phosphate-Buffered Saline (PBS)) was added to each well and the plates were incubated at 37°C for 4 h. After the supernatant had been removed and shaken with 200 μL of dimethyl sulfoxide (Jersey Lab Supply, Livingston, NJ, USA) for 30 min, absorbance was measured at 570 nm. All experiments were repeated at least 5 times.

Caspase assay: Caspase-3 and 9 assay kits (Cellular Activity Assay Kit Plus) were purchased from BioMol (Plymouth, PA, USA). After experimental treatment, cells were centrifuged (10000xg, 4°C, 10 min) and washed with PBS. Cells were re-suspended in ice-cold cell lysis buffer and incubated on ice for 10 min. Sample were centrifuged at 10000xg (4°C, 10 min) and the supernatant was removed. Supernatant samples (10 μL) were incubated with 50 μL of substrate (400 μM Ac-DEVD-pNA) in 40 μL of assay buffer at 37°C. The absorbance at 405 nm was read at several time points. The pNA concentrations in samples were extrapolated from a standard created using the absorbances of sequential pNA concentrations.

Assessment of mitochondrial membrane depolarization: Mitochondrial membrane depolarization was evaluated using a JC-1 fluorescence probe according to the manufacturer's instructions (Santa Cruz). The AGS cells were labeled with 2 μM JC-1 for 30 min at 37°C and then analyzed by using flow cytometry with 488 nm excitation and 530/30 or 585/42 nm bypass emission filters. The cells without red fluorescence were regarded as the cells manifesting mitochondrial membrane depolarization.

Patch-clamp experiments: A whole-cell configuration of the patch-clamp technique experiment was performed at room temperature (22-25°C). The AGS cells were transferred to a small chamber on an inverted microscope stage (IX70, Olympus, Japan) and were constantly perfused with a solution containing 2.8 mmol L^-1 KCl, 145 mmol L^-1 NaCl, 2 mmol L^-1 CaCl₂, 10 mmol L^-1 glucose, 1.2 mmol L^-1 MgCl₂ and 10 mmol L^-1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), adjusted to a pH of 7.4 with NaOH. The pipette solution contained 145 mmol L^-1 Cs-glutamate, 8 mmol L^-1 NaCl, 10 mmol L^-1 Cs-2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and 10 mmol L^-1 HEPES-CsOH, adjusted to a pH of 7.2 with CsOH. Axopatch I-D (Axon Instruments, Foster City, CA, USA) was used to amplify the membrane currents and potentials. For data acquisition and the application of command pulses, pCLAMP software v.9.2 and a Digidata 1322A units (Axon Instruments) were used. Results were analyzed using pClamp and Origin software (Microcal Origin version 6.0).

TRPM7 expression in human embryonic kidney 293 cells: Human Embryonic Kidney (HEK)-293 cells were transfected with the Flag-murine long transient receptor potential channel 7 (LTRPC7)/pCDNA4-TO construct and grown on glass coverslips in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, blasticidin (5 μg mL^-1) and zeocin (0.4 mg mL^-1). TRPM7 (LTRPC7) expression was induced by adding 1 μg mL^-1 of tetracycline to the culture medium. Whole-cell patch-clamp experiments were performed at 21-25°C with cells that had been grown on the glass coverslips.

Statistical analysis: Data is expressed as Mean±Standard error of the mean (SEM). Differences between the data were evaluated by using the student’s t-test. A p-value of 0.05 was taken to indicate a statistically significant difference.

RESULTS

DS induced cell death in AGS cells: To ascertain whether DS kills AGS cells, the MTT assays were performed. The viable cell population was gradually reduced with increasing concentrations of DS for 72 h in AGS cells (Fig. 1). Thus, the present results demonstrate that DS induces cell death in AGS cells.

DS triggered apoptosis in AGS cells: To determine whether AGS cell death occurs by apoptosis, sub-G1 analysis was conducted (Hotz et al., 1994; Vermes et al., 2000). In this protocol, cells were incubated with DS and stained with a fluorescent DNA stain (PI). The action of endogenous endonucleases in apoptotic cells cleaves DNA into endonucleosomal fragments of typical size, which are extracted from the cells. The loss of DNA is detected by FACS analysis, as the reduced nuclear staining in apoptotic cells, which results in a novel (sub-G1) fluorescence peak to the left of the regular fluorescence peak. Flow cytometric analysis showed that the percentage of sub-G1 phase cells markedly increases in the cells treated with DS in a dose-dependent manner in AGS cells (Fig. 2). In addition, DS elevated mitochondrial membrane depolarization, an early event of an intrinsic apoptosis signaling (Fig. 3).

Fig. 1:	DS induces cell death in AGS cells, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)-based viability assay. The AGS cells were treated with increasing concentrations of DS for 72 h. Distilled water was used as a vehicle. Cell viability is expressed as a value relative to that of the untreated cells which is set to 100%. The figures show Mean±SEM, **p<0.01

Fig. 2:	Increases in activity of sub G1 peak in AGS cells due to DS. Sub-G1 peak measured by FACScan. The figures show Mean±SEM, **p<0.01

Thus, the present findings suggest that DS induces apoptosis via intrinsic apoptotic mechanisms. Caspase-3 and -9 activation is one of the hallmarks of apoptotic cell death. It was also measured that the enzyme activity in AGS cells after DS incubation. Using a synthetic substrate, the caspase-3 and 9 activity in AGS cells can be detected. DS increased the activity of caspase-3 and 9 (Fig. 4).

Involvement of mitogen-activated protein kinases (MAPKs) in DS-induced apoptosis in AGS cells: To investigate the signaling pathway of DS-induced apoptosis in AGS cells, we assessed the effect of DS on MAPKs, because they play critical roles in the apoptosis-related signaling pathway.

Fig. 3:	Increases in mitochondrial membrane depolarization potentials in AGS cells due to DS. Mitochondria membrane depolarization is expressed as a value relative to that of untreated cells which is set to 100%. The figures show Mean±SEM, *p<0.05, **p<0.01

Fig. 4:	Increases in caspase activity in AGS cells due to DS. The AGS cells were cultured with DS at the indicated concentrations for 24 h prior to caspase assays. Caspase activity from untreated cells is expressed as 100%. Pan-caspase inhibitor zVAD-fmk (zVAD) at 20 μM was used to validate the analytical method employed. The figures show Mean±SEM, *p<0.05, **p<0.01

As shown in Fig. 5, exposure to DS with c-jun NH₂-terminal kinase (JNK) II inhibitor (Fig. 5a) or SB203580 (a p38 MAPK inhibitor) (Fig. 5b) resulted in increases in viable cell populations.

Fig. 5(a-b):	Effects of JNK II inhibitor and SB203580 on DS-induced AGS cells. MTT-based viability assay, (a) C-jun NH2-terminal kinase (JNK) II inhibitor or (b) SB203580 (a p38 MAPK inhibitor) were administered to the AGS cells pretreated by DS. The figures show Mean±SEM, *p<0.05, **p<0.01

Effects of DS in TRPM7 currents in AGS and TRPM7 overexpressed HEK293 cells: TRPM7 has been proposed to be required for cell survival on the basis of experiments on genetically-engineered DT-40 B-cells (Nadler et al., 2001). Also, recently it was suggested that as in previous reports, that AGS cells express the TRPM7 channel and that suppression of the TRPM7 channel induced cell death (Kim et al., 2008). Therefore, it as investigated that whether DS influences TRPM7 currents in AGS cells. To confirm the effect of DS in TRPM7 currents, the effects of DS in AGS cells using patch-clamp techniques were investigated. Whole cell voltage-clamp recordings was performed to investigate the effect of DS in TRPM7-like current in AGS cells. A voltage ramp of from +100 mV to -100 mV evoked small inward currents at negative potentials, whereas larger outward currents were evoked at positive potentials, showing that they were outward-rectifying cation currents (n = 4; Fig. 6a). However, in the presence of 500 μg mL^-1 DS, the amplitudes of these currents were inhibited outwardly by 92.3±2.4% and inwardly by 95.1±2.1% (n = 7; Fig. 6a). Also similar results were obtained in HEK293 cells overexpressing TRPM7 (Fig. 6b). To provide additional evidence that supports the contribution of the TRPM7 channel to DS toxicity, the changing expression levels of TRPM7 channel and its influences on DS-mediated cell death was investigated. We used HEK293 cells with inducible TRPM7 channel expression (Nadler et al., 2001). In the absence of induced TRPM7 channel expression [TRPM7(-) cells, Tet(-)], HEK293 cells incubation with DS-induced cell death in the MTT assay (n = 5; Fig. 6c). However, when TRPM7 channel overexpression was induced by adding tetracycline [TRPM7(+) cells, Tet(+)], HEK293 cells incubation with DS induced cell death at an increased rate in the MTT assay, which suggests that increased expression of TRPM7 channels leads to increased rate of DS-induced cell death.

DISCUSSION

DS, an herbal extract, is widely used in traditional herbal medicine in Korea to treat traumatic ecchymosis and pain by promoting blood circulation and relieving blood stasis. However, the effects of DS in cancer has not been examined. In this study, it was demonstrated that DS suppresses AGS cell proliferation and shows sub G1 increase and mitochondrial membrane depolarization. Additionally, DS-induced apoptosis is inhibited by MAPK inhibitors and TRPM7 channels is involved in these effects. These results suggested that DS may be a useful agent for pharmacological approaches for future development of anticancer drugs. Therefore, DS would be a useful drug tool for approaches to identify novel therapeutic targets for gastric cancer.

Sophorae radix (SR), Orostachys japonicas (OJ) and quercetin inhibited the growth and survival of gastric and breast cancer cells due to a blockade of the TRPM7 channel activity (Hwang et al., 2012; Kim, 2012; Kim et al., 2014). Many ion channels are involved in regulation of pathophysiological role in cancer cells. Voltage-gated potassium ion channels were overexpressed in colon cancer (Abdul and Hoosein, 2002a) and voltage-gated sodium ion channels were involved in the growth of prostate cancer (Abdul and Hoosein, 2002b). Volume-regulated Cl-channels were found in a human prostate cancer cell line and in lung cancer cells (Jirsch et al., 1993; Shuba et al., 2000).

Fig. 6(a-c):

Effects of DS on AGS and TRPM7 overexpressed HEK293 cells, representative I-V relationships of the effect of DS on TRPM7 currents in (a) AGS and (b) HEK293 cells while (c) TRPM7 channels were treated or not treated with tetracycline for 1 days. Cells were incubated with DS, followed by MTT assay. A voltage ramp from +100 to -100 mV was applied from a holding potential of -60 mV. The figures show Mean±SEM, **p<0.01

Additionally, TRP channels also might have an important role in apoptosis and the involvement of TRP channel in cancer cells was also investigated. Among the TRP families, TRPC, TRPV and TRPM are mainly related to the growth and progression in cancer cells. Depending on the type of the cancer, regulation of TRP mRNA and protein expression have been changed. These ion channel changes are related with cell growth and apoptotic-induced cell death in cancer cells. Therefore, the regulations of ion channels in cancer cells are the most promising strategy and considerable efforts should be done to fight cancer cells (Santoni and Farfariello, 2011). In line with these studies, the present studies show that DS induces apoptosis in gastric adenocarcinoma cells, which may be due to a blocking of the TRPM7 channel activity and MAPK signaling.

DS represents a mixture of nine herbal medicines, consisting of Angelicae gigantis Radix, Paeoniae Radix, Linderae Radix, Sappan Lignum, Cyperi Rhizoma, Carthami Flos, Persicae Semen, Cinnamomi Cortex and Glycyrrhizae Radix et Rhizoma. Most traditional therapeutic formulations consist of a combination of several drugs. Bioactivity from each drug may collectively act to block multiple targets underlying apoptosis, although little is known about the mechanisms for their pharmacological activities (Lyu et al., 2012; Kim et al., 2011; Qiu, 2007; Wang et al., 2008). The combination of multiple drugs is thought to maximize therapeutic efficacy by facilitating synergistic actions and preventing potential adverse effects. However, little is known concerning the compounds responsible for the apoptotic effect of DS. In future, to perform additional experiments to identify the efficient compounds from DS need.

Taken together, DS induced the apoptosis in AGS cells and both MAPK signaling and TRPM7 channel activity are involved in DS-induced effects. Therefore, DS may be a potential drug for treatment of gastric cancer.

ACKNOWLEDGMENT

This study was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIP) (2014R1A5A2009936).