Ginsenoside-Rh2 Inhibits Proliferation and Induces Apoptosis of Human Gastric Cancer SGC-7901 Side Population Cells

Objectives: To observed the effects of ginsenoside -Rh2 (GS-Rh2) on proliferation and apoptosis of side population (SP) human gastric cancer SGC-7901 cells. Materials and Methods: SGC-7901 SP and Non-SP cells were sorted by flow cytometry and assessed using the cck-8 method. Expression of apoptosis-related proteins Bax and Bcl-2 of SP before and after the intervention was determined by Western-blotting. Results: It was found that the proliferation of SP was significantly faster than that of NSP ( P <0.05). In addition, GS-Rh2 inhibited proliferation of gastric cancer SP cells, induced cell cycle arrest and cell apoptosis, and changed the expression of BAX/Bcl-2 proteins in a time-dependent and concentration-dependent manner ( P <0.05). Conclusions: With increase of GS-Rh2 dose, GS-Rh2 gradually inhibit the proliferation of SGC-7901 SP cells, which have high proliferation rate, through G1/G0 phase arrest, followed by apoptosis which involves the up-regulation of Bax and the down-regulation of Bcl-2.


Introduction
Gastric cancer is the most commonly diagnosed gastrointestinal cancer with high recurrence rate, high metastatic rate and resistance to chemotherapeutic drugs (Saricanbaz et al., 2014). Today, the biological characteristics of gastric cancer cells are investigated mainly through studying proliferation (Kato et al., 2012), apoptosis (Akagi et al., 2013), nude mice tumorigenic (Zheng et al., 2010) and protein molecular level of gastric cancer cells (Wang et al., 2014).
Ginseng is one of the commonly used Chinese traditional medicines that showed anti-cancer activity in clinical studies (Nakata H et al., 1998). The research proved that the mainly active ingredients of anti-tumor in ginseng is ginsenosides (Choi S et al., 2011;Tang XP et al., 2013) , through the comparison of various kinds of ginsenoside found that GS-Rh2 has the strongest ability to inhibited the proliferation of cancer cells (Li B et al., 2011). SP was proved to be a cell subset with low fluorescence as a result of the Hoechst 33342 staining, which is the founding belongs to Goodel et al (Goodell et al., 1996), who used Hoechst 33342 and flow cytometry to stain and sort bone marrow cells in mice. SP can be sorted through ABCG-2 (adenosine triphosphate binding cassette super family G member-2 of transport protein, ABCG- 2) because ABCG-2 has the feature of Hoechst 33342 excretion and expresses in high level in SP (Shaharuddin et al., 2014). Recent studies have shown that SP cells have a faster proliferation rate than NSP cells (Kong et al., 2014), so studying the biological characteristics of malignancies from the SP cells is great significance.
The aim of this study was to investigate effects of GS-Rh2 on the proliferation and apoptosis of SP isolated from human gastric cancer SGC-7901 cells.

Cell line
Human gastric cancer cell line SGC-7901 was purchased from the Chinese Academy of Sciences (Shanghai Cell Bank, Shanghai, China).

Reagents and instruments
Cell culture medium RPMI-1640 and 0.25% trypsin were purchased from Hyclone (Logan, UT, USA); Penicillin-Streptomycin, Liquid was purchased from Invitrogen (Carlsbad, CA, USA); fetal bovine serum (FBS) was purchased from Tianhang Biological Technology, (Hangzhou, Zhejiang, China); Hoechst 33342 and verapamil were obtained from Sigma (Saint Louis, MO, USA); GS-Rh2 was purchased from Sigma; the CCK-8 reagent was purchased from Beyotime (Nantong, Jiangsu, China); and AV kit and PI were obtained from BD (Franklin Lakes, NJ, USA). Antibodies against BAX and Bcl-2 antibodies were obtained from Boster (Wuhan, Hubei, China) and β-actin antibody was obtained from Sigma Company; horseradish peroxidase-labeled goat anti-rabbit IgG was purchased from Zhongshan, (Beijing, China); the substrate reagent was purchased from Millipore Company, (Boston, MA, USA). flow cytometers BD FACS ARIA II SORP and BD FACSCalibur were purchased from BD (Franklin Lakes, NJ, USA), electrophoresis apparatus and gel imaging system were obtained from BioTeK Synergy 2 (BioTeK, VT, USA), and power supply and gel imaging system ChemiDox XRS were from Bio-Rad (Hercules, CA, USA).

Flow cytometry and cell sorting
Cells were digested with 0.25% trypsin-EDTA, centrifuged at 1, 000 rpm for 5 min at 4°C, and resuspended in PBS with 2% FBS. Live cell number was counted at least twice and adjusted to a final cell density of 1 × 10 6 cells/ml, incubated at 37°C for 120min with 5 µg/mL Hoechst 33342, either alone or in combination with 50 µmol/L verapamil (100 mg/L), centrifuged for 5 min, and resuspended in cold PBS with 2% FBS at 1 × 10 6 cells/ml. SP cells were identified and sorted using Hoechst 33342; the signal was collected as follows: blue light, 402-446 nm, and red light, 650-670 nm, SP and NSP cells were separated using a flow cytometer.

Cell Culture
SGC-7901 human gastric cells were maintained in RPMI-1640 medium containing 100 u/ml of penicillin, 100ug/ml of streptomycin and 10% FBS, at 37°C in a humidified and 5% CO 2 atmosphere.

Cell Sorting
The cells were harvested by detachment with 0.25% trypsin-EDTA and resuspended in PBS with 2% FBS, added hoechst 33342 (final concentration is 5 ug/ml) into the cell suspension, and control group was supplemented with 10 ul/ml verapamil additionally. Incubated all the cells at 37°C in 5% CO 2 atmosphere for 90 min. Cells were collected and then resuspended in ice-cold PBS with 2% FBS at 1×10 6 cells/ml. Then SP and NSP cells were identified and sorted by a flow cytometry, and the signal was collected as follows: blue light, 402-446 nm; red light, 650-670 nm.

Cell proliferation assay
Cells were cultured at 37°C in a humidified and 5% CO 2 atmosphere. Cell proliferation was analyzed as detailed in CCK-8 kit instructions. All tests were done in triplicate and the experiments were repeated three times independently. The values were converted to cell numbers using the standard curve. The data were presented as ± standard error (x _ ±s).

Cell Cycle analysis
Cells were collected and washed with PBS twice, and resuspended in 1 ml of cold 70% ethanol, incubated at -20 °C overnight. The cells were pelleted by centrifugation and washed by PBS, resuspended in 0.5 ml PBS with 50 ug/ ml of PI, and incubated in dark at 37°C for 30 min, then cell cycle progression was analyzed by flow cytometry.

Apoptosis assay
The cells were collected and washed with PBS twice, then the cells were resuspended in 250 ul binding buffer, 3 ul AV and 3 ul PI, incubated in dark for 15 min, then cell apoptosis rate was analyzed through flow cytometry.

Protein analysis
Cell total protein was extracted by lysates (PMSF: RIPA=1:100). Protein concentration was adjusted to 60 ug/ ul by RIPA. Protein was boiled at 99°C for 5 min, stored at -80 °C for use. Proteins were separated by electrophoresis in 12% of separating Gel and 5% of stacking gel. Then the protein was transferred onto PVDF membrane. PVDF membrane was blocked with 5% non-fat milk at room temperature for 1-3 h. The PVDF membrane was incubated in primary antibody solution at 4°C overnight. The PVDF membrane was washed by TBST, then the PVDF membrane was incubated in secondary antibody solution at 4 °C for 1 h. Protein bands exposure were performed using chromogenic substrate. The protein bands were analyzed by densitometry using Quantity One software (Bio-Rad). β-actin was used as internal control.

Statistical analysis
All experiments were repeated 3 times independently, and the data were presented as ± standard error (±s). Statistical significance (p<0.05) was assessed by the analysis of variance (ANOVA) followed by Student's t-test.

Ratio of SP in SGC-7901 Cell Line
The ratio of SP in SGC-7901 cell line was (3.53±0.79)%, Meanwhile, the ratio of SP in control group (Verapamil group) was 0.1% ( Figure 1).

Proliferation of SP and NSP in SGC-7901 Cell Line
Compared with NSP, the proliferation ability of SP was significantly higher (p<0.05). (Figure 2).

Proliferation of SP cells treated with GS-Rh2
After treated by the different dose of GS-Rh2 or normal saline respectively, cell proliferation of SGC-7901 SP were determined. As can be see in Figure 3, compared with the normal saline group (control group) at corresponding time point, different dose of GS-Rh2 treatments inhibited cell proliferation in a time-dependent and concentrationdependent manner (p<0.05). (Figure 3).

Cell cycle of SP treated with GS-Rh2
As can be seen in Figure 4 and Table 1, The treatment with GS-Rh2 caused significant changes in SP cell cycle compared to the untreated control group. The percentage of G0/G1 cells increased, whereas that of the cells in the S and G2/M phases decreased. The effect showed a marked dependence on GS-Rh2 concentration (p<0.05) and the Ginsenoside -Rh2 Inhibits Proliferation and Induces Apoptosis of Human Gastric Cancer SGC-7901 Side Population Cells time of treatment: it was more obvious at 48 h than at 24 h (p<0.05). Based on these results, we can conclude that GS-Rh2 restricts SP cell cycle to G0/G1 phase in a time-dependent and dose-dependent manner. (Figure 4 and Table 1).

Apoptosis of SP treated with GS-Rh2
As can be seen in Figure 5 and Table 2, after the treatment with GS-Rh2, SGC-7901 SP demonstrated a significant increase in apoptosis, which depended on  SP were seeded into 96-well plates at 1×10 3 cells per well and were treated by cell culture medium containing GS-Rh2 5ug/ ml, GS-Rh2 10ug/ml or GS-Rh2 20ug/ml or normal saline (control) respectively. Cell proliferation was analyzed after treatments of 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h as detailed in "Cell proliferation assay". A. The number of SGC-7901 SP after intervention (GS-Rh2 5ug/ml, GS-Rh2 10ug/ ml or GS-Rh2 20ug/ml) for 168 hours; B. After intervention for 168 hours, proliferation inhibition rates of SGC-7901 SP, proliferation inhibition rates were determined using following formula. Proliferation inhibition rate (%)= (1-Drug group/ Control group)×100%   dependent and dose-dependent manner. (Figure 5 and Table 2).

BAX and Bcl-2 protein expression in SP treated with GS-Rh2
Based on above results, we investigated the effects of different dose of GS-Rh2 (5ug/ml, 10ug/ml or 20ug/ml) on regulators of apoptosis (main inhibitor or promoter ) as molecular targets for the apoptotic death of SGC-7901 SP. Accordingly, we focus our attention on the effects of different dose of GS-Rh2 (5ug/ml, 10ug/ml or 20ug/ml) on pro-apoptotic protein Bax and Bcl-2 of SGC-7901 SP line because the protein play a key role in cell apoptosis. As shown in Figure 6 and Table 3, treatment of SGC-7901 SP with different dose of GS-Rh2 (5ug/ml, 10ug/ml or 20ug/ ml) resulted in increased expression of Bax and inhibited the expression of Bcl-2 in a time-dependent and dosedependent manner. (p < 0.05). ( Figure 6 and Table 3).

Discussion
Gastric cancer is one of the world's most deadly malignancies, and effective treatment has been the focus of medical research for many years. As a subtype of cells, SP have a faster proliferation rate than NSP, so SP may play a crucial role in tumor progression Nowadays, the effects of using Ginseng to against cancer are increasingly discussed in the reports ( Qian et al., 2015). In the present work, SP of SGC-7901 cell line, which in our research had determined that there is a faster proliferation rate than NSP, and GS-Rh2, which extract from Ginseng , are employed as research object.
Firstly, the effects of different dose of GS-Rh2 (5ug/ ml, 10ug/ml or 20ug/ml) on proliferation of SGC-7901 SP were assessed. From the experimental results , we found that the proliferation of SP were inhibited in a time-dependent and concentration-dependent manner .
We next detected the effects of different dose of GS-Rh2 on cell cycle of SGC-7901 SP. It is found that treatment of SP cells with different dose of GS-Rh2 result in G1/G0 arrest in cell cycle progression of SP followed by apoptosis in a time-dependent and concentrationdependent manner. It was confirmed by subsequent apoptosis rate test.
In order to further investigate apoptosis induction effect of different dose of GS-Rh2 on SGC-7901 SP, Bax and Bcl-2 were selected as the apoptotic cell death studies in the research, because they are the major, possibly causative apoptotic and anti-apoptotic indicators of the multifactorial mechanisms of uncontrolled gastric cancer  divided into 4 groups, then 2 ml of SP cells suspension (5×10 5 cells/ml) were seeded into 60-mm Petri dish respectively, and were treated by culture medium containing GS-Rh2 5ug/ml, GS-Rh2 10ug/ml or GS-Rh2 20ug/ml or normal saline (control) respectively. After 24 h and 48 h of treatment, cell apoptosis rates were analyzed through flow cytometry as detailed in "Apoptosis assay". groups, then SP were seeded into cell culture flasks respectively, and were treated by culture medium containing GS-Rh2 5ug/ml, GS-Rh2 10ug/ml or GS-Rh2 20ug/ml or normal saline (control) respectively. After 24 h and 48 h of treatment, Bax/Bcl-2 was analyzed as detailed in "Protein analysis ". A. western blot for Bax/Bcl-2 protein expression after treated with different dose of GS-Rh2; B. The change of Bax protein expression in SGC-7901 SP c after treated with different dose of GS-Rh2 ; C. The change of Bcl-2 protein expression in SGC-7901 SP after treated with different dose of GS-Rh2 GS-Rh2 dose (p<0.05) as well as treatment time (p<0.05).
These data indicate that GS-Rh2 can effectively induce apoptosis of gastric cancer SGC-7901 SP in a time-growth respectively. Apparently, the results showed that different dose of GS-Rh2 induced the activity of proapoptotic protein Bax and inhibited anti-apoptotic protein Bcl-2 in a time-dependent and concentration-dependent manner in SP. Based on the findings, it is easy to conclude that: GS-Rh2 can inhibit the proliferation of SP of SGC-7901 cell line followed by apoptosis which is partly mediated via promoting Bax but inhibiting Bcl-2 in a time-dependent and concentration-dependent manner.
The experiment did indicate that treatment of SGC-7901 SP with different dose of GS-Rh2 can Inhibit proliferation and induced apoptosis with prolonged treatment, meanwhile, and the dose of GS-Rh2 is a reliable index for effect evaluation.