20(S)-ginsenoside Rg3 promotes senescence and apoptosis in gallbladder cancer cells via the p53 pathway

Gallbladder cancer (GBC), the most frequent malignancy of the biliary tract, is associated with high mortality and extremely poor prognosis. 20(S)-ginsenoside Rg3 (20(S)-Rg3) is a steroidal saponin with high pharmacological activity. However, the anticancer effect of 20(S)-Rg3 in human GBC has not yet been determined. In this study, we primarily found that 20(S)-Rg3 exposure suppressed the survival of both NOZ and GBC-SD cell lines in a concentration-dependent manner. Moreover, induction of cellular senescence and G0/G1 arrest by 20(S)-Rg3 were accompanied by a large accumulation of p53 and p21 as a result of murine double minute 2 (MDM2) inhibition. 20(S)-Rg3 also caused a remarkable increase in apoptosis via the activation of the mitochondrial-mediated intrinsic caspase pathway. Furthermore, intraperitoneal injection of 20(S)-Rg3 (20 or 40 mg/kg) for 3 weeks markedly inhibited the growth of xenografts in nude mice. Our results demonstrated that 20(S)-Rg3 potently inhibited growth and survival of GBC cells both in vitro and in vivo. 20(S)-Rg3 attenuated GBC growth probably via activation of the p53 pathway, and subsequent induction of cellular senescence and mitochondrial-dependent apoptosis. Therefore, 20(S)-Rg3 may be a potential chemotherapeutic agent for GBC therapy.


Introduction
Gallbladder cancer (GBC) is the most common malignancy of the biliary tract, representing 80%-95% of biliary tract cancers worldwide, characterized by high lethality, late diagnosis and chemoresistance, it ranks sixth among gastrointestinal cancers. [1][2][3] Surgery is the only curative treatment for patients with GBC. 4 The majority of patients are diagnosed with advanced GBC, a stage at which treatment by resection is not possible. 5 Moreover, most patients have frequent recurrences following surgery and dismal outcomes after chemotherapy or radiotherapy. 6 The prognosis of patients with GBC in stage 0 and stage 1 is optimistic with 5-year survival rates ranging from 50% to 80%, but the prognosis in stage II-IV is dismal with 5-year survival rates ranging from 2% to 28%. 7 5-Fluorouracil has been shown to have only a 20% response rate, and gemcitabine had a limited response rate of 36%. 8 Therefore, there is an urgent need to develop novel and effective therapy regimens for GBC patients.
Ginseng, such as American ginseng (Panax quinquefolius L.) and Asian ginseng (Panax ginseng CA Meyer), is the root of different Panax species (Araliaceae) and is one of the most commonly used traditional medicines. 9 Ginsenoside Rg3, one of the active ingredients in ginseng, has been reported to exhibit various  10,11 Stereospecific effects have been observed from this compound, with the 20(R) enantiomer, for instance, being more active as an antioxidant and in its promotion of the immune response, 12,13 and the 20(S) having a greater potential antidiabetic activity. 14 The 20(S) enantiomer is more suitable for pharmaceutical development because of its superior solubility compared with the 20(R) enantiomer. The 20(S)ginsenoside Rg3 has also been shown to be remarkably nontoxic and is well-tolerated in mice, rats, and dogs. [15][16][17] Rg3 may increase the efficacy of cancer chemotherapy, possibly through inhibitory effects on NF-κB and AP-1 activity, 17 and downregulation of angiogenesis associated with VEGF expression. 18 Recently, 20(S)-Rg3 has been found to affect growth and survival in several human cancers, including colon cancer, leukemia, and ovarian cancer. [19][20][21] There is currently no published data showing the involvement of 20(S)-Rg3 in human GBC.
In the present study, we investigated the effect of the 20(S)-Rg3 on cell growth and survival in human GBC cell lines to evaluate its antitumor activity. Our data show that 20(S)-Rg3 is capable of inhibiting GBC cell growth via facilitating cellular senescence and apoptosis. Effect of 20(S)-Rg3 on GBC growth was confirmed in vivo using a mouse xenograft model. Our data therefore suggests that the inhibitory effect of 20(S)-Rg3 on growth is functionally related to its promotion effect on cell senescence and apoptosis and that 20(S)-Rg3 could serve as a novel strategy in treating GBC.

Materials and methods
Drugs and reagents 20(S)-ginsenoside Rg3 was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, People's Republic of China), and the purity was at least 95% as determined by HPLC (high performance liquid chromatography). 20(S)-Rg3 was dissolved in dimethyl sulfoxide (DMSO) in a 400 mM stock solution and stored at -20°C, and diluted with fresh complete medium immediately before use. An equal volume of DMSO (final concentration 0.1%) was added to the controls.

cell culture
Human GBC cell lines NOZ, GBC-SD, SGC-996, EH-GB-1, and EH-GB-2 were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, People's Republic of China). NOZ cells were maintained in William's medium (Gibco, Grand Island, NY, USA) supplemented with 100 U/mL penicillinstreptomycin (Hyclone, Logan, UT, USA) and 10% fetal bovine serum (FBS; Gibco). GBC-SD cells were maintained in DMEM (Dulbeccos' Modified Eagle's Medium) medium (Gibco) containing 10% FBS. These cell lines were cultured at 37°C in a humidified incubator containing 5% CO 2 .
cell viability assay NOZ and GBC-SD cells were seeded into 96-well plates at a density of 5×10 3 cells per well, incubated overnight, and then treated with 20(S)-Rg3 at different concentrations (0, 25, 50, 100, 200, and 400 μM) for 24, 48, and 72 hours. In vitro cytotoxicity of the GBC cells was measured by using MTT assay, as previously described. 22 colony formation assay NOZ and GBC-SD cells were seeded into 6-well plates at a density of 500 cells per well, and treated with different concentrations (0, 25, 50, 100, 200, and 400 μM) of 20(S)-Rg3 for 48 hours. After removal of the supernatant, the cells were allowed to form colonies in complete medium for 12 days. Next, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich). After washing, the plates were air-dried, and stained colonies were photographed using a microscope (Leica, Wetzlar, Germany). 20(S)-Rg3 for 48 hours. Senescent cells were analyzed using a senescence-associated β-galactosidase (SA-β-gal) staining kit (Beyotime, Nantong, People's Republic of China) according to the manufacturer's instructions. The staining results were recorded as "positive" or "negative", according to a previous report. 23 cell cycle analysis NOZ and GBC-SD cells were treated with different concentrations (0, 25, 50, 100, 200, and 400 μM) of 20(S)-Rg3 for 48 hours. The cells were harvested by trypsinization, washed twice in cold PBS (phosphate buffered saline), and fixed in 70% ethanol overnight at 4°C. Cell cycle was then detected by flow cytometry with PI (propidium iodide) staining according to a previous report. 24 At least 50,000 stained cells were analyzed on a FACS Caliber system for each determination.

hoechst 33342 staining
Apoptotic cells were identified on the basis of morphological changes in their nuclear assembly by observing chromatin condensation and fragment staining with Hoechst 33342, as previously described. 24 annexin V/Pi analysis NOZ and GBC-SD cells were treated with different concentrations (0, 25, 50, 100, 200, and 400 μM) of 20(S)-Rg3 for 48 hours. Apoptosis was analyzed using an Annexin V/PI apoptosis kit according to the manufacturer's instructions, as previously described. 25 Mitochondrial membrane potential (ΔΨm) assay The ΔΨm was analyzed by fluorescence microscopy using the Rhodamine 123 probe. 26 After treatment with different concentrations of 20(S)-Rg3 for 48 hours, the supernatant was removed and the cells were washed with PBS twice and then stained in Rhodamine 123 staining solution (5 μg/mL) at 37°C for 30 minutes. The samples were analyzed by using a flow cytometer (BD Biosciences).

In vivo efficacy of 20(S)-Rg3
Six-week-old BALB/c homozygous (nu/nu) nude mice (~18 g body weight) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, People's Republic of China). All animal treatments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Institutional Animal Care and Use Committee of Shanghai Jiaotong University. The mice were maintained in a specific pathogen-free environment. After 1 week, NOZ cells (2×10 6 ) in 100 μL PBS were injected into the right flank of nude mice. After another week, these mice were randomly divided into five groups. Three groups (seven mice/group) received an intraperitoneal (IP) injection of PBS, 20 mg/kg of 20(S)-Rg3, and 40 mg/kg of 20(S)-Rg3 every day, respectively, and were sacrificed to detect tumor weight after 3 weeks. The other two groups (10 mice/group) was administered an IP injection of PBS and 20(S)-Rg3 at 40 mg/kg every day, respectively, for survival analysis. Immunohistochemical analysis was performed as described previously. 27

Immunofluorescence staining
The expression of PCNA and cleaved caspase-3 was detected by immunofluorescence. Deparaffinized 4-μm tissue sections were cultured with PCNA or cleaved caspase-3 antibody. Subsequent antibody detection was carried out with Alexa Fluor 488 goat anti-mouse IgG secondary antibody. Sections were examined with a fluorescence microscope (Leica), and merged images were formed using Adobe Photoshop CS4.

statistical analysis
All data are expressed as mean values ± standard errors from at least three independent experiments. Statistical Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com

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Zhang et al significance was calculated using the Student's t-test, and a P-value less than 0.05 was considered significant in all tests. All analyses were performed using the SPSS software version 19.0 (SPSS Inc, Chicago, IL, USA).

20(s)-rg3 decreases viability and proliferation of gBc cells in a dose-dependent manner
To evaluate the toxic effect of 20(S)-Rg3 on GBC, five human GBC cell lines were treated with 20(S)-Rg3 at different concentrations (0, 0.1, 1, 10, 100, and 1,000 μM) for 24 and 48 hours. MTT assay showed that 20(S)-Rg3 exhibited a concentration-dependent and time-dependent killing of diverse GBC cell lines, with an IC 50 (half maximal inhibitory concentration) value of around 100 μM ( Figure S1A and B). Therefore, GBC cells were treated with 20(S)-Rg3 in the range of the drug concentrations (25-400 μM) in vitro. GBC-SD cell line was more sensitive to 20(S)-Rg3 than other cell lines, while NOZ cell line was less sensitive compared to other cell lines ( Figure 1A-D). Besides, the proliferative rate of GBC-SD cell line was much lower than other cell lines, while that of NOZ cell line was much higher ( Figure S1C). Therefore, NOZ and GBC-SD cell lines were chosen as optimal cell models for subsequent functional analyses.
The ability of GBC cells to form colonies in the presence of 20(S)-Rg3 was assessed by colony formation assay ( Figure 1E). 20(S)-Rg3 induced a dose-dependent decrease in both the number and the size of colonies formed in NOZ and GBC-SD cells ( Figure 1F and G). The results indicate that 20(S)-Rg3 may have a long-term inhibitory effect on the proliferation of GBC cells.

20(s)-rg3 accelerates senescence of gBc cells
Morphological changes of NOZ and GBC-SD cells could be seen in Figure 2A and B. Both cell lines became enlarged and flattened, had more cytoplasmic vacuoles in a concentration-dependent manner, representing a senescencelike state. SA-β-gal is of lysosomal origin and appears as the result of their increased biogenesis, 28 paralleled by increased expression of the GLB1 gene encoding acidic lysosomal β-galactosidase, 29 making it a widely used marker of senescent cells in vitro and in vivo. As shown in Figure 2C and D, the higher the concentration of 20(S)-Rg3, the more SA-β-gal positive cells in both cell lines was observed. Moreover, the protein amounts of p53 and p21 Cip1 increased in senescent cells but not the p16 and pRB protein ( Figure 2E). These results suggest that the growth arrest in GBC cells results from an increase in p53-mediated p21 expression as cells enter senescence.

20(s)-rg3 blocks cell cycle progression of gBc cells
Cellular senescence, which can be defined as a stress response preventing the propagation of cells that have accumulated potentially oncogenic alterations, is invariably associated with a permanent cell cycle arrest. To determine whether cell cycle arrest is responsible for 20(S)-Rg3-induced decrease in cell viability, flow cytometry was performed, which showed that the percentage of cells in G 0 /G 1 phase was significantly increased in both cell lines following 20(S)-Rg3 treatment, while cell populations in S phase and G 2 /M phase were simultaneously reduced ( Figure 3A and B). These results indicate that 20(S)-Rg3 could arrest the cell cycle at G 0 /G 1 phase in a dose-dependent manner. Western blot further confirmed that the levels of the S-related protein Cyclin A and the G 2 /M-related protein Cyclin B1 were down-regulated in NOZ and GBC-SD cells following 20(S)-Rg3 treatment ( Figure 3C).

20(s)-rg3 triggers mitochondrial-related apoptosis of gBc cells
We could also see from flow cytometric DNA-histogram that cells were accumulated in the sub-G 1 phase following 20(S)-Rg3 treatment. Hoechst 33342 staining was thus performed to examine the nuclear morphology. In control cells, the nuclei were stained weakly, and homogeneously blue, whereas, in cells treated with 20(S)-Rg3 some bright chromatin condensation and nuclear fragmentation were observed ( Figure 4A). The numbers of apoptotic nuclei containing condensed chromatin increased significantly as the 20(S)-Rg3 concentration increased ( Figure 4B). PI staining also showed a dose-dependent increase in necrosis in both cell lines after 20(S)-Rg3 treatment ( Figure S2). A marked dose-dependent increase in both the early and late stages of apoptosis was observed in NOZ and GBC-SC cells following 20(S)-Rg3 treatment by flow cytometry analysis with Annexin V-FITC [fluorescein isothiocyanate]/PI staining ( Figure 4C and D). These results from different apoptosis assays reveal significant features of apoptosis, which strongly suggest that 20(S)-Rg3-mediated inhibition of cell growth in GBC is closely correlated with the enhanced apoptosis.
To determine whether the cell apoptosis was dependent or independent of mitochondria, we tested whether   Apoptosis is a type of programmed cell death that is caspase-dependent. 30 When NOZ and GBC-SD cells were treated with 20(S)-Rg3 at different concentrations, significant proteolytic cleavage of caspase-3 was detected using Western blot. The levels of pro-and antiapoptotic mitochondrial proteins Bad, Bax, Bcl-2, and Bcl-XL were also visualized by Western blot. 20(S)-Rg3 increased the expression of Bad and Bax, and conversely decreased Bcl-2 and Bcl-XL expression in both cell lines ( Figure 4G). These results indicate that 20(S)-Rg3 could induce apoptosis in GBC cells through mitochondrial signaling pathways.   p53 is a key player in tumor suppression, as it regulates cellular senescence, cell cycle arrest, and apoptosis. Thus, understanding the mechanism of p53 regulation is very important for cancer therapy. 31 Real-time PCR analysis revealed that 20(S)-Rg3 had no influence on the transcription level of p53 ( Figure S3). We next investigated the involvement of the transcription-independent function of p53 using a protein synthesis inhibitor, (CHX), and found the suppressive effect of CHX on p53 protein accumulation was neutralized by 20(S)-Rg3 ( Figure 5A and B). Western blot further showed that MDM2 protein levels were decreased by 20(S)-Rg3 treatment, in a dose-and time-dependent manner ( Figure 5C and D). These results suggest that p53 protein levels were elevated as a result of MDM2 inhibition in GBC cells.

20(s)-rg3 inhibits tumor growth in xenografted nude mice model by causing apoptotic cell death
To further evaluate whether 20(S)-Rg3 had an effect on inhibition of tumor growth in vivo, we measured the tumor volume in a xenograft tumor model in which NOZ cells were injected IP into nude mice. As shown in Figure 6A, we found that the drug concentration in the serum of mice with 40 mg/kg 20(S)-Rg3 administered IP daily for 1 week reached a peak at about 200 μM by a HPLC assay, which just fell within the range of the drug concentrations (25-400 μM) in vitro. So, 20 and 40 mg/kg of 20(S)-Rg3 were selected for in vivo experiments. When transplant tumors reached a mean group size of approximately 100 mm 3 , mice were treated every day for 3 weeks with various dose of 20(S)-Rg3. 20(S)-Rg3 showed a significant inhibitory effect on tumor volume and weight ( Figure 6B-D). The survival of xenografted nude mice was prolonged after 40 mg/kg 20(S)-Rg3 treatment ( Figure 6E). Moreover, SA-β-gal staining of tumor sections indicated that 20(S)-Rg3 caused tumor cell senescence. 20(S)-Rg3 dose-dependently induced a significant increase in p53 and p21 Waf1 expression ( Figure 6F). In addition, tumor cell density and nuclear amount were both reduced. These results indicate that 20(S)-Rg3 inhibit tumor growth by causing cell senescence and apoptosis. Furthermore, PCNA expression was decreased, and caspase-3 activity was increased in 20(S)-Rg3 treatment groups ( Figure 6G and H).

Discussion
Ginsenoside Rg3, which is the active component of ginseng, has various medical effects, such as antitumorigenic, antiangiogenesic, and antifatiguing activities. 32   that the impaired cell viability was probably partially dependent on the inducement of cellular senescence, which is an irreversible growth arrest characterized by decreased cell proliferation, combined with accumulation of mitotic defects and chromosomal instability, 33 representing an important tumor suppression mechanism. Classically, the p16/RB and p53/p21 axes are two major senescence-associated pathways in response to various stressors. 34 Increased p53 and p21 Cip1 expression were found in senescent cells induced by 20(S)-Rg3. p21 is a cell cycle inhibitor and tumor suppressor downstream of p53. A previous study reported that p53 and p21 act together to inactivate the Cyclin-Cdk complex in damaged cells, which could mediate G 0 /G 1 and G 2 /M arrest. 35 In this study, induction of G 0 /G 1 arrest by 20(S)-Rg3 was accompanied by a large accumulation of p53 and p21. It is well known that p53 can lead to induction of the apoptotic cascade. 36 To determine whether apoptotic cell death is responsible for the toxic effect of 20(S)-Rg3 on GBC cells, flow cytometry analysis with Annexin V/PI staining was performed and showed that apoptotic cells were significantly elevated following 20(S)-Rg3 treatment. The nuclear morphology observed by Hoechst 33342 staining also confirmed that 20(S)-Rg3 could cause apoptosis of GBC cells.
The related ginseng-derived 20(S)-ginsenoside Rh2 has been shown to induce apoptosis in leukemia Reh cells through mitochondrial signaling pathways. 37 As expected, 20(S)-Rg3 induced mitochondrial-related apoptosis through activation of caspase-3, upregulation of Bad and Bax expression, and downregulation of Bcl-2 and Bcl-XL expression in NOZ and GBC-SC cells. MDM2, a product of a p53-inducible gene, binds directly with the transactivation domain of p53 and assists in ubiquitin-proteasomal degradation of p53, thereby acting as a negative regulator. 38 20(S)-Rg3 neutralized the ability of MDM2 to promote p53 degradation, leading to the stabilization and accumulation of p53. Previous studies have shown that 20(S)-Rg3 induces apoptosis in several human cancers. For instance, 20(S)-Rg3 promotes apoptosis in colon cancer cells through AMPK pathway. 19 20(S)-Rg3-induced apoptosis in ovarian cancer cells is associated with PI3K/Akt and XIAP pathways. 21 Our data provide a novel mechanism for 20(S)-Rg3-induced apoptosis in GBC cells that involves the activation of p53 pathway through a mitochondria-dependent apoptotic cascade. Therefore, a model depicting our findings is presented in Figure 7. 20(S)-Rg3 down-regulates MDM2 expression in GBC cells, leading to the accumulation of p53. On one hand, p53-mediated p21 expression accelerates cellular senescence accompanied by a cell cycle arrest. On the other hand, the activation of p53 leads to induction of the mitochondria-dependent apoptotic cascade.
Furthermore, 20(S)-Rg3 potently inhibit tumor growth by inducing cell senescence and apoptosis in vivo. In addition, there was no significant difference in body weight

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Zhang et al ( Figure 6A), blood routine, and hepatorenal function between the mice IP injected with 20(S)-Rg3 daily for 3 weeks and the mice treated with PBS (data not shown), suggesting 20(S)-Rg3 was not toxic for administration to mice in vivo and had potential clinical applications.
In conclusion, we suggest 20(S)-Rg3 as a potent growth inhibitor of GBC in vitro and in vivo. The mechanisms of 20(S)-Rg3 attenuating GBC growth may be via activation of the p53 pathway and subsequent induction of cellular senescence, which is a stress response that accompanies stable exit from the cell cycle, as well as activation of the mitochondrialmediated intrinsic caspase pathway. Therefore, 20(S)-Rg3 has potential as a novel chemotherapeutic agent for GBC therapy.

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