Solasonine induces apoptosis of the SGC‐7901 human gastric cancer cell line in vitro via the mitochondria‐mediated pathway

Abstract Solasonine, a steroidal glycoalkaloid isolated from the herbal plant Solanum nigrum Linn., has shown active against multiple human cancers; however, there is little knowledge on the activity of solasonine against gastric cancer until now. This study aimed to examine the effect of solasonine on the biological behaviours of human gastric cancer SGC‐7901 cells. The results showed that solasonine suppressed SGC‐7901 cell proliferation in a dose‐dependent manner. Solasonine treatment mainly induced the cell cycle arrest at G2 phase in SGC‐7901 cells. Treatment with solasonine resulted in significant down‐regulation of Bcl‐2 and Caspase‐3 protein expression and reduced Bax and Bcl‐xL protein expression in SGC‐7901 cells. Solasonine shows a comparable inhibitory effect on the proliferation of human gastric cancer SGC‐7901 cells with cisplatin, and solasonine induces of SGC‐7901 cell apoptosis through triggering the endoplasmic reticulum stress pathway and the mitochondrial pathway. Our data indicate that solasonine may be a promising agent for the treatment of gastric cancer.

as a supplement to currently available treatments, is therefore of great significance to improve the prognosis of gastric cancer patients. 7 Solanum nigrum Linn. is a herbal plant that frequently grows in temperate climate regions. 8 As a traditional Chinese medicine, S. nigrum has shown antitumour, anti-infective, antiphlogistic, febrifuge, sedative, analgesic, lipid-lowering, hepato-protective, antitussive and expectorant, antiviral, antimicrobial and anti-shock actions. 9,10 Solasonine, a steroidal glycoalkaloid isolated from S. nigrum, 11 has shown active against hepatocellular carcinoma (HCC), glioma, lung cancer and colorectal cancer. [12][13][14][15][16] However, there is little knowledge on the activity of solasonine against gastric cancer until now. 17,18 This study was therefore designed with aims to examine the effect of solasonine on the biological behaviours of human gastric cancer SGC-7901 cells.

| Extraction of solasonine
Solanum nigrum was ground into powder measuring approximately 0.1 mm 3 . Then, 50 g powder was immersed in 250 ml ethanol for 6 hours, followed by ultrasonic-assisted extraction for 30 min. The leaching was repeated in triplicate until the leaching solution was colourless. The triplicate leaching solutions were merged together, and dried under reduced pressure distillation at 40°C to yield the ethanol extracts of S. nigrum. Solanum nigrum powder (50 g) was immersed in 250 ml purified water for 2 hours, followed by boiling for 3 h. The boiling solution was filtered, and the filter residue was boiled twice in 250 ml purified water. All boiling solutions were merged together, and dried under reduced pressure distillation and vacuum freezing to yield the water extracts of S. nigrum.
The ethanol and water extracts of S. nigrum were dissolved in 30% methanol to prepare into the test sample at 50 mg/ml. The concentration of solasonine was determined using high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS).

| Qualitative and quantitative detection of solasonine
Solasonine standard sample was dissolved in chromatographic pure methanol to prepare into stock solutions at a concentration of 10 mg/ml, and diluted into solutions at 10, 20, 30,   Logan, UT, USA) for 1 min digestion to allow cells to separate from the wall. Subsequently, the petri dish was added with 3 ml 37°C pre-warmed RPMI 1640 medium, and cells were passaged at a ratio of 1:2. After two passages, cells were harvested for the subsequent experiments.
Briefly, SGC-7901 cells were seeded onto 96-well plates (Corning, Inc.; Corning, NJ, USA) at a density of 1.5 × 10 5 cells/ml, and each well was added with 100 μL cells, and incubated at 37°C containing 5% CO 2 for 24 hours. Then, the medium was removed, and fresh RPMI 1640 medium containing solasonine or cisplatin at final concentrations of 1, 2.5, 5, 10, 20, 30 and 50 μg/ml, with 5 replicate wells assigned for each drug concentration, while fresh medium served as a negative control. After cells were incubated at 37°C containing 5% CO 2 for 24 h, each well was transferred with 10 μl

| Analysis of cell cycle of SGC-7801 cells
The cells cycle of SGC-7901 cells was analysed using flow cytometry. Briefly, SGC-7901 cells were seeded onto 6-well plates (Corning, Inc.; Corning, NJ, USA) at a density of 8 × 10 5 cells/well, and each well was added with RPMI 1640 medium to 2 ml. Cells were incubated at 37°C containing 5% CO 2 for 24 h, and then, the medium was discarded and 2 ml fresh RPMI 1640 medium containing solasonine, cisplatin or solasonine-cisplatin combinations were added, while fresh RPMI 1640 medium served as a control. Three replicate wells assigned for each treatment. After cells were incubated at 37°C containing 5% CO 2 for 24 h, the medium was removed and the plate wall-adherent cells were washed once with PBS and digested with 1 ml 0.25% pancreatin for 1 min.
Cells were gently separated with a pipette, transferred to 15 ml centrifugation tubes and centrifuged at 800g for 5 min at room temperature. The supernatant was removed, and cells were resuspended in 2 ml 4°C pre-cooled PBS and centrifuged at 800g for 5 min at room temperature. The supernatant was discarded, and cells were re-suspended in 1 ml ice-water bath pre-cooled PBS, slowly added with -20°C pre-cooled absolute ethanol to yield a final concentration of 70% and incubated in water bath at 4°C for 12 hours. Subsequently, cells were centrifuged at 800g for 5 min at 4°C, and the supernatant was removed. Cells were washed in 1 ml 4°C pre-cooled PBS, added with propidium (PI) staining so-

| Detection of SGC-7901 cell apoptosis
The apoptosis of SGC-7901 cells was detected using flow cytometry. Briefly, SGC-7901 cells were seeded onto 6-well plates at a density of 8 × 10 5 cells/well, and each well was added with RPMI 1640 medium to 2 ml. Cells were incubated at 37°C containing 5% CO 2 for 24 h, and then, the medium was discarded and 2 ml fresh RPMI 1640 medium containing solasonine, cisplatin or solasoninecisplatin combinations were added, while fresh RPMI 1640 medium served as a control. Three replicate wells assigned for each treatment. After cells were incubated at 37°C containing 5% CO 2 for 24 h, the medium was removed, and the plate wall-adherent cells were washed once with PBS and digested with 1 ml 0.25% pancreatin for 1 min. Cells were gently separated with a pipette, trans-

| Western blotting assay
The expression of apoptotic regulatory proteins was quantified using a Western blotting assay. Briefly, SGC-7901 cells were seeded onto 6-well plates at a density of 8 × 10 5 cells/well, and each well was added with RPMI 1640 medium to 2 ml. Cells were incubated at 37°C containing 5% CO 2 for 24 h, and then, the medium was dis-

| Statistical analysis
All data were the representatives of at least three independent experiments, and described as mean ± standard deviation (SD).
Differences of means between groups were tested for statistical significance with Student's t test, and a P value of < 0.05 was considered statistically significant.

| Effect of solasonine on the cell cycle of SGC-7901 cells
Flow cytometry revealed that the SGC-7901 cells exposed to fresh RPMI 1640 medium were evenly distributed at G1/G1, S and G2/M phases ( Figure 3A), and cisplatin-treated SGC-7901 cells were predominantly arrested at G0/G1 phase, with a few cells at S and G2/M phases ( Figure 3B), which in in agreement with cisplatin-induced DNA replication. 19 Following exposure to solasonine for 24 h, SGC-7901 cells were predominantly arrested at G2 phase ( Figure 3C). These data indicate that solasonine does not suppress the SGC-7901 cell proliferation through inhibiting DNA replication, but through suppressing the diploid cell division, which is remarkably different from the mode of action of cisplatin. In addition, the combined treatment with solasonine and cisplatin resulted in cell cycle arrest at G0/G1 phase in most SGC-7901 cells, and a few cells were detected at G2 phase ( Figure 3D).
These findings demonstrate that cisplatin-induced inhibition of DNA replication may exhibit a predominant role, and solasonine may promote the cisplatin activity. Our data suggest that the solasonine-cisplatin combination may achieve a higher therapeutic efficacy against gastric cancer.

| DISCUSS ION
As a traditional Chinese medicinal herb, S. nigrum is widespread in China 8 and has shown a wide range of pharmacological actions. 9,10 In this study, a HPLC-MS/MS-based assay was established for the qualitative and quantitative assessment of solasonine, and the solosonine concentration was approximate 0.68 mg/g in the traditional Chinese medicinal herb S. nigrum. In this study, quantitative and qualitative analyses showed that solasonine content was not high in the medicinal herb Solanum nigrum, which may be associated with Currently, there are three main apoptosis-associated signalling pathways, including mitochondrial apoptotic pathway, cell death receptor pathway and endoplasmic reticulum stress pathway. [27][28][29] Previous studies have demonstrated that solasonine induces the reactive oxygen species (ROS)-mediated apoptosis through triggering the endoplasmic reticulum stress apoptotic pathway. 12 In the current study, solasonine treatment significantly suppressed Bcl-2 protein expression in SGC-7901 cells, which was consistent with previous findings in HCC HepG2 cells and prostate cancer PC-3 cells. 30,31 Bcl-2 family proteins, a critical factor that determines the opening and closure of the mitochondrial permeability transition pore (mPTM), are a key protein that triggers the mitochondrial apoptotic pathway. 32 Down-regulation of Bcl-2 protein expression causes mitochondrial damages and a reduction in mitochondrial Ca 2+ concentrations, and this induces a rise in intracellular Ca 2+ concentrations and a reduction in mitochondrial membrane potential, which activates Caspase-3 and reduces the Bcl-2/Bax ratio, thereby inducing the cell cycle arrest at M phase. [33][34][35] Solasonine was found to reduce the level of both Bax and Bcl-2 in gastric cancer cells simultaneously, and decreased the Bax/Bcl-2 ratio (about 2.1 folds). It is therefore considered that solasonine induce gastric cancer cell apoptosis through the mitochondria-mediated pathway.
Previous studies have shown that solasonine induces apoptosis of liver cancer, breast cancer and prostate cancer cells via the mitochondrial signalling pathway; however, there is little knowledge on the effects of solasonine on gastric cancer, and there is no knowledge pertaining to the in vivo activity of solasonine against gastric cancer until now. Since solasonine is major active ingredient of the medicinal herb Solanum nigrum, most studies pertaining to the pharmaceutical actions of solasonine have been performed by Chinese scientists. It has been shown that solasonine may increase the erythrocyte membrane fluidity to restore the immune functions of erythrocytes in tumour-bearing mice, thereby resulting in antitumour activity. 36 Zhang 18 suggested that solasonine against the gastric cancer via modulation miR-486-5p/PI3KR1 axis.
In summary, the results of the present study demonstrate that solasonine shows a comparable inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells with cisplatin, and solasonine induces of SGC-7901 cell apoptosis through triggering the endoplasmic reticulum stress pathway and the mitochondrial pathway. In addition, the cisplatin-solasonine combination may improve the efficacy against gastric cancer. Our data indicate that solasonine may be a promising agent for the treatment of gastric cancer.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are available on request from the authors.