Inhibition of Proliferation of SGC7901 and BGC823 Human Gastric Cancer Cells by Ursolic Acid Occurs Through a Caspase-Dependent Apoptotic Pathway.

<strong>BACKGROUND</strong> Worldwide, gastric cancer is one of the most common malignant tumors. Ursolic acid is a plant metabolite and pentacyclic triterpenoid used in traditional Chinese medicine. This study aimed to investigate the effects of ursolic acid the growth and apoptosis of SGC7901 and BGC823 human gastric cancer cells <i>in vitro</i>. <strong>MATERIAL AND METHODS</strong> SGC7901 and BGC823 human gastric cancer cells and normal GES-1 gastric epithelial cells were cultured with increasing doses of ursolic acid at 50, 60, and 100 µM. Cell viability and proliferation were assessed using an MTT assay. Flow cytometry was used to assess cell apoptosis. Western blot was used to measure procaspase-8, procaspase-9, procaspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) expression. The expression of receptor interaction protein 3 (RIP3) was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Morphological changes in the gastric cancer cells were determined using Hoechst 33342 staining following ursolic acid treatment. <strong>RESULTS</strong> Ursolic acid inhibited the viability of SGC7901 and BGC823 cells but not GES-1 cells. Ursolic acid treatment significantly induced apoptosis in SGC7901 and BGC823 cells when compared with GES-1 cells (P<0.05), and significantly increased the activation of caspase-3, caspase-8, caspase-9, poly ADPribose polymerase (PARP), and the production of reactive oxygen species (ROS). Treatment of SGC7901 and BGC823 cells with ursolic acid for 72 h did not induce necroptosis. <strong>CONCLUSIONS</strong> Ursolic acid inhibited the proliferation of SGC7901 and BGC823 human gastric cancer cells <i>in vitro</i> through a caspase-dependent apoptotic pathway.

Background

Worldwide, gastric cancer is one of the most commonly diagnosed malignant tumors of the gastrointestinal tract [1]. Gastric cancer ranks third for mortality and fourth for morbidity from cancer [1,2]. Although the incidence of gastric cancer has recently shown a slight reduction, effective treatment strategies for gastric cancer are still required [3]. Current treatments for gastric cancer are limited by its complex etiology [3]. The primary treatment for gastric cancer involves surgical resection, but the 5-year survival rate for patients remains low, and recurrence following surgery is common [4]. Patients with advanced gastric cancer are also treated with systemic chemotherapy [5]. However, the efficacy of chemotherapeutic agents is limited by the drug resistance [6,7]. Therefore, the development of novel chemotherapeutic agents is still required.

Apoptosis is a complex cellular process that results in the elimination of cells and is regulated by several genes [8]. Studies have shown that the signaling pathways associated with the induction of apoptosis are linked with the activation of protease enzymes, known as caspases [9]. In the initial stages of apoptosis, caspases activate a well-organized cell signaling program that results in cell death without direct cell killing [10]. Reactive oxygen species (ROS) are metabolites produced in the cells, which have a crucial role in the development of cancer [11]. Disruption of the equilibrium between ROS production and anti-oxidative defense system in the cells leads to oxidative stress. Production of ROS in higher concentration acts as signaling for the induction of apoptosis and catalyzes DNA damage [12]. Therefore, upregulation of ROS by chemotherapeutic agents is a significant part of cancer treatment.

Necroptosis is a process of programmed cell death that is characterized by the development of cell necrosis, including cell swelling, lysis of the plasma membrane, and chromatin condensation. Cell death by necroptosis requires the activation of serine/threonine kinase by receptor interaction protein 3 (RIP3) [13,14]. The expression of RIP3 in cancer cells is important in the progression of cancer [13,14].

Ursolic acid is a plant metabolite and pentacyclic triterpenoid that has been used in traditional Chinese medicine. Ursolic acid has been shown to inhibit hematopoietic tumor growth [15,16], to suppress tumor metastasis [17,18], and inhibit tumor progression and angiogenesis [19]. Ursolic acid has been shown to exhibit its effect through inhibition of NF-kappaB [20]. Therefore, this study aimed to investigate the effects of ursolic acid the growth and apoptosis of SGC7901 and BGC823 human gastric cancer cells in vitro.

Material and Methods

Reagents

Ursolic acid was purchased from the Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) and other chemicals were supplied by Merck Millipore (Burlington, MA, USA).

Cell culture

The gastric cancer cell lines, SGC7901 and BGC823, and normal gastric epithelial cell line, GES-1, were supplied by the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cell culture was performed in RPMI-1640 medium (Gibco, Thermofisher Scientific, Waltham, MA, USA) which contained 10% fetal calf serum (FCS) (Gibco, Thermofisher Scientific, Waltham, MA, USA). The medium was mixed with penicillin (100 unit/ml) and streptomycin (100 μg/ml). Cells were cultured in an incubator at 37°C in a humidified atmosphere containing 5% CO2. Cells were also pre-treated with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (Z-VAD-FMK).

MTT assay

The MTT assay determined the changes in viability of SGC7901 and BGC82 cells following treatment with ursolic acid for 72 h. Briefly, the cells placed into the 96-well plates at 2×105 cells/well density and were cultured for 24 h. The cells were then treated with 10, 20, 30, 40, 50, 60, and 100 μM concentrations of ursolic acid for 72 h. After incubation, MTT solution (20 μl) was added to each well and incubation was continued for a further 4.5 h. The medium was then discarded and 150 μl of DMSO was placed into the wells for dissolving any insoluble material formed. A microplate reader performed the measurement of absorbance at a wavelength of 485 nm.

Analysis of cell morphological changes

SGC7901 and BGC82 cells were incubated in 12well plates at a density of 2×106 cells/well for 72 h with 40, 50, 60, and 100 μM concentrations of ursolic acid. The cells were then washed twice with PBS and subsequently fixed at room temperature with 4% paraformaldehyde for 15 min. Again, cells were washed with PBS and then underwent Hoechst 33342 staining at 37°C for 20 min. A fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) was used to examine the changes in the morphology of the cells following treatment with ursolic acid.

Detection of cell apoptosis by Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining

SGC7901 and BGC82 cells were treated with 72 h to 40, 50, 60, and 100 μM concentrations of ursolic acid. The cells were harvested and subsequently washed with PBS. The cells at 1×106 cells/ml concentration in binding buffer underwent to Annexin-V FITC and PI staining in the dark for 25 min at room temperature. The flow cytometry (Beckman Coulter, Inc., Brea, CA, USA) was used for the examination of apoptosis in the cultured cells.

Determination of release of lactate dehydrogenase (LDH)

SGC7901 and BGC82 cells were distributed in 96-well plates at 1.5×105 cells/well density. The cells were treated with 40, 50, 60, and 100 μM concentrations of ursolic acid for 72 h. A commercial LDH kit (Beyotime, Shanghai, China) was used to measure LDH release, according to the manufacturer’s instructions.

Reactive oxygen species (ROS) production assay

Treatmnent of SGC7901 and BGC823 cells with 40, 50, 60, and 100 μM concentrations of ursolic acid or N-acetylcysteine (NAC) for 72 h was followed washing in PBS. The cells were then resuspended in the medium containing 10 μM solution of dichloro-dihydro-fluorescein diacetate (DCFH-DA) as an assay for oxidative stress. The cells incubated with 50 μg/ml solution of ROSup (Solarbio Science & Technology Co., Ltd., Beijing, China) were used as a positive control. The ROS production in the cells was detected using flow cytometry.

Western blot

SGC7901 and BGC823 cells treated with 40, 50, 60, and 100 μM concentrations of ursolic acid were subjected to lysis on treatment with RIPA buffer. The cell lysis was performed on ice for 10 min, followed by centrifugation at 18,000×g at 4°C for 20 min. The quantity of proteins in the lysates was analyzed by commercially available bicinchoninic acid protein kit (Thermofisher Scientific, Waltham, MA, USA). The protein samples were separated on 8–12% of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and then transferred to the polyvinylidene fluoride (PVDF) membranes. The membrane non-specific sites were blocked on treatment with 5% dried skimmed milk powder. Incubation of the membranes was carried out overnight with primary antibodies at 4°C. After incubation, membranes were washed and then treated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at room temperature. The band visualization was performed by the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). The primary antibodies were used to caspase-3 (dilution 1: 1000) (catalog no. AC030), caspase-8 (dilution 1: 1000) (catalog no. AC056), caspase-9 (dilution 1: 1000) (catalog no. AC062), poly (ADP-ribose) polymerase (PARP) (dilution 1: 1000) (catalog no. AP102), LC3 (dilution 1: 1000) (catalog no. NB100-2220) and RIP3 (dilution 1: 1000) (catalog no. GTX107574).

Reverse transcription-polymerase chain reaction (RT-PCR) assay

Total RNA from SGC7901 and BGC823 cells treated with 40, 50, 60, and 100 μM concentrations of ursolic acid was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then 1 μg of total RNA was used for the synthesis of cDNA for 20 min at 37°C using Primescript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). A LightCycler®96 real-time PCR system linked to SYBR Premix EX Taq II kit (Takara, Biotechnology Co., Ltd.) was used to perform the RT-PCR assay. The reaction was performed using a 20 μl volume consisting of 10 μl of SYBR Premix EX Taq II, 0.8 μl of the forward primer, 0.8 μl of the reverse primer, 2 μl of cDNA and 6.4 μl of the sterilized H2O. The conditions used for amplification consisted of initial pre-degeneration for 3 min at 94°C, which was followed by 39 cycles of denaturation for 15 sec at 94°C and annealing for 25 sec at 58°C. The expression of GAPDH protein was used as an internal control.

Statistical analysis

The data were presented as the mean ± standard deviation (SD) of experiments independently performed in triplicate. Data were analyzed using SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). Determination of the significance of differences was carried out using one-way analysis of variance (ANOVA). A P-value <0.05 was considered to be statistically significant.

Results

Cell viability of SGC7901 and BGC823 human gastric cancer cells was inhibited by ursolic acid

The MTT assay was used to determine the effect of ursolic acid on the viability of GES-1 normal gastric epithelial cells and SGC7901 and BGC823 human gastric cancer cells (Figure 1A). No change in the viability of GES-1 cells was observed following treatment with 10, 20, 30, 40, 50, 60, and 100 μM concentrations of ursolic acid for 72 h. Ursolic acid treatment of SGC7901 and BGC823 cells resulted in a significant decrease in cell viability in a dose-dependent manner. The viability of SGC7901 cells was reduced to 93%, 86%, 69%, 57%, 38%, 22%, and 17%, respectively on treatment with 10, 20, 30, 40, 50, 60, and 100 μM concentrations of ursolic acid for 72 h.

Figure 1

Effect of ursolic acid on the viability of SGC7901 and BGC823 human gastric cancer cells. (A) SGC7901 and BGC823 human gastric cancer cells and GES-1 normal gastric epithelial cells were treated with 10, 20, 30, 40, 50, 60, and 100 μM of ursolic acid. Changes in cell viability were examined by MTT assay after 72 h. (B) Ursolic acid treated cells were examined under microscopy. Magnification ×250. * P<0.05, ** P<0.002 and *** P<0.001 vs. untreated cells.

Following treatment with 10, 20, 30, 40, 50, 60, and 100 μM concentrations of ursolic acid, the viability of BGC823 cells was decreased to 91%, 82%, 65%, 54%, 31%, 19%, and 15%, respectively. The effect of ursolic acid on the morphology of SGC7901 and BGC823 cells was also examined by light microscopy (Figure 1B). Treatment with 50, 60, and 100 μM of ursolic acid markedly changed the morphology of SGC7901 and BGC823 cells. Microscopic examination showed that ursolic acid caused rounding of gastric cancer cells and decreased the number of cells.

Ursolic acid treatment of SGC7901 and BGC823 human gastric cancer cells induced cell apoptosis

Apoptosis was induced by ursolic acid in SGC7901 and BGC823 cells and was analyzed using Hoechst 33342 staining (Figure 2). The control cells showed very weak blue fluorescence, and the nuclei were normal in structure. The cells treated with ursolic acid showed condensation of chromatin material, presence of apoptotic bodies, and intense blue fluorescence. The ursolic acid induced apoptosis in SGC7901 and BGC823 cells were also examined by Annexin-V/PI staining assay following 72 h of treatment with 50, 60, and 100 μM concentrations (Figure 3). A significant increase in the population of apoptotic cells was observed in SGC7901 and BGC823 cell cultures on treatment with ursolic acid in comparison to the control cells.

Figure 2

Ursolic acid treatment induced apoptosis of SGC7901 and BGC823 human gastric cancer cells. SGC7901 and BGC823 cells after treatment with 50, 60, and 100 μM of ursolic acid were stained with Hoechst 33342. The images of the stained cells were captured by fluorescence microscopy. Magnification ×250.

Figure 3

Ursolic acid treatment induced apoptosis of SGC7901 and BGC823 human gastric cancer cells. SGC7901 and BGC823 cells after treatment with 50, 60, and 100 μM of ursolic acid were stained with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining. Stained cells were examined by flow cytometry.

Ursolic acid increased the release of lactate dehydrogenase (LDH) from SGC7901 and BGC823 human gastric cancer cells

Treatment of SGC7901 and BGC823 cells with ursolic acid caused a marked increase in the release of LDH in comparison to the untreated cells (Figure 4A). In both the gastric cancer cell lines, treatment with ursolic acid caused an increase in LDH release in a dose-dependent manner. To investigate whether ursolic acid inhibited SGC7901 and BGC823 cell viability through a caspase-dependent pathway, the cells were pre-treated with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) (Figure 4.B). The MTT assay showed that treatment of SGC7901 and BGC823 cells with Z-VAD-FMK prevented ursolic acid induced suppression of cell viability.

Figure 4

The effects of ursolic acid on the release of lactate dehydrogenase (LDH) from SGC7901 and BGC823 human gastric cancer cells. (A) The SGC7901 and BGC823 human gastric cancer cells were treated with ursolic acid for 72 hours, and LDH levels were measured by Western blot. (B) The SGC7901 and BGC823 human gastric cancer cells following 3 h of pre-treatment with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (Z-VAD-FMK) followed by treatment with 40, 50, 60 and 100 μM concentrations of ursolic acid. The cell viability was determined by MTT assay. * P<0.05 and ** P<0.002 vs. untreated cells.

Ursolic acid upregulated the expression of apoptotic proteins in SGC7901 and BGC823 human gastric cancer cells

The expression of caspase-8, caspase-9, and caspase-3 following 72 h of ursolic acid treatment was analyzed in SGC7901 and BGC823 cells by Western blotting (Figure 5). Treatment with 40, 50, 60, and 100 μM concentrations of ursolic acid resulted in a dose-dependent increase in caspase-8, caspase-9, and caspase-3 levels. Treatment with ursolic acid increased the expression of cleaved poly (ADP-ribose) polymerase (PARP) in both SGC7901 and BGC823 cells.

Figure 5

The effects of ursolic acid on expression of apoptosis-associated proteins in SGC7901 and BGC823 human gastric cancer cells. The cells after treatment with 40, 50, 60, and 100 μM of ursolic acid underwent Western blot for determination of procaspase-8, procaspase-9, procaspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) expression.

Ursolic acid increased the production of reactive oxygen species (ROS) in SGC7901 and BGC823 human gastric cancer cells

The effect of ursolic acid on ROS production was analyzed in SGC7901 and BGC823 cells by DCFHDA probe (Figure 6). A significant increase in the ROS production was caused by ursolic acid in SGC7901 and BGC823 cells at 72 h. The ROS production increased significantly with the increase in the concentration of ursolic acid, from 40 μM to 100 μM.

Figure 6

The effects of ursolic acid on the production of reactive oxygen species (ROS) production by SGC7901 and BGC823 human gastric cancer cells. The SGC7901 and BGC823 cells after 72 h of treatment with 50, 60, and 100 μM of ursolic acid were assessed for ROS production.

Ursolic acid did not activate necroptosis in SGC7901 and BGC823 human gastric cancer cells

To investigate whether ursolic acid treatment activated necroptosis in SGC7901 and BGC823 cells, the cells were treated for 3 hours with necrostatin-1 or the necroptosis inhibitor, NSA, were treated with 40, 50, 60, and 100 μM concentrations of ursolic acid (Figure 7A). The MTT assay showed that necrostatin-1 prevented the ursolic acid-mediated suppression of SGC7901 and BGC823 cell viability. The effect of ursolic acid on level of RIP3 in SGC7901 and BGC823 cells was analyzed by Western blot (Figure 7B). The ursolic acid did not induce the expression of RIP3 in SGC7901 and BGC823 cells.

Figure 7

The effects of ursolic acid on necroptosis of SGC7901 and BGC823 human gastric cancer cells. (A) The cells following pre-treatment with 30 μM of necrostatin-1 were then treated with 40, 50, 60, and 100 μM concentrations of ursolic acid. The MTT assay analyzed changes in cell viability. (B) The expression of receptor interaction protein 3 (RIP3) was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR) assays. * P<0.05 and ** P<0.002 vs. untreated cells.

Discussion

The findings from this study demonstrated the effect of ursolic acid on the growth of gastric cancer cells in vitro. The study showed that ursolic acid suppressed the viability of SGC7901 and BGC823 cells in a dose-dependent manner. There was no significant change in the GES-1 normal gastric cell viability following treatment with ursolic acid. These findings showed that ursolic acid specifically suppressed the gastric cancer cell viability.

The growth inhibitory effect of many natural products on cancer cells involves the induction of apoptosis, which is a type of programmed cell death. In the present study, ursolic acid also suppressed gastric cancer viability through induction of apoptosis, as shown by Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining. The effects of ursolic acid-induced apoptosis in SGC7901 and BGC823 cells was also confirmed by Hoechst 33342 staining. These findings supported that ursolic acid suppressed gastric cancer cell viability by inducing apoptosis.

The process of cancer cell apoptosis is mediated by the activation of caspases [21]. Depending on the stage of entry into the process of apoptosis, caspases are classified as apoptosis initiators and executors. Caspase-2, caspase-8, caspase-9, and caspase-10, which are expressed at the initial stage of apoptosis, lead to the activation of initiator caspases, caspase-3, caspase-6, or caspase-7 [22]. In the present study, treatment of SGC7901 and BGC823 human gastric cancer cells with ursolic acid resulted in a dose-dependent reduction in the expression of procaspase-8, procaspase-9, and procaspase-3. However, the expression of cleaved poly (ADP-ribose) polymerase (PARP), which is an indicator of caspase activation, was significantly increased in the ursolic acid-treated SGC7901 and BGC823 cells. To confirm whether ursolic acid caused the induction of apoptosis in gastric cancer cells through the activation of caspases, the cells were pre-treated with the caspase inhibitor, Z-VAD-FMK. The results showed that pre-treatment of SGC7901 and BGC823 cells with Z-VAD-FMK prevented ursolic acid-induced suppression of cell viability. These findings showed that ursolic acid suppressed gastric cancer cell viability in vitro through activation of caspases.

Production of reactive oxygen species (ROS) in the cells acts as the messenger for inducing apoptosis through signaling cascades [23]. Production of ROS in high concentrations leads to DNA damage and death of cells, either by apoptosis or necrosis [24]. In leukemia cells, increased production of ROS has been shown to cause apoptosis of the cells through a ROS-dependent pathway [25]. The results from the present study showed that ursolic acid treatment of SGC7901 and BGC823 cells significantly increased the production of ROS. Inhibition of ROS production by pre-treatment of gastric cancer cells with N-acetylcysteine (NAC) prevented ursolic acid-induced suppression of cell viability. Therefore, the production of ROS by ursolic acid may have a role in the suppression of SGC7901 and BGC823 cell viability.

In addition to apoptosis, another process that occurs in programmed cell death is necroptosis, which is activated by several factors, including tumor necrosis factor-α (TNF-α). The main regulator of necroptosis that is mediated by TNF-α in cancer cells is receptor interaction protein 3 (RIP3) [26]. In the present study, treatment of SGC7901 and BGC823 cells with ursolic acid did not induce the expression of RIP3. Also, pre-treatment of SGC7901 and BGC823 cells with necrostatin-1 did not affect cell viability. These results showed that ursolic acid did not activate necroptosis in SGC7901 and BGC823 cells. Therefore, ursolic acid inhibited gastric cancer cell viability by inducing apoptosis.

Conclusions

The viability of SGC7901 and BGC823 human gastric cancer cells was significantly suppressed by treatment with ursolic acid in vitro, without inhibiting normal epithelial gastric cells. Ursolic acid induced apoptosis through activation of caspase-8, caspase-9, and caspase-3 and by cleavage of poly (ADP-ribose) polymerase (PARP). Apoptosis in gastric cancer cells was also facilitated by increased production of reactive oxygen species (ROS). Further in vitro and in vivo studies are required to investigate the role of ursolic acid in gastric cancer further.