AGS, SNU1 and Hs746T cell lines were purchased from Corbioer Company (Nanjing, China). Shanghai Winherb Medical S&T Development Co. Ltd. (Shanghai, China) provided purified CuD. All the primary antibodies including those for iNOS (diluted at 1:1,000; cat. no. 13120), Bax (diluted at 1:1,000; cat. no. 2722), B-cell lymphoma 2 (Bcl-2; cat. no. 2870), C-caspase-9 (diluted at 1:1,000; cat. no. 9509P), cytochrome c (diluted at 1:1,000; cat. no. 4272), phosphorylated and total Akt (diluted at 1:1,000; cat. no. 4060/4691), mechanistic target of rapamycin (mTOR, cat. no. 2971/2983), sr6 (diluted at 1:1,000; cat. no. 9204/2708) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; diluted at 1:1,000; cat. no. 5174) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The Akt activator SC79 was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany.) The iNOS inhibitor L-canavanine was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Cell proliferation and viability were assessed by Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology, Haimen, China). Cells were seeded into a 96-well plate. After the cells were treated with CuD, CCK-8 solution (10 µl) was added to each well. After 4 h of incubation, an enzyme-linked immunosorbent assay (ELISA) (Synergy HT; BioTek Instruments, Inc., Winooski, VT, USA) was used to the determine absorbance at 450 nm.

Apoptosis was detected by a terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labelling (TUNEL) assay (ApopTag Plus Fluorescein In Situ Apoptosis Detection kit; Millipore, Darmstadt, Germany). After washing with phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA), the cells were fixed with 1% paraformaldehyde in PBS. TUNEL reagents (EMD Millipore, Billerica, MA, USA) were used to stain the apoptotic cells and 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen; Life Technologies Carlsbad, CA, USA) was used to stain the DNA. A microscope (Olympus BX51TRF; Olympus Corp., Tokyo, Japan) was used to analyse and count positive cells.

Reactive oxygen species (ROS) were detected with a ROS Assay kit (Cell Biolabs, Inc., San Diego, CA, USA). A cell-permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) was used to label ROS. A fluorometric plate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to quantify fluorescence at 480/530 nm.

The levels of ATP were detected by an ATP assay kit (S0026; Beyotime Institute of Biotechnology). After the cells were treated with CuD, cells lysates were collected and incubated with ATP detection solution (100 µl). A fluorometric plate reader was used to determine the levels of ATP. Standard curve method was used to calculate the ATP concentration in each group.

The level of intracellular Ca²⁺ was assessed using Fluo-3/AM (S1056; Beyotime Institute of Biotechnology). After treatment, D-Hanks balanced salt solution (D-HBBS; Jinuo Co., Ltd., Shanghai, China) (without Ca²⁺, Mg²⁺ and phenol red) was used to wash cells. Then, cells were incubated with Fluo-3/AM (5 µM) for 60 min at 37°C. After being washed in D-HBBS for 30 min, a microplate reader was used to determine the fluorescent absorbance value, with representative intracellular Ca²⁺ levels.

NO production was detected using Griess method kit (S0021; Beyotime Institute of Biotechnology) according to the instructions of the NO assay kit.

Cell lysates were collected using RIPA lysis buffer. Total protein (50 µg) was used for SDS-PAGE. After being transferred into immobilon-FL transfer membranes (IPFL00010; Millipore, Billerica, MA, USA), proteins were incubated with the primary antibody overnight at 4°C, followed by incubation with secondary antibodies for 1 h. A two-coloured infrared imaging system (Odyssey; LI-COR Biosciences, Lincoln, NE, USA) was used to scan. GAPDH protein was used as the reference protein.

All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 8023, revised 1978). All animal experiments were approved by the Committee on the Use of Live Animals of The First Affiliated Hospital of Zhengzhou University. Male BALB/c-nu/nu nude mice (6–8 weeks) were purchased from the Beijing HFK Bioscience and housed in specific pathogen-free conditions under approved institutional animal care and use protocols. Mice received injection of 1×10⁶ AGS cell suspension. After reaching ~200 mm³ tumour volume, the mice were treated with CuD (intraperitoneal injection, 1 mg/kg, once daily), SC79 (intraperitoneal injection, 20 mg/kg, once daily) or combination of CuD with SC79 for 20 days. The tumor size was measured by vernier caliper measurement of the tumor size and the short diameter. Tumour volumes were calculated using the following formula: Length × width² × 0.5236.

We used the SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) for statistical analysis. All data were expressed as the mean ± standard deviation. The difference among groups was assessed by analysis of variance (ANOVA). The difference between groups was assessed by Student's t-test. We considered P<0.05 to indicate a statistically significant difference.

Under normal conditions, human gastric cancer cell lines (AGS, SNU1 and Hs746T) have a fast growth rate. In the present study, cancer cell proliferation was inhibited in a dose- and time-dependent manner when the cells were incubated with CuD (0.25, 0.5, 1, 1.5 and 2 µM) for 12, 24, 48 and 72 h (Fig. 2). The anti-growth effect of CuD was more significant in the AGS cell line, indicating that the AGS cell line was more sensitive to CuD.

Increasing the generation of ROS is an effective antitumour property of the cucurbitacin family. We detected ROS generation after cells were treated with CuD (0.5, 1 and 2 µM) for 24 h. We found that CuD increased the ROS levels of gastric cancer cell lines in a dose-dependent manner (Fig. 3).

Subsequently, TUNEL staining was used to determine the effect of CuD on gastric cancer cell apoptosis. After incubation with CuD (0.5, 1 and 2 µM) for 24 h, the gastric cancer cells had a high ratio of apoptosis-positive to apoptosis-negative cells, which was dose-dependent (Fig. 4).

Increased Ca²⁺ fluxion and generation of ATP are closely related to cell apoptosis. In the present study, intracellular Ca²⁺ and ATP generation were assessed by their fluorescence absorbance values. Our results revealed that incubation of AGS cells with CuD (0.5, 1, 1.5 and 2 µM) for 12, 24, 48 and 72 h increased the levels of intracellular Ca²⁺ (Fig. 5A) and ATP (Fig. 5B) in a dose- and time-dependent manner. Intracellular nitric oxide (NO) production in the culture media was assessed as nitrite concentration. We observed that incubation of AGS cells with CuD (0.5, 1, 1.5 and 2 µM) for 12, 24, 48 and 72 h markedly increased NO production both in a dose- and time-dependent manner (Fig. 5C and D).

NO production is triggered by NO synthase (NOS). Apoptosis is triggered by intrinsic and extrinsic apoptotic pathways. Therefore, we evaluated the expression of iNOS and the mitochondrial pathway in AGS cells after treatment with CuD (0.5, 1 and 2 µM) for 24 h. Our results revealed that CuD increased the expression level of iNOS, which triggered the generation of NO. CuD also increased Bax levels and reduced the expression of Bcl-2, triggering the activation of caspase-9, as well as the release of cytochrome c in AGS cells (Fig. 6).

The Akt pathway is involved in many processes associated with the poor prognosis of cancer, such as pro-survival, anti-apoptosis, metastases and chemotherapy resistance (11). CuD has been reported to inhibit Akt in schwannoma and meningioma cells (13). We found that incubation of AGS cells with CuD (0.5, 1 and 2 µM) for 24 h significantly reduced the levels of phosphorylated Akt, mTOR (a downstream protein of the Akt pathway) and ribosomal S6 protein (rS6) (Fig. 7). These data indicated that CuD targeted the Akt signalling pathway in gastric cancer cells.

To confirm that the effects of CuD were dependent on Akt and iNOS, AGS cells were treated with SC79 (4 µg/ml) or L-canavanine (1 mM) and CuD (2 µM) for 24 h. We observed that both SC79 and L-canavanine reversed the antiproliferative and pro-apoptotic effects of CuD in AGS cells (Fig. 8). This indicated that CuD targeted Akt and iNOS.

The effects of CuD on gastric cancer were confirmed by an in vivo xenograft-mouse model. After tumours became palpable, the mice were treated with either CuD and/or SC79 for 20 days. We found that CuD induced tumour cell apoptosis and arrested tumour growth in vivo. In contrast, SC79 inhibited tumour cell apoptosis and augmented tumour growth. In addition, SC79 reversed the anti-growth effect of CuD (Fig. 9).