SGC-7901 cells were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Invitrogen™; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator containing 5% CO₂. Hypoxia was induced by adding CoCl2 (final concentration, 100 µM) in the culture medium. Cells with satisfactory growth were selected for subsequent experiments.

SGC-7901 cells were seeded onto 6-well plates (Corning Inc., Corning, NY, USA) at a density of 1×10⁶ cells/well, and incubated in a 37°C incubator in a humidified atmosphere containing 5% CO₂. One day later, the cells were treated with 1 and 3 mM MLT (Sigma-Aldrich, St. Louis, MO, USA), and cell morphology was visualized 24 h post-treatment with a TE2000 inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan).

The SGC-7901 cells were seeded onto 96-well plates (Corning Inc.) at a density of 5,000 cells/well), and treated with 0, 0.01, 0.1, 1 and 3 mM MLT one day later. Following a further culture for 0.2, 2, 16 and 24 h, the viability and proliferation of the SGC-7901 cells was detected using a cell counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. Cell density was determined by measuring the absorbance value (A value) at 450 nm with a Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific, Inc.).

A total of 60 4-week-old male nude mice of the BALB/c strain [BALB/cA-nu (nu/nu)] were purchased from Shanghai Laboratory Animal Center (Shanghai, China), and were maintained in a specific pathogen-free facility at 20–25°C in a relatively humidity of 40–60% under a 12 h-light/12 h-dark cycle, with free access to food and water. The mice were subcutaneously injected in the flank with SGC-7901 cells at a density of 3×10⁶ cells per mouse, and were randomly assigned into four groups by concentration of MLT that the mice were treated with: Control (0 mg/kg), low (50 mg/kg), medium (100 mg/kg) and high (150 mg/kg). The indicated amounts of MLT, which were intraperitoneally injected into the mice, were not administered until the diameter of the xenografts reached 2 mm. The long (L) and short (W) axes of the tumors were measured with a caliper, and the tumor size (V) was calculated using the following formula: V = 4/3π × l/2 × (W/2)².

Mice were sacrificed using the cervical dislocation method, and tumors were excised 7 days following MLT treatment. The excised tumors were cut into 5-µm sections, and transferred to gelatin-coated slides (Sigma-Aldrich). Following a 30-min incubation in phosphate-buffered saline (PBS) containing 0.3% Triton X-100 (Sigma-Aldrich), the sections were incubated with a primary antibody against RZR/ROR receptor [rabbit anti-RZR/RORα polyclonal antibody (1:500; catalog number ab60134; Abcam, Cambridge, MA, USA), rabbit anti-RZR/RORβ polyclonal antibody (1:500; catalog number ab78007; Abcam) or rabbit anti-RZR/RORγ polyclonal antibody (1:500; catalog number ab188756; Abcam) or vascular endothelial growth factor (VEGF) (rabbit anti-VEGF polyclonal antibody (1:200; catalog number ab46154; Abcam) at 37°C overnight. After washing with 1X-PBS three times, the sections were incubated with a polyclonal goat anti-rabbit biotinylated antibody (1:100; catalog number BA1003; Boster Biological Technology Co., Pleasanton, CA, USA) for 1 h, and subsequently incubated in VECTASTAIN^® Elite ABC Reagent (Vector Laboratories, Inc., Burlingame, CA, USA). Subsequently, the sections were stained with 3,3′-diaminobenzidine (DAB; Sigma-Aldrich). Images were taken with a Leica DM4000B photo microscope (Leica Microsystems GmbH, Wetzlar, Germany; magnification, ×400).

SGC-7901 cells were seeded onto dishes (60 mm diameter) and incubated at 37°C for 24 h. Subsequently, the cells were incubated in darkness with MLT at various concentrations (0, 50, 100 and 150 mM) and 100 µM CoCl2 (Sigma-Aldrich) at 37°C for 24 h. The cell cultures were transferred to centrifuge tubes and centrifuged at 4°C, 18,000 × g for 15 min on a 5417 R refrigerated microcentrifuge (Eppendorf, Hamburg, Germany). The supernatants were collected and the VEGF level in the supernatant was determined using a Human VEGF Quantikine ELISA kit (R & D Systems, Inc., Minneapolis, MN, USA) by measuring the A value at 450 nm. The cell count was estimated with a hemocytometer at least three times, and the standard curve was plotted.

Total protein was isolated from the tumor tissues using a Total Protein Extraction kit (Beijing Transgen Biotech Co., Ltd., Beijing, China), and the protein concentration was quantified with a Bicinchoninic Acid Protein Assay kit (Sigma-Aldrich). The protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (110 V; 1.5 h), and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore; Billerica, MA, USA) at 4°C (110 V; 1.5 h) using a wet transfer technique. Subsequently, the membranes were blocked in 5% skimmed milk in PBS, incubated with a primary antibody against SUMO-specific protease 1 (SENP1) (rabbit anti-SENP1 polyclonal antibody; 1:1,000; catalog number SAB2102107; Sigma-Aldrich) or hypoxia-inducible factor-1α (HIF-1α) (rabbit anti-HIF-1α monoclonal antibody; 1:1,000; catalog number ab51608; Abcam) at 4°C overnight, washed in Tris-buffered saline-Tween 20 (TBST), and incubated with a monoclonal goat anti-mouse IgG secondary antibody (1:4,000; catalog number, A3562; Sigma-Aldrich) at room temperature for 1.5 h. Subsequently, the PVDF membranes were washed in TBST and the immunoreactive bands were visualized through exposure to X-ray film for 1–15 min. The protein purity was evaluated by densitometric analysis using the Quantity One software version 4.4.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The quantity of target protein was calibrated with respect to β-actin, and control values and relative intensities were obtained.

Total RNA was extracted from the tumor tissues using TRIzol reagent (Invitrogen™; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The RNA was reverse-transcribed into first-strand cDNA using the GoScript™ Reverse Transcription System (Promega Corporation, Madison, WI, USA), which contained M-MLV reverse transcriptase. The primers used for RT were designed using the Primer Express version 5.0 software (Applied Biosystems^®; Thermo Fisher Scientific, Inc.), and were synthesized by BioSune Biotechnology Co. (Shanghai, China). The cycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. The reaction was conducted on a ABI PRISM 7500 Real-time PCR System (Applied Biosystems^®; Thermo Fisher Scientific, Inc.). The relative expression of RZR/ROR receptor, SENP1, HIF-1α, VEGF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes was determined using qPCR with Brilliant III SYBR Green QRT-PCR Master Mix (Agilent Technologies, Inc., Santa Clara, CA, USA) on a StepOnePlus^® Real-Time PCR system (Applied Biosystems^®; Thermo Fisher Scientific, Inc.). qPCR was performed using the following primers: RZR/RORγ, forward 5′-GAGGCCATTCAGTACGTGGT-3′ and reverse 5′-GCAATCTCATCCTCGGAAAA-3′; SENP1, forward 5′-GAGGATGGATGCTGGAGAAG-3′ and reverse 5′-TGTCTGAGGAAGGATTATCTGAG-3′; HIF-1α, forward 5′-ACTCAGGACACAGATTTAGACTTG-3′ and reverse 5′-ATCAGTGGTGGCAGTGGTAG-3′; VEGF, forward 5′-CTTGCCTTGCTGCTCTAC-3′ and reverse 5′-ACCACTTCGTGATGATTCTG-3′; GAPDH, forward 5′-CCGAGAATGGGAAGCTTGTC-3′ and reverse 5′-TTCTCGTGGTTCACACCCATC-3′. The cycling conditions were as follows: 40 cycles of 95°C for 30 sec, and 60°C for 1 min. Relative mRNA expression levels were calculated using the 2^−∆∆Cq method (16). Melting curve analysis was employed to check the specificity of the amplification reaction.

All experiments were repeated at least three times. All data are expressed as the mean ± standard error of the mean, and all statistical analyses were performed with SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance, followed by Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

To examine the effects of MLT treatment on SGC-7901 cells, cell morphology and cellular viability in response to MLT stimuli was examined. Microscopy revealed a significantly reduced number of SGC-7901 cells in the MLT treatment groups compared to the control cells. In addition, MLT treatment inhibited the growth of SGC-7901 cells in a time- and dose-dependent manner (Fig. 1A and B). Subsequently, whether angiogenesis was impaired following MLT treatment was assessed by measuring VEGF protein content in the supernatant of the cells. The present findings demonstrated that treatment with low concentrations of MLT had no remarkable effect on VEGF secretion, while 3 mM MLT inhibited VEGF production in SGC-7901 cells (P=0.023), which was in agreement with the growth inhibitory effects (Fig. 1C).

Since the antitumor activity of MLT has been extensively described previously, the present study sought to determine the effective concentration of MLT that was required to inhibit the in vivo xenograft tumor growth of gastric cancer in nude mice. Tumor weight and volume (Fig. 2A and B, respectively) were decreased in tumor-bearing mice treated with low (50 mg/kg) doses of MLT, and significantly decreased in tumor-bearing mice treated with medium (100 mg/kg; P=0.043 and 0.041 in Fig. 2A and B, respectively) and high (150 mg/kg; P=0.039 and 0.031 in Fig. 2A and B, respectively) doses of MLT compared with untreated mice. These results additionally confirmed the antitumor activity of MLT against gastric cancer cells.

Immunohistochemistry staining results revealed that RZR/RORγ was expressed in gastric cancer tissues (Fig. 3A), while the expression of RZR/RORα or RZR/RORβ was not detected (data not shown). To investigate the effect of effective concentrations of MLT on VEGF expression in gastric cancer tissue, immunohistochemical staining was performed to detect VEGF protein expression. The present findings demonstrated that there was a significantly reduced VEGF protein expression in the tumor tissues collected from MLT-treated tumor-bearing mice compared with control mice (P=0.009 and 0.003 for 100 and 150 mg/kg MLT, respectively; Fig. 3B and C).

To explore the underlying mechanisms of the anti-tumor activity of MLT in gastric cancer tissue, the present study sought to determine the expression of RZR/RORγ, SENP1, HIF-1α and VEGF following treatment with MLT. The transcriptional and translational expression of these molecules was determined using qPCR and western blot analysis, respectively. The present findings revelaed significantly reduced mRNA and protein levels of RZR/ROR receptor, SENP1, HIF-1α and VEGF in the tumor tissues derived from the MLT-treated tumor-bearing mice compared with untreated mice (Fig. 4A and B). The P-values obtained for 100 mg/kg MLT in regards to mRNA expression were as follows: P=0.038 for RZR/RORγ, P=0.035 for SENP1, P=0.033 for HIF-1α and P=0.034 for VEGF, while for 150 mg/kg MLT, the results revealed P=0.005 for RZR/RORγ, P=0.007 for SENP1, P=0.006 for HIF-1α and P=0.004 for VEGF (Fig. 4A). In terms of protein expression, 100 mg/kg MLT resulted in P=0.040 for RZR/RORγ, P=0.042 for SENP1, P=0.038 for HIF-1α and P=0.028 for VEGF, while 150 mg/kg MLT led to P=0.006 for RZR/RORγ, P=0.009 for SENP1, P=0.005 for HIF-1α and P 0.004 for VEGF (Fig. 4B).