The human GC SGC7901 cell line was donated from the Shanghai Tumor Research Institute (no. 01842; Shanghai, China). The primers of PI3K and HIF-1α were synthesized by the Shanghai Biological Engineering Technology Co., Ltd. (Shanghai, China). The PI3K rabbit-anti-human polyclonal antibody (sc-1637) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); p-Akt rabbit-anti-human polyclonal antibody (D9E) and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); and HIF-1α rabbit-anti-human polyclonal antibody (BA0912) was obtained form Wuhan Boster Biological Engineering Co., Ltd (Wuhan, China). The GC tissue samples treated with paclitaxel were obtained from severe combined immune deficiency (SCID) mice orthotopically implanted with human GC cells from the Gastrointestinal Disease Laboratory of Shanghai Sixth People’s Hospital (Shanghai, China).

Paclitaxel was donated by Professor G-Y Fan from the Kunming Botany Institute (Yunnan, China); the Cell Counting Kit-8 (CCK-8) was purchased from Tongren Chemical Institute (Japan); Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA); TRIzol^® reagent and LY294002 were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA); M-MLV Reverse Transcriptase was purchased from Promega Corporation (Madison, WI, USA); SYBR Green master mix was purchased from Takara Bio, Inc., (Otsu, Japan); the Annexin V-fluorescein isothiocyanate cell apoptosis detection kit was obtained from KeyGen Biotech., Co., Ltd. (ab14085; Nanjing, China); and the Enhanced Chemiluminescence (ECL)-PLUS™ western blotting reagents were purchased from GE Healthcare (Piscataway, NJ, USA).

A total of 45 patients with GC and 36 patients with chronic gastritis were enrolled in this study at the General Surgery and Digestive Endoscopy Room from December, 2007 to June, 2008. The pathological staging was determined according to the American Joint Committee on Cancer tumor-node-metastasis (TNM) staging system. The use of the tissue samples and clinical data was approved by the Medical Ethics Committee of Shanghai Jiao Tong University (Shanghai, China).

The protein expression of PI3K, p-Akt and HIF-1α were analyzed by immunohistochemical staining. The anti-PI3K, p-Akt and HIF-1α antibodies were used at 1:100 dilutions. Endogenous peroxidase was inhibited by incubation with freshly prepared 3% hydrogen peroxide with 0.1% sodium azide. Non-specific staining was blocked with 0.5% casein and 5% normal serum. The tissues were incubated with biotinylated antibodies and horseradish peroxidase (Cell Signaling Technology, Inc.). Staining was developed with diaminobenzidine substrate and sections were counterstained with hematoxylin and eosin. Normal serum or phosphate-buffered saline (PBS) replaced the antibodies in negative controls. The images were analyzed with Image-Pro Plus 4.5 System (Media Cybernetics, Inc., Rockville, MD, USA). The total optical density value and area of intracellular fluorescence for each section was measured.

The SGC7901 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were stored in a humidified atmosphere of 5% CO₂ at 37°C for 30 min. Under normal oxygen, the cells were incubated in 20% O₂ and 5% CO₂ with saturated humidity at 37°C for 30 min. The hypoxic cells were incubated in an AnaeroPack™ containing 20% CO₂ and <1% O₂ at 37°C for 30 min. The cells pretreated under hypoxic conditions were further treated with the PI3K inhibitor, LY294002, (40 Mm) for 30 min, followed by paclitaxel (0.1 Mm).

To quantitatively determine the mRNA expression levels of PI3K and HIF-1α in GC SGC-7901 cells, qPCR was used. Total RNA of each clone was extracted with TRIzol reagent according to the manufacturer’s instructions. Reverse-transcription using M-MLV, and cDNA amplification using SYBR Green master mix kit, were performed according to the manufacturer’s protocol. The genes were amplified using specific oligonucleotide primers and a human β-actin gene was used as an endogenous control. The PCR primer sequences were as follows: Forward, 5′-AAGAAGCAAGCAGCTGAG-3′ and reverse, 5′-CTACAGAGCAGGCATAG-3′ for PI3K; forward, 5′-TCAAAGTCGGACAGCCTC-3′ and reverse, 5′-CCAGCAGTCTACATGC-3′ for HIF-1α; forward, 5′-CTTCGAGCAAGAGATGGC-3′ and reverse, 5′-CTCCTTCTGCATCCTGTC-3′ for β-actin. Data were analyzed using the comparative Ct method (2^−ΔΔCt). Three separate experiments were performed for each clone.

The hypoxic SGC-7901 cells treated with LY294002 and/or paclitaxel were harvested and extracted using lysis buffer [Tris-HCl, sodium-dodecyl sulfate (SDS), mercaptoethanol and glycerol] purchased from Santa Cruz Biotechnology, Inc. Cell extracts were heated to boiling point for 5 min in loading buffer and equal amounts of cell extracts were separated on 15% SDS-PAGE gels. Separated protein bands were transferred into polyvinylidene fluoride membranes, which were blocked in 5% skimmed milk powder. The primary antibodies against PI3K, p-Akt and HIF-1α were diluted according to the manufacturer’s instructions and incubated overnight at 4°C. Horseradish peroxidase-linked secondary biotinylated antibodies were added at a dilution ratio of 1:1,000 and incubated at room temperature for 2 h. The membranes were washed with PBS three times and the immunoreactive bands were visualized using the ECL-PLUS/Kit according to the manufacturer’s instructions. The relative protein levels in different cell lines were normalized to the GAPDH concentration. Three separate experiments were performed for each clone. Finally, the immune complexes were developed using an ECL detection kit according to the manufacturer’s instructions (ECL GST western blotting detection kit, Pierce Biotechnology, Inc., Waltham, MA, USA) and the GelGDoc2000 imaging system (Bio-Rad Laboratories GmbH, Munich, Germany) was employed to analyze the bands, and the protein levels by the relative optical density.

Cell proliferation following treatment with LY294002 and/or paclitaxel was measured by the CCK-8 assay. The GC SGC-7901 cells were seeded at a density of 2×10⁴ cells/100 μl/well in 96-well plates and left to attach overnight. The medium was then removed and 200 μl of FBS was added followed by LY294002 and/or paclitaxel. The cells were incubated under these conditions for 24, 48, 72, 96, 120 and 148 h at 37°C in a humidified atmosphere of 5% CO₂. After the designated time, CCK-8 was added to each well containing 200 μl of the culture medium and the oligopeptide mixture, and further incubated for 4 h at 37°C. The amount of formazan dye was measured at 450 nm using the Multi-Mode Microplate Reader (Nanchang Biotek Medical Device Co., Ltd., Nanchang, China). All the experiments were performed in triplicate and repeated three times.

Cell apoptosis detection was performed using the BD Accuri™ C6 Flow cytometer (BD Biosciences, San Jose, CA, USA). The exposure of PBS on the extracellular side of the cell membrane was quantified by propidium iodide (PI) staining (Invitrogen Life Technologies). The SGC-7901 cells were placed in six-well plates and, after 24 h of incubation, the cells were treated with LY294002 and/or paclitaxel for 24 h and then harvested. Following centrifugation, cell pellets were washed twice with cold PBS. The cells were then incubated with 5 μl of PI at room temperature for 15 min in the dark. Following incubation, 400 μl of 1X binding buffer was added to each tube. The cells were immediately analyzed by flow cytometry.

Data are expressed as the means ± standard deviation where applicable. Statistically significant differences in each assay were determined by SPSS software, version 20.0 (SPSS, Inc., Chicago, IL, USA). Differences in each group were tested for significance using Student’s t-test or one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The GC tissue sections were analyzed by immunohistochemistry (IHC) and Image-Pro Plus 4.5 software (Media Cybernetics, Inc.). As shown in Fig. 1 and Table I, the positive expression of PI3K, p-Akt and HIF-1α was predominantly localized in the cytoplasm of the GC tissue cells, but was not identified in the chronic gastritis cells. The expression intensities of PI3K, p-Akt and HIF-1α were significantly increased in GC tissues compared with chronic gastritis tissues (each P<0.01).

The correlations of PI3K, p-Akt and HIF-1α expression and various clinical and pathological characteristics were analyzed. As summarized in Table II, no significant correlation was found between PI3K, p-Akt and HIF-1α expression and gender, age, tumor size and peripheral nerve infiltration in patients with GC (each P>0.05), while their expression was significantly correlated with TNM staging, lymph node metastases, lymphatic infiltration and vascular infiltration (each P<0.01), but inversely correlated with tumor differentiation (P<0.01).

qPCR and western blot analysis were performed to detect the effects of LY294002 and paclitaxel on the expression of PI3K, p-Akt and HIF-1α in the GC SGC-7901 cells. LY294002 combined with paclitaxel markedly inhibited the mRNA (Fig. 2A and B) expression levels of PI3K and HIF-1α (it was not necessary to detect the expression of p-Akt as it is downstream of PI3K) and protein (Fig. 3A and B) expression levels of PI3K, p-Akt and HIF-1α in GC SGC-7901 cells compared with the single treatment of LY294002 or paclitaxel (each P<0.01). LY294002 or paclitaxel decreased the expression of PI3K, p-Akt and HIF-1α at the transcriptional and translational levels compared with the hypoxic group (each P<0.01).

To determine whether LY294002 and/or paclitaxel affect the proliferative activity of hypoxic GC cells, the CCK-8 assay was performed to detect cell viability. As summarized in Table III, LY294002 combined with paclitaxel significantly decreased the cell viability in SGC-7901 cells compared with the single treatment of LY294002 or paclitaxel (each P<0.01). In addition, LY294002 or paclitaxel reduced cell viability compared with the hypoxic group (each P<0.01).

Cell apoptosis was measured by flow cytometry using PI staining. Following treatment with LY294002 and/or paclitaxel for 24 h, LY294002 combined with paclitaxel significantly increased the percentage of early cell apoptosis compared with the single treatment of LY294002 or paclitaxel (each P<0.01) (Fig. 4A and B). LY294002 or paclitaxel increased the percentage of early cell apoptosis compared with the hypoxic group (both P<0.01).

SCID mice orthotopically implanted with human GC tissues were treated with paclitaxel (intraperitoneal administration of 5 mg/kg) and the effects of paclitaxel on the expression of PI3K, p-Akt and HIF-1α were assessed by IHC. Paclitaxel decreased the expression of PI3K, p-Akt and HIF-1α (Fig. 5) compared with the GC and normal control groups (each P<0.01).