The effects of trastuzumab, Oxa, DDP, 5-fluorouracil (FU) and taxol administration (alone and in combination) on malignant cell growth were studied in a panel of five human GC cell lines (AGS, NCI-N87, MGC-803, HGC and MKN45) obtained from the Shanghai Institute of Cell Biology (Shanghai, China). Of these, NCI-N87 is an HER2-amplified cell line (19). All of the cell lines were cultured in RPMI-1640 medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% bovine serum (Invitrogen Life Technologies, Carlsbad, CA, USA), 100 mg/ml streptomycin (Sichuan Pharmaceutical Co., Ltd., Sichuan, China), 100 U/ml penicillin (Sichuan Pharmaceutical Co., Ltd.), insulin (Tonghua Dongbao Pharmaceutical Co., Ltd., Jilin, China), glutamine and pyruvate (Invitrogen Life Technologies) at 37°C in a 5% CO₂ water-saturated atmosphere.

Prior to each experiment, the dilutions of all of the reagents were freshly prepared. Trastuzumab was obtained from the University of California Pharmaceutical Services (Los Angeles, CA, USA) and was prepared from a stock concentration of 20 mg/ml. Oxa, DDP, 5-FU and Taxol were supplied by the Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China). A CellTiter 96^® AQueous One Solution Cell Proliferation Assay kit was purchased from Promega Corporation (Madison, WI, USA) and the Annexin-V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit was purchased from Invitrogen Life Technologies.

The CellTiter 96 kit (Promega Corporation) was used to determine cytotoxicity, according to the manufacturer’s instructions. Briefly, the five human GC cell lines were grown to the log phase, trypsinized, seeded into 96-well plates at a density of 2×10³ cells/well and incubated overnight to allow cell adherence. Subsequently, the medium in each well was replaced with fresh (platinum agent-free) medium or medium containing various concentrations (1×10⁻⁴, 1×10⁻³, 1×10⁻², 1×10⁻¹, 1, 10 and 100 μg/ml) of platinum agents (Oxa and DDP) and was incubated for an additional 48 h. CellTiter 96 AQueous One solution was added to each well at one fifth of the mixture volume and the plates were incubated for 3 h. Absorbance was determined at an wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA), with blank control wells to zero the absorbance. For each experiment, 10 control wells were allocated for platinum agent-free medium and a minimum of six replicate wells were allocated for each concentration of platinum agent-containing medium. Using the background-corrected absorbance values, the inhibition rate [I (%)] was calculated by the following equation: I (%) = 100 × (A_{untreated control well} − A_{experimental well})/A_{untreated control well}. The half maximal inhibitory concentration (IC₅₀) was defined as the concentration of platinum agent required for 50% inhibition of cell growth.

The number of apoptotic cells was quantified using an Annexin V-FITC Apoptosis Detection kit (Invitrogen Life Technologies), according to the manufacturer’s instructions. Briefly, the NCI-N87 cells were grown to 75–80% confluence in 60-mm Petri dishes, exposed to trastuzumab (1.0 μg/ml) and platinum agents (Oxa, 5 μg/ml; DDP, 2.5 μg/ml) alone or in combination for 48 h, and compared with the untreated control cells. To quantify the apoptosis, the cells were collected, resuspended in 500 μl binding buffer, treated with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI), and analyzed using a FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Briefly, the three human GC cell lines, NCI-N87, HGC27 and MKN45, were grown to the log phase, trypsinized, washed with phosphate-buffered saline (PBS) and collected by performing centrifugation for 5 min at 174 × g. Total RNA was extracted from each cell line using the SV Total RNA isolation system (Promega Corporation), according to the manufacturer’s instructions. The purity and quality of the extracted mRNA were determined using a Bio-visible spectrophotometer at 260 and 280 nm (Eppendorf, Hamburg, Germany); and the integrity of the extracted mRNA was determined by performing agarose gel electrophoresis on a 1% gel. Reverse transcriptase from the reverse transcription system was used according to the manufacturer’s instructions (Promega Corporation) to synthesize a total volume of 20 μl complementary DNA for each cell line, and the iCycler iQ™ Multi-Color Real Time PCR detection system (Bio-Rad Laboratories, Inc.) was used to perform RT-qPCR of the target genes and the internal control (β-actin). Applied Biosystems Life Technologies (Foster city, CA, USA) supplied the primers [1X; Assay IDs: Hs00819517_mH (TRF1); Hs01554305_g1 (TIN2); Hs00194619_m1 (TRF2); Hs00368526_g1 (TPP1); Hs00430292_m1 (TRF2IP); Hs00209984_m1 (POT1); and Hs99999903_m1 (β-actin)] and probe mixture, and AbGene Ltd. (Surrey, UK) supplied the ABsolute qPCR mix (1X) used to produce the 20-μl PCR reaction mixture. The PCR conditions were as follows: 50°C for 2 min, 95°C for 15 min, followed by 45 cycles at 95°C for 15 sec and 60°C for 1 min. Using β-actin as an endogenous control and commercial human total RNA samples (Clontech Laboratories, Inc., Mountainview, CA, USA) as calibrators, the relative gene expression levels were quantified according to the comparative Ct method, employed the formula 2^−ΔΔCt to determine the final results (20). Following PCR, the 10-μl product was loaded onto a 1.5% agarose gel and visualized by ethidium bromide staining (Sigma-Aldrich, Munich, Germany).

Total cell lysates were prepared in RIPA lysis buffer (Pierce Biotechnology, Inc., Rockford, IL, USA). Following determination of protein concentration using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc.), an aliquot of lysate containing 50 μg of each protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, blocked with blocking buffer (PBS Tween-20 containing 5% non-fat milk) for 2 h at room temperature and incubated overnight at 4°C with the following specific primary antibodies: monoclonal mouse anti-human TPP1 (ACD; cat. no. TA504406), monoclonal rabbit anti-human POT1 (cat. no. TA310771), polyclonal rabbit anti-human TRF1 (cat. no. TA322887), rabbit anti-human monoclonal TRF2 (cat. no. TA307200), rabbit anti-human polyclonal TIN2 (cat. no. TA315321), rabbit anti-human polyclonal TRF2IP (cat. no. TA324532 ) and rabbit anti-human polyclonal GAPDH (cat. no. TA308884) (1:10,000, all primary antibodies; OriGene Technologies, Inc., Rockville, MD, USA). Subsequent incubation with the appropriate horseradish peroxidase-conjugated goat anti-rabbit POT1, TRF1, TRF2, TIN2, TRF2IP, GAPDH (cat. no. SP 9001) and goat anti-mouse TPP1 (cat. no. SP9002) secondary antibodies (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was performed for 2 h at room temperature. Signals were then detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, MA, USA), and a FluorChem SP imaging system (Alpha Innotech, San Leandro, CA, USA) was used for image capture.

Values are expressed as the mean ± standard deviation. All statistical analyses were performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Statistical comparison was performed using Student’s t-test and P<0.05 was considered to indicate a statistically significant difference.

The cytotoxicity of trastuzumab to five GC cell lines was initially analyzed using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit. It was identified that trastuzumab (6.28, 12.56, 25.12, 50.24 and 100.48 μg/ml)rendered more significant cytotoxicity to the NCI-N87 cell line with HER2 amplification compared with the four other cell lines (P<0.05). At 0.81–100.48 μg/ml trastuzumab, the inhibition rates to NCI-N87 cells were not >30% and 0.81–1.62 μg/ml trastuzumab was not obviously cytotoxic to cancer cells (survival rate, >90%; Fig. 1). Thus, as treatment of the cells with 1.0 μg/ml trastuzumab exhibited no significant effect on cell viability, this concentration was used for subsequent analyses.

Additionally, 1.0 μg/ml trastuzumab was administered to five GC cell lines and the effect on the sensitivity of the cell lines to various platinum agents was investigated. It was identified that pretreatment with trastuzumab significantly increased the sensitivity of only the NCI-N87 cell line to platinum agents. As indicated in Table I, the IC₅₀ of Oxa and DDP was decreased to ~3.29 (P=0.001) and 6.91 times (P=0.002) in NCI-N87 cells, respectively; however, trastuzumab did not alter the IC₅₀ of Oxa or DDP in the other four cell lines. Subsequently, it was identified that simultaneous treatment with low-dose trastuzumab and platinum agents may significantly increase the sensitivity of NCI-N87 cells to platinum agents; for example, the IC₅₀ of Oxa and DDP were reduced by ~2.67 (P=0.001) and 4.56 times (P=0.003), respectively. Thus, these data indicate that low-dose trastuzumab may increase platinum sensitivity in NCI-N87 cells.

To further investigate whether low-dose trastuzumab increases the sensitivity of NCI-N87 cells to platinum agents, GC cells were double-stained with Annexin V-FITC and PI to detect early apoptosis induced by treatment with platinum agents and trastuzumab, alone or in combination. The doses of Oxa and DDP selected were 5 μg/ml and 2.5 μg/ml, respectively, as they were close to the 20% inhibitory concentrations (IC₂₀) of NCI-N87 cells. The dose of trastuzumab used was 1.0 μg/ml, as previously determined. For the groups treated with two agents in combination, the cells were pretreated with trastuzumab for 48 h, followed by the addition of platinum agents for 48 h. As indicated in Fig. 2, the percentage of early apoptosis induced by independent Oxa and DDP administration in NCI-N87 cells was 8.0 and 7.3%, respectively; however, this increased to 13.9 and 20.7% upon treatment with trastuzumab in combination with Oxa and DDP, respectively. Furthermore, treatment with trastuzumab alone (1.0 μg/ml) did not induce significant apoptosis in the cancer cells. Thus, it appears that low-dose trastuzumab administration may increase platinum agent-induced apoptosis in NCI-N87 cells and subsequently result in the increased cytotoxicity of platinum agents (Fig. 1).

To understand the mechanisms by which trastuzumab may increase the sensitivity of NCI-N87 cells to platinum agents, we hypothesized that trastuzumab may affect the expression levels of telomere-associated genes or proteins in NCI-N87 cells, thus, influencing their sensitivity to platinum agents. Following incubation with 1.0 μg/ml trastuzumab alone, 2.5 μg/ml DDP alone or the two agents in combination, the mRNA expression of various telomere-associated genes and their proteins were assessed in NCI-N87 cells by performing RT-qPCR and western blot analysis. As demonstrated in Fig. 3A, the mRNA expression levels of TRF1, TRF2, POT1, TPP1 and TRF2IP were significantly downregulated following treatment with trastuzumab alone or DDP in combination with trastuzumab in NCI-N87 cells (P<0.05), and DDP alone only downregulated the expression level of TRF2 (P<0.05). However, in the HGC27 and MKN45 cell lines, treatment wth low dose trastuzumab alone or in combination with DDP did not significantly downregulate the mRNA expression levels of any of the telomere-associated genes (P>0.05) (data not shown). Western blot analysis indicated that treatment with trastuzumab alone and in combination with DDP significantly downregulated the protein expression levels of TRF2, POT1 and TPP1 in NCI-N87 cells, and DDP alone did not downregulate the protein expression level of any of the telomere-associated genes investigated (data not shown). The results indicated that low-dose trastuzumab administration may downregulate the mRNA and protein expression levels in an area of the telomere-associated genes that may be involved in chemosensitivity NCI-N87 cells to platinum agents.