The SGC-7901 (expressing mutant p53) and the MKN45 (expressing wild-type p53) GC cell lines were obtained from the Central Laboratory of Weifang Medical College (Weifang, China). Cell lines were passaged four times prior to harvesting for RNA isolation. All human cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% foetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C/5% CO₂.

MKN-45 and SGC-7901 cells were seeded in 96-well plates at a density of 5×10⁴ cells/ml and incubated at 37°C/5% CO₂ for 24 h, in order to achieve the exponential phase of cell growth. The supernatant was then discarded, and fresh culture medium was added to the wells. Treatments were performed by adding to the culture medium the following: i) 4 µg/ml cisplatin (Deyao Pharmaceutical Co., Ltd., Dezhou, China); ii) 50 IU/ml rmhTNF (Shanghai Weike Biopharmaceutical Co., Ltd., Shanghai, China); iii) 100 IU/ml rmhTNF; iv) 200 IU/ml rmhTNF; v) 4 µg/ml cisplatin and 50 IU/ml rmhTNF; and vi) 4 µg/ml cisplatin and 100 IU/ml rmhTNF. Control wells contained medium alone (untreated cells). Following culture for 24 h at 37°C/5% CO₂, the medium was replaced with 110 µl10% Cell Counting Kit-8 medium (Yesen Biotechnology Scientific Inc., Shanghai, China), and cells were incubated for another 2 h at 37°C/5% CO₂. The absorbance at 450 nm (A₄₅₀) was determined using a Bio-Tek PowerWave XS microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). The mean absorbance values were determined from four wells for each treatment group, and the growth inhibition rate was calculated using the following formula:

IC denotes the growth inhibition rate (%), A_Exp the mean absorbance for the treatment group, A_C the mean absorbance for the control group, and A_Emp the mean absorbance for wells without cells or reagents added.

4×10⁴ MKN45 and SGC7901 cells in the exponential phase of growth were seeded in per well in 6-well plates and cultured for 24 h in medium supplemented with 4 µg/ml cisplatin, 50 U/ml rmhTNF, 100 U/ml rmhTNF, 200 U/ml rmhTNF, 4 µg/ml cisplatin and 50 U/ml rmhTNF, or 4 µg/ml cisplatin and 100 U/ml rmhTNF. As a control, normal growth medium without supplements was used (untreated cells). TRIzol^® RNA isolation, M-MuLV first-chain synthesis and PCR amplification kits were all bought from Shanghai Sangon Biotech Corporation Ltd. (Shanghai, China). The concentration of total RNA in each sample was determined with a 2000c UV-Vis spectrophotometer (Thermo Fisher Scientific, Inc.). Each 20 µl reverse transcription reaction contained 4 µl 25 mM MgCl₂, 2 µl 10xPCR Buffer II, 1 µl double-distilled water, 8 µl premixed deoxyribonucleoside triphosphates, 1 µl RNA inhibitor solution (20 U/µl), 1 µl random hexamers, 1 µl MuLV reverse transcriptase, and 2 µl total RNA (≤1 µg), as per the kit's protocols (Shanghai Sangon Biotech Corporation Ltd.). Total RNA was added just prior to the start of the reaction. Reactions were incubated at 25°C for 10 min, then at 42°C for 30 min, followed by 95°C for 5 min and then cooled at 5°C for 5 min. Samples were then diluted 1:4 to obtain a final concentration of 10 ng/µl cDNA. The PCR step was carried out on a PE-5700 MyCycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) and involved 35 amplification cycles at 94°C for 1 min, 58°C for 50 sec and 72°C for 1 min. Specific oligonucleotide primers were used as listed in Table I. Amplicons were subjected to 1% (w/v) agarose gel electrophoresis, and gels were analysed with a BioSpectrum AC Gel Imaging System (Alpha Innotech Corp., San Leandro, CA, USA). The relative expression levels of target genes were calculated using the following formula:

SPSS 20.0 (IBM SPSS, Armonk, NY, USA) was used in order to analyse experimental data, with values presented as the mean ± standard deviation. The difference among groups was assessed by one-way analysis of variance. Comparisons between two groups were conducted using the Student-Newman-Keuls method or Student's t-test. The relationship between two variables was analysed using Pearson's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

Following treatment with rmhTNF for 24 h, a significant inhibition of cell growth was observed in MKN45 cells in a dose-dependent manner (P<0.05; Fig. 1). However, no significant effect was observed on the cell growth of the mutant p53-expressing SGC7901 cells with rmhTNF treatment (Fig. 1). Combination treatment of cisplatin and rmhTNF acted synergistically to further enhance the inhibitory effect on the growth of MKN45 cells, compared with either cisplatin or rmhTNF treatments alone (P<0.01; Fig. 1). A synergistic effect of cisplatin and rmhTNF was not observed in the mutant p53-expressing SGC7901 cells (Fig. 1). The combination treatment of cisplatin was performed with two different doses of rmhTNF (50 and 100 IU/ml) and the growth inhibition observed was dose-dependent in MKN45 cells (P<0.01; Fig. 1).

The mRNA expression of bcl-2 and p53β in MKN45 cells (Fig. 2A) and bcl-2 in SGC7901 cells (Fig. 2B) were determined. In MKN45 cells, p53β mRNA expression levels were significantly increased by cisplatin alone compared with untreated cells (P<0.01; Figs. 2A and 3). Treatment of MKN45 cells with rmhTNF alone had no effect on p53β mRNA expression compared with untreated cells (Figs. 2A and 3). However, when cisplatin was used in combination with rmhTNF, p53β mRNA expression levels were further increased compared with cells treated with cisplatin alone (P<0.01; Figs. 2A and 3), suggesting again that rmhTNF and cisplatin act synergistically in MKN45 GC cells.

In MKN45 cells, bcl-2 mRNA expression levels were significantly downregulated by cisplatin alone, or by combined cisplatin and rmhTNF treatment, compared with untreated cells (P<0.01; Figs. 2A and 4). However, no significant effect on bcl-2 mRNA expression levels was detected with rmhTNF treatment alone in MKN45 cells, compared with untreated cells (P>0.05; Figs. 2A and 4). In the mutant p53-expressing SGC7901 cells, bcl-2 mRNA levels were not significantly altered in either of the various treatment groups (P>0.05; Figs. 2B and 4).

Correlation analysis was performed to assess whether bcl-2 and p53β mRNA expression are associated with each other and with the growth inhibition phenotype. The results indicated that bcl-2 mRNA expression in MKN45 cells was negatively correlated with mRNA expression of p53β (r=−0.897; P<0.01; Fig. 5), and negatively correlated with the cell growth inhibition rate (r=−0.906; P<0.01; Fig. 6).