Gastric cancer cell lines AGS, MGC-803 and MKN-45 were purchased from the Cell Bank of China (Shanghai, China), and all these three cell lines were grown in RPMI-1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gemini Scientific, West Sacramento, CA, USA) at 37°C in a humidified atmosphere with 5% CO₂, and the culture medium was routinely changed every 3 days.

Cell were seeded in 96-wells plate with a density of 5,000 cells/well, treated with cisplatin (Sigma-Aldrich, Inc., St. Louis, MO, USA) for 24 h, followed by the Akt inhibitor, MK-2206 (Selleck Chemicals, Houston, TX, USA), for another 48 h. The monotherapy control groups were treated with cisplatin for 24 h followed by dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Inc.) for another 48 h, or were treated with MK-2206 alone for 72 h, and the culture medium in each well was discharged and 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Inc.) was added. The cells were cultured for another 4 h, MTT in each well was removed and 150 µl DMSO was added to each well and was cultured for another 5 min to dissolve the crystal at 37°C. The absorbance was measured by microplate reader at 490 nm (EXI800; BioTek, WInooski, VT, USA).

Subsequent to seeding the AGS gastric cancer cells in 6-well plates with a density of 2×10⁵ cells/well, the cells were treated with cisplatin for 12 h, followed by MK-2206 for 24 h, and the monotherapy groups were treated with cisplatin for 12 h followed by DMSO for another 24 h or MK-2206 alone for 24 h. The control group was treated with DMSO alone. Following the harvesting of cells in each well, staining was performed with the Annexin V/propidium iodide (PI) kit (Nanjing Keygen Biotechnology, Nanjing, China) according to the manufacturer's protocol, and PI/fluorescein isothiocyanate two-dimensional flow cytometric analysis was performed.

The gastric cancer AGS cells were treated with cisplatin for 12 h, followed by MK-2206 for 24 h, and the cells were washed by phosphate-buffered saline twice, harvested and lysed in radioimmunoprecipitation assay buffer (Sigma-Aldrich, Inc.) with protease inhibitors. Cell lysates containing 50 µg of protein were resolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a polyvinylidene fluoride filter, and probed with the antibodies of total Akt [rabbit monoclonal antibody (mAb); 1:1,000; Cell Signaling Technology, Danvers, MA, USA), p-Akt (Ser473; rabbit mAb; 1:1,000, Cell Signaling Technology), cleaved polyadenosine diphosphate ribose polymerase (PARP) (rabbit mAb; 1:1,000, Cell Signaling Technology) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (rabbit mAb, 1:4,000; Sanying Biotechnology, Wuhan, China). Primary antibodies were detected with the secondary antibodies of goat anti-rabbit, using enhanced chemiluminescence (1:3,000; Beijing Beyotime Biology, Beijing, China).

Results were calculated as mean ± standard deviation. Two-sided t-test was used to detect statistical significance, and P<0.05 was considered to indicate a statistically significant difference.

To detect whether Akt phosphorylation has a role in drug resistance to cisplatin in gastric cancer, western blot analysis was performed to detect the level of p-Akt (Ser473) in three gastric cancer cell lines, AGS, MGC-803 and MKN-45. The level of p-Akt in AGS cells was higher compared to MGC-803 and MKN-45 cells, and the MTT assay was used to determine the effect of cisplatin in these three cell lines (Fig. 1A). The half maximal inhibitory concentration (IC₅₀) of cisplatin for each cell line was calculated. The IC₅₀ of AGS was almost twice as high as for MGC-803 and MKN-45 (Fig. 1B), which may indicate that Akt phosphorylation can increase the resistance to cisplatin in gastric cancer cells.

As Akt phosphorylation may have a role in the resistance to cisplatin, we hypothesized that Akt inhibition may increase the effect of cisplatin in gastric cancer cells. To confirm this hypothesis, the Akt inhibitor MK-2206 was used, and in a previous study, three sequences have been used to detect the combination effect of MK-2206 and traditional chemotherapy drugs in cancer: Traditional drugs followed by MK-2206, MK-2206 prior to traditional drugs, and MK-2206 and traditional drugs simultaneously, and it has been reported that traditional drugs followed by MK-2206 showed a good effect, and the effect of other two sequences were limited (18,19). MK-2206 can increase the effect of cisplatin in high p-Akt activation gastric cancer AGS cells, but the combination effect was poor in the other two gastric cancer cells with lower p-Akt (Fig. 2).

The combination of MK-2206 and cisplatin lead to a synthetic cytotoxic effect in the higher Akt phosphorylation gastric cancer cell line, AGS; however, the combination showed a slight effect in the other two gastric cancer cell lines, which have a low level of Akt phosphorylation. To investigate whether MK-2206 can also increase the apoptosis induced by cisplatin, AGS cells were treated as aforementioned, and were subjected to Annexin V/PI staining. Sequential treatment of AGS cells with 10 µM cisplatin for 12 h followed by 10 µM MK-2206 for a further 24 h induced more apoptosis compared to the monotherapy groups (16.17% in the combination group, 3.17% in the MK-2206 group and 5.33% in the cisplatin group) (Fig. 3).

MK-2206, as a potent Akt inhibitor, significantly decreased the Akt phosphorylation and activation. Subsequently, the effect of the potential inhibition of the Akt pathway by cisplatin and MK-2206 combination was analyzed. The results of western blot analysis (Fig. 4) showed that the combination can markedly decrease the p-Akt (Ser473) in AGS cells, compared with control and cisplatin monotherapy groups; however, the total Akt remained stable in all groups. PARP is recognized as a marker of apoptosis, so therefore the effect of the combination on the expression of cleaved PARP was examined. In the untreated group, the expression of cleaved PARP was limited, and the combination group induced more expression of cleaved PARP in contrast to the cisplatin or MK-2206 alone groups, and this was in accordance with the results of the MTT and apoptosis assays.