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Introduction

Gastric cancer is a type of common and fatal digestive malignant disease, particularly in eastern Asia (1), which has a tendency to occur as metastasis in the early stage. Despite the progress in earlier diagnosis and new therapeutic strategies, which improved the overall prognosis for gastric cancer patients, advanced gastric cancer remains a rigorous challenge for clinical staff, and the median overall survival is <1-year, and 5-year survival is ~7% for metastasis gastric cancer patient (2). Chemotherapy is an extremely important therapeutic strategy for advanced gastric cancer; however, chemotherapy resistance is a severe obstacle (3), particularly for patients who make progression or reoccur subsequent to finishing their first-line treatment, and these patients are rarely suitable for additional treatment as tumor cells show a poor response to any cytotoxic drugs or drug combinations (4).

To overcome the dilemma induced by chemotherapy resistance, numerous molecular-targeted agents have been designed and considered as new therapeutic strategies for several malignant diseases (5,6), such as trastuzumab for Her-2 positive in breast cancer and gastric cancer (7,8), and a number of other attractive molecular-targeted agents have also been merged and have shown promising data in preclinical trials (9–11).

The phosphoinositide 3-kinase (PI3K)/Akt pathway dysregulation has been widely reported in different types of malignant diseases (12,13), such as the PIK3CA gene amplification or activation mutation, overactivation of the downstream molecule Akt, and loss of tumor suppressor phosphatase and tensin homolog (14). In gastric cancer, the abnormalities of the PI3K/Akt pathway are considered to have an important role in carcinogenesis, proliferation and resistance to chemotherapy drugs (15,16). Additionally, as reported in a previous study, the treatment of conventional chemotherapy drugs can induce further activation of the PI3K/Akt pathway in a number of solid cancers (17), which may lead to the occurrence of acquired drug resistance. Therefore, we hypothesize that Akt inhibition may increase the efficiency of chemotherapy and reverse the drug resistance.

MK-2206 is a highly selective oral Akt inhibitor, which has a higher inhibition effect on Akt1 and Akt2, compared to Akt3. In vitro, MK-2206 is found to have a synergistic effect with conventional chemotherapy agents in ovarian cancer and osteosarcoma (18,19). However, to the best of our knowledge, there are no previous studies evaluating the combination effect of cisplatin and MK-2206 in gastric cancer.

Materials and methods

Cell culture

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.

Drug sensitivity assay

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).

Apoptosis assay by flow cytometry

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.

Western blot analysis

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).

Statistical analysis

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.

Results

Gastric cancer cell line with the higher Akt phosphorylation is more resistant to cisplatin

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.

Figure 1. Expression of p-Akt (Ser473) and half maximal inhibitory concentration (IC50) of cisplatin in gastric cancer cell lines. (A) The results of western blot analysis showed that the expression of p-Akt (Ser473) was highest in the gastric cancer AGS cell line. (B) From the results of the MTT assay, the IC50 of cisplatin in the AGS cells was also the highest in the three cell lines. The mean IC50 for AGS, MKN-45 and MGC-803 cells were 8.48, 1.89 and 0.89 µM.

Akt inhibitor MK-2206 increases the effect of cisplatin with a sequence-dependant manner 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).

Figure 2. Effect of the cisplatin and MK-2206 combination in the gastric cancer cell lines, AGS, MGC-803 and MKN-45. Following treatment with cisplatin for 24 h, followed with MK-2206 for another 48 h, the results of the MTT assay showed that MK-2206 can significantly increase the effect of cisplatin in AGS cells (P<0.05), but MK-2206 showed a slight combination effect in the other two cell lines.

MK-2206 can increase the apoptosis induced by cisplatin in gastric cancer cell line AGS

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).

Figure 3. Effect of the cisplatin and MK-2206 combination on the apoptosis in AGS cells. The mean apoptosis rate in the combination groups was 16.17%, which was higher compared to the MK-2206 and cisplatin monotherapy groups (3.17 and 5.33%).

MK-2206 inhibits the expression of p-Akt and increases the expression of cleaved PARP in the gastric cancer AGS cell line

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.

Figure 4. Effect of cisplatin and MK-2206 combination on the expression of total Akt, p-Akt and cleaved poly adenosine diphosphate ribose polymerase (PARP). MK2206 can significantly inhibit the expression of p-Akt (Ser473), and there was no difference in the level of total Akt in these four groups. In the combination group, the level of cleaved PARP was higher compared to the other three groups. DMSO, dimethyl sulfoxide.

Discussion

The limited effectiveness of chemotherapy drugs can be attributed to several reasons, such as checkpoints that arrest the cell cycle to allow DNA damage repair, or activation of certain signaling pathways to help the cancer cell escape from the cytotoxicity induced by numerous drugs (20,21). Therefore, targeting survival pathways may be logically considered as a good strategy for increasing the chemotherapeutic effect in cancer cells (22,23).

The PI3K/Akt pathway has been widely investigated, and was reported to participate in several biological and pathological responses in numerous types of cancer, including gastric cancer. Numerous molecules targeting the PI3K/Akt pathway have been designed (24), MK-2206 is one of these agents, although the monotherapy effect of MK-2206 is limited in a number of cancers, and certain studies have reported that it showed a good therapeutic effect when combined with other traditional chemotherapy drugs (25).

The present study evaluated the benefit of the combination of cisplatin and MK-2206 in gastric cancer cell lines, and detected the effect of the Akt inhibitor MK-2206 on gastric cancer cell lines alone and in combination of cisplatin. The monotherapy effect of MK-2206 was not significant in the three cell gastric cancer cell lines; however, when treating the AGS cells with cisplatin followed by MK-2206, an increase in apoptosis and proliferation inhibition was induced in gastric cancer cells. Western blot analysis also showed that the expression of p-Akt (Ser473) was significantly blocked in the combination group, and consistently, the level of the apoptosis marker, cleaved PARP, was higher compared to the monotherapy groups.

The present study has certain limitations. The number of gastric cancer cell lines used in the experiments is limited, there is no in vivo experiment evaluation and the mechanisms for the effect remain to be elucidated. However, the data appear promising, and may be helpful for high Akt phosphorylation gastric cancer patients.

Acknowledgements

The present study was sponsored by a grant from the National Nature Science Foundation of China (no. 81172294).

References