MK-2206 co-treatment with 5-fluorouracil or doxorubicin enhances chemosensitivity and apoptosis in gastric cancer by attenuation of Akt phosphorylation

Abstract

The anticancer effect of MK-2206, an Akt inhibitor, has been explored in some types of cancers, but its effect on gastric cancer is unclear. In this study, we aimed to investigate its anticancer effect in gastric cancer cells. Cell viability and colony formation assays showed that MK-2206 effectively inhibited the proliferation of SGC-7901 and MKN45 cells. The 50% inhibitory concentration values after 24, 48, and 72 hours' treatment were 22.92, 13.68, and 8.55 μM in SGC-7901 cells and 19.21, 13.10, and 9.11 μM in MKN45 cells, respectively. Treatment with MK-2206 induced apoptosis in SGC-7901 cells as indicated by flow cytometry assay. The combination indexes of MK-2206 and doxorubicin were 0.59 in SGC-7901 cells and 0.57 in MKN45 cells, whereas for 5-fluorouracil (5-FU) the indexes were 0.17 in SGC-7901 cells and 0.73 in MKN45 cells, indicating that MK-2206 could work synergistically with doxorubicin or 5-FU to inhibit cell growth. Furthermore, a small dose (1 μM) of MK-2206 co-treatment with doxorubicin or 5-FU was sufficient for complete inhibition of chemotherapeutic alteration of phosphorylated Akt expression and significant enhancement of pro-apoptosis effect through the activation of caspase pathway. Therefore, MK-2206 effectively inhibits gastric cancer cell growth by attenuation of Akt phosphorylation and synergistically enhances the antitumor effect of doxorubicin and 5-FU via caspase-dependent apoptosis.

Keywords: Akt pathway; MK-2206; apoptosis; caspase; chemotherapy; gastric cancer.

Figure 1

MK-2206 attenuated proliferation of gastric cancer cells. Notes: (A) MK-2206 significantly inhibited proliferation of SGC-7901 and MKN45 cells. Gastric cancer cells (3,000 per well) were treated with MK-2206 at various concentrations (2–32 μM) or vehicle for 24, 48, and 72 hours, respectively. Cell viability was determined using CellTiter Cell Proliferation Assay. (B) Effect of MK-2206 on the colony formation of SGC-7901 cells. Treatment with MK-2206 significantly suppressed the colony formation ability of SGC-7901 cells. Photographs are representative of three independent experiments (left panel); enlarged photographs are ×40 magnification images of representative colonies (middle panels). The scale bar represents 100 μm. Right panel shows quantitative analysis of colony formation efficiencies (*P<0.01).

Figure 2

MK-2206-induced apoptosis in SGC-7901 cells. Notes: (A) Representative microscopic images of SGC-7901 cells treated with vehicle or MK-2206 (1, 2, 4, 8, and 16 μM) for 72 hours. Morphological changes such as shrinkage, aggregation, and spherical and micronucleation were observed as the concentrations of MK-2206 increased. The scale bars represent 50 μm. (B and C) Cells were treated with MK-2206 (0, 1, 2, 4, 8, and 16 μM) for 72 hours and apoptosis was evaluated with Annexin-V–PI assay. The upper right quadrant (UR; Annexin-V⁺/PI⁺) indicates late apoptosis, and the lower right quadrant (LR; Annexin-V⁺/PI⁻) indicates early apoptosis. The experiment was performed in triplicate and representative data are shown. Cell percentages were indicated in quadrants. *P<0.05. Abbreviation: PI, propidium iodide.

Figure 3

Cell viability difference between the combination treatment and two corresponding single treatments. Notes: Cells were simultaneously treated with MK-2206 and doxorubicin (or 5-FU) at a fixed concentration ratio for 72 hours. Cell growth was evaluated by CellTiter Cell Proliferation Assay. CI values were calculated according to the equation of median-effect method of Chou. (A) SGC-7901 and MKN45 cells were treated with MK-2206 alone, doxorubicin alone, or MK-2206 and doxorubicin combination. (B) SGC-7901 and MKN45 cells were treated with MK-2206 alone, 5-FU alone, or MK-2206 and 5-FU combination. Abbreviations: 5-FU, 5-fluorouracil; CI, combination index.

Figure 4

Effect of Akt inhibitor MK-2206 on Akt phosphorylation (p-Akt) in gastric cancer cells. Notes: (A) Dose-dependent response. SGC-7901cells were treated with MK-2206 at the indicated concentration for 12 and 48 hours, respectively. Cell lysates were subjected to Western blotting using anti-phospho-Akt (specific to Ser473, p-Akt) and anti-Akt1/2. GAPDH was used as loading control. (B) MK-2206 inhibited the activation of Akt Ser473 induced by doxorubicin. Cells were treated with doxorubicin (0.1, 0.2, 0.8 μM) in the presence of MK-2206 (1 μM) for 24 hours. Cell lysates were prepared, and p-Akt expression level was determined by Western blotting. (C) Time-dependent effect of MK-2206 and doxorubicin on p-Akt in SGC-7901 cells. Cells were treated with 1 μM MK-2206 alone, 0.2 μM doxorubicin alone, or both agents for 12, 24, 36, and 48 hours. Expression levels of p-Akt and total Akt were determined by Western blotting. Abbreviations: Dox, doxorubicin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Effect of MK-2206 on induction of caspase cascade by doxorubicin or 5-FU. Notes: SGC-7901 cells were treated with (A) doxorubicin (0, 0.1, 0.2, 0.8 μM) or (B) 5-FU (0, 12, 24, 96 μg/mL) in the presence of MK-2206 (1 μM) or DMSO vehicle for 24 hours. Cell lysates were prepared, and the amount of caspase-3, -7, -9, PARP and cleaved caspase-3, -7, -9, PARP were determined by Western blotting. Abbreviations: Dox, doxorubicin; 5-FU, 5-fluorouracil; DMSO, dimethylsulfoxide; PARP, poly ADP ribose polymerase.