MK-2206, an Akt inhibitor, enhances carboplatinum/paclitaxel efficacy in gastric cancer cell lines

Abstract

Background: Several molecularly-targeted agents are being evaluated in gastric cancer cell lines. In this study we evaluated the synergistic potential of MK-2206, an oral potent allosteric Akt inhibitor, in combination with chemotherapeutic agents in human gastric cancer cell lines.

Materials and methods: We evaluated effects of MK-2206 on cell growth and cell signaling using a panel of gastric cancer cell lines AGS, SNU-1 and SNU 16. The analysis of drug combinations was conducted by using CellTiter-Blue™ Cell Viability Assay which yielded the combination index (CI). MK-2206 and representative chemotherapy agent were further evaluated regarding their effects on Akt inhibition and downstream targets using western blots probed with the appropriate antibodies. We assessed the combination of MK-2206 and chemotherapy in three different treatment sequences.

Results: We demonstrated in vitro synergistic efficacy of MK-2206 when combined with carboplatinum and paclitaxel in the three cell lines examined. Efficacy was dose dependent. We assessed the combination of MK-2206 and carboplatinum/paclitaxel in three different treatment sequences; 24 h of exposure to combination chemotherapy followed by a 48 h exposure to MK-2206 resulted in the highest synergistic antiproliferative effect in all cell lines. On the other hand, the reverse sequence (MK-2206 followed by chemotherapy) and the concurrent treatment schedule were slightly synergistic or additive as well. The effects of MK-2206 on p-Akt and other downstream targets was reported.

Conclusions: Our findings suggest that Akt inhibition augments the efficacy of existing gastric cancer therapeutics (carboplatinum and paclitaxel); thus, MK-2206 is a promising agent to treat gastric cancer patients who receive these cytotoxic agents. The magnitude of synergy depended on the treatment sequence; a schedule of MK-2206 dosed before or concurrently with chemotherapy was not as effective as being dosed after chemotherapy. Further experiments addressing MK-2206's mechanism of action in combination with chemotherapy are needed.

Keywords: MK-2206; carboplatinum; gastric cancer; paclitaxel.

Figure 1. Effect of MK2206 plus carboplatinum (carbo) paclitaxel (Taxol) combinations on cell viability in three different gastric cancer cell lines: AGS, SNU-1, and SNU-16. The growth inhibition produced by titrated concentrations of MK2206 (blue lines; concentrations indicated on the bottom horizontal axis) and Carbo/Taxol (400:1 ratio) (red lines; concentrations indicated on top horizontal axis) is plotted against viability (% of untreated control). (A) Concurrent treatment (72 h) MK-2206 and Carbo/Taxol 1:8 ratio. (B) Sequential treatment; cells were treated with MK-2206 for 24 h then Carbo/Taxol combination was added for 48 h 1:8 ratio. (C) Sequential treatment; cells were treated with Carbo/Taxol for 24 h then MK-2206 was added for 48 h 1:2 ratio.

Figure 2. Effect of MK-2206 with and without chemotherapy on the PI3K pathways. Gastric cells were treated with carboplatinum/paclitaxel in the presence or absence of MK-2206. Cell lysates were prepared, and the levels of Akt and pAkt, were determined by western blot probed with the indicated antibodies. MDA-MB 468 cell lines were used as positive control.

Figure 3. PARP cleavage analysis in AGS cells by western blotting with anti-PARP. PARP proenzyme (116 kD) and cleaved subunit (85 kD) are indicated. AGS cell treated with MK-2206 and chemotherapy showed higher cleaved/uncleaved PARP.

Figure 4. MK-2206 synergizes with CarboTaxol in reducing anchorage-independent growth in three gastric cancer cell lines. AGS cells treated with 100 µM MK-2206, 100 nM CarboTaxol (1000:1 molar ratio of carboplatinum and paclitaxel respectively) or combination for 2 weeks. MK-2206 was re-dosed twice weekly. *The CarboTaxol and MK-2206 combination significantly reduced colony counts relative to CarboTaxol alone (unpaired t test P values were 0.002, 0.001, and 0.03 for AGS, SNU-1, and SNU-16 cell lines, respectively).