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. 2014 Oct 10:8:1827-37.
doi: 10.2147/DDDT.S67961. eCollection 2014.

Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo

Yuan-Yuan Zhao  1 , Ying Tian  1 , Jing Zhang  2 , Fei Xu  1 , Yun-Peng Yang  1 , Yan Huang  1 , Hong-Yun Zhao  3 , Jian-Wei Zhang  4 , Cong Xue  1 , Michael H Lam  5 , Li Yan  5 , Zhi-Huang Hu  1 , Xiao-Xiao Dinglin  6 , Li Zhang  7
Affiliations

Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo

Yuan-Yuan Zhao et al. Drug Des Devel Ther. .

Abstract

Aim: Protein kinase B (AKT) signaling frequently is deregulated in human cancers and plays an important role in nasopharyngeal carcinoma (NPC). This preclinical study investigated the effect of MK-2206, a potent allosteric AKT inhibitor, on human NPC cells in vitro and in vivo.

Methods: The effect of MK-2206 on the growth and proliferation of CNE-1, CNE-2, HONE-1, and SUNE-1 cells was assessed by Cell Counting Kit 8 and colony formation assay. Flow cytometry was performed to analyze cell cycle and apoptosis. The effects of MK-2206 on the AKT pathway were analyzed by Western blotting. Autophagy induction was evaluated via electron microscopy and Western blot. To test the effects of MK-2206 in vivo, CNE-2 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with MK-2206 or placebo. Tumors were harvested for immunohistochemical analysis.

Results: In vitro, MK-2206 inhibited the four NPC cell line growths and reduced the sizes of the colonies in a dose-dependent manner. At 72 and 96 hours, the half maximal inhibitory concentration (IC50) values of MK-2206 in CNE-1, CNE-2, and HONE-1 cell lines were 3-5 μM, whereas in SUNE-1, IC50 was less than 1 μM, and MK-2206 induced cell cycle arrest at the G1 phase. However, our study found no evidence of apoptosis. MK-2206 induced autophagy in NPC cells, as evidenced by electron microscopy and Western blot, and inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of downstream phosphorylation through the PRAS40 and S6 pathways seems to be the main mechanism for the MK-2206-induced growth inhibition.

Conclusion: Our preclinical study suggests that MK-2206's antiproliferative effect may be useful for NPC treatment; however, strategies for reinforcing this effect are needed to maximize clinical benefit.

Keywords: AKT inhibitor; MK-2206; nasopharyngeal carcinoma.

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Figures

Figure 1
Figure 1
Growth-inhibitory effect of MK-2206 on nasopharyngeal carcinoma cell lines. Notes: (A) CNE-1. (B) CNE-2. (C) HONE-1. (D) SUNE-1. Cells were cultured at 2,500–4,000 cells per well in a 96-well plate, exposed to different concentrations of MK-2206, and incubated for 72 and 96 hours. Points, average of three experiments; bars, standard error; solid line, 96 hours test; dashed line, 72 hours test. (E) Average half maximal inhibitory concentration for cell growth in nasopharyngeal carcinoma cell lines after exposure to MK-2206 for 72 and 96 hours. (F) Cells at a density of 500 cells per well were seeded in six-well plates. On the second day, cells were treated with the indicated concentrations of MK-2206. The same treatments were repeated every 3 days. After 10 days, the plates were stained for the formation of cell colonies with methylene blue trihydrate. The picture of the colonies was then taken using a digital camera. One representative experiment is shown (HONE-1). Values are presented as mean ± standard deviation. Abbreviations: IC50, half maximal (50%) inhibitory concentration; CON, control.
Figure 2
Figure 2
MK-2206 induces cell cycle arrest at G1 in a dose-dependent manner in CNE-2 and HONE-1 cells. Notes: #P<0.05 compared with vehicle control. (A) CNE-2 (24 hours). (B) CNE-2 (48 hours). (C) HONE-1 (24 hours). (D) HONE-1 (48 hours). Cell lines were incubated with 0–10 μM MK-2206 for 24 or 48 hours, respectively, and were thereby labeled with propidium iodine, followed by analysis with flow cytometry. Values are presented as mean ± standard deviation. (E) One representative experiment is shown (HONE-1 24 hours). Abbreviation: CON, control.
Figure 3
Figure 3
No apoptosis was induced by MK-2206 in the four nasopharyngeal carcinoma cell lines. Notes: (A) No characteristic apoptotic cells were found in diamidino-2-phenylindole nuclear staining assay. Magnification, ×40. The experiments are repeated twice with similar results. (B) No evidence of apoptosis in any of the four cell lines was seen. Testing was by Annexin V assays, which were carried out 72 hours after a single treatment of cells with MK-2206 at doses as high as 10 μM. One representative experiment is shown (CNE-2 72 hours). Abbreviation: CON, control.
Figure 4
Figure 4
MK-2206 inhibited phosphorylation of AKT downstream targets. Notes: SUNE-1 and CNE-2 cells were treated with different concentrations of MK-2206 for 24 hours. Then the cells were collected and lysed. Western blot was conducted and probed with anti-AKT, anti-phospho-AKT, anti-phospho-PRAS40, anti-phospho-S6, and anti-phospho-GSKα/β. Abbreviations: AKT, Protein kinase B; Con, control; GSK, glycogen synthase kinase; PRAS40, proline-rich-Akt substrate, 40kDA.
Figure 5
Figure 5
Effect of MK-2206 on autophagy in human nasopharyngeal carcinoma cells. Notes: (A) CNE-2 cells cultured in medium supplemented with 10% fetal bovine serum were treated with MK-2206 for 24 hours; the level of light chain 3 was examined by Western blot. Glyceraldehyde 3-phosphate dehydrogenase was used as a loading control. (B) CNE-2 cells treated with MK-2206 (5 μmol/L, 2.5 μmol/L) or vehicle were harvested by trypsinization, fixed, and embedded in spur resin. Ultrathin sections were cut and examined by transmission electron microscope. Arrows indicate autophagic vacuoles. Abbreviation: CON, control.
Figure 6
Figure 6
Effects of MK-2206 on tumor growth of human CNE-2 xenografts in nude mice. Notes: (A) The treatments began on day 1 after grouping (day 0), including 30% Captisol 10 mL/kg once a week, MK-2206 480 mg/kg once a week, and MK-2206 240 mg/kg three times a week for 2 weeks. During treatment, tumor volumes were measured every other day. Points, mean of tumors; bars, standard deviation. #P<0.05 compared with vehicle control; *P<0.01 compared with vehicle control. (B) After 18 days of grouping, the mice were killed and the tumors were removed and weighed. *P<0.01 compared with vehicle control. (C) Body weight was measured every other day and used to assess toxicity of treatment. (D) Tumors were resected, fixed, and paraffin embedded. The sections were analyzed by hematoxylin-eosin (HE) staining and by immunohistochemistry, using p-PRAS40 and p-S6 antibodies. The representative photographs in tumor sections are shown (×400). Abbreviations: tiw, three times per week; qw, once a week; PRAS40, proline-rich-Akt substrate, 40kDA.
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