Inhibition of esophageal cancer growth through the suppression of PI3K/AKT/mTOR signaling pathway

Abstract

Background: The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is implicated in several cancers. AKT allosteric inhibitor MK2206 and dual PI3K and mTOR inhibitor BEZ235 are promising drug candidates with potential anti-tumor effects.

Purpose: In this study, we aimed to detect the activation of PI3K/AKT/mTOR pathway and assess the efficacy of MK2206 and BEZ235 in inhibiting esophageal cancer growth.

Materials and methods: We used three different systems including carcinogen-induced animal model, human esophageal squamous cell carcinoma (SCC) cell lines, and xenograft mouse model.

Results: Our data indicated that components of the PI3K/AKT/mTOR pathway were overexpressed and activated in esophageal SCC. MK2206 and BEZ235 inhibited cell proliferation, enhanced apoptosis, and induced cell-cycle arrest through downstream effectors SKP2, MCL-1, and cyclin D1 in esophageal SCC cells. MK2206 and BEZ235 also inhibited tumor growth in xenograft mice through the inhibition of AKT phosphorylation. MK2206/BEZ235 combination showed greater anti-tumor effect than MK2206 or BEZ235 alone. The enhanced efficacy of the combination was associated with the inhibition of phosphorylation ATK on both Thr308 and Ser473.

Conclusion: The combination of MK2206 and BEZ235 exhibits potent antitumor effects and may have important clinical applications for esophageal SCC treatment.

Keywords: BEZ235; Esophageal squamous cell carcinoma; MK2206; PI3K/AKT/mTOR.

Figure 1

Activation of AKT, PI3K, and mTOR in human esophageal cancer cell lines and NMBA-induced rat esophageal SCC. (A) Phosphorylation of AKT, PI3K, and mTOR in normal esophageal epithelial cell line HET-1A and esophageal SCC cell lines KYSE 70, KYSE 150, KYSE 270, and KYSE 410; (B) phosphorylation of AKT, PI3K, and mTOR in rats treated with NMBA.

Figure 2

MK2206 and BEZ235 inhibit cell proliferation and phosphorylation of AKT, PI3K, and mTOR in KYSE 150. (A) MK2206 inhibits cell proliferation in a dose-dependent manner; (B) BEZ235 inhibits cell proliferation in a dose-dependent manner; (C) MK2206 inhibits phosphorylation of AKT, PI3K, and mTOR; and (D) BEZ2356 inhibits phosphorylation of AKT, PI3K, and mTOR. The values are expressed as mean; bars, ±SE.

Figure 3

MK2206 and BEZ235 induce apoptosis and apoptosis-related proteins in KYSE 150 in a dose-dependent manner. (A) MK2206 induces apoptosis; (B) BEZ235 induces apoptosis; (C) MK2206 reduces MCL-1 expression and induces cleaved-caspase 3; and (D) BEZ235 reduces MCL-1 expression and induces cleaved-caspase 3. The values are expressed as mean; bars, ±SE. *P<0.05; **P<0.01; ***P<0.001.

Figure 4

MK2206 and BEZ235 induce G2/M accumulation and cell cycle-related proteins in KYSE 150 in a dose-dependent manner. (A) MK2206 induces G2/M accumulation; (B) BEZ235 induces G2/M accumulation; (C) MK2206 reduces SKP2, CDK2, and cyclin D1; and (D) BEZ235 reduces SKP2, CDK2, and cyclin D1. The values are expressed as mean; bars, ±SE. *P<0.05; ***P<0.001.

Figure 5

Combination of MK2206 and BEZ235 has better antitumor effects than MK2206 and BEZ235 alone. (A) The combination of MK2206 and BEZ235 at lower dose inhibits cell proliferation than MK2206 or BEZ235 alone at higher dose; (B) the combination of MK2206 and BEZ235 at lower dose inhibits AKT phosphorylation at Thr308 and Ser473 better than MK2206 or BEZ235 alone at higher dose; (C) the combination of MK2206 and BEZ235 at lower dose induces apoptosis better than MK2206 or BEZ235 alone at higher dose; and (D) the combination of MK2206 and BEZ235 at lower dose induces G2/M accumulation better than MK2206 or BEZ235 alone at higher dose. The values are expressed as mean; bars, ±SE. **P<0.01; ***P<0.001; ^##P<0.01; ^###P<0.001.

Figure 6

Combination of MK2206 and BEZ235 has better antitumor effects than MK2206 or BEZ235 alone in xenograft animal model. (A and B) The images of the tumors in nude mice and (C) the tumor volume in control and treatment groups. The values are expressed as mean; bars, ±SE. *P<0.05; **P<0.01; ***P<0.001.