Dual inhibition of autophagy and PI3K/mTOR pathway as a potential therapeutic strategy against laryngeal squamous cell carcinoma

Abstract

Background: New and effective chemotherapy or targeted therapy strategies are needed against laryngeal squamous cell carcinoma (LSCC). We aimed to explore the antitumor effect of dual PI3K/mTOR inhibitor combined with autophagy suppression on LSCC and its underlying mechanism.

Methods: Hep-2 and AMC-HN-8 cell lines were treated with the Akt inhibitor LY294002, mTOR inhibitor rapamycin, and dual inhibitor NVP-BEZ235 separately. The biological characteristics of in vitro proliferation, cell cycle, apoptosis, migration, invasion, and autophagy were analyzed, and the expression levels of PI3K/Akt/mTOR pathway-related proteins were also measured. The in vivo effects of NVP-BEZ235 combined with inhibition of autophagy using pharmacological inhibitor was further assessed.

Results: Compared with Akt or mTOR inhibitor, NVP-BEZ235 had the most significant biological effects on LSCC cells. When combined with various autophagy inhibitors, along with siRNA against ATG7, NVP-BEZ235 showed a synergic antitumor effect in LSCC through increasing cell apoptosis and death both in vitro and vivo.

Conclusions: NVP-BEZ235 exerted potent antitumor effects on LSCC, especially when combined with the autophagy inhibitor both in vitro and vivo, providing convincing experimental data for new molecular targeted therapy for LSCC.

Keywords: Laryngeal squamous cell carcinoma (LSCC); PI3K/mTOR inhibitors; autophagy.

Figure 1

Cell cycle is affected by inhibitors of PI3K/mTOR pathway. (A) FCM analysis of cultured Hep-2 and AMC-HN-8 cells. (B) Cell cycle analysis of the Hep-2 and AMC-HN-8 cell lines. Cells in G2/M phase were increased by NVP-BEZ235 in both cell lines. Columns, means of duplicate measurements; bars, ± SD. (C) Western blotting analysis of the expression levels of cyclin D1 and cyclin E2 in two cell lines treated with three different PI3K/mTOR inhibitors. Cyclin D1 and cyclin E2 decreased significantly after treatment with NVP-BEZ235. (D,E) The expression levels were evaluated by ImageJ and analyzed with 2way ANOVA; bars, ± SD; **, P<0.001. FCM, flow cytometric.

Figure 2

Cell apoptosis is increased by inhibitors of PI3K/mTOR pathway. (A) FCM analysis of the cell apoptosis of cultured Hep-2 and AMC-HN-8 cell lines. (B) Cell apoptosis was increased by all three inhibitors in both cell lines; ****, P<0.0001. The apoptosis increase was similar with Ly294002 and rapamycin but was more pronounce with NVP-BEZ235; ****, P<0.0001. Columns, means of duplicate measurements; bars, ± SD. (C) Caspases 8, 9, and 3 expression in the Hep-2 and AMC-HN-8 cell lines increased after NVP-BEZ235 treatment. (D,E) The expression levels were evaluated by ImageJ and analyzed with 2way ANOVA; bars, ± SD; *, P<0.05; **, P<0.001. FCM, flow cytometric.

Figure 3

Protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway of Hep-2 and AMC-HN-8 cell lines, assessed by western blotting. (A) Expression of molecules in PI3K/mTOR signaling pathway in the Hep-2 and AMC-HN-8 cell lines after treated with inhibitors. (B,C) The expression levels were evaluated by ImageJ and analyzed with 2way ANOVA; bars, ± SD; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure 4

Cell migration and invasion is modulated by PI3K/mTOR inhibitors. (A,B) Representative images of the Transwell migration and invasion assay at 24 h in different treatment groups (LY294002, rapamycin, NVP-BEZ235 and DMSO as control group) in Hep-2 and AMC-HN-8 cell lines (HE stain). (C,D) Cell quantification showed a significant decrease in migration and invasion by all three inhibitors in both cell lines, and NVP-BEZ235 showed a stronger effect. (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; scale bar = 100 µm). HE, haemotoxylin and eosin.

Figure 5

Cell autophagy and of PI3K/Akt/mTOR pathway inhibition. (A) FCM analyses of the cell apoptosis of cultured Hep-2 cells or AMC-HN-8 cells. (B,C) Cell apoptosis increased in both cell lines treated with a combination of NVP-BEZ235 and autophagy inhibitors. Columns, means of duplicate measurements; bars ± SD. (D,E) Cell death increased in both cell lines treated with a combination of NVP-BEZ235 and autophagy inhibitors. (F,H) Western blotting of LC3II and LC3I expression in two cell lines treated with three PI3K inhibitors. (G,I) Western blotting analysis showed LC3II/I ratio decreased significantly following treatment of both cell lines with Ly294002, rapamycin, and NVP-BEZ235. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. FCM, flow cytometric.

Figure 6

Anti-tumor efficacy of the combination of NVP-BEZ235 with autophagy inhibitor in vivo. (A) Tumors obtained from mice and their immunohistochemical results. (B,C) Tumor volumes increased with days, and their analysis results. Points, mean of four replicate determinations; bars ± SD. (D) Differences in tumor weights between the four groups. Points and columns, mean of four replicate determinations; bars ± SD. (E) Western blotting testing for LC3II and LC3I expression of tumors from the four groups and the corresponding analysis. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure 7

Mechanism of the anti-tumor affect following the treatment of PI3K/mTOR and autophagy inhibitors. Inhibitors of PI3K/mTOR pathway resulted in decreased Akt and mTOR phosphorylation, subsequently regulates downstream molecules and influence cell apoptosis, autophagy (blue arrow). The combination of autophagy inhibitors weakens the promoting effect of autophagy caused by the inhibition of mTOR, therefore exerts synergistic antitumor effect.