Ophiopogonin B-induced autophagy in non-small cell lung cancer cells via inhibition of the PI3K/Akt signaling pathway

Abstract

Ophiopogonin B (OP-B) is a bioactive component of Radix Ophiopogon Japonicus, which is often used in Chinese traditional medicine to treat pulmonary disease. However, whether or not OP-B has any potential antitumor activity has not been reported. Here, we show that the non-small cell lung cancer (NSCLC) cell lines NCI-H157 and NCI-H460 treated with OP-B grow more slowly and accumulate vacuoles in their cytoplasm compared to untreated control cells. Flow cytometric analysis showed that the cells were arrested in G0/G1 phase. Nuclear morphology, Annexin-V/PI staining, and expression of cleaved caspase-3 all confirm that OP-B does not induce apoptosis. Instead, based on results from both transmission electron microscopy (TEM) and the expression of microtubule-associated protein 1 light chain 3-II (LC3-II), we determined that OP-B treatment induced autophagy in both cell lines. Next, we examined the PI3K/Akt/mTOR signaling pathway and found that OP-B inhibited phosphorylation of Akt (Ser473, Thr308) in NCI-H157 cells and also inhibited several key components of the pathway in NCI-H460 cells, such as p-Akt(Ser473, Thr308), p-p70S6K (Thr389). Additionally, insulin-mediated activation of the PI3K/Akt/mTOR pathway provides evidence that activation of this pathway may correlate with induction of autophagy in H460 cells. Therefore, OP-B is a prospective inhibitor of PI3K/Akt and may be used as an alternative compound to treat NSCLC.

Figure 1

Dose-dependent effect of OP-B on cell viability in NSCLC cell lines. (A) Structural formula of OP-B. (B) Cell viability of eleven NSCLC cell lines 3 days after exposure to increasing doses of OP-B, actual cell numbers were counted with Alamar blue dye. Results shown are the means of three independent experiments; bars, SD.

Figure 2

Effects of OP-B on NCI-H157 and H460 cell cycle and apoptosis. (A and B) NCI-H157 and H460 cells incubated with varying concentrations of OP-B for 24 h: (A) cell images were taken by phase-contrast microscope; (B) cell number percentage in each phase (sub-G1, G0/G1, S, and G2/M) was calculated and expressed. (C) NCI-H157 and H460 cells treated with 10 μmol/l OP-B for 24 h were analyzed by immunoblotting with antibodies against caspase-3, Bcl-2 and actin. (D) NCI-H460 cells treated with 10 μmol/l OP-B for 12, 24, 48 h or 1 μmol/l Staurosporine for 12 h were analyzed by immunoblotting with antibodies against cleaved caspase-3 and actin. (E) NCI-H157 and H460 cells treated with 10 μmol/l OP-B or 1 μmol/l Staurosporine for 24 h were stained by Hoechst 33258 and observed by fluorescence microscopy. (F) Cells were incubated with OP-B for 24 h and then fixed and stained with Hoechst 33258 and Alexa Fluor 488 Annexin-V/Dead cell apoptosis kit. Images of cells were taken by the high content screening (HCS) KineticScan Reader (x200).

Figure 3

OP.B dose- and time-dependently induces the formation of LC3-II, and induced autophagic vacuole in NCI-H157 and H460 cells. (A) Transmission electron microscopic examination of NCI-H157 and H460 cells treated with 10 μmol/l OP-B for 48 h. Numerous autophagical vacuoles with typical double-layer membrane containing organelle remnants are highlighted by arrows. (B and D) NCI-H157 and H460 cells treated with various concentrations of OP-B for 24 h or 10 μmol/l OP-B for 0, 1, 3, 6, 12 h were analyzed by immunoblotting with antibodies against LC3 and actin. (C and E) Densitometry analysis of LC3- II levels relative to actin was performed using three independent experiments. Error bars, SD; ^**p<0.01; ^***p<0.001.

Figure 4

Effect of OP-B on PI3K/Akt/mTOR/ p70s6k signaling pathway in NCI-H157 and H460 cells. (A and B) The NCI-H157 and H460 cells treated with 0, 2.5, 5, 10 μmol/l OP-B for 1.5 h or 10 μmol/l of OP-B for 0, 0.5, 1 or 2 h were analyzed by immunoblotting with antibodies against p-Akt (Ser473), p-p70S6K (Thr389), p-4EBP1 (Thr37/46), Akt, p70S6K, 4EBP1, and actin. (C and D) The NCI-H157 and H460 treated with 10 μmol/l OP-B, 10 μmol/l LY294002 or 10 μmol/l Rapmycin for 4 h were analyzed by immunoblotting with antibodies against p-PDK1 (Ser241), p-Akt (Thr308), p-Akt (Ser473), p-p70S6K (Thr389), LC3, and actin. (E) Densitometry analysis of LC3-II levels relative to actin in H460 cells was performed using three independent experiments. Error bars, SD; ^**p<0.01; ^***p<0.001.

Figure 5

Correlation between inhibition of PI3K/Akt/mTOR/p70S6K and the induction of autophagy in H460 cells. (A) cells treated with 10 μmol/l OP-B for 4 h followed by treatment with or without 200 nM insulin for 30 min were analyzed by immunoblotting for levels of p-Akt (Ser473), p-p70S6K (Thr389), LC3 or actin. (B) Densitometry analysis of LC3-II levels relative to actin in H460 cells was performed using three independent experiments. Error bars, SD; ^**p<0.01.