Hyperoside induces both autophagy and apoptosis in non-small cell lung cancer cells in vitro

Abstract

Aim: Hyperoside (quercetin-3-O-β-D-galactopyranoside) is a flavonol glycoside found in plants of the genera Hypericum and Crataegus, which exhibits anticancer, anti-oxidant, and anti-inflammatory activities. In this study we investigated whether autophagy was involved in the anticancer mechanisms of hyperoside in human non-small cell lung cancer cells in vitro.

Methods: Human non-small cell lung cancer cell line A549 was tested, and human bronchial epithelial cell line BEAS-2B was used for comparison. The expression of LC3-II, apoptotic and signaling proteins was measured using Western blotting. Autophagosomes were observed with MDC staining, LC3 immunocytochemistry, and GFP-LC3 fusion protein techniques. Cell viability was assessed using MTT assay.

Results: Hyperoside (0.5, 1, 2 mmol/L) dose-dependently increased the expression of LC3-II and autophagosome numbers in A549 cells, but had no such effects in BEAS-2B cells. Moreover, hyperoside dose-dependently inhibited the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, but increased the phosphorylation of ERK1/2 in A549 cells. Insulin (200 nmol/L) markedly enhanced the phosphorylation of Akt and decreased LC3-II expression in A549 cells, which were reversed by pretreatment with hyperoside, whereas the MEK1/2 inhibitor U0126 (20 μmol/L) did not blocked hyperoside-induced LC3-II expression. Finally, hyperoside dose-dependently suppressed the cell viability and induced apoptosis in A549 cells, which were significantly attenuated by pretreatment with the autophagy inhibitor 3-methyladenine (2.5 mmol/L).

Conclusion: Hyperoside induces both autophagy and apoptosis in human non-small cell lung cancer cells in vitro. The autophagy is induced through inhibiting the Akt/mTOR/p70S6K signal pathways, which contributes to anticancer actions of hyperoside.

Figure 1

Hyperoside induces autophagy in human non-small cell lung cancer cell lines A549. (A) Conversions of LC3-Ι to LC3-II were determined by Western blotting with an antibody against LC3A/B after A549 cells were treated with various concentrations of hyperoside for 48 h. β-Actin was a loading control. The bar chart shows semiquantitative analysis of LC3-II levels relative to actin using three independent experiments. (B) A549 cells treated with 2 mmol/L hyperoside for 6–48 h were analyzed by Western blotting with antibodies against LC3 A/B and β-actin. The bar chart shows semiquantitative analysis of LC3-II levels relative to actin using three independent experiments. (C) Monodansycadaverine (MDC) staining. A549 cells treated with hyperoside (2 mmol/L) for 48 h were incubated with MDC (0.05 mmol/L) for 20 min and observed under a fluorescence microscope. Inserts are high magnification micrographs of the boxed regions (scale bar, 10 μm). (D) A549 cells treated with various concentrations of hyperoside for 48 h were analyzed by immunoblotting with antibodies against p62 and β-actin. The bar chart shows semiquantitative analysis of p62 levels relative to actin using three independent experiments. (E) A549 cells transfected with GFP-LC3 expression vector for 4 h were subsequently treated with hyperoside (2 mmol/L) for 48 h. The GFP-LC3 dots induced by hyperoside in A549 cells were observed with a confocal microscope. (F) A549 cells treated with 2 mmol/L hyperoside for 48 h were stained with antibodies against LC3A/B. They were analyzed with confocal microscopy. Inserts are high magnification micrographs of the boxed regions (scale bar, 10 μm). The data are expressed as the mean±SEM of 3 independent experiments. ^cP<0.01 compared with the control group.

Figure 2

Hyperoside induced autophagic flux in a human non-small cell lung cancer cell line. (A) A549 cells treated with hyperoside (2 mmol/L) with or without E64d (10 μg/mL) and pepstatin A (10 μg/mL) were analyzed by immunoblotting with antibodies against LC3A/B and β-actin. The bar chart shows semiquantitative analysis of LC3 levels relative to β-actin using three independent experiments. (B) A549 cells transfected with GFP-LC3 expression vector for 24 h were treated with (2 mmol/L) hyperoside for 48 h. Conversions of GFP-LC3 and endogenous LC3 were determined by Western blotting. β-Actin was a loading control. The data are expressed as the mean±SEM of 3 independent experiments. ^cP<0.01 compared with the control group. ^eP<0.05 compared with the E64d and pepstatin A group.

Figure 3

Hyperoside inhibits the Akt/mTOR/p70S6 signaling pathway and activates ERK1/2 signaling in A549 cells. A549 cells treated with hyperoside (0.5, 1, and 2 mmol/L) for 24 h were analyzed by Western blotting against phospho-p70S6K (Thr389) and total p70S6K (A), phospho-4E-BP1 (Thr37/46) and total 4E-BP1 (B), phospho-mTOR (Ser2448) and total mTOR (C), phospho-Akt (Ser473) and total Akt (D), and phospho-ERK1/2 and total ERK1/2 (E). β-Actin was a loading control. Group data showing normalized phosphorylated or total kinases to β-Actin were determined in each group of three experiments. (F) A549 cells treated with 2 mmol/L hyperoside for 6, 12, or 24 h were analyzed by immunoblotting for levels of phospho-mTOR and phospho-p70S6K. β-actin was a loading control. The data are expressed as the mean±SEM of 3 independent experiments. ^bP<0.05, ^cP<0.01 compared with the control group.

Figure 4

Hyperoside-induced autophagy is dependent on the PI3K/Akt/mTOR pathway. (A and B) A549 cells treated with hyperoside (2 mmol/L) for 4 h followed by treatment with or without 200 nmol/L insulin for 30 min were analyzed by Western blotting against phospho-Akt (A) or LC3A/B (B). Group data showing normalized phosphorylated Akt or LC3-II to β-actin were determined in each group of three experiments. The data are expressed as the mean±SEM. ^cP<0.01 compared with the control group. ^fP<0.01 compared with insulin group. (C and D) A549 cells treated with hyperoside (2 mmol/L), 30 μmol/L triciribine, or hyperoside (2 mmol/L) with 30 μmol/L triciribine for 12 h were analyzed by immunoblotting against phospho-Akt (C) or LC3A/B (D). Group data showing normalized phosphorylated Akt or LC3-II to β-actin were determined in each group of three experiments. The data are expressed as the mean±SEM. ^bP<0.05, ^cP<0.01 compared with the control group. ^fp<0.01 compared with triciribine group. (E and F) A549 cells treated with 2 mmol/L hyperoside with or without 20 μmol/L U0126 for 12 h were analyzed by immunoblotting using antibodies against phospho-ERK (E) or LC3A/B (F). Group data showing normalized phosphorylated ERK or LC3A/B to β-actin were determined in each group of three experiments. The data are expressed as the mean±SEM. ^cP<0.01 compared with the control group. ^fP<0.01 compared with hyperoside group.

Figure 5

Rapamycin treatment with hyperoside shows an enhancement of autophagy. (A) A549 cells treated with rapamycin (1 μmol/L) with or without 2 mmol/L hyperoside for 48 h were analyzed by Western blotting with antibodies against LC3A/B and β-actin. The bar chart shows the LC3-II/actin ratio using three independent experiments. The data are expressed as the mean±SEM of 3 independent experiments. ^cP<0.01 compared with the control group. ^eP<0.05 compared with the single treatment of hyperoside. (B) A549 cells treated with hyperoside (2 mmol/L) with or without rapamycin (1 μmol/L) for 48 h were incubated with MDC (0.05 mmol/L) for 20 min and observed under a fluorescence microscope.

Figure 6

Autophagy is involved in hyperoside-induced A549 cell death. (A) A549 cells (5×10³ cells/well in 96-well plate) were treated with different concentrations of hyperoside (0.1, 0.5, 1, and 2 mmol/L) for 24 h or 48 h. Cell viability was analyzed by the MTT assay. (B) After A549 cells (5×10³ cells/well in 96-well plate) were treated with different concentrations of hyperoside (0.1, 0.5, 1, and 2 mmol/L) for 48 h, cell proliferation was measured using the BrdU incorporation assay. (C and D) A549 cells were pretreated with 2.5 mmol/L 3-MA for 1 h before the addition of 2 mmol/L hyperoside for an additional incubation of 24 or 48 h. Cellular proteins of the 48 h treatment sample were lysed and subjected to Western blot analysis with an anti-LC3A/B antibody. The bar chart showed LC3-II/β-actin ratio using three independent experiments (C). Cell viability was analyzed by the MTT assay (D). The data are expressed as the mean±SEM of 3 independent experiments. ^bP<0.05, ^cP<0.01 compared with the control group. ^fP<0.01 compared with the single treatment with hyperoside (2 mmol/L).

Figure 7

Inhibition of autophagy reduced hyperoside-induced apoptotic cell death. (A) A549 cells were treated with hyperoside (0.5, 1 or 2 mmol/L) for 48 h and analyzed with immunoblotting for levels of caspase-3 and cleaved caspase-3. The bar chart shows semiquantitative analysis of caspase-3 or cleaved caspase-3 to β-actin using three independent experiments. (B) A549 cells were treated with hyperoside (0.5, 1 or 2 mmol/L) for 48 h and analyzed with immunoblotting for cleaved PARP. The bar chart shows semiquantitative analysis of cleaved PARP. (C) A549 cells were treated with hyperoside (2 mmol/L) with or without 3-MA (2 mmol/L) for 48 h and analyzed with immunoblotting for cleaved caspase-3 and cleaved PARP. The bar chart shows cleaved caspase-3 or cleaved PARP/β-actin ratio. The data are expressed as the mean±SEM of 3 independent experiments. ^bP<0.05, ^cP<0.01 compared with the control group. ^fP<0.01 compared with the single treatment with hyperoside (2 mmol/L). (D) A549 cells treated with hyperoside (2 mmol/L) with or without 3-MA (2 mmol/L) for 48 h were incubated with MDC (0.05 mmol/L) for 20 min and observed under a fluorescence microscope.

Figure 8

Effect of hyperoside on autophagy in human bronchial epithelial cells BEAS-2B. (A) Conversions of LC3-Ι to LC3-II were determined by Western blotting with an antibody against LC3A/B after BEAS-2B cells were treated with various concentrations of hyperoside for 48 h. β-actin was a loading control. The bar chart shows semiquantitative analysis of LC3-II levels relative to β-actin using three independent experiments. The data are expressed as the mean±SEM of 3 independent experiments. (B) BEAS-2B cells treated with hyperoside (2 mmol/L) for 48 h were incubated with MDC (0.05 mmol/L) for 20 min and observed under a fluorescence microscope.