Inhibition of Non-Small Cell Lung Cancer Proliferation and Survival by Rosemary Extract Is Associated with Activation of ERK and AMPK

Abstract

Non-small cell lung cancer (NSCLC) represents an aggressive form of lung cancer which often develops resistance to chemo- and radiotherapy emphasizing a need to identify novel treatment agents to combat it. Many plants contain compounds with anti-inflammatory, antimicrobial, antidiabetic, and anticancer properties and some plant-derived chemicals are used in the treatment of cancer. A limited number of in vitro and in vivo animal studies provide evidence of anticancer effects of rosemary (Rosmarinus officinalis) extract (RE); however, no studies have explored its role in H1299 NSCLC cells, and its underlying mechanism(s) of action are not understood. The current study examined the effects of RE on H1299 cell proliferation, survival, and migration using specific assays. Additionally, immunoblotting was used to investigate the effects of RE treatment on signalling molecules implicated in cell growth and survival. Treatment with RE dose-dependently inhibited H1299 proliferation with an IC₅₀ value of 19 µg/mL. Similarly, RE dose-dependently reduced cell survival, and this reduction correlated with increased levels of cleaved poly (ADP-ribose) polymerase (PARP), a marker of apoptosis. RE was also able to inhibit cell migration as assessed with a wound healing assay. These cellular effects of RE were associated with an increase in phosphorylated levels of extracellular signal-regulated kinase (ERK), AMP-activated protein kinase (AMPK), and its downstream targets ACC, the mTORC1 protein raptor, and decreased p70S6K phosphorylation. More studies are required to fully examine the effects of RE against NSCLC.

Keywords: AMPK; ERK; apoptosis; lung cancer; mTOR; p70S6K; polyphenolics; rosemary extract.

Figure 1

Effect of RE on H1299 cell proliferation. H1299 cells were seeded in triplicate and treated without (control, 0) or with the indicated concentrations of RE (A–C), resveratrol (RSV; B) or DMSO (C) for 72 h followed by fixing and staining with 0.5% crystal violet. The stain was solubilized, and absorbance was read at 570 nm. Data are the mean ± SEM of 3–4 separate experiments expressed as % of control. * p < 0.05, *** p < 0.001, ns, no significance compared to control. #### p < 0.0001 compared to DMSO vehicle control.

Figure 2

Effect of RE on clonogenic survival of H1299 human lung cancer cells. Cells were seeded in triplicate at a low density and incubated without (control) or with the indicated concentrations of RE for 7 days followed by fixing and staining with 0.05% methylene blue. Colonies of more than 50 cells were counted. The data are expressed as percent of control and are the mean ± SEM of 3–4 separate experiments. ** p < 0.01, *** p < 0.001, compared to control.

Figure 3

Effect of RE on PARP. Whole cell lysates were prepared from H1299 cells treated without (control) or with the indicated concentrations of RE for 24 h or 48 h. Cell lysates (20 µg) were resolved by SDS-PAGE and immunoblotted with specific antibodies against PARP or β-actin. Upper panel: A representative immunoblot is shown. Lower panel: The densitometry of the bands, expressed in arbitrary units, was measured using ImageJ software. The data are expressed as percent of control and are the mean ± SEM of 3–5 separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to control. Original western blot figure can be found in Figure S1.

Figure 4

Effect of RE on H1299 human lung cancer cell migration. Confluent H1299 cells were exposed to 1 μg/mL mitomycin-C (MMC) for one hour, followed by a wound/ scratch injury (black lines) and treatment without (control) or with 5 μg/mL, 25 μg/mL, 50 μg/mL RE. The data shown are the mean ± SEM of 3 separate experiments. ** p < 0.01, compared to control.

Figure 5

Effect of RE on ERK. Whole cell lysates were prepared from H1299 cells treated without (control) or with the indicated concentrations of RE for 24 h or 48 h, or 50 nM PTX for 48 h. Cell lysates (20 µg) were resolved by SDS-PAGE and immunoblotted with specific antibodies against total or phosphorylated ERK or β-actin. Upper panel: A representative immunoblot is shown. Lower panel: The densitometry of the bands, expressed in arbitrary units, was measured using ImageJ software. The data are expressed as percent of control and are the mean ± SEM of 4–5 separate experiments. ** p < 0.01 *** p < 0.001, compared to control. Original western blot figure can be found in Figure S2.

Figure 6

Effect of RE on AMPK. Whole cell lysates were prepared from H1299 cells treated without (control) or with 5, 25, or 50 µg/mL RE for 24 or 48 h, or 10 µM RSV for 48 h. Cell lysates (20 µg) were resolved by SDS-PAGE and immunoblotted with specific antibodies against total or phosphorylated AMPK or β-actin. Upper panel: A representative immunoblot is shown. Lower panel: The densitometry of the bands, expressed in arbitrary units, was measured using ImageJ software. The data are expressed as percent of control and are the mean ± SEM of 3–6 separate experiments. ** p < 0.01, *** p < 0.001, compared to control. Original western blot figure can be found in Figure S4.

Figure 7

Effect of RE on ACC. Whole cell lysates were prepared from H1299 cells treated without (control) or with 5, 25, or 50 µg/mL RE for 24 or 48 h, or 10 µM RSV for 48 h. Cell lysates (20 µg) were resolved by SDS-PAGE and immunoblotted with specific antibodies against total or phosphorylated ACC or β-actin. Upper panel: A representative immunoblot is shown. Lower panel: The densitometry of the bands, expressed in arbitrary units, was measured using ImageJ software. The data are expressed as percent of control and are the mean ± SEM of 3–6 separate experiments. * p < 0.05, *** p < 0.001, compared to control cells. Original western blot figure can be found in Figure S5.

Figure 8

Effects of RE on mTORC1. Whole cell lysates were prepared from H1299 cells treated without (control) or with 50 µg/mL RE for 24 or 48 h. Cell lysates (20 µg) were resolved by SDS-PAGE and immunoblotted with specific antibodies against total or phosphorylated Raptor (A), p70 S6K (B), mTOR, (C) or β-actin. The densitometry of the bands, expressed in arbitrary units, was measured using ImageJ software. The data are expressed as percent of control and are the mean ± SEM of 7 separate experiments. ** p < 0.01, *** p < 0.001, compared to control cells. Original western blot figure can be found in Figure S6.

Figure 9

Effects of RE on Akt. Whole cell lysates were prepared from H1299 cells treated without (control) or with 50 µg/mL RE for 3–48 h. Cell lysates (20 µg) were resolved by SDS-PAGE and immunoblotted with specific antibodies against total or phosphorylated Akt with β-actin used as a loading control. The densitometry of the bands, expressed in arbitrary units, was measured using ImageJ software. The data are expressed as percent of control and are the mean ± SEM of 4 separate experiments. ** p < 0.01, *** p < 0.001, compared to control cells. Original western blot figure can be found in Figure S7.

Figure 10

Summary of the effects of RE on H1299 cells. Rosemary extract decreased proliferation, survival, and migration; and enhanced apoptosis of H1299 lung cancer cells. These effects were associated with increased PARP cleavage; increased ERK, Akt, AMPK, ACC, and Raptor phosphorylation; and decreased p70 S6K phosphorylation.