Enterolactone Induces G1-phase Cell Cycle Arrest in Nonsmall Cell Lung Cancer Cells by Downregulating Cyclins and Cyclin-dependent Kinases

Abstract

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anticancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study, we investigated the anticancer effects of EL for several nonsmall cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The antiproliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G₁-phase cell cycle arrest. Molecular studies revealed that EL decreased mRNA or protein expression levels of the G₁-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21^WAF1/CIP1, a negative regulator of the G₁ phase. The results suggest that EL inhibits the growth of NSCLC cell lines by downregulating G₁-phase cyclins and CDKs, and upregulating p21^WAF1/CIP1, which leads to G₁-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.

Figure 1

EL inhibits growth of lung cancer cell lines. A. The short-term growth of A549, H441, and H520 was measured by the percentage of alamarBlue® reduction in the presence of EL (0–100 μM) over a period of 4 days. The data represent the average ± standard deviation of eight replicate wells for three independent experiments for every cell line. In A549 and H520 cells, EL-treatment (≥50 μM) and in H441 cells, EL (100 μM) resulted in a significant inhibition of growth in a time-dependent manner. B. Cell viability of Hs888Lu cells was examined by a trypan blue exclusion assay following EL (0, 10, and 100 μM) treatment for 24 and 48 h. The bar graph represents the average cell count for two independent experiments. The error bars represent standard deviation from two independent experiments. C. The long-term growth of lung cancer cells in the presence of EL (10 and 100 μM) was determined by a clonogenic survival assay. The fraction of cells surviving EL treatment was examined after 14 days. The image shown is representative of one typical experiment. The data shown in the bar graph represent the average ± standard deviation for the fraction of surviving cells for three independent experiments for each cell line. (* designates p ≤ 0.05 as compared to control).

Figure 2

EL exposure results in G₁-phase cell cycle arrest, but not cell death. A. Cell viability of A549, H441, and H520 cells was examined by a trypan blue exclusion assay following EL (0–100 μM) treatment for 24, 48, and 72 h. The bar graph represents the average total cell count, inclusive of live (black) and dead (white) cells, for three independent experiments for each cell line. The error bars represent standard deviation of the total number of dead cells from three independent experiments for each cell line. B. An Annexin V-FITC/PI apoptosis assay was performed to further assess the potential cytotoxic effects of EL. A549 cells were treated with EL (0, 10, 50, and 100 μM) for 24, 48, and 72 h. The treated cells were then stained with Annexin V/PI and flow cytometric analysis was performed. The representative data from three independent experiments are shown in dot plots. C. EL-induced G₁-phase cell cycle arrest in lung cancer cells was examined by flow cytometry. The histograms show the cell cycle distribution for A549, H441, and H520 treated with the vehicle control or EL (10 and 100 μM) for 48 h. The data represent the average percentage of cells in each phase of the cell cycle for three independent experiments for each cell line.

Figure 3

EL regulates the expression of G₁-phase related genes. A549, H441, and H520 cells were treated with EL (100 μM) for 24 h. Relative mRNA expression levels for cyclin D1, CDK2, CDK4, cdc25A, p21^WAF1/CIP1, and p27^KIP1 were determined by quantitative PCR. The relative mRNA levels for all genes were normalized to 18S rRNA. The data shown in the bar graphs represent the average ± standard deviation of relative mRNA expression for each gene for three independent experiments for each cell line. (* designates p ≤ 0.05 as compared to control).

Figure 4

EL induces cell cycle arrest by modulating the expression of G₁-phase related proteins. A. A549 and H520 cells were treated with EL (100 μM) for 0, 1, 3, 6, 12, and 24 h. H441 cells were treated with EL (100 μM) for 0, 6, 12, and 24 h. The cells were lysed to collect proteins for western blotting. 30 μg protein were loaded on the gels, and blots were probed with p-pRb. B. Quantification of western blotting results was done using ImageJ. The data shown are the average of three independent experiments and are represented as mean ± S.E.M. A549 (C), H441 (E), and H520 (G) cells were treated with EL (100 μM) for 0, 6, 12, and 24 h. The cells were then lysed to collect proteins for western blotting. 30 μg protein were loaded on the gels, and blots were probed with cyclin D1, cyclin E, p-cdc25A, CDK2, CDK4, CDK6, p21^WAF1/CIP1, and p27^KIP1 G₁-phase cell cycle regulatory proteins. Total cdc25A (t-cdc25A) protein levels were used to normalize phosphorylated cdc25A (p-cdc25A) protein levels. Quantification of western blotting results for A549 (D), H441 (F), and H520 (H) was done using ImageJ. The data shown are the average of three independent experiments and are represented as mean ± S.E.M. GAPDH was used as a loading control for the remaining western blots. (* designates p ≤ 0.05 as compared to control).

Figure 5

Proposed molecular mechanism of EL-induced G₁-phase cell cycle arrest. EL suppresses expression of cyclin D1-CDK4/6 and cyclinE-CDK2 proteins by up-regulating expression of the cell cycle inhibitor p21^WAF1/CIP1. EL-mediated down-regulation of cyclin-CDKs complexes suppresses phosphorylation of pRb. Hypo-phosphorylated pRb binds to transcription factor E2F, and represses its transcriptional activity and the activation of genes required for the G₁-S phase transition, such as cdc25A. This leads to G₁-phase cell cycle arrest and growth inhibition.