Nutr Cancer.2017 69(4):652-662.

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

Chikara S1*, Lindsey K1, Dhillon H1, Mamidi S2, Kittilson J1, Christofidou-Solomidou M2, Reindl KM1

1Department of Biological Sciences, North Dakota State University , Fargo , North Dakota , USA.

2Department of Plant Sciences, North Dakota State University , Fargo , North Dakota , USA.

3Department of Medicine, University of Pennsylvania , Philadelphia , Pennsylvania , USA.

*Correspondence should be addressed to: Shireen Chikara, Department of Biological Sciences, North Dakota State University, 1340 Bolley Drive, 201 Stevens Hall, Tel: 701-231-9427, Fax: 701-231-7149, Email: shireen.chikara@ndus.edu

 

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 anti-cancer 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 anti-cancer effects of EL for several non-small 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 anti-proliferative 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 p21WAF1/CIP1, a negative regulator of the G1-phase. The results suggest that EL inhibits the growth of NSCLC cell lines by down-regulating G1-phase cyclins and CDKs, and up-regulating p21WAF1/CIP1, which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500210/

 

Supplement

Lung cancer accounts for approximately 38% of all cancer-related deaths in the US [1]. The conventional cancer treatment options inflict toxicity to healthy tissues, while, bioactive compounds isolated from natural food sources have been shown to selectively target cancer cells. Therefore, it is interesting to study the anticancer properties of bioactive compounds in lung cancer.

 

The lignans, naturally occurring phytoestrogens, exhibit health-promoting effects in several diseases including cancer. Flaxseed, one of the richest sources of lignans has shown chemopreventive and chemotherapeutic benefits in an observational study of breast cancer [2], human clinical trials [3-4], and animal models of breast [5-7], colon [8-9], and prostate [10] cancers. On ingestion, flaxseed lignan secoisolariciresinol diglucoside (SDG) is metabolized by intestinal flora into mammalian lignan, enterolactone (EL) [11]. We evaluated the anticancer effects of EL in non-small lung cancer (NSCLC) cell lines having varied genetic backgrounds.

 

We observed that EL suppressed the growth of NSCLC cells in a concentration and time-dependent manner. The in vitro anti-proliferative effect was due to arrest of lung cancer cells in G1-phase of the cell cycle and not due to increased apoptosis. Progression of cells through the G1, S, G2, and M cell cycle phases is positively regulated by cyclins and cyclin dependent kinases (CDK), and is negatively regulated by dephosphorylation and activation of CDKs to facilitate G1-S phase transition [12]. We found that EL reduced cyclin D and E, CDK-2, 4 and 6, and cdc25A expression and upregulated p21WAF1/CIP1 expression, at certain time points, in lung cancer cells. In addition, the tumor suppressor protein retinoblastoma (pRb) plays a critical role in cell cycle progression. In quiescent cells, pRb exists in an activated/hypo-phosphorylated state, and sequesters members of the E2F gene family of transcription factors, which suppresses cell-cycle progression [13]. Phosphorylation of pRb leads to the disruption of the pRb-E2F transcription complex, and the release of active E2F, which promotes cell-cycle progression [13]. We observed that EL-treatment caused a significant reduction in phosphorylated p-pRb protein in lung cell lines.

 

We concluded that EL inhibited cell proliferation by inducing G1-phase cell cycle arrest (Figure 1). EL treatment resulted in down-regulation of cell cycle promoting cyclins and CDKs and up-regulation of their inhibitors, leading to decreased phosphorylation of pRb protein and thereby, decreased expression of genes that promote G1 to S-phase transition.

 

 

Figure 1. EL-mediated induction of G1-phase cell cycle arrest. EL-mediated induction of cell cycle inhibitor p21 leads to downregulation of cyclin D1-CDK4/6 and cyclinE-CDK2 proteins complexes. Inhibition cyclin-CDK complexes suppresses phosphorylation (activation) of pRb, which then remains bound to transcription factor E2F. Therefore, E2F is unavailable to drive activation of genes, such as cdc25A, required for G1-S phase transcription. This results in growth inhibition via G1-phase cell cycle arrest

  

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