Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in Non-Small Cell Lung Cancer Cells

RESEARCH ARTICLE Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in NonSmall Cell Lung Cancer Cells Ji In Kang☯, Ji-Young Hong☯, Hye-Jung Lee, Song Yi Bae, Cholomi Jung, Hyen Joo Park, Sang Kook Lee* College of Pharmacy, Seoul National University, Seoul, 151–742, Republic of Korea ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Kang JI, Hong J-Y, Lee H-J, Bae SY, Jung C, Park HJ, et al. (2015) Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in NonSmall Cell Lung Cancer Cells. PLoS ONE 10(12): e0144368. doi:10.1371/journal.pone.0144368 Editor: Cong Cao, Suzhou University, CHINA Received: February 10, 2015 Accepted: November 17, 2015 Published: December 11, 2015 Copyright: © 2015 Kang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2013R1A1A2012389 and No. 20100024361). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Yuanhuacine (YC), a daphnane diterpenoid from the flowers of Daphne genkwa, exhibited a potential growth inhibitory activity against human non-small cell lung cancer (NSCLC) cells. YC also suppressed the invasion and migration of lung cancer cells. However, the precise molecular mechanisms remain to be elucidated. In the present study, we report that YC significantly activated AMP-activated protein kinase (AMPK) signaling pathway and suppressed mTORC2mediated downstream signaling pathway in H1993 human NSCLC cells. AMPK plays an important role in energy metabolism and cancer biology. Therefore, activators of AMPK signaling pathways can be applicable to the treatment of cancer. YC enhanced the expression of pAMPKα. The co-treatment of YC and compound C (an AMPK inhibitor) or metformin (an AMPK activator) also confirmed that YC increases p-AMPKα. YC also suppressed the activation of the mammalian target of rapamycin (mTOR) expression, a downstream target of AMPK. Further study revealed that YC modulates mTORC2-associated downstream signaling pathways with a decreased expressions of p-Akt, p-protein kinase C alpha (PKCα), p-ras-related C3 botulinum toxin substrate 1 (Rac1) and filamentous actin (F-actin) that are known to activate cell growth and organize actin cytoskeleton. In addition, YC inhibited the tumor growth in H1993 cell-implanted xenograft nude mouse model. These data suggest the YC could be a potential candidate for cancer chemotherapeutic agents derived from natural products by regulating AMPK/mTORC2 signaling pathway and actin cytoskeleton organization. Introduction Lung cancer is one of the most common diseases in the world and the leading cause of cancerrelated death [1]. There are two main forms of the disease, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), where NSCLC is approximately 85% of all lung cancer cases. Along with surgery and radiotherapy, chemotherapy is one of the most common PLOS ONE | DOI:10.1371/journal.pone.0144368 December 11, 2015 1 / 17 Anti-Tumor Activity of Yuanhuacine treatments for lung cancer therapy. However, chemotherapy is still not effective enough for patients with advanced NSCLC with a median survival rate of around one year [2, 3]. Therefore, the development of novel potent anticancer agents is still needed to treat the disease. Daphne genkwa (Thymelaeaceae) is a well-known traditional medicinal plant distributed mainly in Korea and China, and its flower has been reported to exhibit abortifacient, anti-cancer, anti-tussive, diuretic, and expectorant activities [4]. Several compounds including daphnane-type diterpenes have been isolated from D. genkwa [5]. Daphane-type diterpenoids showed various biological effects including anti-cancer, transient receptor potential cation channel subfamily V member 1 (TRPV1) activating, anti-fertility, pesticide, neurotrophic, cholesterol-lowering, anti-hyperglycemic, irritant, tumor-promoting, and anti-human immunodeficiency virus (HIV) activities [6]. Yuanhuacine (YC) is a major component of daphnane-type diterpenoids isolated from the flower buds of Daphne genkwa. YC showed the anti-cancer activity in various cancer cell lines in vitro [7]. We also reported that YC exhibits the relatively selective growth inhibitory activity against human A549 lung cancer cells compared to the MRC-5 normal lung epithelial cells [8]. However, the underlying molecular mechanism of YC in human lung cancer cells has not been elucidated yet. AMP-activated protein kinase (AMPK) is a ubiquitous serine/threonine protein kinase constituted of a catalytic α subunit and two regulator subunits (β and γ) [9], and is known to regulate cellular energy metabolism [10, 11]. Activation of AMPK is caused by cellular stress such as oxidative stress, hypoxia, and hypoglycemia, and it leads to increased ratio between cellular adenosine monophosphate (AMP) and adenosine triphosphate (ATP). AMPK also controls cell growth, proliferation and autophagy through the modulation of mammalian target of rapamycin (mTOR) activity, which is consistently deregulated in cancer cells [12]. There are two types of mTOR, mTORC1 and mTORC2 that are structurally and functionally different multi-protein complexes [13]. Generally, mTORC1 controls cell growth in response to nutrient availability and growth regulators. In contrast, mTORC2 is a key regulator of actin cytoskeleton that is correlated with cancer metastasis, and controls the phosphorylation of Akt at Ser-473 through the interaction between rapamycin-insensitive companion of mTOR (rictor) and mTOR [14, 15]. In the present study, the anti-tumor activity of YC and its underlying molecular mechanisms of action were investigated both in human H1993 lung cancer cells in vitro culture and in H1993-implanted xenograft nude mouse model in vivo. Materials and Methods Reagents Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsinEDTA solution (1X), antibiotic-antimycotic solution (100X) and phosphate-buffered saline (PBS) (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-pAMPKα, anti-AMPKα, anti-p-ACC, anti-ACC, anti-p-mTOR, anti-mTOR, anti-p-4EBP1, anti (...truncated)