Octyl Ester of Ginsenoside Rh2 Induces Apoptosis and G1 Cell Cycle

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Octyl Ester of Ginsenoside Rh2 Induces Apoptosis and G1 Cell Cycle Arrest in Human HepG2 Cells by Activating the Extrinsic Apoptotic Pathway and Modulating the Akt/p38 MAPK Signaling Pathway Fang Chen, Shi-Lian Zheng, Jiang-Ning Hu, Yong Sun, Yue-Ming He, Han Peng, Bing Zhang, David Julian McClements, and Ze-Yuan Deng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03519 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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Journal of Agricultural and Food Chemistry

Octyl Ester of Ginsenoside Rh2 Induces Apoptosis and G1 Cell Cycle Arrest in Human HepG2 Cells by Activating the Extrinsic Apoptotic Pathway and Modulating the Akt/p38 MAPK Signaling Pathway Fang Chen,†,‡ Shi-Lian Zheng,†,‡ Jiang-Ning Hu,*,†,‡ Yong Sun,†,‡ Yue-Ming He,†,‡ Han Peng,†,‡ Bing Zhang,† David Julian McClements,& Ze-Yuan Deng*,†,‡ †

State Key Laboratory of Food Science and Technology, Nanchang University,

Nanchang, Jiangxi 330047, China ‡

College of Food Science, Nanchang University, Nanchang, Jiangxi 330047, China

&

Department of Food Science, University of Massachusetts Amherst, Amherst, MA

01003, USA

Corresponding authors: *Jiang-Ning Hu: Telephone No: +86 88304449-8226, E-mail address: [email protected] *Ze-Yuan Deng: Telephone/ Fax No: +86 791 88304402, E-mail address: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Ginsenoside Rh2 is a potential active metabolite of ginseng that has antitumor activity

3

against a variety of tumor cells. Previously, we reported that Rh2-O, an octyl ester

4

derivative of ginsenoside Rh2, had a higher anti-cancer activity than Rh2 through

5

activating the intrinsic apoptotic pathway. In this study, we found that the extrinsic

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apoptotic pathway was also involved in Rh2-O-induced HepG2 cells apoptosis as

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evidenced by the up-regulation of Fas, FasL, TNFR1 and TNF-α, as well as the

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cleavage of caspase 8. Moreover, flow cytometric analysis demonstrated that Rh2-O

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induced G1 cell cycle arrest in HepG2 cells. Rh2-O-induced G1 phase arrest was

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accompanied by the down-regulation of cyclin D3 and cyclin E and cyclin-dependent

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kinase (CDK) 4 and 6, and the up-regulation of p21WAF1/CIP1 and p27KIP1. In addition,

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Rh2-O down-regulated the phosphorylation of Akt, and its inhibitor LY294002

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promoted Rh2-O-induced G1 phase arrest. Rh2-O treatment also activated p38

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MAPK, JNK and ERK expression. Inhibitors of p38 MAPK (SB203580), but not

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those of JNK (SP600125) or ERK (PB98095), promoted Rh2-O-induced G1 phase

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arrest in HepG2 cells. These results indicated that the disruption of Akt and p38

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MAPK cascades played a pivotal role in Rh2-O-induced G1 phase arrest.

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KEYWORDS: Ginsenoside Rh2, octyl ester derivative, cell cycle, HepG2 cells, Akt,

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MAPKs


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INTRODUCTION

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Ginseng, well known as “renshen” in Chinese, is one of the most popular tonics in

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oriental countries. Ginsenoside Rh2 is a major bioactive ingredient in ginseng that has

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been shown to have antitumor activities on a variety of tumor cells, including human

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hepatoma SK-HEP-1 cells, human astroglioma U87MG cells, and human cervical

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carcinoma Hela cells.1-4 However, Rh2 currently has a limited application as a

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bioactive ingredient in the food, supplement, and pharmaceutical industries because

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of its poor oral bioavailability, which is associated with its low membrane

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permeability.5-7 The main reason for the low permeability of Rh2 is the fact that it is a

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hydrophilic substance that is not easily solubilized and transported through the

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phospholipid bilayers in cell membranes.

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has been increased by synthesizing an octyl ester (Rh2-O) from Rh2 and octanoyl

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chloride.8 Structural analysis indicated that the fatty acid ester substituent connected

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to the hydroxyl group at C-12 of Rh2.8 A recent study in our laboratory showed that

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Rh2-O improved the absorption of Rh2 in Caco 2 cells, which suggested that

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esterification of Rh2 might lead to an improvement in its bioavailability.9 Moreover,

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it was confirmed that Rh2-O had a higher anti-cancer activity than Rh2.8 However,

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the exact molecular mechanisms responsible for this increased activity need to be

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elucidated before Rh2-O can be used as an effective anticancer agent.

Recently, the lipophilic character of Rh2

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Apoptosis, a process of programmed cell death, is a highly regulated form of cell

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death.10, 11 Two main cysteine-aspartate proteases (caspase) activation cascades have

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been characterized.12,

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receptors, such as Fas, TNFR, and DR4.14, 15 The death receptors are activated when

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they bind with their corresponding natural ligands, which is followed by activation of

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The extrinsic apoptotic pathway is mediated by death



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caspase 8 to induce cell apoptosis.16 The intrinsic apoptotic pathway, driven by the

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Bcl-2 family proteins, involves the depolarization of the mitochondrial outer

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membrane, which subsequently leads to the release of cytochrome c and the activation

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of caspase 9.17, 18 Our previous study demonstrated that Rh2-O induced HepG2 cells

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apoptosis by stimulating the production of reactive oxygen species (ROS),

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mitochondrial dysfunction, and caspase activation.8 However, the signal transduction

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pathways involving the extrinsic apoptotic pathway in Rh2-O-induced HepG2 cells

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apoptosis are not yet fully understood.

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Controlling the growth and proliferation of cancer cells is another main

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molecular mechanism responsible for the anti-tumor effects of phytochemicals.

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Eukaryotic

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cyclin-dependent kinases (CDKs), which is dependent upon association with

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cyclins.19 In addition, some CDK inhibitors (CDKIs) are also involved in Eukaryotic

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cell-cycle progression.20 Rh2 has been shown to induce G1 cell cycle arrest by

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down-regulations of the protein levels and kinase activities of cyclin D1, cyclin E, and

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CDK 6, and up-regulation of pRb2/p130 in human lung adenocarcinoma A549 cells.21

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Another study has shown that Rh2-induced G1 cell cycle arrest was accompanied by

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the down-regulation of CDK4, CDK6, cyclin D1/2/3, and cyclin E at protein level and

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the gradual up-regulation of p21WAF1/CIP1 and p27KIP1 in HL-60 promyelocytic

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leukemia and U937 human histocytic lymphoma cells.22 However, the specific

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mechanisms of action and underlying signaling pathways in the inhibition of the

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proliferation of HepG2 cells by Rh2-O have not yet been elucidated.

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cell-cycle

progression

involves

the

sequential

activation

of

A network of interacting proteins mediates the transmission of signals in cells a

large

number

of cellular processes.23

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that govern

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3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases (MAPKs)


Phosphatidylinositol

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play critical roles in controlling the growth and proliferation of cancer cells through

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different extracellular stimulations.24,

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MAPKs has been reported in several cancers, including H1299 lung cancer cell lines,

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HSC-3 oral carcinoma cell lines, and MDA-MB-468 breast cancer cell lines.26-28

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Therefore, it is important to verify the relationship between Rh2-O and PI3K/Akt or

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MAPKs in carcinogenesis for cancer prevention or treatment. The primary objective

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of the current study was therefore to identify the molecular mechanisms responsible

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for the anticancer activity of Rh2-O using HepG2 as a representative cell line model.

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The results obtained are expected to support the development of a strong scientific

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foundation for using Rh2-O as a chemopreventive and/or chemotherapeutic agent

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against liver cancer.

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MATERIALS AND METHODS

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Materials and Reagents. Rh2-O was synthesized using the method described in our

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recent study.8 Normal growth media (MEM) medium and fetal bovine serum (FBS)

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were

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diphenyltetrazolium bromide (MTT), proteinase K, phenylmethanesulfonyl fluoride

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(PMSF) as well as all inhibitors utilized in this study were procured from Sigma

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Chemical Co. (St. Louis, MO, USA). The cell cycle detection kit was obtained from

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B.D. Clontech Laboratories (Mountain View, CA, USA). Antibodies against cyclin

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D3, cyclin E, CDK2, CDK4, CDK6, p21WAF1/CIP1, p27KIP1, Fas, FasL, TNF-α, TNFR1,

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DR4, cleaved-caspase 8, pro-caspase 8, ERK1/2, p-ERK1/2, p38 MAPK, p-p38

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MAPK, JNK, p-JNK, Akt and p-Akt were obtained from Cell Signaling Technology

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(Beverly, MA, USA). Antibodies against β-actin and all the second antibodies were

obtained

from

25

Gibco-BRL

The proliferative role of PI3K/Akt and

Co.

3-(4,5-dimethylthiazole-2-yl)-2,5



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purchased from Trans Transgen Biotechnology Co. (Beijing, China). All other

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compounds were of analytical grade.

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Cell Culture and Treatment. The human liver hepatocellular carcinoma cell line

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HepG2 was obtained from the National Centre for Cell Sciences (NCCS, Shanghai,

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China). HepG2 cells were grown in MEM medium supplemented with 10% (v/v) FBS,

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100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were incubated in a

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humidified incubator with 95% air and 5% CO2 at 37 °C. In additional experiments,

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cells were pretreated with different inhibitors: PI3K inhibitor (LY294002, 10-30 µM),

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p38 MAPK inhibitor (SB203580, 10-30 µM), ERK inhibitor (PD98059, 50 µM), or

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JNK inhibitor (SP600125, 50 µM) for 1 h followed by incubation with Rh2-O

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(dissolved in DMSO) or the vehicle (DMSO, < 0.1%) for 12 h.

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Cell Viability Assay. The effects of inhibitors on Rh2-O-induced HepG2 cell death

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were monitored using MTT assays.29 Briefly, 0.2 mL of cells (1 × 104 cells per well)

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was placed onto 96-well microassay plates. Following incubation for 12 h, cells were

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pretreated with various inhibitors (LY294002, SB203580, PD98059, or SP600125)

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for 1 h and then treated with 17.5 µM of Rh2-O. After treatment, 0.2 mL of MTT

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solution (2 mg/mL in culture media) was added into each well of the plates and then

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incubated for 4 h in the dark. The supernatant was then removed, and incubated with

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0.15 mL of DMSO to dissolve the purple MTT-formazan crystals with shaking for 10

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min. The absorbance was determined at 490 nm in a microplate reader. Cells

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incubated with 0.1% DMSO were used as controls. In MTT assays, every sample was

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performed at least 3 times.

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PI Staining Assay. Cell cycle distribution was measured by flow cytometric

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analysis.27 HepG2 cells were seeded on culture dishes for 12 h, and a double

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thymidine block procedure was used to synchronize the cell cycle. The cell cycle


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synchronized HepG2 cells were cleansed twice with PBS and then re-stimulated to

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enter the G1 phase together by treatment with Rh2 (17.5 µM) or various

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concentrations of Rh2-O (10-17.5 µM) for 12 h. The cells were collected and washed

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with pre-cooling PBS. One mL 70% ethanol was added to fix the collected cells at

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-20 °C overnight. After centrifugation, the collected cells were incubated with 50.1

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µg/mL PI (1.21 mg/mL Tris, 700 U/mL RNase, pH 8.0) for 1-2 h. The DNA contents

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were further determined by flow cytometry (FACSCalibur; Becton Dickinson

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Biosciences, San Jose, CA). The number of cells for each sample was at least 20,000.

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RNA Isolation and Real-Time Quantitative Reverse Transcription-PCR. Total

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RNA of the cells was extracted using total RNA extraction reagent (TaKaRa)

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according to the manufacturer’s instruction. RNA was confirmed by running on a 1.5%

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agarose gel. Reverse transcription was performed with 1 µg of total RNA using

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TransScript first-strand cDNA synthesis supermix kit (TaKaRa) according to the

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supplier’s instruction. The mRNA expression levels of genes were determined by

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real-time PCR using SYBR premix ex TagTM (TaKaRa) in the ABI 7900HT

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real-time PCR system (Applied Biosystems, USA). Simultaneously, the quantitative

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analysis of mRNA levels was normalized to the endogenous reference gene GAPDH.

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The reaction was carried out under the following conditions: 95 °C for 30 s, 40 cycles

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at 95 °C for 5 s, and 58 °C for 30 s. The primers used are shown in Table 1. Values of

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each group mRNA level was calculated as 2-∆∆Ct levels and performed at least 3 times.

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Preparation of Whole-Cell Lysates. HepG2 cells (1 × 105 cells) were plated in a 100

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mm dish and treated with 17.5 µM of Rh2 or various concentrations of Rh2-O

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(10-17.5 µM) for 12 h. After treatment, cells were cleansed twice with cold PBS and

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then lysed with iced-cold radio-immunoprecipitation assay (RIPA) buffer. PMSF (10

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mg/mL), leueptin (17 mg/mL), and sodium orthovanadate (10 mg/mL) were then



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added. After incubation for 30 min on ice, the lysates were centrifuged at 1000g for

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10 min, and then the supernatants were collected as whole-cell lysates.

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Western Blot Analysis. The concentrations of protein in the samples were quantified

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using a BCA Protein Assay kit (Beyotime Biotechnology, Shanghai, China). Forty µg

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of cell lysate protein was mixed with SDS loading buffer (6 ×) and then treated by

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boiling for 10 min. The obtained protein samples were separated on the basis of their

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size using 12% gradient SDS-PAGE gel, and then transferred onto a nitrocellulose

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membrane. After that, the membrane was blocked with 5% nonfat milk containing 1%

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(v/v) Tween-20 for 1 h at ambient temperature, and then incubated with specific

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primary antibodies at 4 °C for more than 16 h. After washing 5 times with

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Tris-buffered saline containing 1% (v/v) Tween-20 (TBST), the membrane was

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incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse

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secondary antibodies at ambient temperature for 1 h. Finally, protein bands were

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detected using an enhanced chemiluminescence (ECL) detection system (GE

156

Healthcare).

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Statistical Analysis. Study data are reported as means ± the standard deviations.

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Statistical analyses were carried out by the student’s t-test or the one-way analysis of

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variance (ANOVA) using a statistical software package (SPSS, USA). p-values of

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Octyl Ester of Ginsenoside Rh2 Induces Apoptosis and G1 Cell Cycle

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