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Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.


Article history:

Received 24 April 2013

Received in revised form 5 July 2013

Accepted 2 August 2013


Arctigenin (ARG)





The lignan arctigenin (ARG) from the herb Arctitim lappa L possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the [G.sub.D]/[G.sub.1] phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cycl in H/CDK7, while apoptosis was induced through the modulation of the Ala-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level.

[C]2013 Elsevier GmbH. All rights reserved.


Lung cancer is a major worldwide health problem and has accounted for approximately 16% of global cancer deaths (Pisani et at., 1999). There are 2 main types of lung cancer: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). Lung adenocarcinoma, which belongs to the NSCLC group, constitutes approximately 75-85% of all lung cancers (Greenlee et al., 2001).

Chemotherapy has been established as a standard treatment for NSCLC: however, despite the success of chemotherapy, it has a limited ability to improve a patient's symptoms and quality of life (Ten Bokkel Huinink et al., 1999). While some lung cancer therapies have shown promising results for the control of disease progression, the toxicity of this therapeutics often restricts the completion of the recommended dose. Therefore, the identification of an anticancer agent with high selectivity against NSCLC merits further investigation and is preferable than searching for agents with high toxicity.

Increasing attention has been paid to the pharmaceutical value and the biological activity of herbal plant medicines over the last decade. Numerous bioactive compounds isolated from herbal plants exhibit selective toxicity toward tumorigenic tissues, and thus display cancer-targeting properties (Quasney et al., 2001). The lignans compose one group of chemical compounds found in plants, and include arctigenin (ARG), a dibenzylbutyrolactone lignan isolated from Arctium lappa L. ARG has been reported to possess many important pharmacological or biological properties, including anti-oxidative, anti-tumorigenic, and anti-inflammatory activities (Awale et al., 2006; Matsumoto et al., 2006). Our previous study showed that ARG specifically inhibited the proliferation of lung adenocarcinoma (A549) cells, which might consequently lead to the induction of apoptosis (Susanti etal., 2012). Although the anticancer activity of ARC! appears to be promising, the mechanism of its action is not yet fully understood. Several studies have reported that ARG blocks the unfolded protein response and activates Akt in glucose-deprived solid tumors (Kim et al., 2010; Awale etal., 2006). However, the mechanism proposed for the glucose-deprived cancer cells may not necessarily be applicable to our current study. It is known that the inhibition of cell proliferation is involved in the anti-cancer mechanism of ARC; however, the signal transduction pathways leading to cell cycle arrest are entirely unknown. ARG has been shown to induce cell cycle arrest at the [G.sub.0]/[G.sub.1] phase in gastric cancer cells by modulation of regulatory proteins such as Rb and cyclin D1 (Jeong et al., 2011), and at the [G.sub.2]/IVI phase in colorectal cancer cells via the regulation of the Wnt/[belta]-catenin signaling pathway (Yoo et al., 2010). Furthermore, ARG was detoxified by glutathione (GSH) in liver cells, indicating that the expression level of GSH synthase may be an additional factor controlling cell susceptibility to this agent (Moritani et al., 1996). Therefore, the mechanism of action of ARG is likely to vary among tissues and cancer cell types.

The current study aims to gain insight into the mechanisms involved in ARG-mediated inhibition of proliferation and induction of apoptosis in lung adenocarcinoma. Furthermore, this study aims to delineate the specificity of ARG via comparative studies on various normal cell lines. The results of this investigation will provide valuable new information on the usefulness of ARG as a promising chemotherapeutic agent with few adverse effects.

Materials and methods

Isolation and purification of ARC

The isolation and purification of ARG from A. lappa L. extract were performed as described previously in Susanti etal. (2012).

Cell cultures

Various human normal embryo fibroblast (OUMS-36, OUMS-36T-2F, and OUMS-36T-5F) and lung adenocarcinoma (A549) cell lines were purchased from the Japanese Cancer Research Resources Bank (JCRB, lbaraki, Japan). Cells were cultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum) in a humidified 5% C[O.sub.2] atmosphere at 37 C.

Cell viability assay

Cells suspended in DMEM were seeded at 1 x [10.sup.4] cells (100 [mu]l) per well in 96-well plates and pre-incubated overnight in a humidified atmosphere of 5% C[O.sub.2] at 37 [degrees]C. After a 24-h incubation with varying concentrations of ARG, cell viability was determined using MTS assay kits according to the manufacturer's instructions (Cell Titer 96[R] AQueous non-radioactive cell proliferation assay, Promega Co., Madison, USA). All experiments were performed in triplicate, and cell viability was expressed as the relative viability of the treated cells compared to untreated cells (control).

Cell cycle analysis

Cells (3 x [10.sup.5]) were seeded in 60-mm culture dishes and preincubated for 24h at 37[degrees]C. Cells were washed with PBS before the replacement of the medium, and then cultured in DMEM supplemented with or without ARG (6 [micro]g/m1). After a 24h incubation, cells were harvested using ICT AccutaseTm cell detachment solution (Innovative Cell Technologies, Inc, San Diego, CA, USA), washed with PBS, fixed with 70% cold ethanol, and incubated overnight at 4 [degrees]C. Ethanol was removed by decantation, and cells were re-suspended in PBS on ice for 10 min. Cell suspensions were centrifuged at 1000 rpm for 10 min, re-suspended in a 250 U/m1 RNase solution, and incubated for 20 min at room temperature. After 200 [micro]g/ml propid ium iodide solutions was added, the cell suspensions were transferred and filtered through a 35 mM nylon filter into flow cytometer tubes. Data acquisition and analysis were performed by a FACS Calibur flow cytometer system (BD Biosciences).

Western blot analysis

Proteins were isolated using PRO-PREPTm Protein Extraction Solution (iNtRON Biotechnology, Korea) from the lysate of 2.5 x [10.sup.6] cells that had been treated with 6[micro]g/m1ARG for 24 h. The protein concentration was measured using Quant-iT[TM] Protein Assay Kits (Invitrogen, USA) according to the manufacturer's instructions. Protein samples were dissolved in an equal amount of sample buffer solution (EzApply, Atta, Osaka, Japan). Proteins (10 [micro]g) were separated on 12.5% SDS-PAGE gels (e-PAGEL, E-R12.5L, ATTO, Tokyo, Japan) run at 15-20 mA in running buffer (EzRun, ATTO, Tokyo, Japan). The proteins were transferred to PVDF membranes by using the iBlotTm Dry Blotting System (lnvitrogen, Tokyo, Japan). The PVDF membranes were treated with blocking buffer (Blocking One, Nacalai Tesque, Kyoto, Japan) for 1 h and washed with TBS (1 x PBS in 0.1% Tween-20). Subsequently, the membranes were incubated with primary antibody [belta]-actin, CDK2, p-CDK, CDK7, Cyclin E, Cyclin Rb, P-Rb, and NPAT) with gentle shaking overnight. After washing, the membranes were incubated with an HRP-linked anti-rabbit IgG secondary antibody for 1 h and then washed again. The detection of target proteins was performed using a luminescent image analyzer (ImageQuant LAS 4000 mini, GE Healthcare, Uppsala, Sweden) and Amersham ECL Advance Western Blotting Detection Kits (GE Healthcare, Buckinghamshire, UK). The relative intensities (%) of protein bands were measured using Image J software version 1.47d.

Total RNA isolation and quantitative real-time PCR analysis

Total RNA was extracted from either untreated or treated cells (1 x [10.sup.6]) by using the AquaPure RNA Isolation Kit (Bio-Rad Laboratories) according to the manufacturer's instructions. The quality of the isolated RNA was checked using MultiNA Reagent Kits on a microchip electrophoresis system (MultiNA-Biotech, Shimadzu, Japan) according to the manufacturer's instructions. Total RNA (2 lig) was reverse transcribed to produce cDNA by using High Capacity RNA-to-cDNA Kits according to the manufacturer's instructions (Applied Biosystems, USA). cDNA samples (20 aliquots) were stored at -20 [degrees]C. For real-time PCR analy- sis, the cDNAs were amplified in a StepOnePlus Real-Time PCR system (Applied Biosystems, CA, USA) by using Fast SYBR Green Master Mix (Applied Biosystems, CA, USA), according to the manufacturer's instructions. Table 1 lists the sequences of the primers that were used for quantitative real-time RT-PCR analysis. All analyses were performed in triplicate, and the gene expression levels were normalized to the housekeeping gene ACTB. Fold changes in gene expression were calculated on the basis of the standard curve, which was constructed using the calibration data produced by the StepOne software.

Table 1 Primer sequences used for quantitative
real time RT-PCR.

Gene   Description         Primer sequence                 Product

Fas    Fas (TNF receptor   5-AAGAATGCTCTCAATCAACCCA-3'         103
       superfamily,        (forward primer. F) 5'
       member 6)          -GAAGTTGATGCC A ATTACG A AG C-
                           3 (reverse primer, R)

Bcl-2  B-cell lymphoma 2   5-TTCTACGACAGCAAATTGCCC-3'         102
                           (forward primer. F) 5
                           (reverse primer, R)

Bax    BCL2-associated X   5'-TCTGACGGCAACrTCAACTGG-3'         115
       protein             (forward primer. F) 5-AG
                           CCCATG ATG GTTCTG ATC A- 3'
                           (reverse primer, R)

Bad    BCL2-associated     5-CAGTGACCTTCGCTCCACATC-3'         123
       agonist of cell     (forward primer. F) 5'-AAG
       death               G AG ACAG CACG GATCCTC-3'
                           (reverse primer, R)

Akt1   v-akt murine        5'-AG AG AAG C CAC                102
       thymoma viral       GCTGTCCTCT-3' (forward primer.
       oncogene homolog    F) 5-CCGCAGGATAGTTTTCTTCCC-3
       1                   (reverse primer, R)

AIF    Mitochondrial       5-ACTAGTTTGCCCACAGTTGGTGTT-31      101
       apoptosis inducing  (forward primer, F)
       factor              5-TCACTCTCTGATCGGATACCAGTTC-3
                           (reverse primer, R)

Casp   Caspase 8.          5-CATATATCCCGCATCAGGCTGAC-3        101
8      apoptosis-related   (forward primer, F) 5'
       cysteine            TGACTCGATGTACCAGGTTCCC-3'
       peptidase           (reverse primer, R)

Casp   Caspase 3.          5'-CCCTGAGCAGAGACATGACTCA-3         106
3      apoptosis-related   (forward primer, F)
       cysteine            5'-TCATCCACACATACCAGTGCC-3'
       peptidase           (reverse primer, R)

Casp   Caspase 9.          5'-TCACTTGTCTCCCATGATCCC-3'         108
9      apoptosis-related   (forward primer. F) 5'-
       cysteine            AATGT AC AC G AC AG CCTC AC AG
       peptidase           C-3' (reverse primer, R)

ACTB   Actin, beta         5-TCACCGAGCCCCGCT-3 (forward        60
                           primer. F) 5
                           (reverse primer, R)

Gene   Accession no.

Fas    NM_152871J2

Bcl-2  NM_004019.3

Bax    NM_138764.4

Bad    NM_004322.3

Akt1   NM_001014432.1

AIF    NM_AL049703.1

Casp   NM_001080124.1

Casp   NM_004346.3

Casp   NM_001229.3

ACTB   NM_001101.3

Effect of GSH depletion on ARG cytotoxicity

The effect of GSH depletion on the cytotoxicity of ARG was studied using the GSH synthase inhibitor L-buthionine-(S,R)-sulfoximine (BSO). Cells suspended in DMEM were seeded at 1 x 104 cells (100 [micro]l) per well in 96-well plates and pre-incubated in a humidified 5% CO2 atmosphere at 37 C for 10 h. The cells were incubated with 50M of BSO for 14 h, and subsequently treated with varying concentrations of ARG for 24 h. Cell viability was then determined using MTS assay kits according to the manufacturer's instructions (Cell Titer 96[R] AQueous non-radioactive cell proliferation assay, Promega Co., Madison, USA). All experiments were performed in triplicate, and the cell cytotoxicity was expressed as the relative viability of treated cells compared to that of untreated control cells. In the case of BSO pretreatment, control cells were treated with BSO only. The ED50 of ARG with or without BSO pretreatment was estimated by fitting the following formula to the titration curve:

y=[[belta].sub.3] + [[belta].sub.4] / (1 + exp([[belta].sub.1] = [[belta].sub.2]x) where y is the cell viability; x is the concentration of the test substance in the medium; and [[belta].sub.1] - [[belta].sub.3] is the constant. Fitting the formula after transformation to the linear model was performed using a simple regression analysis program (R Version 2.11.1).

Statistical analysis

The data are expressed as means [+ or -] standard deviation (SD). Statistical significance between pairs of means was evaluated using the Student's t test (p <0.05 or p <0.01).


Specific cytotoxicity of ARG in various cell lines

Fig. 1 shows the effect of ARG concentration on the cell viability of human lung cancer (A549) and human normal (OUMS-36T-2F) cells. OUMS-36 and OUMS-36T-5F human normal cells were found to be resistant to ARG, whereas A549 cancer cells and OUMS-36T-2F normal cells were susceptible. Among the cells studied, the A549 cancer cells were found to be most susceptible to ARG treatment, reinforcing the concept of cancer-specific ARG cytotoxicity.

The effect of ARG on the cell cycle in various cell lines

The flow cytometric histogram in Fig. 2 illustrates that ARG treatment increased the proportion of cells in the [G.sub.0]/[G.sub.1] phase from 55.8% (control) to 81.4% in A549 cells, and from 48.5% (control) to 74.8% in OUMS-36T-2F normal cells. This result indicates that ARG (6 pg/ml) arrested the cell cycle of both cell types at the [G.sub.0]/[G.sub.1] phase. In contrast, there were no significant changes in the proportion of cells in the G0/G1 phase for the 2 other normal cell lines (OUMS-36 and OUMS-36T-5F).

The effect of ARG on the expression of cell cycle regulatory proteins

ARG has been shown to inhibit the proliferation of A549 lung cancer cells without any adverse effect on normal cells (Susanti et al., 2012). In order to explore the molecular mechanism of ARG-mediated cell cycle arrest, the expression of several cell cycle regulatory proteins was analyzed by western blotting.

ARG (6 [micro]/m1) lowered the level of regulatory proteins involved in the [G.sub.1]/S checkpoint signaling, including cyclin CDK7, cyclin E, CDK2, p-CDK, and NPAT (Fig. 3). The effects of ARG appeared to depend on the malignancy of the cells (Fig. 3). In A549 lung cancer cells, ARG significantly decreased the concentration of the cyclin H, CDK7, cyclin E, CDK2, p-CDK, and NPAT proteins. Similarly, ARG treatment lowered the concentration of these proteins in OUMS-36T-2F normal cells, except for cyclin H. In contrast, no effect was observed in the 2 other types of normal cells (OUMS-36 and OUMS-36T-5F). On the other hand, ARG increased the concentration of p-CDK in both OUMS-36 and OUMS-36T-5F normal cells, and cyclin E in OUMS-36 cells only. These data are consistent with our previous observation that ARG specifically arrested the cell cycle of cancer cells at the [G.sub.0]/[G.sub.1] phase. Furthermore, no significant difference was observed in the concentration of Rb and P-Rb proteins between ARG-treated and untreated cells.

The effect of ARG on the expression of apoptosis-related genes

ARG has been reported to induce apoptosis through caspase-3 activation in A549 lung cancer cells (Susanti et al., 2012). In the current study, we aimed to gain more insight into the signaling pathway of apoptosis in ARC-treated cells. Thus, we analyzed the expression of caspase-3 and other pro-apoptosis (Fas, Box, Bad, AIF, Caspase-8, and Caspase-9) and anti-apoptosis (BcI-2 and Akt-1) genes.

Fig. 4 shows the effect of ARG on the expression of apoptosis-related genes. ARG treatment up-regulated the expression of the pro-apoptosis genes in A549 lung cancer cells, except for Bad gene. ARG also up-regulated the expression of the anti-apoptosis gene BcI-2 and down-regulated another anti-apoptosis gene Akt-1. In contrast to these observations in A549 cells, ARG had no effect on the anti-apoptotic genes in OUMS-36 and OUMS-36T-5F normal cells and largely decreased the expression of some pro-apoptotic genes.

Furthermore, ARG treatment had no differential effect on the expression of apoptosis-related genes in OUMS-36T-2F normal cells, as ARG treatment significantly up-regulated both pro- and anti-apoptosis genes in this cell type.

The effect of GSH depletion on the cytotoxicity of ARG

Our previous study noted that the responses of normal cell lines (OUMS-36, OUMS-36T-5F, and OUMS-36T-2F) to ARG were not necessarily consistent. OUMS-36 and OUMS-36T-5F cells were resistant to ARG, whereas OUMS-36T-2F cells were sensitive to ARG and showed a response that was comparable to that of A549 lung cancer cells. The clarification of the mechanism underlying these different responses to ARG is highly important. Thus, we employed BSO, a glutathione synthase inhibitor agent, to investigate the effect of glutathione depletion on the sensitivity of various cell lines to ARG treatment. The [ED.sub.50] of ARG was significantly reduced by BSO pre-treatment in A549 lung cancer cells and OUMS-36T-2F normal cells, whereas no changes in ED50 were observed in OUMS-36 and OUMS-36T-5F normal cells (Fig. 5).


We screened 365 Kampo Medicine and found the occurrence of lung cancer-specific cytotoxicity in 9 plant species. Among them, A. lappa L. was found to contain the cancer-specific agent ARG (Susanti et al., 2012), the selectivity of which was reinforced in this study (Fig. 1).

Our current study furthermore showed that ARG selectively induced apoptosis via the up-regulation of pro-apoptosis genes (Bax, Fas, Caspase-3, 8, and 9) and the down-regulation of an anti-apoptosis gene (Akt-1) (Fig. 4). However, the pro-apoptosis gene Bad and anti-apoptosis gene BcI-2 were down- and up-regulated, respectively, by ARG in A549 cancer cells. Bad activity has been shown to be regulated by phosphorylation via the action of Ala-1. Bad binds with BcI-2/13cl-xL in the cytosol and represses their anti-apoptotic activity. Phosphorylation of Bad by Akt-1 promotes its dissociation from the complex and relocates Bc1-2/Bc1-xL to the mitochondria, thereby inactivating the apoptotic process (Alberts et al., 2002). Thus, the induction of apoptosis by ARG may be mediated via the suppression of Akt-1. However, some of the gene expression profiles in A549 cancer cells appeared to argue against this explanation: ARG increased the expression of the anti-apoptotic gene BcI-2 and decreased the expression of the apoptotic gene Bad. These data indicate that the role of Akt-1 signaling might have limited significance in the induction of apoptosis by ARG. Further protein profile studies are needed to determine which signal transduction pathways are involved in ARG-induced apoptosis.

In addition to its role in apoptosis induction, Akt is known to play a role in the control of the cell cycle (Fig. 6). Under various circumstances, the activation of Akt overcomes the GI cell cycle arrest, thus enabling cell survival and proliferation (Ramaswamy et al., 1999). Inhibition of PI3K/Akt signaling has been shown to arrest the cell cycle at the [G.sub.1] phase and induce apoptosis in Hodgkin lymphoma (Georgakis et al., 2005). Similarly, the suppression of Akt-1 in the ERBB-2/Akt signaling pathway can inhibit E2F-1 (a protein with a crucial role in controlling cell cycle progression) and induce [G.sub.0]/[G.sub.1] cell cycle arrest (Guo et al., 2010). Thus, the ARG-mediated decrease in Akt-1 expression may contribute in part to the cell cycle arrest at the [G.sub.0]/[G.sub.1] phase observed in this study (Fig. 2).

Cell cycle arrest and apoptosis are closely linked in mammalian cells. The sensitivity of cells to certain types of apoptosis induction often depends on the cell cycle, and inhibition of the cell cycle has been considered as a target for cancer treatment (Sherr, 1994; Kornberg and Lorch, 1999). A previous study reported that ARG blocked progression of the cell cycle from the GI phase to the S phase by down-regulation of P-Rb in human gastric cancer (Jeong et al., 2011). Similarly, ARG increased the proportion of cells in the G1 phase of the cell cycle in A549 cancer cells and induced [G.sub.1]/S arrest. The investigation of cell cycle regulators showed that ARG decreased the levels of cycl in H, CDK-7, cyclin E, CDK-2, P-CDK, and NPAT proteins and had no effect on Rb and P-Rb proteins (Fig. 3). It is therefore likely that ARG arrests the cell cycle of lung cancer cells via a mechanism that is different from that for of human gastric cancer.

A previous study showed that in human gastric cancer, ARG arrested the cell cycle at the G0/G1 phase thorough the phosphorylation state of Rb protein, probably via the down-regulation of cyclin E/CDK2 or cyclin D/CDK4 (Jeong et al., 2011). ARG has been shown to arrest the cell cycle of human colorectal cancer cells in the G2/M phase by the modulation of the Wnt/[belta]-catenin signaling pathway (Yoo et al., 2010). These observations collectively indicate the variation in the cell cycle arrest point and relevant signaling pathways in cancer cells treated with ARG. It is noteworthy that ARG decreased the level of cyclin H/CDK-7. In addition to cyclin binding, CDK activity is also regulated by phosphorylation of conserved threonine and tyrosine residues. Thus, the full activation of CDK-2 requires phosphorylation of threonine 160 mediated by a cyclin H/CDK-7 complex termed CDK activating kinase (CAK). Our observation provided the first indication that ARG affected all cell cycle phases through the down-regulation of a cyclin H/CDK-7 complex (Fig. 6).

The modulation of signaling pathways by ARG appeared to converge into the regulation of the NPAT final effector proteins to control the cell proliferation process. NPAT is involved in histone biosynthesis, and the inhibition of NPAT expression impedes expression of all histone subtypes (Zhao et al., 2000). The phosphorylation of NPAT by cyclin E/CDK-2 has been shown to lead to its association with histone gene promoters. Therefore, it is likely that inhibition of cyclin E/CDK2 led to the down-regulation of NPAT observed in this study (Fig. 3).

A previous study reported that the cytotoxicity of A. lappa L. extract is reduced by intracellular GSH (Moritani et al., 1996). In the present study, the inclusion of a GSH synthase inhibitor enhanced the sensitivity of A549 and OUMS-36-2F cells to ARG (Fig. 5). This finding suggested that the GSH content of cancer cells may also determine their susceptibility to ARG. It is suggested that GSH sequestered ARG from the system through the conjugation reaction (illustrated in Fig. 7) and consequently reduced its cytotoxicity. Thus, in addition to cell cycle control, the expression of GSH synthase may become of a new target for the development of anticancer agents.

Our previous study, among others, suggested that the anticancer activity of ARG is quite cell-specific: the cytotoxicity of ARG affects cancer cells, while normal cells are resistant. However, the current study also showed variation in the cytotoxic effects of ARG on different types of normal cells. OUMS-3T-2F normal cells were sensitive to ARG, as was the case for A549 cancer cells, whereas OUMS-36 and OUMS-36T-5F normal cells were resistant to this agent. Both OUMS-3T-2F and OUMS-3T-5F were generated via transformation of OUMS-36 primary cells with the hTRT gene (human telomerase reverse transcriptase gene in pMX) (Kouchi and Namba, 2000). Restriction Fragment Length Polymorphism (RFLP) analysis or (short tandem repeat (STR)-PCR) of the genomic DNA found that OUMS-3T-2F only differs from other OUMS-36 series cell lines in the length of the vWA microsatellite markers; the STR 20 in OUMS-3T-2F was replaced with STR 21 in OUMS-3T-5F. Therefore, it is clear that the difference in the genomic DNA sequence close to this marker was responsible for the susceptibility to ARG, and investigation of this notion may shed light on the mechanism underlying the cancer-specific cytotoxicity of ARG. Furthermore, continued research into this topic will contribute to the development of novel anticancer agents with fewer adverse effects.


We are grateful to the council of the United Graduate School of Agricultural Sciences for the funding for this project through the Rendai-Student Supporting Program (2012).

Abbreviations: ARG, arctigenin; GSH, glutathione; [G.sub.0]/[G.sub.1] , Gap 0/Gap 1; NPAT, nuclear protein of the ataxia telangiectasia locus; CDK2, cyclin-dependent kinase 2; CDK7, cyclin-dependent kinase 7: akt-1, alpha serine/threonine-protein kinase.

0944-7113/$--see front matter [C] 2013 Elsevier GmbH. All rights reserved.


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Siti Susanti (a), (b), (c) Hironori Iwasaki (b), Masashi Inafuku (b), Naoyuki Taira (a), (b), Hirosuke Oku (b), *

(a) United Graduate School of Agricultural Sciences. Kagoshima University, 1-21-24. Korimoto, Kagoshima 890-0065, Japan

(b) Center of Molecular Biosciences, Tropical Biosphere Research Center. University of the Ryukyus, Nishihara, Okinawa 903-0213. Japan

(c) Department of Animal and Agricultural Sciences, Diponegoro University, Central Java, Indonesia

CDK7, cyclin-dependent kinase 7; akt-1, alpha serine/threonine-protein kin

* Corresponding author. Tel.: +81 98 895 8972; fax: +81 98 895 8944. E-mail address: okuhiros@comb.u-ryukyu.acjp (H. Oku).

GO/G1 phase  80.1%
S phase       8.5%
G2/M phase   10.6%

GO/G1 phase  82.4%
S phase       4.5%
G2/M phase   12.4%

GO/G1 phase  53.1%
S phase      16.2%
G2/M phase   28.6%

GO/G1 phase  50.5%
S phase      17.3%
G2/M phase   30.3%

GO/G1 phase  48.5%
S phase      26.0%
G2/M phase  23.5%

GO/G1 phase  74.8%
S phase       3.7%
G2/M phase   21.0%

GO/G1 phase  55.7%
S phase      24.7%
G2/M phase   19.4%

GO/G1 phase  81.4%
S phase      1O.1%
G2/M phase   8.1%
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Author:Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Tairaa, Naoyuki; Oku, Hirosuke
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9JAPA
Date:Dec 15, 2013
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