Acta Biochim Biophys Sin 2014, 46: 441 – 449 | ª The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu025. Advance Access Publication 16 April 2014

Original Article

Target genes involved in antiproliferative effect of modified ginseng extracts in lung cancer A549 cells Keun-Hong Kim1†, Ilsan Choi2†, Yeon-Weol Lee1, Chong-Kwan Cho1, Hwa-Seung Yoo1 *, Seung-Bae Lee3, Suk Ho Choi3, Ki-Rok Kwon4, and Jun-Hyeog Jang2* 1

East-West Cancer Center, Dunsan Oriental Hospital of Daejeon University, Daejeon 302-122, Korea Department of Biochemistry, Inha University School of Medicine, Incheon 400-712, Korea 3 Division of Animal Resources and Life Science, Sangji University, Wonju 220-702, Korea 4 Research Center of Pharmacopuncture Medicine, Korean Pharmacopuncture Institute, Seoul 157-801, Korea † These authors contributed equally to this work. *Correspondence address. Tel: þ82-31-890-0933; Fax: þ82-32-882-1877; E-mail: [email protected] (J.J). Tel: þ82-42-470-9456; Fax: þ82-42-470-9006; E-mail: [email protected] (H.Y.) 2

Keywords apoptosis

ginseng;

Received: January 10, 2014

saponins;

cancer;

ginsenoside;

Accepted: February 20, 2014

Introduction Lung cancer is the leading cause of cancer-related deaths worldwide, and 1.61 million new cases are reported every

year [1]. Non-small-cell lung cancer, which is the most common type of lung cancer, accounts for 85%–90% of all lung cancer cases and is strongly associated with smoking [2]. The root of Panax ginseng C.A. Meyer (Korean ginseng) has traditionally been used as a herbal medicine in East Asia for the treatment of various diseases, including cancer [3–7]. The major biologically active components of Panax ginseng are a series of saponin glycosides collectively known as ginsenosides, which are a group of steroidal saponins. To date, over 50 ginsenosides have been isolated and identified from ginseng saponins [8]. Ginsenosides have a steroid-like skeleton consisting of four trans-rings with modifications that depend on the type (e.g. glucose, maltose, and fructose) and number of sugar moieties, as well as the sites of attachment of the hydroxyl group (e.g. C-3, C-6, or C-20). Based on the chemical structural characteristics, ginseng saponins can be divided into two groups, namely protopanaxadiol and protopanaxatriol (PPT), with the exception of ginsenoside Ro, which is derived from an oleanolic group. In the protopanaxadiol group, sugars are attached to the b-OH at C-3 and another –OH at C-20, as found in Rb1, Rb2, Rc, Rd, Rg3, and Rh2. In the PPT group, sugar residues are attached to the a-OH at C-6, with another –OH at C-20, as found in Re, Rg1, Rg2, Rh1, and Rf. Recent studies have reported the cytotoxic effects of minor saponins such as Rh1 and Rh 2 on the growth of various cancer cells, and the inhibitory effects of human intestinal bacterial saponin metabolites such as Compound K and PPT on the growth, invasion, and migration of tumor cells [9–13]. Human intestinal bacteria have recently been shown to cleave the oligosaccharide connected to the aglycon stepwise from the terminal sugar of ginsenosides to form the metabolite 20S-protopanaxadiol 20-O-b-D-glucopyranoside (IH-901, Compound K) after oral administration of Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 6 | Page 441

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Lung cancer is the most common cancer and the leading cause of cancer-related deaths. Panax ginseng has long been used to treat cancer and other diseases worldwide. Most of the pharmacological actions of ginseng are attributed to a variety of ginsenosides, which are often metabolized by intestinal bacteria into more effective forms. In this study, we found that the antiproliferative activity of ginseng was increased after enzymatic processing of ginseng saponin (50% inhibitory concentration, >70 mg/ml). To elucidate the mechanism by which modified ginseng extract (MGX) induced cell death in human lung cancer cells, the gene expression profiles of A549 cells regulated by MGX were assayed using Agilent PrimeView Human Gene Expression Arrays. The expression of 17 genes involved in the regulation of cell signaling, cell metabolism, transport, and cytoskeleton-regulation was up-regulated, whereas the expression of 16 genes implicated in invasion and metastasis and cellular metabolism was down-regulated in MGXtreated A549 cells. Moreover, nuclear staining with 40 ,6-diamidino-2-phenylindole revealed that MGX clearly caused nuclear condensation and fragmentation which are observed in apoptosis cell. These results elucidate crucial anticancer mechanisms of MGX and provide potential new targets for the assessment of anticancer activity of MGX.

Anticancer mechanisms of modified ginseng extracts

Materials and Methods Preparation of enzyme-MGX The root of regular ginseng (4 years old) was purchased from G&V (Wonju, Korea). A total of 20 g of pulverized ginseng root powder was suspended in 380 ml of distilled water and then sterilized by heating at 1218C for 15 min. To reduce the complexity of the components in the ginseng root, the extract was fractionated via extraction with water, methanol, and butanol. Among the fractions, the ginseng butanol extract (GBX) contained the compounds with specific anticancer activity; therefore, GBX was used as a control for the MGX. In addition, the suspension was treated with aliquots of filter-sterilized commercial enzymes (100 mg laminarinase and 100 mg pectinase) at an equimolar ratio (1 : 1, specific activity unit). For these treatments, the mixture was incubated at 408C for 2 days and then evaporated to dryness at 608C. The enzyme-modified ginseng powders were then suspended in 400 ml of 80% (v/v) methanol and subject to ultrasonication for 5 min followed by filtration through Whatman No. 2 filter paper. The wet powder on the filter paper was then collected, suspended, treated in an ultrasound bath, and filtered in the same manner once again, after which the two filtrates were combined and evaporated to dryness at 508C. The methanol extract was then dissolved in 200 ml of distilled water, washed with 200 ml of ethyl acetate in a separation funnel, and then extracted with 200 ml of butanol. Next, the butanol extract was evaporated to dryness at 508C and then dissolved to a concentration of 10% (w/v) in 70% ethanol. The final sample was designated as the MGX. HPLC analysis of ginsenosides Standard ginsenosides, including Rg1, Re, Rf, Rh1, Rb1, Rc, Rb2, f1, Rd, Rg3(S), Rg3(R), PPT, Compound K, Rh2, Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 6 | Page 442

and PPD, were obtained from ChromaDex, Inc. (Irvine, USA) and analyzed using an Acquity UPLC system (Waters, Milford, USA) with an Acquity BEH C18 HPLC column. The mobile phase consisted of Solution A (CH3CN, HPLC-grade, JT Baker, Phillipsburg, USA) and Solution B (Milli-Q H2O, Millipore, Billerica, USA). The flow rate was set at 0.6 ml/min and the injection volume was fixed at 2 : l. The separation of ginsenosides was performed using an isocratic gradient of 100% Solution A for 27 min. The column was maintained at room temperature during the separation, and the UV diode array detection was set at 203 nm. To confirm a reliable retention time of sample ginsenosides, the individual ginsenosides were identified and quantified based on comparison with the retention times of standard ginsenosides.

Cell culture Human non-small-cell cancer cell line A549 was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). A549 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum (Invitrogen, Carlsbad, USA), 100 units/ml penicillin G sodium, 100 mg/ml streptomycin sulfate, and 0.25 mg/ml amphotericin B (Invitrogen) under 5% CO2 at 378C. Confluent cells were detached with 0.25% trypsin-EDTA for 5 min, after which the aliquots were subcultured. Cell viability assay The effects of MGX on cell viability were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which quantifies the number of viable cells. Cells were seeded (2  104 cells/well) in 24-well flatbottomed plates (Nunc, Thermo Scientific, Waltham, USA). After incubation at 378C for 24 h, the medium was replaced with MGX at the appropriate concentrations. Control cells were treated with DMSO at a final concentration of ,0.2%. After incubation for an additional 24 h, cells were washed three times with Dulbecco’s phosphate-buffered saline, and 500 ml of MTT solution (5 mg/ml in PBS) was added to each well. After incubating for 4 h, the medium was removed and 200 ml of formazan (crystals dissolved in DMSO) was added to the cells. The absorbance of each well was then read at 540 nm using a microplate reader. RNA extraction and cDNA synthesis Total RNA was extracted using an Easy-spin RNA Extraction kit (iNtRON, Seoul, Korea) according to the manufacturer’s instructions. The purity of the RNA was assessed by measuring the absorption at 260 and 280 nm (A260/A280 ratios of 1.9–2.1 were considered acceptable) and by ethidium bromide staining of 18S and 28S RNA by gel electrophoresis. RNA concentrations were determined from the A260. Next, 2 mg of total RNA were reverse-transcribed in a 20-ml reaction mixture containing 50 units of

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ginseng extract [14]. Transformed 20S-protopanaxadiol 20-O-b-D-glucopyranoside from ginsenosides Rb1, Rb2, and Rc induces an antimetastatic or anticarcinogenic effect [15]. Wakabayashi et al. [16] revealed that the antitumor activities of ginsenosides following oral administration are attributable to metabolites formed by colonic bacteriamediated deglycosylation. In addition, Compound K is known to exhibit cytotoxicity via the induction of apoptosis and cell cycle arrest in G1 phase through a caspasedependent pathway involving mitochondrial disruption of tumor cells and reversal of multidrug resistance in tumor cells [17–19]. Furthermore, combined treatment with Compound K and radiation enhances human lung cancer cell death [17,20]. Therefore, in this study, we investigated the antiproliferative effects of modified ginseng extract (MGX) after transformation by enzyme treatment. To this end, we used human non-small-cell lung A549 cells and further investigated the possible mechanisms of action.

Anticancer mechanisms of modified ginseng extracts

SuperScript II reverse transcriptase (Invitrogen), 5 mM DTT, 40 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), 0.5 mM random hexanucleotide primers, and 500 mM of dNTP mixture. The reverse transcription reaction was performed at 508C for 60 min, after which the reaction was terminated by heating the mixture at 708C for 15 min. The complementary DNA (cDNA) was then stored at 2208C until use.

Microarray assay Gene expression was analyzed using the Agilent PrimeView Human Gene Expression Arrays containing 36,000 gene transcripts and variants from the UniGene resource (Affymetrix, Santa Clara, USA). This array is composed of over 45,000 probe sets representing 38,500 well-characterized human genes. For each gene, 11 pairs of oligonucleotide probes

DAPI staining To detect the apoptotic cells, the cells were first washed twice with cold PBS and then fixed with 4% paraformaldehyde (Sigma, St Louis, USA) for 30 min. The fixed cells were then washed again with PBS and stained with 1 mg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma) solution for 30 min. Finally, the cells were washed two more times with PBS and analyzed by fluorescence microscopy (Axiovert 200 inverted microscope; Zeiss, Oberkochen, Germany).

Results Effects of MGX on cell growth and viability in human non-small lung cancer A549 cells Ginsenosides have been shown to inhibit tumor cell proliferation and tumor growth, as well as to induce differentiation

Table 1. Primer sequences for qRT-PCR experiments

Genes

Sense (50 – 30 )

Anti-sense (50 – 30 )

FHL1 CYP1B1 ACAT2 CXCL1 AQP3 MMP7 GAPDH

GCATGCTTTTCTGAGCCTTC TGATAAGCACGTTGCAGGAG CGTGGGCTTACACCTTTAGC CCGAAGTCATAGCCACACTC CCTTCTTGGGTGCTGGAATA AAAGAGATCCCCCTGCATTT TGGAAGGACTCATGACCACA

TTCCTCTGAAACAGGCTCGT CAAAGCAAATGGCACAGATG AGCTTGCTTTATGGCTGGAA GGTGCTCCCCTTGTTCAGTA CACACGATAAGGGAGGCTGT GTGAGCATCTCCTCCGAGAC TTCAGCTCAGGGATGACCTT

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Quantitative real-time reverse transcriptase polymerase chain reaction Overexpression of the target genes was confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). All real-time PCR analyses were performed using an ABI Step One Real-time PCR system (Applied Biosystems, Foster City, USA). Each reaction contained 0.1 mM of each primer, 10 ml of the 2 SYBR Green PCR master mix (including AmpliTaq Gold DNA polymerase with buffer, dNTPs mix, SYBR Green I dye, ROX dye, and 10 mM MgCl2), and 1 ml of the template cDNA in a 20-ml total reaction volume. The typical amplification program included activation of the enzyme at 948C for 10 min, followed by 40 cycles of denaturation at 948C for 15 s, then annealing and extension at 608C for 1 min. The CT (cycle threshold) value for each gene was determined using the automated threshold analysis function of the ABI instrument and normalizing to CT(G3PDH) to obtain dCT ( ¼ CT(G3PDH) 2 CT(test)). The difference in n between the 2 CT or dCT values indicates a 2n-fold difference in the amount of the target sequence between the two cDNA samples being compared. The primers used for quantitative PCR are shown in Table 1.

were synthesized in situ on the arrays. Biotinylated cRNA was then prepared according to the standard Affymetrix protocol from 100 ng of total RNA (Expression Analysis Technical Manual, 2001, Affymetrix). Following fragmentation, 12 mg of RNA was hybridized for 16 h at 458C on a GeneChip Human Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450, after which they were scanned using an Affymetrix GeneChip Scanner 3000 7G. The data were then analyzed using the Microarray Suite version 5.0 and the Affymetrix default analysis settings, and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The normalized and log-transformed intensity values were then analyzed using GeneSpring GX 11.5.1 (Agilent Technologies, CA). Fold-change filters included the requirement that the genes be present in at least 200% of the controls for genes showing up-regulated expression and ,50% of controls for genes showing down-regulated expression. Hierarchical cluster analysis using GeneSpring GX 11.5.1 (Agilent technologies) identified clustered groups as those that behaved in a similar manner in all experiments. The clustering algorithm was Euclidean distance, with average linkage.

Anticancer mechanisms of modified ginseng extracts

and apoptosis [21]. To determine the antiproliferative effects of MGX on human non-small lung cancer A549 cells, we conducted an MTT assay. MGX inhibited A549 cell growth in a dose-dependent manner at 14.8 22.2, 33.3, 50, and 100 mg/ml at 24 h. The concentration at which 50% growth inhibition occurred was 70 mg/ml (Fig. 1). However, exposure of A549 cells to 100 mg/ml non-treated ginseng saponin extract showed less growth inhibition (10%). Therefore, these results suggest that enzymatic processing of ginseng saponin increased its antiproliferative effects on A549 cells.

Real-time RT-PCR validation of microarray data To confirm the results obtained using microarrays, we investigated the expression of six target genes in MGX treated A549 cells by qRT-PCR analysis. These genes included four that showed highly up-regulated expression, namely FHL1, CYP1B1, AQP3, and ACAT2 and two that was highly down-regulated, namely, CXCL3 and MMP7. To accurately quantify the expression of these six genes, the data were

Figure 1. Antiproliferative effects of MGX on human A549 lung cancer cells (A) Cell viability at the indicated concentrations of MGX at 24 h was assessed by the MTT assay. (B) Microscopic images of A549 cells treated with MGX (0, 50, and 100 mg/ml) (100-folds). Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 6 | Page 444

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Identification of differentially expressed genes in A549 human lung adenocarcinoma cells treated with MGX Although ginseng has been long used as a traditional medicine for the treatment of cancer, the underlying mechanisms for the varying activities observed following exposure to ginseng are largely unclear. To identify relevant alterations in gene expression associated with MGX treatment, we analyzed the gene expression profiles of A549 cells by using Affymetrix Gene Chip arrays containing more than 38,500 genes. Of a total of 38,500 genes, we identified known 33 genes whose expression was significantly up- or downregulated with magnitude exceeding a cutoff of 2-fold change following treatment with MGX. Among these, 17 had significantly up-regulated expression, and 16 had significantly down-regulated expression when compared with the control treatment (Tables 2 and 3).

Several genes involved in the regulation of cell signaling (FHL1, IGF-2, BPTF2, LUZP1, and MYEF2) were expressed at higher levels following treatment with MGX. Furthermore, several genes implicated in cell metabolism (ACAT2, RDH10, and NAPEPLD), transport (AQP3 and SLC11A2), and cytoskeleton regulation (DST and KIF21A) showed increased expression in A549 human non-small-cell lung cancer cells that were treated with MGX (Table 2). In contrast, 16 genes showed reduced expression in A549 human non-small-cell lung cancer cells treated with MGX. There was an over-representation of members of genes involved in invasion and metastasis (CXCL1, CXCL3, Rhotekin 2, TFPI2, NPTX1, EFNA1, and MMP7) and cellular metabolism (MT1, ARSI, and YWHAE). Table 3 lists the names of genes exhibiting reduced expression where the difference was greater than 2-folds. This differently expressed group of genes may serve as an important target in MGX-mediated anticancer activity in human non-small-cell lung cancer cells.

Anticancer mechanisms of modified ginseng extracts

Table 2. Genes with highly up-regulated expression due to MGX in A549

Symbol

FHL1

Gene description

Fold GenBank ID change

Symbol

4.46

NM001159699

CXCL1

3.6

NM000612

3.2 3.2

NM000104 NM032582

2.8

NM005891

2.6 2.6 2.6

NM001723 NM004925 NM004459

2.6

NM001173463

2.5 2.4 2.3 2.2 2.2 2.2

NM001142546 NM000617 NM001025356 NM014646 NM001122838 NM001122838

2.1 2.1

NM016132 NM001145354

normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The expression levels of FHL1, CYP1B1, AQP3, and ACAT2 increased by 3-folds, 18-folds, 2.7-folds, and 2-folds, respectively, upon treatment with MGX (Fig. 2). Conversely, the expressions of CXCL1 and MMP7 were decreased by 4-folds and 2-folds, respectively. These results indicate that the qRT-PCR results are consistent with the microarray data.

MGX-mediated cell death in A549 non-small-cell lung cancer cells To determine whether MGX has an effect on lung cancer cells, A549 cells treated with DMSO or MGX for 24 h were subjected to fluorescence microscopic analysis after DAPI staining. In the control cells, the nuclei showed uniform staining, thereby indicating that cells were healthy and the nuclei were intact. However, after treatment with 50–100 mg/ml MGX for 24 h, some nuclei exhibited typical apoptotic characteristics such as nuclear condensation and fragmentation.

Gene description

Chemokine (C– X – C motif) Ligand 1 CXCL3 Chemokine (C– X – C motif) Ligand 3 IL8 Interleukin 8 RTKN2 Rhotekin 2 MT1F Metallothionein 1F TFPI2 Tissue factor pathway inhibitor 2 NPTX1 Neuronal pentraxin I ARSI Arylsulfatase family, Member I SCLT1 Sodium channel and clathrin linker 1 EFNA1 Ephrin-A1 MAP3K8 Mitogen-activated protein kinase kinase kinase 8 YWHAE Tyrosine 3-monooxygenase IGFBP3 Insulin-like growth factor binding protein 3 MMP7 Matrix metallopeptidase 7 TPM1 Tropomyosin 1 IL15 Interleukin 15

Fold change

GenBank ID

28.3

NM_001511

26.1

NM_002090

24.1 22.8 22.7 22.7

NM000584 NM145307 NM005949 NM006528

22.6 22.5

NM002522 NM001012301

22.4

NM144643

22.3 22.2

NM004428 NM005204

22.2

NM006761

22.1

NM000598

22.1 22.1 22.0

NM002423 NM000366 NM000585

Some floating dead cells were also observed at this time (Fig. 3). These results suggest that MGX induces cell death in A549 cells.

Discussion Recent studies including ours have reported the cytotoxic effects of minor saponins such as Rh1 and Rh 2 on the growth of various cancer cells, and the inhibitory effects of human intestinal bacterial saponin metabolites such as Compound K and PPT on the growth, invasion, and migration of tumor cells [9–13,22]. In this study, we used cDNA microarray analysis to identify novel target genes involved in the anticancer activity of MGX (Fig. 4). We found that several genes involved in the regulation of cell signaling (FHL1, IGF-2, BPTF2, LUZP1, and MYEF2), cell metabolism (ACAT2, RDH10, and NAPEPLD), transport (AQP3 and SLC11A2), and cytoskeleton regulation (DST and KIF21A) were expressed at higher levels following treatment with MGX. Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 6 | Page 445

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Four and a half LIM domains 1 IGF2 Insulin-like growth factor 2 CYP1B1 Cytochrome P450 USP32 Ubiquitin-specific peptidase 32 ACAT2 Acetyl-CoA acetyltransferase 2 DST Dystonin AQP3 Aquaporin 3 BPTF2 Bromodomain PHD finger transcription factor KIF21A Kinesin family member 21A LUZP1 Leucine zipper protein 1 SLC11A2 Solute carrier family 11 ANO6 Anoctamin 6 LPIN2 Lipin 2 RDH10 Retinol dehydrogenase 10 NAPEPLD N-acyl phosphatidylethanolamine phospholipase D MYEF2 Myelin expression factor 2 MKLN1 Muskelin 1

Table 3. Genes with highly down-regulated expression due to MGX in A549

Anticancer mechanisms of modified ginseng extracts

In this study, FHL1 proteins, which are a family of LIM domain-only proteins implicated in transcriptional regulation and suppression of tumor growth, were significantly up-regulated by MGX. The FHL1 protein has been shown to interact with Smad proteins, increase the expression of growth inhibitor genes such as the CDK inhibitor p21, and decrease the expression of the growth-promoting gene c-myc [23]. Furthermore, FHL1 inhibits hepatocellular carcinoma cell growth, both in vitro and in nude mice. Cytochrome P450 is a member of the cytochrome P450 monooxygenase superfamily that plays an important role in xenobiotic metabolism as well as in carcinogenesis [24]. In this study, the CYP1B1 levels were significantly increased after treatment with MGX. CYP1B1 enzymes mediate the bioactivation of carcinogens found in both the human diet and the environment, including compounds such as polycyclic aromatic hydrocarbons, which include 3-methylcholanthrene, benzo(a)pyrene, polyhalogenated aromatic hydrocarbons, and certain congeners of polyhalogenated biphenyls [25]. Our finding that MGX increased the expression of CYP1B1 is Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 6 | Page 446

consistent with the results of previous studies that showed the induction of CYP1B1 expression by ginsenosides Rg1 and Rb1 [26]. A similar result was shown in our previous study with MGX in HepG2 hepatocarcinoma cell [22]. However, several genes implicated in invasion and metastasis (CXCL1, CXCL3, Rhotekin 2, TFPI2, NPTX1, EFNA1, and MMP7) and cellular metabolism (MT1, ARSI, and YWHAE) exhibited decreased expression in A549 human lung adenocarcinoma cells treated with MGX. Chemokine (C–X– C motif) ligand (CXCL) is a chemokine that enhances cancer cell survival and chemoresistance. Furthermore, CXCl1 blocker increases the effectiveness of chemotherapy against breast cancer, particularly, against metastasis [27]. Rhotekin is a scaffold protein that interacts with S100A4 to facilitate invasion and metastasis [28]. Rhotekin is over-expressed in metastatic colon cancer cells [29] and gastric adenocarcinoma cells, where it confers resistance to apoptosis through activation of nuclear factor-kB [30]. Matrix metalloproteinase 7 (MMP7), also known as matrilysin, is a member of a family of zinc-dependent

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Figure 2. qRT-PCR validation of microarray data (A) qRT-PCR analysis of the four up-regulated genes: ACAT2, CYP1B1, FHL1, and AQP3. (B) qRT-PCR analysis of the two down-regulated genes: CXCL3 and MMP7. Values are -fold change+SE (n ¼ 3).

Anticancer mechanisms of modified ginseng extracts

Figure 4. A schematic diagram of microarray data analysis Microarray data analysis used the Agilent PrimeView human Gene expression array containing 38,500 gene transcripts and variants via UniGene annotations. Gene expression profiles of the A549 cells regulated by MGX were assayed. Scatter plot was shown for global comparison of gene expression. Only mRNAs with a cutoff of 2-fold change in expression were reported.

endopeptidases [31]. MMP7 is over-expressed in pancreatic, gastric, and colorectal cancers [32,33], and MMP levels correlate with clinical stages of malignancy, including lymph node involvement and distant metastasis [34–36]. Our

results showing the decreased expression of several genes involved in invasion and metastasis in A549 cells treated with MGX may facilitate elucidation of the anticancer mechanisms of MGX. Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 6 | Page 447

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Figure 3. MGX induces cell death in A549 cells A549 cells were exposed to MGX (50 and 100 mg/ml) for 24 h, after which the DAPI-stained nuclei were analyzed for apoptotic morphology by fluorescence microscopy. A549 cells were also photographed by inverted phase-contrast microscopy (upper panel, original magnification 400).

Anticancer mechanisms of modified ginseng extracts

Moreover, nuclear staining with DAPI revealed that MGX clearly caused nuclear condensation and fragmentation observed in apoptosis in A549 human lung adenocarcinoma. Taken together, these results elucidate crucial anticancer mechanisms of MGX and provide potential new targets for the assessment of anticancer activity of MGX.

Funding This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MOE) (2010–0022628, 2013R1A1A2011375) and an Inha University Grant.

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Acta Biochim Biophys Sin (2014) | Volume 46 | Issue 6 | Page 449

Target genes involved in antiproliferative effect of modified ginseng extracts in lung cancer A549 cells.

Lung cancer is the most common cancer and the leading cause of cancer-related deaths. Panax ginseng has long been used to treat cancer and other disea...
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