Anticancer activity and mechanism of Scutellaria barbata extract on human lung cancer cell line A549

Life Sciences 75 (2004) 2233 – 2244 www.elsevier.com/locate/lifescie

Anticancer activity and mechanism of Scutellaria barbata extract on human lung cancer cell line A549 Xiaolu Yina, Jiangbing Zhoua, Chunfa Jieb, Dongming Xingc, Ying Zhanga,* a

Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD 21205, USA b McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, United States c Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China Received 17 November 2003; accepted 12 May 2004

Abstract Scutellaria barbata (S. barbata), a traditional Chinese herbal medicine native to southern China, is widely used as an anti-inflammatory and a diuretic in China. Several studies have indicated that extracts of S. barbata have growth inhibitory effects on a number of human cancers. Treatment of lung cancer, digestive system cancers, hepatoma, breast cancer, and chorioepithelioma by S. barbata extracts was reported. However, the mechanism underlying the antitumor activity was unclear. In this study, we studied the growth inhibitory effect of S. barbata and determined its mechanism of antitumor activity using human lung cancer cell line A549. Our results showed that ethanol extracts of S. barbata greatly inhibited A549 cell growth, with IC50 of 0.21 mg/ml. The major mechanisms of inhibition included cell apoptosis and cytotoxic effects. cDNA microarray analysis showed that 16 genes, involved in DNA damage, cell cycle control, nucleic acid binding and protein phosphorylation, underwent more than 5-fold change. These data indicated that these processes are involved in S. barbata-mediated killing of cancer cells. A surprising finding is that CD209, related to dendritic cell (DC) function, was dramatically downregulated by 102-fold. Further functional studies are needed to assess the role of the array-identified genes in S. barbata mediated anticancer activity. D 2004 Elsevier Inc. All rights reserved. Keywords: Cancer; Herbal medicine; Scutellaria barbata

* Corresponding author. Tel: +1 410 614 2975; fax: +1 410 955 0105. E-mail address: [email protected] (Y. Zhang). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.05.015

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Introduction Lung cancer is now the most common cause of cancer-related death among both men and women, accounting for 28 percent of all cancer deaths in the U.S (Non-Small Cell Lung Cancer Collaborative Group, 1995; Cardenal et al., 1999). Each year an estimated 171,600 Americans will be diagnosed with lung cancer (Non-Small Cell Lung Cancer Collaborative Group, 1995; Cardenal et al., 1999). Lung cancer is also very serious in some other countries, including China. Currently chemotherapy is still the standard treatment method. Although chemotherapy significantly improves symptoms and the quality of life of patients with lung cancer, only modest increase in survival rate can be achieved (Ten Bokkel Huinink et al., 1999; Sandler et al., 2000; Bonomi et al., 2000; Chou et al., 2003). Faced with palliative care, many cancer patients use alternative medicines, including herbal therapies (Eisenberg et al., 1998; Risberg et al., 1998; Sadava et al., 2002). Among these therapies, traditional Chinese medicine is probably the best established and codified, dating back several thousand years. Specific herbal extracts, and combinations, have been devised to treat specific diseases including cancers (Huang, 1999; Tang and Eisenbrand, 1992). Scutellaria barbata (S. barbata), a plant native to southern China, has been used in traditional Chinese medicine as an anti-inflammatory and anti-tumor agent and also a diuretic (Qian, 1987). Extracts of S. barbata have been shown to have in vivo growth inhibitory effects on a number of cancers (S180 mouse sarcoma, U14 cervical carcinoma, solid hepatoma, W256 rat sarcoma. and EhrlichTs sarcoma) (Qian, 1987). In the clinic, the herb has been used in the treatment of lung cancer, digestive system cancers, hepatoma, breast cancer, and chorioepithelioma in China (Qian, 1987). In particular, sixty-two percent of patients suffering from hepatoma were completely cured when treated with S. barbata (Qian, 1987). However, the effect of S. barbata on human lung cancer cells in vitro and its mechanism of action are unknown and need further investigation. Two major pathways leading to apoptosis exist in cells: the extrinsic pathway, which involves the activation of the TNF/Fas death receptor family and the intrinsic pathway, which involves the mitochondria. In both pathways, an apoptotic death stimulus results in the activation of Caspases, the major executioners of this process, either directly or via activation of the mitochondrial death program (Nicholson, 1999; Yacobi et al., 2004). The Caspase family includes initiator Caspases (i.e. Caspase-8 and -9), which activate effector Caspases (i.e. Caspase-3, -6, and -7). All Caspases are expressed as inactive enzymes (zymogens) and their activation involves two cleavage events. These cleavages result in the generation of a large and small subunit, which associate to form an active heterotetrameric enzyme (Yacobi et al., 2004). Caspase-3 is the most characterized effector Caspase, and its activation leads to the final stages of cellular death by proteolytic dismantling of a large variety of cellular components on the one hand, and activation of proapoptotic factors on the other (Nicholson, 1999; Yacobi et al., 2004). Caspase-7 is another effector Caspase that is very similar to Caspase-3 in terms of substrate specificity and was suggested to play a role together with Caspase-3 in apoptosis (Yacobi et al., 2004; Matikainen et al., 2001; Cohen, 1997). In this study, we demonstrated that the S. barbata ethanol extract showed anti-tumor activity in vitro and could inhibit the growth of human lung cancer cell line A549. The basic mechanism of inhibition was due to cell apoptosis and cytotoxic effects. cDNA microarray experiment was also performed to reveal the molecular basis of S. barbata induced killing of the lung cancer cell line.

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Materials and methods Preparation of S. barbata extract The herb was obtained from Tong Ren Tang Chinese herbal medicine store, Beijing. The original plant was collected in Henan Province, China (August, 2002) and identified as S. barbata by Dr. Xing. Voucher specimens were deposited in the Herbarium of the Biological Sciences and Technology Department in Tsinghua University, Beijing, China. The whole plant was minced and extracted with 30% ethanol. The S. barbata ethanol extract was used to derive a series of concentrations used in this paper. Cell culture Human lung cancer cell line A549 was obtained from ATCC (American Type Culture Collection). Cells were grown in DMEM medium (GIBCO) supplemented with 10% fetal bovine serum, 100 units/ ml penicillin and 100 Ag/ml streptomycin at 378C in an incubator containing 5% CO2. MTT assay MTT assay was performed as described (Wang et al., 2001). Briefly, cells were seeded at a concentration of 1.5104 cells/ml in a 96-well plate. After overnight incubation, serial concentrations of S. barbata ethanol extract (0.10, 0.25 and 0.50 mg/ml), were added. Since 1% ethanol was added to drug treatment group, control group was added with 1% ethanol. Each concentration was repeated three times. These cells were incubated in a humidified atmosphere with 5% CO2 for 3 days. Then, 20 Al MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) solution (4.14 mg/ml) was added to each well and incubated at 378C for 4 hours. The medium was removed and formazan was dissolved in DMSO and the optical density was measured at 590 nm using a Bio-assay reader (BioRad, USA). The growth inhibition was determined using: Growth inhibition = (control O.D.-sample O.D.)/control O.D. Cell viability assay Cells were seeded on 24-well tissue culture plates and S. barbata extract was added to a series of concentrations as indicated. The cultures were maintained at 378C in a tissue culture incubator containing 5% CO2 for 3 days. The cells were collected by trypsinization and stained with trypan blue. The dead cells and the total cells were counted. The percentage of viable cells (%) was calculated as [(total cells-dead cells) / total cells]  100% (Wu et al., 2002). Cytotoxicity assay The cytotoxic potential of the S. barbata extract was determined by the cytotoxicity detection kit (Roche Molecular Biochemicals), which was based on the detection of lactate dehydrogenase (LDH) released from dead cells as a result of cytotoxicity. Cell-free culture supernatants from S. barbata treated A549 cells were collected and transferred to microtiter plates. Substrate mixture containing tetrazolium salts was added, and then incubated for 3 h. The formazan dye was quantitated by measuring the absorbance at 490 nm.

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Caspase 3/7 assay Caspase 3/7 activities were assayed by using Apo-ONEk Homogeneous Caspase-3/7 Assay kit (Promega). Briefly, 100 Al S. barbata extract treated A549 cells, control cells and blank (no cell medium) were transferred into a 96 well plate and 100 Al of Homogeneous Caspase-3/7 Reagent was added. The plate was covered with a plate sealer. After incubation for 12 h, fluorescence of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Fluorescent microscopic analysis of apoptosis by Annexin-V staining Annexin-V-FLUOS Staining Kit (Roche Diagnostics) was performed as described by the manufacturer. Apoptotic cells were visualized by fluorescence microscopy and expressed as a percentage of apoptotic cells. Briefly, 100 Al labeling solution (20 Al Annexin-V-FLUOS-labeling reagent in 1000 Al HEPES buffer with 20 Al propidium iodide (50 Ag/ml) added) was added to control and drug treated cells. Samples stained only with propidium iodide or Annexin-V-FLUOS were used as single staining control. All samples were incubated at room temperature for 10-15 min in the dark. A control without staining was also prepared. Microarray and data analysis A549 cells including both treated and non-treated were collected after 3 day culture. After washing with PBS, cells were lysed with Lysing Matrix D (Qbiogene) and Trizol LS by rapid agitation in a FastPrep 120 Instrument (Qbiogene) for 15 seconds at 12,000 rpm. Subsequent to lysis, RNA extraction was performed according to the InVitrogen Trizol LS protocol, with the following minor modifications. In brief, two microliters of 5 mg/ml glycogen was used as a carrier for the isopropanol precipitation, and the duration of isopropanol precipitation was increased to overnight, and all centrifugation times were increased to 15 min. RNA pellets were resuspended in 55 Al Nuclease-free water. Quantitation was performed using a Beckman spectrophotometer, and quality assessment was determined by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. A Qiagen RNeasy total RNA cleanup protocol was performed and the purified RNAs were subsequently re-quantitated by spectrophotometry. Microarray experiment was performed according to the manufacture protocol, which is available from Affymetrix (Santa Clara, CA). For each experimental sample, 7.5 ug of purified RNA was reverse transcribed by Superscript II reverse transcriptase (Life Technologies) using T7-(dT)24 primer containing a T7 RNA polymerase promoter to obtain the complementary DNA (cDNA) strand. The cDNA was then used in an in vitro transcription reaction to generate biotinylated complementary RNA (cRNA) using the BioArray High Yield RNA Transcript Labeling Kit (ENZO) according to the manufacturerTs recommended protocol. Ten microgram of cRNA of each sample was hybridized to a Human Genome Focus Array GeneChip (Affymetrix) for 16 h at 458C with constant rotation at 60 rpm according the Affymetrix protocol. This high-density oligonucleotide-based array containing 8793 human genes was selected from the National Center for Biotechnology Information (NCBI) Gene Bank database. After hybridization, the genechip was washed and stained on an automated Affymetrix fluidics station. The arrays were then transferred to a confocal scanner (Affymetrix) and scanned twice at an emission wavelength of 570 nm at 6 um resolution. Intensity of hybridization for each probe pair was computed by Microarray Suite 5.0 software.

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The image data from the probe array scan was created and stored as data files. Primary analysis consisted of a quality assessment of the hybridization of each sample. The eukaryotic hybridization controls that were added to the hybridization cocktail consisted of a mixture of four biotin-labeled antisense fragmented control cDNA, prepared in staggered concentration, that bind to control probe sets on the array. These allowed for assessment of hybridization quality and array performance. The synthetic control oligo B2, which was also added to the hybridization cocktail, provided alignment signals used by the software to position a grid over the array image. Furthermore, ratios of signal for probe sets at 5V and 3V regions of housekeeping genes were calculated and monitored as an indication of transcript quality for each sample. True expression data analysis commenced with Absolute and Comparison analyses performed with Microarray suite 5.0 software. For Absolute analysis, the hybridization intensity for each probe array was examined to calculate a set of metrics, which were then used to determine an absolute call for each transcript: Present (P), Absent (A) or Marginal (M). In comparison analysis, the hybridization intensity from two arrays was compared, and difference calls were calculated for each transcript: Increased (I), Decreased (D), Marginally Increased (MI), Marginally Decreased (MD), or No Change (NC). A fold change calculation was also computed to indicate the relative change of each transcript represented on the probe array. Further analyses with Data Mining Tool (Affymetrix) and GeneSpring (Silicon Genetics) were performed. Statistical analysis Data regarding caspase activity assays and densitometric analysis were expressed as the mean F SEM of pooled results obtained from at least three independent experiments. Data regarding caspase activity assays needed a root square variance-stabilizing transformation. Statistical analysis was performed by one-way ANOVA test followed by Fisher’s protected least significant difference posttest for multiple comparisons using the StatView Program (Abacus Concepts, Berkeley, CA). Significance level was considered as P b 0.05.

Results S. barbata inhibited cancer cell growth The effect of S. barbata extract on cancer cell growth was examined on A549 human lung cancer cell line. Under the experimental conditions used in 3 day treatment, S. barbata extract exhibited a marked growth inhibitory effect on A549 cells in a dose-dependent manner. The IC50 was approximately 0.21 F 0.04 mg/ml (Fig. 1). Cytotoxic effects of S. barbata on A549 cells We characterized the cytotoxic effects of S. barbata on A549 cells by conducting cell viability assay stained with trypan blue. Cultures of the A549 cells were treated with S. barbata extract at various concentrations for 72 h. As shown in Fig. 2, the results indicated that S. barbata extract had obvious cytotoxicity on A549 cells. After 72 h treatment with S. barbata extract at a concentration of 0.5 mg/ml, 42.59 F 4.59% of A549 cell showed low viability, as compared to only 3.32 F 1.35% in control cells.

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Fig. 1. Growth inhibitory effect of S. barbata extract on A549 lung cancer cells. Cells were treated with different concentrations of S. barbata extract for 3 days when the cell viability was determined by the MTT assay. The growth inhibition was calculated as percentage of inhibition compared with the control.

In order to better characterize the cytotoxic effects, we performed LDH leakage experiment by using the cytotoxicity detection kit (Roche Molecular Biochemicals). After treatment with S. barbata for 24 h, cellfree culture supernatants were collected and incubated with substrate mixture containing tetrazolium salts. The formazan dye formed was quantitated by measuring the absorbance at 590 nm. Results showed that cytotoxicity effect of 0.5 mg/ml S. barbata extract was about 37.43 F 2.34% of the positive control, 1% Triton X-100, which could induced all of A549 cell death through cytotoxic effects (Fig. 3).

Fig. 2. Cytotoxic effects of S. barbata extract on A549 cells. A549 cells were seeded on 24-well tissue culture plates and treated with S. barbata extract as indicated for 72 h. The cells were harvested by trypsinization, stained with trypan blue and the viable and dead cells were counted. Viable cells (%) = [(total cells-dead cells) / total cells]  100%.

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Fig. 3. Cytotoxic effects of S. barbata determined by LDH leakage experiment using the cytotoxicity detection kit.

S. barbata induced A549 cell apoptosis Portions of the A549 cells after S. barbata treatment showed apoptosis phenotype. To confirm the apoptosis, we assayed the changes of Caspase 3/7 activity, critical Caspases in Caspase dependent apoptosis, by using Apo-ONEk Homogeneous Caspase-3/7 Assay kit. As shown in Fig. 4, 72 h treatment with S. barbata extract at concentrations of 0.25 and 0.5 mg/ml increased A549 Caspase 3/7

Fig. 4. Effect of S. barbata extract on A549 cell Caspase 3/7 activity.

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activities by 2.22 F 0.19 and 5.35 F 0.17 fold, respectively. These results indicated that, apart from exerting its cytotoxic effects, S. barbata could also induce apoptosis in a population of A549 cells. Apoptosis of A549 cells inducing by S. barbata was confirmed by fluorescent microscopic analysis of apoptosis by Annexin-V staining by counting 5 different scopes and more than 500 hundred cells. Treatment with 0.5 mg/ml S. barbata extract for 48 h resulted in a rate of cell apoptosis of 57.67%. In contrast, apoptosis cells only account 2.36% of total cells in control group (Fig. 5). Taken together, these results suggest that. S. barbata extract could inhibit significant cell apoptosis. Gene expression by microarray cDNA microarray experiment was performed to characterize the mechanism of S. barbata induced killing. We used the Human Genome Focus Array (Affymetrix) chip to assess the expression profiles in the S. barbata treated cells. After 72 h incubation, 195 functionally related genes were found to change in treated cells. Of them, 55 genes were down-regulated and 140 genes were up-regulated. Table 1 shows the genes whose changes were more than 5 fold (Table 1). As shown in Table 1, two genes related to cell response to DNA damage, GADD45A and GIP, decreased dramatically. They were down-regulated by 5.16 and 19.58 fold. S. barbata treatment altered genes involved in cell cycle activity. Totally, 20 cell cycle genes were found to change after treatment, which indicated that cell cycle was widely involved in S. barbata treatment. Six of them, including GADD45A, STK6, MCM5, MKI67, CDC20 and STK12, were found to change more than 5-fold. Except GADD45A, all other genes were up-regulated. S. barbata treatment also affected some enzyme activity (STK12, DUSP5 and TOPK), cell signal transduction (GIP, BMP2), nucleic acid binding (ATF3, HNRPD and SMARCF1) and gene expression.

Fig. 5. Apoptosis inducing effect of S. barbata. S. barbata extract was to A549 cells and its effect on apoptosis was assessed by fluorescent-annexin staining and fluorescence microscopy (lower panel) along with differential interference contrast (DIC) microscopy (top panel). Apoptotic cells are stained green whereas the control live cells are not stained. Representative data from three independent experiments are presented.

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Table 1 List of genes with more than 5-fold changes in mRNA levels in S. barbata treated A549 lung cancer cells identified by cDNA microarray Probe set ID

Gene name

Bio process

Molecular function

203725_at

GADD45A

Structural constituent of ribosome;

5.16

208079_s_at

STK6

Response to DNA damage; regulation of CDK activity; protein biosynthesis; inferred from electronic annotation; DNA repair; apoptosis; cell cycle arrest; Mitosis; oncogenesis; cell cycle; protein amino acid phosphorylation;

6.43

216237_s_at

MCM5

Protein kinase activity; cAMPdependent protein kinase activity; protein tyrosine kinase activity; transferase activity; ATP binding; protein kinase CK2 activity; protein serine/threonine kinase activity; DNA dependent ATPase activity; DNA binding; ATP binding;

212020_s_at 202870_s_at

MKI67 CDC20

209464_at

STK12

209457_at

DUSP5

Protein amino acid dephosphorylation

202672_s_at

ATF3

Regulation of transcription; DNA-dependent;

200073_s_at

HNRPD

RNA processing; RNA catabolism;

Regulation of transcription; DNA-dependent; regulation of cell cycle; DNA replication; DNA replication initiation; Regulation of cell cycle; Regulation of cell cycle; ubiquitin-dependent protein catabolism; Cell cycle; protein amino acid phosphorylation;

Fold change

7.04

ATP binding ?

8.32 5.27

cAMP-dependent protein kinase activity; protein tyrosine kinase activity; transferase activity; ATP binding; protein kinase CK2 activity; protein serine/threonine kinase activity; pkinase; protein kinase activity; Protein tyrosine phosphatase activity; CTD phosphatase activity; hydrolase activity; protein tyrosine/serine/ threonine phosphatase activity; protein phosphatase type 2A activity; protein phosphatase type 2B activity; protein phosphatase type 2C activity; calciumdependent protein serine/threonine phosphatase activity; magnesiumdependent protein serine/threonine phosphatase activity; myosin phosphatase activity bZIP; DNA binding activity; transcription co-repressor activity; transcription factor activity; rrm; nucleic acid binding activity; RNA binding;

5.59

5.76

8.51

6.45

(continued on next page)

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Table 1 (continued) Probe set ID

Gene name

Bio process

Molecular function

207899_at

GIP

205289_at

BMP2

Signal transduction; energy pathways; Growth factor activity; cytokine activity;

218755_at

KIF20A

219148_at

TOPK

210649_s_at 207277_at 211506_s_at

SMARCF1 CD209

Response to DNA damage; regulation of CDK activity; Cell-cell signaling; cell growth and/or maintenance; skeletal development; Intracellular protein transport; vesicle-mediated transport; microtubule-based process; Protein amino acid phosphorylation; ? ? ?

Fold change 19.58 6.18

Protein transporter activity; vesicle ransport; ATP binding; motor activity;

7.05

Transferase activity; ATP binding; protein kinase activity; DNA binding activity; Lectin_c; sugar binding activity; ?

5.07 5.01 101.65 7.94

STK12 and TOPK, two genes related to protein phosphorylation, were up-regulated by 5.59 fold, while DUSP5, a gene related to protein dephosphoylation was down-regulated by 5.76 fold. In addition, we were surprised to find that CD209, a gene related to dendritic cell (DC) function, was dramatically down-regulated by 102-fold (Table 1), which indicated that CD209 may play an important role in S. barbata treatment. Additionally, we also found from that a gene, whose probe set ID is 211506_s_at in Affymetrix Human Genome Focus Array but with unknown function, was down-regulated by about 8fold (Table 1). This gene may also be involved in cytotoxic effect or apoptosis inducing effect of S. barbata on A549 cells.

Discussion Chinese medicine, which is based on its own theories and philosophy, has been practiced by about one-fifth of the worldTs population for more than 2,000 years (Qian, 1987). In the past few years, a number of Chinese herbal medicines with potent anti-cancer activity were reported, such as Polygonatum Zanlanscianense Pamp (Wang et al., 2001), Hemsleya amabilis (Wu et al., 2002), and Phyllanthus urinaria (Huang et al., 2003). S. barbata is a promising anticancer herb whose mechanism of action was largely unclear. Inhibition of cancer growth has been a continuous effort in cancer treatment. A reduction in cell growth and an induction in cell death are two major means to inhibit tumor growth (Huang et al., 2003). In this study, we demonstrated that S. barbata could cause significant growth inhibition in A549 human lung cancer cell line at low concentrations, with IC50 at about 0.21 mg/ml. Cell death, due to necrosis (caused by cytotoxic effect) and apoptosis, was involved in S. barbata treatment. The cytotoxic effects of S. barbata were demonstrated by LDH production assay and trypan blue exclusion assay. S. barbata at a concentration of 0.5 mg/ml was shown to have about 35% of cytoxicity of Triton X-100 (1%), which can cause almost all cell death through cytotoxic effect. Trypan blue exclusion experiment confirmed this cytotoxic effect indirectly, as shown that about 43% of A549 cell died after treatment with a concentration of 0.5 mg/ml in comparison of 3.3% dead cells in control).

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In addition to inhibiting tumor cell growth, S. barbata also showed a good ability in inducing cell apoptosis. As indicated in results, apoptosis inducing effect of S. barbata was confirmed by increasing Caspase 3/7 activity and staining with Annexin V dyes. Caspase 3/7 are two critical effector Caspases in Caspase-depend apoptosis pathway (Nicholson, 1999; Yacobi et al., 2004; Matikainen et al., 2001; Cohen, 1997). Annexin V is a kind of dyes preferentially binds phosphatidylserine (PS), which is exposed during early apoptosis because of cell surface phospholipid asymmetry disruption (Bedner et al., 1999; van Engeland et al., 1998). After treatment with S. barbata for 72 h, A549 caspase 3/7 activities increased more than 5 fold. About 57.7% of cells were stained by Annexin V after treatment with S. barbata for 48 h. Therefore, Caspase-dependent apoptosis also played an important role in S. barbata induced A549 growth inhibition. cDNA microarray is a powerful tool to identify changes in expression of large numbers of genes simultaneously and quickly. Many drug targets are components of complex signaling pathways, and activation of signaling pathways leads to changes in multiple mRNA expression, thus microarray technology can be used to provide a detailed quantitative assessment of the consequences of this activation and makes it possible to identify the mechanism of drug action efficiently (Lee et al., 2002; Huang et al., 2000; Zanders, 2000). In this experiment, we found that S. barbata could dramatically change gene expression in A549 cell. Altogether, 195 functionally related genes were found to change in treated cells, with 55 genes down-regulated and 140 genes up-regulated. Sixteen genes, whose changes were more than 5-fold, were used to analyze gene expression. The gene expression pattern in S. barbata treated A549 cells indicates that the herb exhibits its inhibitory and killing activity through multiple mechanisms. First, S. barbata treatment was related to A549 cell DNA damage since the expression of GADD45A and GIP, involved in response to DNA damage, decreased dramatically. Second, S. barbata treatment changed the expression profile of 20 cell cycle genes. As cell cycle regulation is complicated and involves many genes, further experiment is needed to clarify the effect of S. barbata on the cell cycle. Third, S. barbata treatment also affected some enzyme activity (STK12, DUSP5 and TOPK), cell signal transduction (GIP, BMP2), nucleic acid binding (ATF3, HNRPD and SMARCF1) and gene expression. Expression of genes STK12, TOPK, and DUSP5 involved in protein phosphorylation was also altered. These changes suggested that cell signaling through protein phosphorylation could be an important process in S. barbata mediated killing of A549 cells. Finally, the observation that the CD209 gene was down-regulated by 102-fold in human lung cancer cells A549 is unexpected. CD209, also called DC-SIGN (dendritic cell-specific ICAM-grabbing non-integrin), has recently been found to be a mannose-specific C-type lectin expressed by immature dendritic cells (DC). DC-SIGN binds to carbohydrates of diverse pathogens and also participates in DC migration and T cell activation (Relloso et al., 2002). The significance of the S. barbata mediated down-regulation of DC-SIGN is unknown, but further studies are needed to clarify its role in S. barbata mediated anticancer activity. As indicated before, S. barbata extract has been used in the clinic to treat several types of cancer (Qian, 1987). Apart from human lung cancer, we also tested the effect of S. barbata on other cell lines and found that it also significantly inhibited human liver cancer cell line HepG2 and human gastric cancer cell line AGS in vitro, with IC50 at 0.2 mg/ml and 0.38 mg/ml. It is likely that active components in S. barbata have good potential to be developed into useful treatment for human cancers. Further work is needed to identify the active components that induce apoptosis and necrosis.

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